- Email: [email protected]
High fat diet consumption results in mitochondrial dysfunction, oxidative stress, and oligodendrocyte loss in the central nervous system
Journal Pre-proof High fat diet consumption results in mitochondrial dysfunction, oxidative stress, and Oligodendrocyte loss in the central nervous system
Monica R. Langley, Hyesook Yoon, Ha-Neui Kim, Chan-Il Choi, Whitney Simon, Laurel Kleppe, Ian R. Lanza, Nathan K. LeBrasseur, Aleksey Matveyenko, Isobel A. Scarisbrick PII:
S0925-4439(19)30358-8
DOI:
https://doi.org/10.1016/j.bbadis.2019.165630
Reference:
BBADIS 165630
To appear in:
BBA - Molecular Basis of Disease
Received date:
20 September 2019
Revised date:
14 November 2019
Accepted date:
2 December 2019
Please cite this article as: M.R. Langley, H. Yoon, H.-N. Kim, et al., High fat diet consumption results in mitochondrial dysfunction, oxidative stress, and Oligodendrocyte loss in the central nervous system, BBA - Molecular Basis of Disease(2019), https://doi.org/10.1016/j.bbadis.2019.165630
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof
1
High Fat Diet Consumption Results in Mitochondrial Dysfunction, Oxidative Stress, and Oligodendrocyte Loss in the Central Nervous System Short running title: High fat diet causes oligodendrocyte loss Authors Monica R. Langleya, Hyesook Yoona,b, Ha-Neui Kima, Chan-Il Choia, Whitney Simona, Laurel Kleppea, Ian R. Lanzab,c, Nathan K. LeBrasseura,b, Aleksey Matveyenkob,c, Isobel
ro
of
A. Scarisbricka,b,*.
Affiliations
Department of Physical Medicine & Rehabilitation, Rehabilitation Medicine Research
-p
a
Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN
lP
b
re
Center, Mayo Clinic, Rochester, MN 55905, USA
Department of Endocrinology, Mayo Clinic, Rochester, MN 55905, USA
Jo
ur
c
na
55905, USA
* Correspondence to: Isobel A. Scarisbrick, Ph.D., Neurobiology of Disease Program, Physical Medicine a& Rehabilitation, Rehabilitation Medicine Research Center, 642B Guggenheim Building, Mayo Clinic, Rochester, 200 First St SW, Rochester, MN 55905, Tel: 507-284-0124, Fax:507-266-4716, Email: [email protected]
Journal Pre-proof
2
Abstract Metabolic syndrome is a key risk factor and co-morbidity in multiple sclerosis (MS) and other neurological conditions, such that a better understanding of how a high fat diet contributes to oligodendrocyte loss and the capacity for myelin regeneration has the potential to highlight new treatment targets. Results demonstrate that modeling metabolic dysfunction in mice with chronic high fat diet (HFD) consumption promotes
of
loss of oligodendrocyte progenitors across the brain and spinal cord. A number of
ro
transcriptomic and metabolomic changes in ER stress, mitochondrial dysfunction, and
-p
oxidative stress pathways in HFD-fed mouse spinal cords were also identified.
re
Moreover, deficits in TCA cycle intermediates and mitochondrial respiration were observed in the chronic HFD spinal cord tissue. Oligodendrocytes are known to be
lP
particularly vulnerable to oxidative damage, and we observed increased markers of
na
oxidative stress in both the brain and spinal cord of HFD-fed mice. We additionally identified that increased apoptotic cell death signaling is underway in oligodendrocytes
ur
from mice chronically fed a HFD. When cultured under high saturated fat conditions,
Jo
oligodendrocytes decreased both mitochondrial function and differentiation. Overall, our findings show that HFD-related changes in metabolic regulators, decreased mitochondrial function, and oxidative stress contribute to a loss of myelinating cells. These studies identify HFD consumption as a key modifiable lifestyle factor for improved myelin integrity in the adult central nervous system and in addition new tractable metabolic targets for myelin protection and repair strategies.
Keywords high fat diet, myelin, oligodendrocyte, mitochondria, apoptosis
Journal Pre-proof Abbreviations 4-HNE= 4-hydroxynonenal aNSCs= adult neural stem cells CLAMS= Comprehensive Lab Animal Monitoring System EDSS= expanded disability status score EAE= experimental autoimmune encephalitis
of
HFD= high fat diet
ro
MetSyn = metabolic syndrome MS= multiple sclerosis
-p
MBP= myelin basic protein (MBP
re
OLC= oligodendrocyte lineage cell
lP
OPCs= Oligodendrocyte progenitor cells
Olig2= oligodendrocyte lineage transcription factor 2
na
PLP= proteolipid protein RER= respiratory exchange rate
ur
RRMS= relapsing remitting MS
Jo
SPMS= secondary progressive MS TCA= tricarboxylic acid
VWMD= vanishing white matter disease
3
Journal Pre-proof
4
1. INTRODUCTION White matter pathology including myelin sheath abnormalities, demyelination, loss of oligodendrocytes, and impaired myelin repair processes are significant pathophysiological characteristics of diverse neurological conditions, including multiple sclerosis (MS), Alzheimer’s disease, neuropsychiatric disorders, and traumatic brain and spinal cord injury [1-6]. Oligodendrocytes not only serve as insulation by
of
ensheathing axons and allowing for increased speed and temporal control of action
ro
potential propagation, but also provide key metabolic and trophic support for neurons [1,
-p
7, 8]. Development of new strategies to prevent oligodendrocyte loss and guide efficient
re
oligodendrogenesis and myelination has the potential to therapeutically benefit individuals affected by white matter-related neurological injury and disease. Since
lP
oligodendrocyte function is intimately linked to neighboring glial cells and neuronal
na
activity, in addition to microenvironmental factors, the most promising interventions will
ur
likely involve a combinatorial approach [1, 9].
Jo
Obesity during childhood or adolescence is associated with an increased risk of developing MS [10-12]. Recent studies support a causal role for elevated body mass index (BMI) in MS etiology which is further increased when interacting with other known genetic and environmental risk factors such as HLA polymorphisms or Epstein Barr virus positivity [10, 13, 14]. Interestingly, metabolic dysfunction resulting from excessive dietary fat intake may also influence the initiation, progression, or disability burden of MS [13, 15, 16]. In terms of conversion from relapsing remitting (RRMS) to secondary progressive (SPMS), elevated BMI is associated with an increased risk of SPMS in smokers, but not non-smokers [15]. There is also a significant positive association
Journal Pre-proof
5
between higher BMI or adverse lipid profile and higher Expanded Disability Status Score (EDSS) [16]. Since obesity and metabolic syndrome are common co-morbidities which impact quality of life for individuals with MS, recent studies have evaluated the utility of drugs and interventions to overcome metabolic syndrome as a strategy to improve patients’ physical and mental health or radiological outcomes [16-18].
of
Disruptions in brain lipid metabolism, serum lipid profiles, and cholesterol levels
ro
have all been reported during active disease in MS [16, 19]. How to manage dietary fat consumption for optimal CNS health is still controversial since accurate myelin
-p
assembly requires large amounts of lipids and fatty acid synthesis, yet overconsumption
re
of saturated fats is detrimental for overall brain function [19, 20]. HFD consumption
lP
exacerbates both autoreactive immune responses and neuroinflammation in experimental autoimmune encephalitis (EAE) [21, 22]. Metabolic derangement resulting
na
from obesogenic diets or excessive saturated fatty acids can lead to oxidative and ER
ur
stress responses and ultimate death of vulnerable cell types [23-25]. Elevated levels of oxidative stress and ER stress-related proteins have been observed in MS lesions [26,
Jo
27]. Oligodendrocytes are particularly sensitive and susceptible to oxidative stress having extremely high energetic and ER demands in order to support myelin membranes [28]. Moreover, our recent findings show that chronic consumption of a Western style diet, containing high fat and high sucrose, causes significant loss of oligodendrocytes and their progenitors in the intact adult murine spinal cord [29]. Therefore, to elucidate mechanisms underlying oligodendrocyte loss observed in the context of a Western diet, here we specifically address whether similar effects occur with consumption of a diet high in saturated fat alone. The brain and spinal cord of mice
Journal Pre-proof
6
consuming a regular or a high fat diet for 4 to 12 wk were analyzed using transcriptomic, metabolomic, functional, and immunohistochemical outcome measures to reveal potential mechanisms of oligodendrocyte loss. These approaches identified perturbations in mitochondrial function among the key mechanisms by which HFDconsumption is likely to impair myelinating cell function in vivo. Parallel disruptions in mitochondrial function and differentiation were also identified when HFD was modeled
of
in cultures of purified oligodendrocytes grown under high saturated fat conditions.
ro
Altogether these findings highlight new pathogenic mechanisms by which excess
-p
dietary fat contributes to myelin injury in the adult brain and spinal cord and uncover
lP
2. MATERIALS AND METHODS
re
new targets for therapy.
2.1 Experimental Method Details
na
2.1.1 Animal husbandry and diet
ur
All diets, procedures, and experiments were approved by The Mayo Clinic
Jo
Institutional Animal Care and Use Committee (IACUC) and performed in accordance with appropriate institutional and regulatory guidelines. Male C57BL6/J mice were obtained from Jackson Labs (Bar Harbor, ME) at 8 wk of age and were randomly assigned to feeding on either a regular diet (RD, 10 % kcal fat, D12450K, Research Diets, New Brunswick, NJ) or a high fat diet (HFD, 60% kcal fat, D12492, Research Diets, New Brunswick, NJ) beginning at 10 wk of age. Saturated fat comprised 22.7 and 32% of the total fat content within the diets, respectively. Carbohydrate comprised 70% kcal for RD and 20% HFD, while both diets had 20% kcal protein. Mice were
Journal Pre-proof
7
provided ad libitum access to the diets along with tap water and marked by tail tattoo for individual identification. Food intake and body weights were monitored weekly. 2.1.2 Glucose tolerance test Following a 6 h fast, baseline glucose levels were measured by needle puncture of the tail of mice consuming RD or HFD for 3 wk (Bayer Breeze2, Bayer, Mishawaka, IN). Next, an intraperitoneal injection of glucose (1g/kg) was administered and blood
of
glucose levels measured again at each time point (0, 15, 30, 60, 90, 120 min) as
ro
previously described [30].
-p
2.1.3 Behavioral Assessment
re
Behavioral analysis was performed at baseline and following 4 or 12 wk of RD or HFD consumption. To examine spontaneous movement, mice were placed in a
lP
chamber (27 x 27 x 20.3 cm, Med Associates Inc., St. Albans, VT) equipped with
na
infrared beams to record horizontal and vertical movements over a 30 min interval during the light phase. Mice were allowed to acclimate to the room conditions for 1 h
ur
prior to recording at 50 ms resolution in Activity Monitor Software. Equipment was
Jo
cleaned with 70% ethanol between mice. The same testing chamber and period was also used to evaluate anxiety-like behavior by quantifying the amount of time a mouse spent in the center zone (4.5, 4.5 to 12, 12 cm) versus the residual area. 2.1.4 Immunohistochemistry 4% Paraformaldehyde-fixed, paraffin-embedded brain and lumbosacral spinal cord tissues were cut to 6 µm and utilized for all immunostaining procedures. Immunoperoxidase and immunofluorescence was performed as previously described [29]. Briefly, sections were washed, steamed in antigen retrieval solution (sodium
Journal Pre-proof
8
citrate,10 mM, pH 6.0), blocked (20 % normal goat serum with 0.25 % Triton-x), then incubated in primary antibodies for Olig2 (Millipore, MABN50, 1:200), CC-1 (Millipore, OP80, 1:50), Nkx2.2 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa city, IA), NG2 (Millipore, ab5320), 4-HNE (Abcam, ab46545, 1:500), Bax (Invitrogen, MA514003,1:200 ), Bcl-2 (Abcam, AB59348, 1:500), cleaved caspase-3 (Cell Signaling, 9664, 1:1000), GFAP (Abcam, ab4647, 1:2000) for 18 h. For
of
immunoperoxidase staining, subsequent incubation with an appropriate biotinylated
ro
secondary antibody (Jackson, 1:200) followed by incubation with an avidin peroxidase
-p
solution (Vector) was used to yield a brown reaction product after incubation with 3,3-
re
diaminobenzidine (DAB, Sigma D5637). Immunostained sections were counter stained with methyl green (Sigma, 67060), dehydrated, cover slipped using DPX Mountant
lP
(Sigma), and imaged using an Olympus BX51 microscope (Olympus, Center Valley,
na
PA).
Immunofluorescence staining was achieved by incubating with appropriate
ur
fluorochrome-conjugated secondary antibodies (Jackson, 1:200) and counterstaining
Jo
with 4’,6’-diamindino-2-phenylindole (DAPI). Cell counting and thresholding of images from the dorsal column of the spinal cord and corpus callosum of the brain was performed in ImageJ (NIH) without knowledge of treatment groups using cropped regions of interest averaged from 2-3 sections per animal with at least n= 4 per group. 2.1.5 Western blotting One half of the spinal cord (cervical and thoracic) was homogenized in radioimmunoprecipitation buffer and 35 µg protein per sample was resolved by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA) and electroblotted as described previously [29].
Journal Pre-proof
9
Membranes were probed for AMPK (Cell Signaling, 2532S), pAMPK (Cell Signaling, 2531S), SIRT1 (Abcam, Ab121193), PGC1α (Abcam, Ab54481), and β-actin (Novus Biological, NB600-501). Detection was accomplished using appropriate HRPconjugated secondary antibodies (GE Healthcare), Pierce chemiluminescent developer (ThermoFisher), and ImageLab 2.0 Software (Bio-Rad). Relative optical density (ROD) for each protein was normalized to the actin of the same membrane.
of
2.1.6 RNA isolation, qPCR, and sequencing
ro
Total RNA was isolated from half of the spinal cords or primary murine cells
-p
using RNA Stat60 (Tel-Test, Friendswood, TX) per manufacturer’s instructions and
re
used for qPCR using BioRad iTaq Universal Probes (ThermoFisher) or primers (Integrated DNA Technologies) and SYBR Green One-step kits. RNA expression of
lP
each gene was normalized to Rn18s.
na
RNA libraries were prepared using 200 ng of total RNA according to the manufacturer’s instructions for the TruSeq RNA Sample Prep Kit v2 (Illumina, San
ur
Diego, CA). The concentration and size distribution of the completed libraries was
Jo
determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). Libraries were sequenced at 100 million reads per sample following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 X 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.20 collection software. Base-calling was performed using Illumina’s RTA version 2.5.2. One regular diet-fed mouse was excluded because of poor sequencing.
Journal Pre-proof
10
mRNA-seq data was processed by the Mayo Bioinformatics Core Facility to identify genes with differential expression among groups. MAP-RSeq [31] workflow (v1.2.1.3) was used to process mRNA-seq data, including read alignment, quality control, gene expression quantification and finally summarizing the data across samples. Paired-end reads were aligned by TopHat (v2.0.12) against the mouse genome build (mm10) using the bowtie1aligner [32], and the gene counts were
of
generated using Subread package [33] (v1.4.4). The workflow also provides detailed
ro
quality control metrics across genes using the RSeQC [34] (v2.3.2).
-p
Differential expression analysis was performed with edgeR [35] package (v2.6.2)
re
to identify genes with altered expression. A cutoff for false discovery rate-adjusted pvalue was set at < 0.05 to determine the genes with significant expression changes
lP
between conditions. The differential genes were further submitted to Ingenuity Pathway
na
Analysis (IPA®) for pathway enrichment analysis and causal network analysis. For causal network analysis (also described as “master regulator analysis”), only directed
ur
and experimentally observed interactions with a false discovery rate of less than 0.1
Jo
were used. The overall heatmap represents RPKM values representing the entire gene profile differences between groups irrespective of FDR organized by hierarchical clustering with the row distance of one minus Pearson correlation, while the scatterplot depicts only significantly differentially expressed genes based on a FDR of less than 0.15. Heatmaps derived from select PantherGo Pathways as well as the scatterplots from 500 gene sets for each cell type [36] were represented regardless of FDR. 2.1.7 Qualitative large-scale metabolite profiling and data analysis
Journal Pre-proof
11
Untargeted metabolomics analysis was performed by the Metabolomics Resource Core Facility at Mayo Clinic in spinal cord tissues of RD or HFD-fed animals by adapting previously described methods [37]. At study endpoints, spinal cords were quickly removed by flushing PBS through the vertebral column, split in half vertically and immediately snap frozen on dry ice for subsequent metabolic and transcriptomic analyses. Briefly, spinal cord samples that were stored at -80 C were pulverized in liquid
of
nitrogen using a custom mortar and pestle into a fine powder. Accurate weight was
ro
obtained using an analytical balance making sure the pulverized sample remained
-p
frozen. Pulverized samples were kept at -80 0C. Before metabolite extraction, samples
re
were thawed on ice for 15 min and 1X PBS was added to make a 100 mg/ml homogenate. An aliquot of 50 µl (~ 5 mg tissue) was used for metabolite extraction. 13
lP
C6-phenylalanine (2 µl at 250 ng/µl) was added as internal standard (IS) to all samples
na
and tubes were sonicated in the ice bath for 2 min (30 sec x 4 pulses). Samples were then deproteinated with cold acetonitrile: methanol (1:6 ratio), kept on ice with
ur
intermittent vortexing and centrifuged at 18000xg for 30 min at 4 C. The supernatant
Jo
was divided into 2 aliquots and dried down using a stream of nitrogen gas for analysis on a Quadruple Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data was acquired under both positive and negative electrospray ionization conditions over a mass range m/z of 100 - 1700 at a resolution of 10,000 (separate runs) in scan mode. Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1
Journal Pre-proof
12
x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters) with gradient described previously [37]. Run time was 18 min for the HILIC and 27 min for the C18 column using a flow rate of 400 µl/min. A total of four runs per sample were performed to give maximum coverage of metabolites. Samples were injected in duplicate, wherever necessary, and a pooled quality control (QC) sample, made up of all of the samples from each study was injected several times
of
during a run. A separate plasma quality control (QC) sample was analyzed with pooled
ro
QC to account for analytical and instrumental variability. Dried samples were stored at -
-p
20 C until analysis. Samples were reconstituted in running buffer and analyzed within
re
48 h of reconstitution. Auto-MS/MS data was also acquired with pooled QC sample to aid in unknown metabolite identification using fragmentation pattern. The tissue pellet
lP
from pulverized samples remaining after the extraction was stored at -80 C.
na
Total protein content from the tissue pellet was determined using Pierce BCA protein assay kit using a Spectramax Plus microplate reader. Briefly, the tissue pellet,
ur
after metabolite extraction, was solubilized in 0.3 M sodium hydroxide and 10 µl lysate
Jo
was used to determine total protein concentration. Protein determination was performed in triplicate and bovine serum albumin (BSA) was used to generate the standard curve. Data alignment, filtering, univariate, multivariate statistical and differential analysis was performed using Mass Profiler Professional (Agilent Inc, USA). Metabolites detected in at least ≥ 80% of one of two groups were selected for differential expression analyses. Metabolite peak intensities and differential regulation of metabolites between groups were determined as described previously [37]. Each
Journal Pre-proof
13
sample was normalized to the protein content and log2 transformed. Unpaired t-test with multiple testing correction p < 0.05 was used to find the differentially expressed metabolites between two groups. Missing values are excluded from the analysis for the calculation of fold change and p-values. Default settings were used with the exception of signal-to-noise ratio threshold (3), mass limit (0.0025 units), and time limit (9 s). Putative identification of each metabolite was done based on accurate mass (m/z)
of
against METLIN database using a detection window of ≤ 7 ppm. The putatively
ro
identified metabolites were annotated as Chemical Abstracts Service (CAS), Kyoto
-p
Encyclopedia of Genes and Genomes (KEGG), Human Metabolome Project (HMP) database, and LIPID MAPS identifiers. The differentially expressed metabolites are
re
analyzed for pathway enrichment using MetaCore (Genego, St. Joseph, MI) – this
lP
identifies pathways using 3 metabolite identifiers – that is, KEGG, CAS and HMDB.
na
2.1.8 Targeted TCA cycle metabolomics
Spinal cord levels of tricarboxylic acid (TCA) cycle metabolites were measured
ur
using the same samples prepared for untargeted metabolomics after the derivatization
Jo
with N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide + 1% tbutyldimethylchlorosilane by gas chromatography/mass spectrometry under electron impact and selected ion monitoring conditions as previously described [37]. All measurements were performed against 12-point calibration curves that underwent the same derivatization with internal standard. 2.1.9 Mitochondrial functional and structural analyses Oroboros oxygraph O2k was used for high resolution respirometry readings from isolated spinal cord mitochondria and Seahorse extracellular flux assay (Agilent) was
Journal Pre-proof
14
used to assess mitochondrial function in cell cultures as described in more detail in our previous publications [38]. Oxygen consumption rates (OCR) were normalized to BCA protein estimation. In brief, fresh whole spinal cord tissue was placed in mitochondria isolation buffer (225 mM Mannitol, 75 mM sucrose, 20 mM MOPS, 1 mM EGTA, 0.5 mM DTT) containing 0.05% Nargase (w/v)/1g tissue and homogenized for 10 min. Next, differential centrifugation was used to isolate mitochondria from other fractions, the
of
mitochondria pellet was resuspended in mitochondria isolation buffer with 0.02%
ro
Digitonin for 10 min, then centrifuged and resuspended again in fresh buffer (125 µl/100
-p
mg tissue) for running the assay. 50 µl of isolated mitochondria suspension was run for
re
each of two technical duplicates. The oxygraph chamber was pre-washed and filled with respiration buffer (120 mM mannitol, 40 mM MOPS, 5mM KH 2PO4, 0.1mM EGTA,
lP
60mM KCl, 5 mM MgCl2) to equilibrate and oxygenate for at least 30 minutes. Oxygen
na
calibrations for ambient air and zero were acquired for each chamber during each run. Oxygen consumption rates were measured in DatLab (Oroboros) after adding the
ur
mitochondria suspension (state I), glutamate (10 mM)/Malate (2 mM)/succinate (10 mM)
Jo
mixture (state II), ADP (14.25 mM), oligomycin (2 µg/ml), FCCP (0.005 mM) titrations, and antimycin A (2.5 mM) using Hamilton syringes. State 3 mitochondrial respiration was assessed following the addition of glutamate, malate, and succinate and ADP to the chamber, giving the basal respiration rate for complexes I and II. Oligomycin injection then inhibited ATP synthase to induce state 4 respiration, attributable to proton leak across the inner mitochondrial membrane. FCCP addition results in an uncoupled state, allowing for determination of maximum capacity, while non-mitochondrial respiration was assessed after antimycin inhibits complex III. State III respiration measurements
Journal Pre-proof
15
were taken from a stable segment following the addition of ADP, while State IV measurements followed oligomycin injection. Maximum respiration was recorded during the highest segment of uncoupled respiration following the FCCP titrations, and nonmitochondrial respiration was measured after anitmycin A addition. All measurements were normalized to protein content of the mitochondria fraction. The Seahorse XF24 (Agilent) extracellular flux assay was used to monitor
of
oxygen consumption rates at various stages of respiration in primary murine
ro
oligodendrocytes as previously described [39]. Following cartridge equilibration with
-p
injection ports filled with oligomycin (1 μg/ml), FCCP (1 μM), and antimycin A (10 μM), a
re
plate pre-treated in serum-free media was placed into the Seahorse analyzer. OCR readouts at each stage were measured in pmol/min and normalized to protein.
lP
For structural analysis of mitochondria, 0.09 x 106 cells primary oligodendrocytes
na
were grown per well on poly-L-lysine (PLL, 10 μg/ml) coated glass coverslips in 24-well plates, treated with 0 or 100 μM palmitate (PA) for 24 h, and then washed twice with
0
ur
fresh media. CMXRos MitoTracker Red (ThermoFisher, 100 nM) was incubated at 37
Jo
C for 30 min. Immunocytochemistry procedures and DAPI counterstaining were then
performed. ImageJ was used to quantify mitochondrial parameters as described previously [39, 40]. Averaged values are reported from analyzed cells within five separate images per coverslip. 2.1.10 Primary glia isolation, OLC culture Mixed glial preparations from postnatal day 0-3 murine cerebral cortex were cultured in DMEM (Fisher 11960-044) with 1 mM sodium pyruvate (Corning 11360070), 20 mM HEPES (Gibco 15630-080), 100 U/ml PenStrep (Life Technologies 15140122),
Journal Pre-proof
16
and 10% fetal bovine serum (FBS, Atlanta Biologicals S11150) for 10-12 d. Oligodendrocytes, astrocytes, and microglia cultures were separated by differential adhesion/shaking overnight at 225 rpm on an Innova 2000 orbital shaker as previously described [41]. For oligodendrocyte treatments and differentiation, 0.09 x 10 6 cells were plated on each well of PLL-coated coverslips or 24 well plates and grown in Neurobasal A
of
media (Life Technologies, 10888022) with N2 (17502048), B27 (17504044), PenStrep,
ro
sodium pyruvate, 2 mM glutamax (35050-061), and 5% BSA (Sigma A4503) for two d
-p
before treating with palmitate (PA, 100 µM, 24 h). This dose was lower than toxic
re
amounts previously reported in other cells types at the same time point [42, 43] and did not result in significant lipotoxicity to oligodendrocytes or aNSCs. RNA levels for MBP,
lP
PLP, and Olig2 were determined in technical duplicates using and an iCycler iQ5
na
system (BioRad) with primers identified within the Key Resources Table. Relative starting quantities were normalized to Rn18S and reported as percent of control. PLP
ur
protein levels were determined based on threshold of immunoreactivity from five fields
Jo
per coverslip quantified in ImageJ. 2.1.11 Adult neural stem cell culture Adult neural stem cells were isolated from the subventricular zone (SVZ) of 8 wk old C57Bl6/J mice as previously described [44]. Briefly, mice were anesthetized with sodium pentobarbital and decapitated to collect coronal brain sections and further dissect stem cells from within the SVZ. Collected cells were dissociated with Accutase and grown in DMEM/F12 with B27, antibiotic-antimycotic, insulin (20 µg/mL), epidermal
Journal Pre-proof
17
growth factor (EGF, 20 ng/mL) and basic fibroblast growth factor (bFGF, 20 ng/mL, PeproTech, Rocky Hill, NJ) in T75 tissue culture treated flasks as neurospheres. For myelin RNA and protein expression experiments, cells were dissociated and plated onto poly-L-ornithine (20 µg/ml) and laminin (10 µg/ml) coated 6 well plates and glass coverslips, respectively. To induce differentiation toward oligodendrocyte lineage, cells were switched to media containing Neurobasal A, antibiotic-antimycotic, B27,
of
insulin (20 µg/mL), T3 (60 ng/ml), glutamax (2 mM) and cultured for an additional 72 h.
ro
To identify effects of PA (100µM) on differentiation to oligodendrocyte lineage, we
-p
compared protein and RNA marker expression to controls by immunofluorescence
re
staining and qRT-PCR. 2.1.12 Statistical analysis
lP
Data were tested for normality using the Shapiro-Wilk test, graphed, and analyzed by
na
Student’s t-test unless noted otherwise in GraphPad Prism 7.0 software. Mann-Whitney nonparametric test was used for histological analysis of 4-HNE immunoreactivity in
ur
brain and spinal cord tissue, while body weight, food intake, and glucose tolerance test
Jo
in mice were analyzed by two-way ANOVA with Bonferroni post-test. p-values ≤ 0.05 were considered significant.
Journal Pre-proof 2.2 KEY RESOURCES TABLE
Abcam
ab28486; AB_776593
AB12193; AB_298923 74.5A5; AB_531794
ab5320; AB_11213678
lP
na
Jo
rabbit anti-4hydroxynonenol rabbit anti-cleaved caspase-3 rabbit anti-Bcl-2 rabbit anti-Neurofilament biotinylated goat anti-rat secondary biotinylated donkey antirabbit secondary biotinylated donkey antimouse secondary goat-anti-rabbit AF488 secondary goat anti-rat Cy3
2531S; AB_330330
Millipore
MAB386; AB_94975
Abcam Novus Biological Abcam
Ab54481; AB_881987 NB600-501; AB_10077656
Cell Signaling Technology Abcam Sigma Aldrich Jackson
9664; AB_10694088
Jackson
711-065-152; AB_2340593
Jackson
715-066-151; AB_2340788
Jackson
111-545-047; AB_2338051
Jackson
112-166-072; AB_2338257
ur
anti-Neural glial antigen2 (NG2) rabbit anti-Proteolipid protein (PLP) rat anti-Myelin basic protein (MBP) rabbit anti-PGC1-α mouse anti-ᵝ-actin
MABN50; AB_10807410 MA514003; AB_10979735 Ab9610; AB_10141047 Ab16794; AB_443473 2532S; AB_330331
re
mouse anti-SIRT1 anti-Nkx2.2
Millipore Invitrogen Millipore Abcam Cell Signaling Technology Cell Signaling Technology Abcam Development al Studies Hybridoma Bank, University of Iowa, Iowa city, IA Millipore
of
rabbit anti-pAMPK
Identifier
ro
Antibodies mouse anti-Olig2 rabbit anti-BAX rabbit anti-Olig2 mouse anti-CC-1 rabbit anti-AMPK
Source
-p
Reagent or Resource
ab46545; AB_722490
AB59348; AB_2064155 N4142; AB_477272 112-066-072; AB_2338185
18
Journal Pre-proof
FIS1
PINK1
SOD2 GFAP
SOD2 Rn18s
Primary isolation (Yoon 2015) Primary isolation (Choi 2017)
N/A
The Jackson Laboratory
N/A
of
N/A
NM_016967; Mm.PT.58.42319010
lP
Applied Biosystems Integrated DNA Technologies Integrated DNA Technologies Applied Biosystems Integrated DNA Technologies Integrated DNA Technologies
ur Jo
PGC1-α
711-166-152; AB_2313568
na
MBP
PLP
Jackson
re
oligonucleotides, (primers/probes) OLIG2
111-545-041; AB_2341130
ro
Experimental models: cell lines Mouse:C57BL/6 oligodendrocyte lineage cells (OLCs) Mouse:C57BL/6 adult neural stem cells (aNSCs) Experimental models: organisms/strains Mouse: C57BL/6J
Jackson
-p
secondary goat anti-rabbit AF488 secondary donkey anti-rabbit Cy3 secondary
19
Integrated DNA Technologies Applied Biosystems Applied Biosystems
NM_001025251; CCAGTAGTCCATTTCTTCAAGAACAT/GC CGATTTATAGTCGGAAGCTC NM_011123.2; TCTTTGGCGACTACAAGACCAC/CACAA ACTTGTCGGGATGTCCTA Mm01208835_m1 Mm.PT.56a.21878911
Mm.PT.58.23711353
Mm01313000_m1 NM_010277.2; GCAGATGAAGCCACCCTGG/ GAGGTCTGGCTTGGCCAC NM_013671.3; Mm01313000_m1 NR_003278.3; Mm03928990_g1
Journal Pre-proof
Chemicals, Peptides, and recombinant proteins palmitic acid dapi Mitotracker Red
20
Sigma Aldrich Cat#P0500 ThermoFisher Cat#D1306; AB_2629482 ThermoFisher Cat#m7512
Commercial kits Seahorse XF Cell MitoStress Test Pierce BCA protein estimation
Agilent
Cat#103015-100
Other Regular diet (10 kcal % fat) High fat diet (60 kcal % fat)
Research Diets Research Diets
https://www.graphpad.com/scientificsoftwar e/prism https://imagej.net/Fiji
D12450K D12492
Jo
ur
na
lP
GraphPad Prism 7
https://www.med-associates.com/
-p
Fuji ImageJ
Med Associates Inc GraphPad Software NIH
re
Software and Algorithms Activity Monitor Software
ro
of
ThermoFisher Cat#23225
2.3 CONTACT FOR REAGENT AND RESOURCE SHARING Additional information and requests for resources can be directed to the corresponding author/Lead Contact, Isobel Scarisbrick ([email protected]).
Journal Pre-proof
21
3. RESULTS 3.1 Chronic high fat diet consumption impairs metabolic parameters and diminishes the number of oligodendrocytes and oligodendrocyte progenitors in the central nervous system Since our prior studies revealed that a diet high in fat and sucrose results in a loss of myelinating cells and their progenitors in the spinal cord [29], we chose to
of
investigate the differential impact of high fat alone and any temporal effects by feeding
ro
10 wk old male C57Bl/6 mice a regular diet (RD, 10% of kcal from fat) or high fat diet
-p
(HFD, 60% kcal from fat) for either 4 or 12 wk (Figure 1A). As expected, the HFD mice appeared noticeably larger (Figure 1B) compared to the RD-fed controls and consumed
re
more kcal of food per day (Figure 1C). Beginning at 3 wk through 12 wk, mice also
lP
gained a significant amount of weight (Figure 1D). After 3 wk, glucose sensitivity was
na
assessed using a glucose tolerance test. As expected, HFD-fed mice demonstrated significantly higher fasting blood glucose levels and impaired glucose tolerance (Figure
ur
1E, F) [30]. A set of RD and HFD mice were monitored in a Comprehensive Lab Animal
Jo
Monitoring System (CLAMS) to reveal significant changes in several metabolic parameters including percent fat/lean mass, VO2, VCO2, respiratory exchange rate (RER), and metabolic rate (Supplemental Figure 1). In a separate cohort, a number of movement measurements were also affected by HFD consumption including anxietylike behavior and decreased vertical motion at 4 wk, and also involving horizontal activity by 12 wk (Supplemental Figure 2). After establishing the systemic and behavioral effects of HFD consumption in mice, we next tested the direct consequences on oligodendrocyte lineage cells (OLCs).
Journal Pre-proof
22
At study endpoints, spinal cord tissues were separated into cervical-thoracic and lumbosacral segments for analysis as depicted in (Figure 1G). The impact of consuming a HFD on myelin producing cells was determined by counting OLC markers in the postfixed lumbosacral spinal cord of each group. Oligodendrocyte progenitor cells (OPCs) were identified using antibodies recognizing NG2, Nkx2.2, and Olig2 (oligodendrocyte lineage transcription factor 2, marks progenitors and early mature cells), while mature
of
oligodendrocytes were considered CC-1+. After 4 wk of HFD, no change in the number
ro
of Olig2+ cells was observed in the spinal cord dorsal column (Figure 1H). RNA
-p
expression of oligodendrocyte markers Olig2 (Figure 1I), myelin basic protein (MBP,
re
Figure 1J), and proteolipid protein (PLP, Figure 1K) remained unchanged after 12 wk of HFD-consumption. By contrast counts of oligodendrocyte progenitors immunoreactive
lP
for Nkx2.2 (Figure 1L, 40x, and Supplemental Figure 3A , 20x), NG2 (Figure 1 M, 40x,
na
and Supplemental Figure 3B, 20x) and Olig2 (Figure 1N, 40x, and Supplemental Figure 3C, 20x) were all reduced at 12 wk. Furthermore, counts of CC-1+ mature
ur
oligodendrocytes (Figure 1O, 40x, and Supplemental Figure 3D, 20x) demonstrated a
Jo
significant depletion. We did not observe a significant change in Olig2 (Supplemental Figure 4A, B), in MBP isoforms (Supplemental Figure 1A, C and Supplemental Figure 5 E-H) or PLP (Supplemental Figure 5 A,D) protein expression by Western blot. Considering we observed a loss of oligodendrocytes and their progenitors in the spinal cord at 12 wk of HFD consumption, we next examined any regional specificity by parallel quantification in the brain. The corpus callosum (Figure 1P) from mice fed a HFD had fewer Olig2+ cells (Figure 1Q) at 12 wk, but staining for MBP (Supplemental Figure 3E), a marker of myelin structural integrity, and mature oligodendrocytes (CC-1+,
Journal Pre-proof
23
Supplemental Figure 3F) were unaffected. Taken together, these findings suggest that oligodendrocyte progenitors in both the brain and spinal cord are vulnerable to death in response to chronic HFD consumption; however, only spinal cord mature oligodendrocytes appeared susceptible to HFD-induced toxicity at the timepoint examined. Interestingly, remaining oligodendrocytes within both central nervous system
Jo
ur
na
lP
re
-p
ro
of
(CNS) regions maintained normal PLP and MBP levels at all time points examined.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
24
Journal Pre-proof
25
Fig 1. Chronic high fat diet consumption impairs metabolic parameters and diminishes the number of oligodendrocytes and progenitors in the central nervous system. A, Schematic depicting experimental design in which 10 wk old male mice were fed a regular diet (RD) or a high fat diet (HFD) for 4 or 12 wk. Representative images show mice consuming a HFD gained weight (B) after consuming more kcal food per day (C, n=3 cages of 5 mice per group). HFD mice gained weight (D, n=10 per
of
group. RD 25.84 g and HFD 24.97 g at study start; RD 27.53 g and HFD 43.02 g at
ro
study end) and had impaired glucose tolerance after 3 wk (E, F; AUC=area under the
-p
curve, n=8 mice per group). Graphical results represented as the mean ± SEM. G, Schematic shows spinal cord regions used in analyses. H, Representative images from
re
immunostained sections of spinal cord dorsal column and counts of the number of
lP
Olig2+ cells at 4 wk (n=5 mice per group). RNA expression of Olig2 (I), MBP (J), and
na
PLP (K) at 12 wk (n=4 mice per group). Representative images of dorsal column and corresponding counts of Nkx2.2+ (L), NG2+ (M), Olig2+ (N), and CC-1+ (O) cells after
ur
12 wk (n=7 mice per group). P, Schematic depicting region of corpus callosum
Jo
examined for immunostaining. Representative images from immunostained sections of the corpus callosum (Q) and counts of Olig2+ cells at 12 wk (n=5 mice per group). Bar graphs shown represent the mean number of cells per area of spinal cord ± SEM and are expressed as percent of RD-fed control mice. (*, p<0.05; **, p<0.01; and ***, p<0.001). Scale bar = 20 μm (L-O) and 50 μm (Q).
Journal Pre-proof
26
3.2 Chronic high fat diet consumption differentially regulates the transcriptional profile of the adult mouse spinal cord To begin to address the mechanisms driving oligodendrocyte depletion in the face of HFD-consumption, we performed RNA sequencing from half of each spinal cord as demonstrated in Figure 2A. An overall heatmap (Figure 2B and Supplemental Figure 4) depicts differential gene expression in the spinal cords of mice consuming a RD (n=3)
of
and HFD (n=4) for 12 wk, where blue is the relative row minimum and red is the relative
ro
row maximum. Using a false discovery rate (FDR) of P < 0.15, a scatter plot of
-p
differentially expressed genes (DEGs, Figure 2C) showed 75 to be transcriptionally
re
distinct between diet groups (32 up and 43 down). Among these, we observed an upregulation of Hba-a2, Sdf2L1, Hmgcs2, Creld2, Cryab, Hspa5/Grp78, Grn, Kdr,
lP
Ddit4l, Fos, Xbp1, Paqr6, Fn14, Gstp1, Ccdc42ep2, and Litaf. Significantly
na
downregulated genes included Lcn2, Ch25h, Sgk1, Slc17a7, Pdyn, Socs3, Plaur, Dbp, Acer2, Pla2g3, Kirrel2, Cp, Fn1, and Klf9.
ur
A number of ER stress induced genes including Creld2, Xbp1, and Hspa5 were
Jo
upregulated in HFD spinal cord. Not surprisingly, ingenuity canonical pathway analysis revealed ER Stress and the Unfolded Protein Response pathways as top pathways affected in spinal cord tissue by HFD consumption (Table 1). Next, to identify transcription factors which may act as master regulators for the observed transcriptional changes, we performed a causal network analysis with only directed and experimentally observed interactions with a FDR less than 0.1 (Table 2). PTK6 (Figure 2D), predicted to be activated, and NFKBIA (Figure 2E), predicted as an inhibited upstream transcriptional regulator, influence expression of Xbp1 and HSPA5, respectively.
Journal Pre-proof
27
Moreover, PDGFRA is a crucial mediator of oligodendrogenesis [45] and was predicted to be inactivated. Loss of Mecp2 in oligodendrocytes down regulates myelin gene expression and was predicted to have lower activation in the HFD mice [46]. We next focused attention on expression of myelin and cholesterol related genes using PantherGO Pathways (Figure 3A), where there was surprisingly a trend for increases in a number of genes, which may reflect a compensatory response in
of
remaining oligodendrocytes and explain why frank loss of myelin was not observed at
ro
the 12 wk endpoint despite the loss of mature myelinating cells and progenitors. Having
-p
characterized overall transcriptomic differences between the spinal cord of mice
re
consuming a RD or HFD, we next used the gene sets described by Zhang et al [36] to profile transcriptional changes enriched at distinct stages of oligodendrocyte lineage
lP
progression (Figure 3B-E), including oligodendrocyte precursors (OPCs, Figure 3C),
na
new OLCs (Figure 3D), and myelinating OLCs (Figure 3E)[36]. Of the OPC-enriched gene set, Slc6a3 (p=0.27), Lox (p=0.18) , Cdc25c (p=0.16), Snora17 (p=0.015), Bglap2
ur
(p=0.79), Gsx1 (p=0.07), Ccnb1 (p=0.18), Alas2 (p=0.10) , Raet1d, and Troap (p=0.19)
Jo
were all increased above 1.5-fold, while Ddx43 (p=0.002) and Cpz were below 0.5-fold in the spinal cord of mice consuming HFD relative to RD (Figure 3C, all genes included regardless of FDR). New OL gene changes included increased Krt28 (p=0.10), S100a14 (p=0.19), Snora26 (p=0.25), 1700001P01Rik (p=0.10), and Sis (p=0.08) and decreased Erp27 (p=0.18), Corin (p=0.09), and Chrng (Figure 3D). In myelinating OLs, Hist1h2bg (p=0.40), 1700001P01Rik (p=0.10), A930003A15Rik (p=0.09), Hist1h2ae (p=0.43), Hist1h4i (p=0.04), and Hist1h2ac (p=0.34) were increased, while Hist1h2bn (p=0.006), Erp27 (p=0.18), and Mpl (p=0.04) were decreased (Figure 3E).
Journal Pre-proof Together, these results suggest that HFD consumption promotes complex transcriptional changes in the spinal cord and in oligodendrocyte lineage cells that include pathways essential for maintaining homoeostasis and highlight several
Jo
ur
na
lP
re
-p
ro
of
important new targets for further investigation.
28
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
29
Journal Pre-proof
30
Fig 2. Transcriptional changes in the spinal cord of adult high fat-fed mice. A, One half of the spinal cord from regular diet (RD, n=3) and high fat diet (HFD, n=4) fed mice were processed whole genome RNA sequencing. B, Heat map of transcriptional changes observed in the spinal cord of male mice fed RD or HFD for 12 wk representing the entire gene profile differences between groups irrespective of FDR, organized by hierarchical clustering with the row distance of one minus Pearson
of
correlation where blue is relative row minimum and red is the row maximum. C, Scatter
ro
plot of differentially expressed genes (DEGs, red dots; 32 up, 43 down with a FDR <
-p
0.15) where the x-axis is false discovery rate (FDR) and y-axis is log fold change (log FC), with annotated gene names for select up (red) and down (blue) regulated genes.
Jo
ur
na
lP
upstream transcriptional regulator.
re
PTK6 (D) was predicted to be activated while NFKBIA (E) was predicted as an inhibited
2.67
2.55E-06
MYOCD
Transcription regulator
3
2.12
31
Target molecules in dataset
Network biascorrected p-value
Predicting activation Z-score
3
0.013
CDC42EP2, CRYAB, GNA12, GSTP1,HBA1/HBA2,H MGCS2,KDR, PDIA6,XBP1 ARL4D, COL8A2,CP, FBN1,FN1, GRN, LCN2,LFNG,
-p
ro
of
Molecule Type Kinase
Exp Log Ratio
PTK6
Master Regulator
Depth
P-value of overlap
Journal Pre-proof
0.012
2
5.69E-04
0.015
HSPA5,PDIA6,SDF2L1, FN1
3
2
1.76E-04
0.022
HSPA5,KDR,SGK1, FN1
2
1.89
3.92E-05
0.026
ARC, GSTP1,KDR,LCN2, COL8A2,FBN1,FN1
transcription regulator
2
1.73
1.06E-03
0.042
KDR,SGK1, FN1
0
transcription regulator
3
1.5
7.52E-05
0.033
ARC, CRYAB, HBA1/HBA2,HMGCS2, HSPA5,KDR, SERPINB1,TNFRSF12 A,XBP1, CH25H,COL8A2, DBP,FBN1,GRN, LAMA4,LRG1
0.04
transcription
1
1.41
6.47E-05
0.0032
HSPA5,XBP1
0.38
Transcription regulator
TAF12
0.04
transcription regulator
SRF
0.11
transcription regulator
NFYC
-0.03
CCNE1
CREB3
1
Jo
ur
na
XBP1
lP
re
8.00E-06
ARC,GSTP1,KDR, XBP1, COL8A2,FBN1,FN1, LCN2,
Journal Pre-proof
32
regulator -0.07
transcription regulator
3
1.41
6.39E-06
0.0094
ARC,CDC42EP2,GSTP 1,KDR,LCN2, COL8A2,FBN1,FN1
ATF6B
0.10
transcription regulator
1
1
6.30E-03
0.0318
XBP1
NFYC
-0.03
transcription regulator
1
1
1.67E-02
0.0449
SGK1
PIK3CA
-0.06
kinase
3
1
1.32E-05
0.049
ARC, CDC42EP2, CRYAB, GNA12,GSTP1,HMGCS 2,HSPA5, PDIA6, XBP1, ARL4D, CIART,CP, DBP, LCN2,LFNG, SGK1
DDIT3
0.13
transcription regulator
1
0.58
PRKAB1
0.00 5
kinase
PRKAG1
0.00 2
RPS6KA 4
-0.04
PRKACA
0.07
2
0.45
1.11E-04
0.0435
HMGCS2, DBP,FN1, LCN2,SGK1
kinase
2
0.45
1.11E-04
0.0435
HMGCS2, DBP,FN1, LCN2,SGK1
kinase
2
0.33
6.49E-06
0.0083
ARC,GSTP1,HSPA5, ,CIART,COL8A2,FBN1, FN1, LCN2,SGK1
chemical endogenous mammalian
3
0.33
3.15E-05
0.0162
ARC,GSTP1,HSPA5, PDIA6, XBP1, CIART,FN1, LCN2, SGK1
kinase
3
0.23
7.34E-06
0.032
ARC, CRYAB,GSTP1,HMGC S2,HSPA5,KDR, SERPINB1, ARL4D,CH25H,CIART, COL8A2,CP,
ur
na
lP
re
heme
0.008
Jo
-p
ro
of
MKL1
1.60E-04
HSPA5, XBP1, LCN2
Journal Pre-proof
33 DBP,FBN1,FN1,GRN, LCN2,LFNG,Podxl
CLOCK
0.02
ARNTL
0.28
PPRC1
-0.05
SMARCA 2
-0.02
kinase
3
0
3.62E-07
0.0008
1
0
3.33E-04
0.0049
HMGCS2, DBP
1
0
0.0184
HMGCS2, DBP
4.05E-03
0.0319
GNG11,LCN2
2.20E-03
0.0344
KDR, CP
0.0153
CDC42EP2, CRYAB, GNA12, HBA1/HBA2,HSPA5,K DR, PDIA6,SERPINB1, XBP1, CH25H,COL8A2,CP, FBN1,FN1, GRN, LCN2,LFNG, SGK1
0.0126
ARC, CDC42EP2, CRYAB,GNA12, GSTP1,HBA1/HBA2,H MGCS2,HSPA5,KDR, PDIA6, SDF2L1,SERPINB1, XBP1, ARL4D, CH25H,CIART,CP, DBP,FN1, GRN, LAMA4,LFNG, PLA2G3, SGK1
re 0
lP 1
na
-0.12
1
kinase
3
2.07E-03
0
-0.23
1.19E-06
Jo
ur
PDGFRA
transcription regulator transcription regulator transcription regulator transcription regulator
ro
of
-0.07
-p
MAP3K3
ARC,CDC42EP2, ,CRYAB,GNA12,HSPA 5,SDF2L1,SERPINB1,T NFRSF12A, ARL4D, ,CH25H,CIART,COL8A 2,CP, ,DBP,FBN1,FN1,GRN, LCN2,LFNG,LRG1,PLA 2G3, SGK1
HTT
-0.04
transcription regulator
3
-0.41
9.99E-07
Journal Pre-proof
MECP2
-0.11
transcription regulator
1
-0.58
1.25E-04
0.0021
HBA1/HBA2,SGK1,SL C17A7
CREB1
0.05
transcription regulator
1
-0.82
2.90E-04
0.0314
ARC,HSPA5, CIART, FN1, LCN2,SGK1
1
-1
8.39E-03
0.0162
SLC17A7
-0.19
transcription regulator transcription regulator transcription regulator
1
-1
1.46E-02
0.0341
DBP
1
-1
1.67E-02
0.0408
LFNG
of
34
ARC, HSPA5, CIART, FN1, GRN, LCN2, SGK1
FEZF2 PER2 MSGN1
0.05
kinase
2
-1.13
1.43E-04
NFKBIA
-0.37
transcription regulator
1
-1.41
liganddependent nuclear receptor
1
-1.73
-p
transcription regulator
re
0.07
CRYAB ,HSPA5, CH25H, CP, FN1,GRN,LCN2, SGK1
2.27E-03
0.0453
GSTP1,HMGCS2,LCN2
3
-2
1.76E-04
0.0222
HSPA5,KDR, FN1, SGK1
6.19E-07
na
TAF11
0.0025
lP
NR1I2
0.0373
ro
PASK
ur
Table 1. Summary of ingenuity canonical pathways significantly affected in spinal cord
Jo
tissue by HFD consumption. Bold indicates increased.
Journal Pre-proof
35
Ingenuity Canonical Pathways
-log(pvalue)
log(B -H pvalue )
Endoplasmic Reticulum Stress Pathway
3.45
1.56
0.0004
0.028
2
XBP1,HSPA5
Aldosterone Signaling in Epithelial Cells
2.86
1.29
0.0014
0.051
3
CRYAB,HSPA5,SGK1
Unfolded protein response
2.63
1.29
0.002
0.051
Glutamate Receptor Signaling
2.58
1.29
0.003
Huntington's Disease Signaling
2.4
1.21
0.004
Ceramide Degradation
2.1
Sphingosine and Sphingosine-1phosphate Metabolism
1.97
#D EG s
Molecules
2
GNG11,SLC17A7
0.062
3
GNG11,HSPA5,SGK1
0.008
0.104
1
ACER2
0.94
0.011
0.115
1
ACER2
1.89
0.94
0.013
0.115
2
GNG11,PLA2G3
0.051
-p
re
lP
ur
0.99
ro
XBP1,HSPA5
na
of
FDR
2
Jo
CCR3 Signaling in Eosinophils
P-value
Ketogenesis
1.88
0.94
0.013
0.115
1
HMGCS2
Mevalonate Pathway I
1.76
0.87
0.017
0.134
1
HMGCS2
Table 2. Master regulator analysis shows significantly affected transcriptional regulators in HFD-fed mouse spinal cord. Bold indicates increased.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
36
Journal Pre-proof
37
Fig 3. Myelin-related transcriptional changes in the spinal cord of adult high fatfed mice. A, Heat map of myelin-related genes in the spinal cord of male mice fed RD or HFD for 12 wk where blue indicates relative minimum value and red is the row maximum value. Genes within groups are sorted by highest to lowest average HFD relative values compared to RD, with red asterisk indicating genes with p<0.05 Schematic depicting differentiation of oligodendrocyte lineage cells (B) and scatterplots
of
of RNA expression of genes enriched in oligodendrocyte progenitors (OPCs, C), new
ro
oligodendrocytes (OL, D) or myelinating OLs (E) in HFD mice normalized relative to RD
Jo
ur
na
lP
re
-p
controls.
Journal Pre-proof
38
3.3 Functional and metabolic changes in spinal cord mitochondria following HFD consumption are time-dependent Prior to initiation of cell death, ER stress pathway activation can have a number of effects on metabolic and bioenergetic processes [28, 47, 48]. Therefore, since the ER Stress Pathway was the top ingenuity canonical pathway identified from transcriptomic profiling of spinal cord tissue, we further explored potential mechanisms for
of
oligodendrocyte loss in this model by comparing metabolite profiles from the spinal
ro
cords of mice fed a RD or HFD using untargeted UPLC-ToF-MS-based metabolomics
-p
as described previously in detail [37].
HFD consumption significantly altered 238 metabolites analyzed using an
re
unpaired t-test with Benjamini-Hochberg multiple testing correction and identified using
lP
the METLIN metabolite database. To determine which pathways were most affected by
na
the metabolites altered due to chronic HFD-consumption, we performed metabolite sets enrichment analysis (MSEA) with MetabaCore depicted with most significant p-values in
ur
red and least significant in white (Figure 4A). These results suggest that protein
Jo
biosynthesis, TCA cycle, glutathione metabolism, and the mitochondrial electron transport chain were among the significantly enriched pathways. Accordingly, we sought to validate these findings by quantifying TCA cycle metabolites at 4 and 12 wk using a targeted GC-MS-based approach [37]. No significant differences were observed at 4 wk (Figure 4B), but intermediates from the TCA cycle including oxaloacetate (p=0.04) , citric acid (p=0.003), and α-ketoglutarate (p=0.03) were strongly depleted in the 12 wk HFD spinal cord when compared to RD controls (Figure 4B, C and Supplemental Figure 7).
Journal Pre-proof
39
A separate cohort of mice was fed a RD or HFD for 4 or 12 wk to assess the functional impact on spinal cord mitochondria using Oroboros oxygraph [38]. Unexpectedly, we observed a significant increase in State 3 Respiration at 4 wk on HFD (Figure 4D), while 12 wk HFD spinal cord mitochondria had diminished State 3 oxygen consumption compared to RD (Figure 4D). Even State 4 respiration was increased at 4 wk, but declined at 12 wk of HFD consumption (Figure 4E). Maximum (Figure 4F) and
of
non-mitochondrial respiration (Figure 4G) were not altered by HFD consumption at 4 or
ro
12 wk. Taken together, these data provide evidence for temporally distinct changes in
-p
TCA cycle metabolites and mitochondrial function, where at 12 wk TCA cycle
Jo
ur
na
lP
re
metabolites are depleted and mitochondrial function is impaired.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
40
Journal Pre-proof
41
Fig 4. Functional and metabolic changes in spinal cord mitochondria following HFD consumption are time-dependent. Metabolite Sets Enrichment Overview (A) summarizing significantly affected pathways in HFD-fed mice as revealed by untargeted LC-MS-MS profiling of spinal cord tissue. Heat map (B) summarizes targeted LC-MSMS profiling of tricarboxylic acid (TCA) cycle metabolites at 4 and 12 wk of RD or HFD consumption with metabolites depleted at 12 wk identified within the schematic by blue
of
arrows (C). Gray indicates metabolite was not detected in samples, red indicated
ro
highest fold expression and blue lowest fold abundance. High resolution respirometry by
-p
Oroboros oxygraph of isolated spinal cord mitochondria indicates oxygen consumption is increased in 4 wk HFD mice (D, E), but decreased by 12 wk (D, E). Injections of
re
glutamate/malate/succinate (GMS), ADP, oligomycin (O), and trifluoromethoxy
lP
carbonylcyanide phenylhydrazon (FCCP) allow for oxygen consumption rates (OCRs) to
na
be determined during various stages of mitochondrial respiration. State 3 mitochondrial respiration was assessed following the addition of glutamate, malate, and succinate to
ur
the chamber, giving the basal respiration rate for complexes I and II. Oligomycin
Jo
injection then inhibited ATP synthase to induce state 4 respiration, attributable to proton leak across the inner mitochondrial membrane. FCCP addition results in an uncoupled state, allowing for determination of maximum capacity, while non-mitochondrial respiration was assessed after antimycin inhibits complex III. OCRs during state 3 (D), state 4 (E), Maximum (F), and non-mitochondrial respiration (G). Graphical results represented as the mean ± SEM (*, p<0.05; **, p<0.01; and ***, p<0.001; n=4-6/group).
Journal Pre-proof
42
3.4 Altered mitochondrial quality control processes, oxidative stress, and apoptosis in the spinal cord following 12 wk of high fat diet consumption. HFD consumption was previously shown to alter the metabolic regulators AMPK and SIRT1 in peripheral tissues and to affect mitochondrial function and quality control processes [49, 50]. Therefore, we next explored this signaling pathway (Figure 5A) in the spinal cord of RD and HFD-fed mice. With 12 wk of HFD, immunoblotting
of
demonstrated reductions in AMPK phosphorylation (Figure 5B, C) and in total AMPK
ro
(Figure 5B, C). SIRT1 protein levels (Figure 5B, C) were also significantly reduced in
-p
the spinal cord of HFD mice compared to controls. PGC1α, a direct target of AMPK and
re
SIRT1 and crucial regulator of mitochondrial biogenesis , showed diminished protein levels (Figure 5B, C) and RNA expression (Figure 5D) in the HFD-fed mice compared to
lP
RD-fed controls. There was also an upregulation of the fission gene, Fis1 (Figure 5D),
na
and a reduction in the mitophagy-related gene PINK1 (Figure 5D), which could indicate an overall accumulation of fragmented mitochondria in the spinal cord of HFD-fed mice.
ur
Oligodendrocytes are particularly vulnerable to cell death mediated by oxidative
Jo
damage due to their relatively low antioxidant levels and high amounts of iron storage [51, 52]. Notably, gene expression of the oxidative defense enzyme SOD2 (Figure 5D) was significantly lower in HFD spinal cords compared to RD. Next, in a heatmap of mitochondrial-related genes (Figure 5E), we noticed a trend for increased Bax gene expression (p=0.03, FDR=0.63), while anti-apoptosis Bcl-2 appeared less (p=0.69, FDR=0.99) in the spinal cords of HFD mice versus RD controls. Free radical production results in oxidative modification of lipids which can damage biological membranes including the myelin sheath. 4-hydroxynonenal (4-HNE) lipid peroxidation products are
Journal Pre-proof
43
found in demyelinating MS lesions and are highly toxic to OLCs [47, 53, 54]. White matter tracts within both the spinal cord dorsal column (Figure 5F) and corpus callosum (Figure 5G) of mice consuming HFD had significantly greater immunoreactivity for 4HNE compared to RD controls. Furthermore, immunofluorescence staining demonstrated increased levels of pro-apoptotic protein Bax (Figure 5H) and decreased levels of anti-apoptotic Bcl-2 (Figure 5I) in the dorsal column of HFD-fed mice.
of
Quantification of the Bax/Bcl-2 ratio (Figure 5H) and double positive Olig2/cleaved-
ro
caspase-3 cells (Figure 5I) suggest apoptotic cell death signaling is underway in
-p
oligodendrocytes chronically fed a HFD. These results point to HFD-related changes in
re
key metabolic regulators such as AMPK and SIRT1, decreased mitochondrial biogenesis and an impaired potential for antioxidant responses as contributing factors to
lP
the increases in oxidative stress markers such as 4-HNE and loss of myelinating cells
Jo
ur
na
observed in mice consuming HFD.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
44
Journal Pre-proof
45
Fig 5. Altered Mitochondrial Quality Control Processes, Oxidative Stress, and Apoptosis in the spinal cord following 12 wk of high fat diet consumption. Schematic (A) and Western blots (B) demonstrate significant reductions in AMPK phosphorylation, SIRT1, and PGC1α. C, Bar graphs show quantification of relative optical density (ROD) highlighting reductions in AMPK phosphorylation and total AMPK, SIRT1, and PGC1α protein expression in the spinal cord of high fat diet (HFD)-
of
consuming mice compared to those fed a regular diet (RD). The first HFD mouse
ro
samples protein level changes were less affected than others due to animal variability
-p
despite consistent conditions between mice. D, Quantitative PCR demonstrated differences in RNA expression of PGC1α, Fis1, PINK1, and SOD2. E, Heat map
re
summarizes RNAseq data for mitochondria-related transcriptional changes.
lP
Representative 4-hydroxynonenol (4-HNE) immunostained sections and corresponding
na
quantification of immunoreactivity normalized to RD control revealing increased lipid peroxidation in the spinal cord (SC) (F) and corpus callosum (G) of HFD-fed mice by 12
ur
wk. Immunofluorescence images and associated bar graphs show increased Bax/Olig2
Jo
double positive cells (H) and an increased Bax/Bcl2 ratio as well as more cleaved caspase-3/Olig2 double positive cells (I) in the white matter of spinal cord tissue from HFD-fed mice versus RD controls, indicating oligodendrocyte apoptosis. Graphical results represented as the mean ± SEM (*, p<0.05, **, p<0.01, and ***, p<0.001 (n=5-7 mice per group)). Scale bar= 50 µm (F), 100 µm (G), and 20 µm (H, I).
Journal Pre-proof
46
3.5 Mitochondrial changes and differentiation in oligodendrocytes treated with saturated fat. To model HFD consumption in vitro and study potential direct effects towards oligodendrocytes, we cultured primary murine oligodendrocytes in media containing the saturated fatty acid palmitate (PA, 100 µM) for 24 h. Seahorse extracellular flux analysis (Figure 6A) was performed to evaluate mitochondrial bioenergetics. PA significantly
of
decreased basal, ATP-linked, and maximal oxygen consumption rates in
ro
oligodendrocytes, but had no effect on proton leak. Since we observed transcriptional
-p
changes in fission and mitophagy genes in the spinal cord of mice consuming a HFD, we also visualized the integrity and structure of oligodendrocyte mitochondria using
re
MitoTracker. Healthy control oligodendrocytes have a long, filamentous network of
lP
mitochondria (Figure 6B), while PA-treated oligodendroglia have mitochondria that are
na
smaller, more compact, and circular. Overall, mitochondrial function and structure are impaired in oligodendrocytes cultured with high levels of saturated fat, in a manner
ur
recapitulating the changes observed in the spinal cord of HFD-fed mice (Figure 4).
Jo
To determine the influence of PA on oligodendrocyte differentiation, PLPimmunoreactivity and RNA transcripts for PLP, MBP and Olig2 were quantified. Addition of PA (100 µM, 24 h) to differentiating primary oligodendrocyte cultures reduced PLP protein (Figure 6C) to half that of controls. Supporting this, PLP, MBP and Olig2 RNA expression were also depleted (Figure 6D). In addition to oligodendrocyte progenitors, neural stem cells also contribute to replacement of oligodendrocytes following injury [44, 55]. Therefore, we next determined if the oligotoxic effects of PA extend to monolayer cultures of neural stem cells (aNSCs) derived from the adult subventricular zone (SVZ).
Journal Pre-proof
47
Supporting deleterious effects of PA across the oligodendrocytes and neural stem cells, the abundance of PLP protein (Figure 6E) and MBP RNA expression (Figure 6F) were significantly less in aNSCs cultured in the presence of PA. Taken together, these findings suggest excess PA impairs the ability of OLCs or aNSCs to differentiate into
Jo
ur
na
lP
re
-p
ro
of
myelinating oligodendrocytes.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
48
Journal Pre-proof
49
Fig 6. Differentiation and mitochondrial changes in oligodendrocyte progenitors cultured in the presence of high saturated fatty acid. High fat consumption was modeled in primary murine oligodendrocyte lineage cell (OLC) cultures by including palmitate in the media (PA, 100µM, 24 h). Bioenergetics characterization (A) of PA in OLCs was measured using a Seahorse XF24 analyzer. Basal, ATP-linked, and maximum capacity oxygen consumption rates were decreased in OLCs treated with PA.
of
Significant changes in proton leak respiration were not observed. MitoTracker staining
ro
(B) showed increased mitochondrial fragmentation in PA-treated OLCs as determined
-p
by mitochondrial parameters including significantly increased solidity and circularity and a smaller average size. Proteolipid (PLP) immunoreactivity of OLCs (C) and qPCR
re
quantification of RNA (D) for PLP, myelin basic protein (MBP) and Olig2 illustrate
lP
impaired differentiation toward a mature myelinating oligodendrocyte when treated with
na
PA. Similarly PA-treated adult SVZ-derived neural stem cell (aNSC) cultures (M) also express less PLP protein (E) and MBP RNA (F). Graphical results represented as the
Jo
ur
mean ± SEM (n=3-6/group; *, p<0.05, **, p<0.01, and ***, p<0.001). Scale bar= 50 µm.
Journal Pre-proof
50
4. Discussion Diet-induced obesity increases the risk for a number of diseases including diabetes, cardiovascular disease, cancer, and cognitive dysfunction and has been steadily growing in prevalence in recent decades [20, 56-59]. The role of oligodendrocytes and myelin plasticity is likewise implicated in a variety of neuropsychiatric and cognitive conditions [1, 2, 6]. Notably, white matter integrity is
of
negatively correlated with BMI and is decreased in the corpus callosum of obese
ro
individuals [4, 14, 60]. Elevated BMI is also linked to an increased incidence and
-p
disease burden in MS, especially in combination with other risk factors [10, 13, 61, 62].
re
Here we explored the potential link between a HFD, myelin integrity and metabolic dysfunction in the CNS. Our findings demonstrate for the first time that a HFD alone
lP
depletes oligodendrocyte progenitors in the brain and spinal cord and high saturated fat
na
impedes oligodendrocyte differentiation in vitro. Overall, we observed changes in genes and pathways involving oligodendrogenesis and differentiation, iron homeostasis,
ur
oxidative stress, ER stress, insulin signaling, and inflammation all of which may
Jo
contribute mechanistically alone or collectively to the loss of myelinating cells in the CNS of mice chronically consuming HFD. In the spinal cords of HFD fed mice, RNA sequencing and untargeted metabolomics point towards ER stress and mitochondrial dysfunction as key mechanisms contributing to oligodendrogliopathy. These findings were validated by results showing depletion of TCA cycle intermediates and reduced oxygen consumption rates in the spinal cords of HFD-consuming mice and in isolated oligodendrocytes. Moreover, deficits in AMPK-SIRT1-PGC1α signaling were present in HFD spinal cords, whereas the oxidative stress marker 4HNE was increased. Overall,
Journal Pre-proof
51
the vast influence of metabolic dysfunction on oligodendrocytes observed here is of high relevance to demyelinating, neuropsychiatric and cognitive disorders and to considerations for strategies to manage myelin protection and repair. The results of the current study suggest that consumption of excess high fat alone is sufficient to trigger oligodendrogliopathy and impairments in oligodendrocyte differentiation across the brain and spinal cord and links these to several disturbances
of
in metabolic status. Oligodendrocytes play critical roles in modulating motor [63, 64] and
ro
psychiatric [5, 65] function by improving the speed and integrity of impulse conduction,
-p
providing neuronal metabolic support, and regulating synapses [8]. In a recent study, we
re
discovered that a diet high in saturated fat (41%) and sucrose (34%), the so-called Western diet, depletes oligodendrocytes and their precursors after 7 wk of consumption
lP
in the adult (4 month) spinal cord [29]. A later study, reported neuroinflammation,
na
cerebrovascular decline, and white matter damage with an increase in Olig2+ progenitors, but a decrease in MBP in the frontal parietal cortex/corpus callosum
ur
following long term (10 month) consumption of a “Western-style” diet (16.4% calories
Jo
from fat with added high-fructose corn syrup) [4]. Importantly, in both prior studies examining the effects of a high fat high sugar diet on myelin, exercise prevented the deleterious effects. The findings of the current study suggest that consumption of high fat alone is sufficient to trigger demyelinating effects in the brain and spinal cord of adult mice. Additional study will be needed to determine if the demyelinating effects of HFD can likewise be prevented by exercise interventions. Many of the transcriptional changes we observed are increased in the spinal cord of mice consuming a HFD are already established to be differentially expressed in
Journal Pre-proof
52
individuals with MS and its experimental models. Fos, Grn, Sdf2l1, Fn14, and Paqr6 RNA accumulation are each separately found in glia of white matter tissue of MS patients, and Grn polymorphisms may influence MS disease course and relapse recovery [66-72]. Down regulated genes in the spinal cord of HFD mice were also affected in MS animal models, include Slc17a7 and Pdyn [73]. Although Cryab was shown to inhibit inflammation and disease in the EAE model, recent studies in the
of
Cuprizone model suggest that Cryab activates astrocytes and exacerbates
ro
demyelination [74]. Moreover, activation of the Sgk1 pathway following oxidative stress
-p
exacerbates EAE [75]. Among many genes identified by Moyon et. al in a study of
re
transcriptional profiling of Cuprizone-“activated” oligodendrocytes, higher expression of Socs3, Ddit4, and lower expression of Kdr and Cdc42ep2 genes may be required to
lP
activate OPCs enabling remyelination [76]. Kruppel like factor 9 (Klf9), down regulated
na
by HFD, is a transcription factor downstream of T3 that regulates oligodendrocyte differentiation and myelin regeneration in the Cuprizone model [77, 78]. Ddit3 is
ur
expressed by activated astrocytes following Cuprizone exposure, while knockout mice
Jo
had less oligodendrocyte apoptosis, demyelination, and microglial and astrocyte activation [79]. HFD also exacerbates disease symptoms and neuroinflammation in EAE models [21, 22]. Notably, the genes identified as having higher expression in HFD-EAE versus RD-EAE did not overlap with those we observed in the spinal cord of adult mice consuming HFD, indicating unique mechanisms contribute to HFD-triggered pathology depending on the disease context [22]. ER stress and inflammation closely follow lipid dysregulation in diabetic neuropathy and obesity-associated allodynia models and cooperate in a feed forward
Journal Pre-proof
53
manner [80-82]. In line with studies showing ER stress in liver, heart, muscle, hypothalamus, and hippocampus in diet-induced obesity models [7, 48, 83, 84], RNA sequencing results in the current study uncovered ER Stress and the Unfolded Protein Response as prominently altered pathways in the adult spinal cord after 12 wk of HFD consumption. Since myelinating cells produce large amounts of plasma membrane, they are particularly susceptible to disruptions in secretory pathways including ER stress and
of
eventual apoptosis if left unchecked [3, 26, 85]. Thus, controlling ER overload may be
ro
beneficial to restoring oligodendrocytes and myelin in various white matter diseases [3,
-p
26, 85, 86]. Xbp1, involved in ER stress responses, is reported to increase in MS
re
lesions as well as in vanishing white matter disease (VWMD) [3, 26]. ER chaperones help control cellular responses to ER stress and some were shown to be required for
lP
oligodendrocyte survival during EAE [85]. ER and mitochondria interact both physically
MS pathology [27].
na
and functionally, and alterations in these connections have recently been described in
ur
Alterations in ER stress are connected to mitochondrial deficits in MS [27].
Jo
Mitochondrial morphology and impaired mitochondrial function and antioxidant responses are also reported in experimental models of demyelinating disease [87-89]. Here we document fragmented mitochondria in cultures of oligodendrocytes grown under high fat conditions. Fis1 is involved in mitochondrial fission and in early events in cell-death signaling, and expression was also upregulated in the spinal cord of mice consuming high fat. Similar changes are likewise engaged in OLCs in the context of oxidative stress or inflammation [40, 89-91]. Moreover, inhibition of fission is protective against demyelination and neuroinflammation, although has little impact on OLC
Journal Pre-proof
54
differentiation in EAE and Cuprizone MS models [89, 92]. Impairments in mitochondria also lead to increased reactive oxygen species and downstream effects on proteins, lipids, and nucleic acids [92-94]. Notably, in both the corpus callosum and spinal cord white matter regions where OLC death occurred following chronic HFD consumption, we observed increased lipid peroxidation. Increased lipid peroxidation was also observed in the spinal cord with consumption of high fat and sucrose [29]. In MS
of
pathogenesis and its animal models, oxidative stress mediates apoptosis of
ro
oligodendrocytes and is commonly documented by an elevated Bax/Bcl2 ratio and
-p
caspase-3 cleavage [95, 96]. Our in vivo data suggest that mitochondrial-dependent
re
apoptosis also occurs in oligodendrocytes in relation to HFD-induced mitochondrial dysfunction and increased oxidative damage.
lP
In the current study, we discovered substantial deficits in TCA cycle
na
intermediates and oxygen consumption rates in the spinal cords of HFD mice. Key substrates at all stages of the TCA cycle, from citric acid to oxaloacetate were reduced
ur
in the spinal cord of mice consuming HF. In addition, these effects were paralleled by
Jo
diminished mitochondrial oxidative respiration. Oligodendrocytes have extremely high energy demands and die hours faster than neurons following nutrient deprivation by arterial occlusion [28, 97]. Impaired energy metabolism has already been proposed as a contributing factor to oligodendrogliopathy in pattern III MS lesions and is described in animal models of the disease [87, 89, 93]. Normally, metabolism of pyruvate and other fuel sources through the mitochondrial TCA cycle leads to ATP synthesis via the electron transport chain. Our findings that the dysmyelinating effects of HFD were associated with significant impairments in TCA cycle function, are of particular interest
Journal Pre-proof
55
given the efficacy of dimethyl fumarate, itself a TCA cycle metabolite, as a first line treatment for relapsing remitting MS [98]. AMPK, SIRT1 and PGC1α were all substantially reduced in the spinal cord of mice consuming HF. AMPK and SIRT1 are crucial metabolic sensors that impact PCG1α and thus mitochondria biogenesis and function. In addition to promoting cell survival, AMPK-SIRT1 activation and mitochondrial function regulate oligodendrocyte
of
differentiation [50, 99-102]. Recently, folate was determined to regulate oligodendrocyte
ro
survival and differentiation through AMPK activity [99]. SIRT1 and SIRT2 activation
-p
redundantly mediate differentiation of neural stem or progenitor cells towards the oligodendrocyte lineage, likely by upregulation of PDGFRα, Sox10, Nkx2.2 and
re
downregulation of p21 [9, 103, 104]. Moreover, SIRT1 activation through
lP
overexpression or agonists is neuroprotective in MS models, while expansion of
na
oligodendrocyte progenitors occurs following SIRT1 inactivation [101, 104]. Exercise and other activators of the AMPK-SIRT1-PGC1α system may be beneficial in reducing
ur
deleterious effects of obesogenic diets on oligodendrocyte lineage cells and myelin
Jo
integrity by promoting mitochondrial homeostasis [4, 29, 49, 101]. Interestingly, we observed a predicted decrease in PDGFRα signaling in the transcriptomic data along with decreased AMPK-SIRT1 signaling, leading to a loss of both progenitors and mature oligodendrocytes in the spinal cord following long term HFD. Whether or not the disruptions we observe in TCA cycle metabolite depletion, impaired mitochondrial function and oligodendrocyte loss observed in HFD-fed spinal cords are a direct consequence of the altered AMPK-SIRT signaling, as specified in other tissues and cell types, should be more directly addressed in future studies.
Journal Pre-proof
56
Mature CC-1+ oligodendrocytes along with Olig2+ progenitors are lost in the spinal cord white matter with HFD consumption, but only the later are impacted in the corpus callosum at the same 12 wk time point. Given their increased energy demands for proliferation and myelin membrane production it is possible that oligodendrocyte progenitors and young oligodendrocytes as labeled by Olig2 are more vulnerable to the stresses associated with HF consumption compared to mature oligodendrocytes. We
of
cannot exclude that mature oligodendroglia in the corpus callosum would be lost with
ro
even more chronic HFD feeding. Region specific and possible temporal differences in
-p
HF-elicited oligotoxicity may also relate to heterogeneity in the local oligodendrocyte
re
populations, region specific effects on neurons, or differential effects across the astrocyte and microglial compartments that so significantly contribute to the
lP
microenvironment [41]. Our studies also demonstrate an anxiety-like phenotype and
na
overall activity level changes in the HFD-fed mice that mirror a number of previous reports [20, 58], however future studies will be required to determine if HFD-induced
ur
behavioral changes are in fact dependent on the oligodendrocyte loss documented.
Jo
Although we show that impaired differentiation and mitochondrial fragmentation occurring with HFD are recapitulated across regions and in cultures of oligodendrocytes, a Ribotag approach or single cell sequencing will be beneficial for the identification of cell-specific transcriptional changes in each tissue, and this is another goal for future experiments.
Journal Pre-proof
57
5. Conclusions The findings of the current study demonstrate significant perturbations in CNS function occur in response to long term consumption of a diet laden with excessive fat, including direct impairment of oligodendrocyte mitochondrial function that ultimately impairs oligodendrocyte survival and differentiation. Previous reports in Western diet-fed mice suggest exercise may mitigate deleterious effects on myelin integrity and
of
behavioral outcomes, and our new data in mice fed a high fat diet suggest that
ro
modulation of mitochondrial function, ER and oxidative stress pathways, in addition to
-p
the TCA cycle may also be important areas to explore as targets to improve myelin
re
protection and regeneration. Accordingly, this work should inspire new research to evaluate ways to mitigate the negative consequences of a chronic high fat diet on
6. Author Contributions
na
lP
oligodendrocytes alone and in the context of white matter injury.
ur
IAS, MRL and AM conceived and designed the study. MRL, HY and AM performed all
Jo
in vivo experiments, necropsies, histological staining and analysis. WS and HNK assisted with necropsies and LK performed some of the histological staining. CC generated heat maps of RNA sequencing data and contributed to neural stem cell cultures. MRL performed and analyzed oxygraph and in vitro experiments. MRL and IAS interpreted the data and wrote the manuscript. All authors reviewed and edited the manuscript.
Journal Pre-proof
58
7. Acknowledgements The work was supported by Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology (CMSAN), the Eugene and Marcia Applebaum Foundation, the Mayo Clinic Center for Biomedical Discovery (CBD) and the Mayo Clinic Metabolomics Core (U24DK100469 and UL1TR000135). Portions of this work were also supported by R01NS052741, RG4958 from the National Multiple Sclerosis Society, a grant from the
of
Craig H. Neilsen Foundation, and the Minnesota State Spinal Cord Injury and Traumatic
ro
Brain Injury Research Program.
-p
We would like to thank Thomas White for his assistance in analyzing the glucose
re
tolerance data and Katherine Klaus for her help the Oroboros oxygraph system. We also thank Xuewei Wang and Tumpa Dutta for their initial assistance with Bioinformatics
lP
for RNAseq and LC-MS-MS measurements, respectively. The diagrams within figures
ur
Declaration of Interests
na
were created using elements from the Biomedical-PPT-Toolkit-Suite (Motifolio).
Jo
The authors have no declarations of interests to disclose.
Journal Pre-proof
59
References
Jo
ur
na
lP
re
-p
ro
of
[1] R. Ohtomo, A. Iwata, K. Arai, Molecular Mechanisms of Oligodendrocyte Regeneration in White Matter-Related Diseases, Int J Mol Sci 19(6) (2018). [2] R.C. Armstrong, A.J. Mierzwa, G.M. Sullivan, M.A. Sanchez, Myelin and oligodendrocyte lineage cells in white matter pathology and plasticity after traumatic brain injury, Neuropharmacology 110(Pt B) (2016) 654-659. [3] B. van Kollenburg, J. van Dijk, J. Garbern, A.A. Thomas, G.C. Scheper, J.M. Powers, M.S. van der Knaap, Glia-specific activation of all pathways of the unfolded protein response in vanishing white matter disease, J Neuropathol Exp Neurol 65(7) (2006) 707-15. [4] L.C. Graham, W.A. Grabowska, Y. Chun, S.L. Risacher, V.M. Philip, A.J. Saykin, I. Alzheimer's Disease Neuroimaging, S.J. Sukoff Rizzo, G.R. Howell, Exercise prevents obesity-induced cognitive decline and white matter damage in mice, Neurobiol Aging 80 (2019) 154-172. [5] K. Roy, J.C. Murtie, B.F. El-Khodor, N. Edgar, S.P. Sardi, B.M. Hooks, M. BenoitMarand, C. Chen, H. Moore, P. O'Donnell, D. Brunner, G. Corfas, Loss of erbB signaling in oligodendrocytes alters myelin and dopaminergic function, a potential mechanism for neuropsychiatric disorders, Proc Natl Acad Sci U S A 104(19) (2007) 8131-6. [6] V. Haroutunian, P. Katsel, P. Roussos, K.L. Davis, L.L. Altshuler, G. Bartzokis, Myelination, oligodendrocytes, and serious mental illness, Glia 62(11) (2014) 1856-77. [7] M. Cai, H. Wang, J.J. Li, Y.L. Zhang, L. Xin, F. Li, S.J. Lou, The signaling mechanisms of hippocampal endoplasmic reticulum stress affecting neuronal plasticity-related protein levels in high fat diet-induced obese rats and the regulation of aerobic exercise, Brain Behav Immun 57 (2016) 347-359. [8] R.E. Pepper, K.A. Pitman, C.L. Cullen, K.M. Young, How Do Cells of the Oligodendrocyte Lineage Affect Neuronal Circuits to Influence Motor Function, Memory and Mood?, Front Cell Neurosci 12 (2018) 399. [9] A. Alizadeh, S. Karimi-Abdolrezaee, Microenvironmental regulation of oligodendrocyte replacement and remyelination in spinal cord injury, J Physiol 594(13) (2016) 3539-52. [10] M.A. Gianfrancesco, B. Acuna, L. Shen, F.B. Briggs, H. Quach, K.H. Bellesis, A. Bernstein, A.K. Hedstrom, I. Kockum, L. Alfredsson, T. Olsson, C. Schaefer, L.F. Barcellos, Obesity during childhood and adolescence increases susceptibility to multiple sclerosis after accounting for established genetic and environmental risk factors, Obes Res Clin Pract 8(5) (2014) e435-47. [11] A. Langer-Gould, S.M. Brara, B.E. Beaber, C. Koebnick, Childhood obesity and risk of pediatric multiple sclerosis and clinically isolated syndrome, Neurology 80(6) (2013) 548-52. [12] K.L. Munger, T. Chitnis, A. Ascherio, Body size and risk of MS in two cohorts of US women, Neurology 73(19) (2009) 1543-50. [13] T. Olsson, L.F. Barcellos, L. Alfredsson, Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis, Nat Rev Neurol 13(1) (2017) 2536.
Journal Pre-proof
60
Jo
ur
na
lP
re
-p
ro
of
[14] T.M. Wassenaar, K. Yaffe, Y.D. van der Werf, C.E. Sexton, Associations between modifiable risk factors and white matter of the aging brain: insights from diffusion tensor imaging studies, Neurobiol Aging 80 (2019) 56-70. [15] A. Manouchehrinia, A.K. Hedstrom, L. Alfredsson, T. Olsson, J. Hillert, R. Ramanujam, Association of Pre-Disease Body Mass Index With Multiple Sclerosis Prognosis, Front Neurol 9 (2018) 232. [16] P. Tettey, S. Simpson, Jr., B. Taylor, L. Blizzard, A.L. Ponsonby, T. Dwyer, K. Kostner, I. van der Mei, An adverse lipid profile is associated with disability and progression in disability, in people with MS, Mult Scler 20(13) (2014) 1737-44. [17] L. Negrotto, M.F. Farez, J. Correale, Immunologic Effects of Metformin and Pioglitazone Treatment on Metabolic Syndrome and Multiple Sclerosis, JAMA Neurol 73(5) (2016) 520-8. [18] A. Sicras-Mainar, E. Ruiz-Beato, R. Navarro-Artieda, J. Maurino, Comorbidity and metabolic syndrome in patients with multiple sclerosis from Asturias and Catalonia, Spain, BMC Neurol 17(1) (2017) 134. [19] P. Dimas, L. Montani, J.A. Pereira, D. Moreno, M. Trotzmuller, J. Gerber, C.F. Semenkovich, H.C. Kofeler, U. Suter, CNS myelination and remyelination depend on fatty acid synthesis by oligodendrocytes, Elife 8 (2019). [20] M. Ogrodnik, Y. Zhu, L.G.P. Langhi, T. Tchkonia, P. Kruger, E. Fielder, S. Victorelli, R.A. Ruswhandi, N. Giorgadze, T. Pirtskhalava, O. Podgorni, G. Enikolopov, K.O. Johnson, M. Xu, C. Inman, A.K. Palmer, M. Schafer, M. Weigl, Y. Ikeno, T.C. Burns, J.F. Passos, T. von Zglinicki, J.L. Kirkland, D. Jurk, Obesity-Induced Cellular Senescence Drives Anxiety and Impairs Neurogenesis, Cell Metab (2019). [21] S. Timmermans, J.F. Bogie, T. Vanmierlo, D. Lutjohann, P. Stinissen, N. Hellings, J.J. Hendriks, High fat diet exacerbates neuroinflammation in an animal model of multiple sclerosis by activation of the Renin Angiotensin system, J Neuroimmune Pharmacol 9(2) (2014) 209-17. [22] M. Hasan, J.E. Seo, K.A. Rahaman, H. Min, K.H. Kim, J.H. Park, C. Sung, J. Son, M.J. Kang, B.H. Jung, W.S. Park, O.S. Kwon, Novel genes in brain tissues of EAE-induced normal and obese mice: Upregulation of metal ion-binding protein genes in obese-EAE mice, Neuroscience 343 (2017) 322-336. [23] R. Ghemrawi, S.F. Battaglia-Hsu, C. Arnold, Endoplasmic Reticulum Stress in Metabolic Disorders, Cells 7(6) (2018). [24] C.M. Mayer, D.D. Belsham, Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation, Endocrinology 151(2) (2010) 576-85. [25] K.S. Gwiazda, T.L. Yang, Y. Lin, J.D. Johnson, Effects of palmitate on ER and cytosolic Ca2+ homeostasis in beta-cells, Am J Physiol Endocrinol Metab 296(4) (2009) E690-701. [26] A.N. Mhaille, S. McQuaid, A. Windebank, P. Cunnea, J. McMahon, A. Samali, U. FitzGerald, Increased expression of endoplasmic reticulum stress-related signaling pathway molecules in multiple sclerosis lesions, J Neuropathol Exp Neurol 67(3) (2008) 200-11.
Journal Pre-proof
61
Jo
ur
na
lP
re
-p
ro
of
[27] Y. Haile, X. Deng, C. Ortiz-Sandoval, N. Tahbaz, A. Janowicz, J.Q. Lu, B.J. Kerr, N.J. Gutowski, J.E. Holley, P. Eggleton, F. Giuliani, T. Simmen, Rab32 connects ER stress to mitochondrial defects in multiple sclerosis, J Neuroinflammation 14(1) (2017) 19. [28] L. Rosko, V.N. Smith, R. Yamazaki, J.K. Huang, Oligodendrocyte Bioenergetics in Health and Disease, Neuroscientist (2018) 1073858418793077. [29] H. Yoon, A. Kleven, A. Paulsen, L. Kleppe, J. Wu, Z. Ying, F. Gomez-Pinilla, I.A. Scarisbrick, Interplay between exercise and dietary fat modulates myelinogenesis in the central nervous system, Biochim Biophys Acta 1862(4) (2016) 545-55. [30] M.E. McGee-Lawrence, T.A. White, N.K. LeBrasseur, J.J. Westendorf, Conditional deletion of Hdac3 in osteoprogenitor cells attenuates diet-induced systemic metabolic dysfunction, Mol Cell Endocrinol 410 (2015) 42-51. [31] K.R. Kalari, A.A. Nair, J.D. Bhavsar, D.R. O'Brien, J.I. Davila, M.A. Bockol, J. Nie, X. Tang, S. Baheti, J.B. Doughty, S. Middha, H. Sicotte, A.E. Thompson, Y.W. Asmann, J.P. Kocher, MAP-RSeq: Mayo Analysis Pipeline for RNA sequencing, BMC Bioinformatics 15 (2014) 224. [32] B. Langmead, C. Trapnell, M. Pop, S.L. Salzberg, Ultrafast and memory-efficient alignment of short DNA sequences to the human genome, Genome Biol 10(3) (2009) R25. [33] S. Anders, P.T. Pyl, W. Huber, HTSeq--a Python framework to work with highthroughput sequencing data, Bioinformatics 31(2) (2015) 166-9. [34] L. Wang, S. Wang, W. Li, RSeQC: quality control of RNA-seq experiments, Bioinformatics 28(16) (2012) 2184-5. [35] M.D. Robinson, D.J. McCarthy, G.K. Smyth, edgeR: a Bioconductor package for differential expression analysis of digital gene expression data, Bioinformatics 26(1) (2010) 139-40. [36] Y. Zhang, K. Chen, S.A. Sloan, M.L. Bennett, A.R. Scholze, S. O'Keeffe, H.P. Phatnani, P. Guarnieri, C. Caneda, N. Ruderisch, S. Deng, S.A. Liddelow, C. Zhang, R. Daneman, T. Maniatis, B.A. Barres, J.Q. Wu, An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex, J Neurosci 34(36) (2014) 11929-47. [37] T. Dutta, Y.C. Kudva, X.M. Persson, L.A. Schenck, G.C. Ford, R.J. Singh, R. Carter, K.S. Nair, Impact of Long-Term Poor and Good Glycemic Control on Metabolomics Alterations in Type 1 Diabetic People, J Clin Endocrinol Metab 101(3) (2016) 1023-33. [38] G.N. Ruegsegger, S. Manjunatha, P. Summer, S. Gopala, P. Zabeilski, S. Dasari, P.M. Vanderboom, I.R. Lanza, K.A. Klaus, K.S. Nair, Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes, FASEB J 33(3) (2019) 4458-4472. [39] M. Langley, A. Ghosh, A. Charli, S. Sarkar, M. Ay, J. Luo, J. Zielonka, T. Brenza, B. Bennett, H. Jin, S. Ghaisas, B. Schlichtmann, D. Kim, V. Anantharam, A. Kanthasamy, B. Narasimhan, B. Kalyanaraman, A.G. Kanthasamy, MitoApocynin Prevents Mitochondrial Dysfunction, Microglial Activation, Oxidative Damage, and Progressive Neurodegeneration in MitoPark Transgenic Mice, Antioxid Redox Signal 27(14) (2017) 1048-1066.
Journal Pre-proof
62
Jo
ur
na
lP
re
-p
ro
of
[40] R.K. Dagda, S.J. Cherra, 3rd, S.M. Kulich, A. Tandon, D. Park, C.T. Chu, Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission, J Biol Chem 284(20) (2009) 13843-55. [41] H. Yoon, G. Walters, A.R. Paulsen, I.A. Scarisbrick, Astrocyte heterogeneity across the brain and spinal cord occurs developmentally, in adulthood and in response to demyelination, PLoS One 12(7) (2017) e0180697. [42] Y.W. Ng, Y.H. Say, Palmitic acid induces neurotoxicity and gliatoxicity in SH-SY5Y human neuroblastoma and T98G human glioblastoma cells, PeerJ 6 (2018) e4696. [43] S. Kim, C. Kim, S. Park, Mdivi-1 Protects Adult Rat Hippocampal Neural Stem Cells against Palmitate-Induced Oxidative Stress and Apoptosis, Int J Mol Sci 18(9) (2017). [44] C.I. Choi, H. Yoon, K.L. Drucker, M.R. Langley, L. Kleppe, I.A. Scarisbrick, The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation, Sci Rep 8(1) (2018) 9360. [45] A. Hall, N.A. Giese, W.D. Richardson, Spinal cord oligodendrocytes develop from ventrally derived progenitor cells that express PDGF alpha-receptors, Development 122(12) (1996) 4085-94. [46] X.R. Jin, X.S. Chen, L. Xiao, MeCP2 Deficiency in Neuroglia: New Progress in the Pathogenesis of Rett Syndrome, Front Mol Neurosci 10 (2017) 316. [47] A.D. Roth, M.T. Nunez, Oligodendrocytes: Functioning in a Delicate Balance Between High Metabolic Requirements and Oxidative Damage, Adv Exp Med Biol 949 (2016) 167-181. [48] K.R. Bohnert, J.D. McMillan, A. Kumar, Emerging roles of ER stress and unfolded protein response pathways in skeletal muscle health and disease, J Cell Physiol 233(1) (2018) 67-78. [49] N.L. Price, A.P. Gomes, A.J. Ling, F.V. Duarte, A. Martin-Montalvo, B.J. North, B. Agarwal, L. Ye, G. Ramadori, J.S. Teodoro, B.P. Hubbard, A.T. Varela, J.G. Davis, B. Varamini, A. Hafner, R. Moaddel, A.P. Rolo, R. Coppari, C.M. Palmeira, R. de Cabo, J.A. Baur, D.A. Sinclair, SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function, Cell Metab 15(5) (2012) 675-90. [50] M. Bonora, E. De Marchi, S. Patergnani, J.M. Suski, F. Celsi, A. Bononi, C. Giorgi, S. Marchi, A. Rimessi, J. Duszynski, T. Pozzan, M.R. Wieckowski, P. Pinton, Tumor necrosis factor-alpha impairs oligodendroglial differentiation through a mitochondria-dependent process, Cell Death Differ 21(8) (2014) 1198-208. [51] O. Baud, R.F. Haynes, H. Wang, R.D. Folkerth, J. Li, J.J. Volpe, P.A. Rosenberg, Developmental up-regulation of MnSOD in rat oligodendrocytes confers protection against oxidative injury, Eur J Neurosci 20(1) (2004) 29-40. [52] H. Lassmann, J. van Horssen, Oxidative stress and its impact on neurons and glia in multiple sclerosis lesions, Biochim Biophys Acta 1862(3) (2016) 506-10. [53] E. McCracken, V. Valeriani, C. Simpson, T. Jover, J. McCulloch, D. Dewar, The lipid peroxidation by-product 4-hydroxynonenal is toxic to axons and oligodendrocytes, J Cereb Blood Flow Metab 20(11) (2000) 1529-36. [54] G.G. Ortiz, F.P. Pacheco-Moises, O.K. Bitzer-Quintero, A.C. Ramirez-Anguiano, L.J. Flores-Alvarado, V. Ramirez-Ramirez, M.A. Macias-Islas, E.D. Torres-
Journal Pre-proof
63
Jo
ur
na
lP
re
-p
ro
of
Sanchez, Immunology and oxidative stress in multiple sclerosis: clinical and basic approach, Clin Dev Immunol 2013 (2013) 708659. [55] M.E. Silva, S. Lange, B. Hinrichsen, A.R. Philp, C.R. Reyes, D. Halabi, J.B. Mansilla, P. Rotheneichner, A. Guzman de la Fuente, S. Couillard-Despres, L.F. Batiz, R.J.M. Franklin, L. Aigner, F.J. Rivera, Pericytes Favor Oligodendrocyte Fate Choice in Adult Neural Stem Cells, Front Cell Neurosci 13 (2019) 85. [56] C.S. Fox, S.H. Golden, C. Anderson, G.A. Bray, L.E. Burke, I.H. de Boer, P. Deedwania, R.H. Eckel, A.G. Ershow, J. Fradkin, S.E. Inzucchi, M. Kosiborod, R.G. Nelson, M.J. Patel, M. Pignone, L. Quinn, P.R. Schauer, E. Selvin, D.K. Vafiadis, L. American Heart Association Diabetes Committee of the Council on, H. Cardiometabolic, C.o.C. Council on Clinical Cardiology, C.o.C.S. Stroke Nursing, C.o.Q.o.C. Anesthesia, R. Outcomes, A. American Diabetes, Update on Prevention of Cardiovascular Disease in Adults With Type 2 Diabetes Mellitus in Light of Recent Evidence: A Scientific Statement From the American Heart Association and the American Diabetes Association, Diabetes Care 38(9) (2015) 1777-803. [57] R.J. Koene, A.E. Prizment, A. Blaes, S.H. Konety, Shared Risk Factors in Cardiovascular Disease and Cancer, Circulation 133(11) (2016) 1104-14. [58] W. Mizunoya, K. Ohnuki, K. Baba, H. Miyahara, N. Shimizu, K. Tabata, T. Kino, Y. Sato, R. Tatsumi, Y. Ikeuchi, Effect of dietary fat type on anxiety-like and depression-like behavior in mice, Springerplus 2(1) (2013) 165. [59] D. Frasca, B.B. Blomberg, R. Paganelli, Aging, Obesity, and Inflammatory AgeRelated Diseases, Front Immunol 8 (2017) 1745. [60] J. Xu, Y. Li, H. Lin, R. Sinha, M.N. Potenza, Body mass index correlates negatively with white matter integrity in the fornix and corpus callosum: a diffusion tensor imaging study, Hum Brain Mapp 34(5) (2013) 1044-52. [61] L.E. Mokry, S. Ross, N.J. Timpson, S. Sawcer, G. Davey Smith, J.B. Richards, Obesity and Multiple Sclerosis: A Mendelian Randomization Study, PLoS Med 13(6) (2016) e1002053. [62] J.J. Guerrero-Garcia, L. Carrera-Quintanar, R.I. Lopez-Roa, A.L. Marquez-Aguirre, A.E. Rojas-Mayorquin, D. Ortuno-Sahagun, Multiple Sclerosis and Obesity: Possible Roles of Adipokines, Mediators Inflamm 2016 (2016) 4036232. [63] I.A. McKenzie, D. Ohayon, H. Li, J.P. de Faria, B. Emery, K. Tohyama, W.D. Richardson, Motor skill learning requires active central myelination, Science 346(6207) (2014) 318-22. [64] S. Schneider, A. Gruart, S. Grade, Y. Zhang, S. Kroger, F. Kirchhoff, G. Eichele, J.M. Delgado Garcia, L. Dimou, Decrease in newly generated oligodendrocytes leads to motor dysfunctions and changed myelin structures that can be rescued by transplanted cells, Glia 64(12) (2016) 2201-2218. [65] X. Chen, W. Zhang, T. Li, Y. Guo, Y. Tian, F. Wang, S. Liu, H.Y. Shen, Y. Feng, L. Xiao, Impairment of Oligodendroglia Maturation Leads to Aberrantly Increased Cortical Glutamate and Anxiety-Like Behaviors in Juvenile Mice, Front Cell Neurosci 9 (2015) 467. [66] M. Luan, Z. Shang, Y. Teng, X. Chen, M. Zhang, H. Lv, R. Zhang, The shared and specific mechanism of four autoimmune diseases, Oncotarget 8(65) (2017) 108355-108374.
Journal Pre-proof
64
Jo
ur
na
lP
re
-p
ro
of
[67] J.S. Yu, T. Hayashi, E. Seboun, R.M. Sklar, T.H. Doolittle, S.L. Hauser, Fos RNA accumulation in multiple sclerosis white matter tissue, J Neurol Sci 103(2) (1991) 209-15. [68] M. Vercellino, C. Fenoglio, D. Galimberti, A. Mattioda, C. Chiavazza, E. Binello, L. Pinessi, D. Giobbe, E. Scarpini, P. Cavalla, Progranulin genetic polymorphisms influence progression of disability and relapse recovery in multiple sclerosis, Mult Scler 22(8) (2016) 1007-12. [69] A. Groves, Y. Kihara, D. Jonnalagadda, R. Rivera, G. Kennedy, M. Mayford, J. Chun, A Functionally Defined In Vivo Astrocyte Population Identified by c-Fos Activation in a Mouse Model of Multiple Sclerosis Modulated by S1P Signaling: Immediate-Early Astrocytes (ieAstrocytes), eNeuro 5(5) (2018). [70] R. Waller, M.N. Woodroofe, S.B. Wharton, P.G. Ince, S. Francese, P.R. Heath, A. Cudzich-Madry, R.H. Thomas, N. Rounding, B. Sharrack, J.E. Simpson, Gene expression profiling of the astrocyte transcriptome in multiple sclerosis normal appearing white matter reveals a neuroprotective role, J Neuroimmunol 299 (2016) 139-146. [71] B. Serafini, R. Magliozzi, B. Rosicarelli, R. Reynolds, T.S. Zheng, F. Aloisi, Expression of TWEAK and its receptor Fn14 in the multiple sclerosis brain: implications for inflammatory tissue injury, J Neuropathol Exp Neurol 67(12) (2008) 1137-48. [72] S. Desplat-Jego, L. Feuillet, R. Creidy, I. Malikova, R. Rance, M. Khrestchatisky, K. Hahm, L.C. Burkly, J. Pelletier, J. Boucraut, TWEAK is expressed at the cell surface of monocytes during multiple sclerosis, J Leukoc Biol 85(1) (2009) 132-5. [73] I. Sevastou, G. Pryce, D. Baker, D.L. Selwood, Characterisation of Transcriptional Changes in the Spinal Cord of the Progressive Experimental Autoimmune Encephalomyelitis Biozzi ABH Mouse Model by RNA Sequencing, PLoS One 11(6) (2016) e0157754. [74] H.F. Kuipers, J. Yoon, J. van Horssen, M.H. Han, P.L. Bollyky, T.D. Palmer, L. Steinman, Phosphorylation of alphaB-crystallin supports reactive astrogliosis in demyelination, Proc Natl Acad Sci U S A 114(9) (2017) E1745-E1754. [75] L. Wang, B. Li, M.Y. Quan, L. Li, Y. Chen, G.J. Tan, J. Zhang, X.P. Liu, L. Guo, Mechanism of oxidative stress p38MAPK-SGK1 signaling axis in experimental autoimmune encephalomyelitis (EAE), Oncotarget 8(26) (2017) 42808-42816. [76] S. Moyon, A.L. Dubessy, M.S. Aigrot, M. Trotter, J.K. Huang, L. Dauphinot, M.C. Potier, C. Kerninon, S. Melik Parsadaniantz, R.J. Franklin, C. Lubetzki, Demyelination causes adult CNS progenitors to revert to an immature state and express immune cues that support their migration, J Neurosci 35(1) (2015) 4-20. [77] M.M. Herrmann, S. Barth, B. Greve, K.M. Schumann, A. Bartels, R. Weissert, Identification of gene expression patterns crucially involved in experimental autoimmune encephalomyelitis and multiple sclerosis, Dis Model Mech 9(10) (2016) 1211-1220. [78] J.C. Dugas, A. Ibrahim, B.A. Barres, The T3-induced gene KLF9 regulates oligodendrocyte differentiation and myelin regeneration, Mol Cell Neurosci 50(1) (2012) 45-57. [79] F. Fischbach, J. Nedelcu, P. Leopold, J. Zhan, T. Clarner, L. Nellessen, C. Beissel, Y. van Heuvel, A. Goswami, J. Weis, B. Denecke, C. Schmitz, T. Hochstrasser,
Journal Pre-proof
65
Jo
ur
na
lP
re
-p
ro
of
S. Nyamoya, M. Victor, C. Beyer, M. Kipp, Cuprizone-induced graded oligodendrocyte vulnerability is regulated by the transcription factor DNA damage-inducible transcript 3, Glia 67(2) (2019) 263-276. [80] K.K. Ryan, S.C. Woods, R.J. Seeley, Central nervous system mechanisms linking the consumption of palatable high-fat diets to the defense of greater adiposity, Cell Metab 15(2) (2012) 137-49. [81] S. Lupachyk, P. Watcho, A.A. Obrosov, R. Stavniichuk, I.G. Obrosova, Endoplasmic reticulum stress contributes to prediabetic peripheral neuropathy, Exp Neurol 247 (2013) 342-8. [82] C.K. Gavini, A.L. Bookout, R. Bonomo, L. Gautron, S. Lee, V. Mansuy-Aubert, Liver X Receptors Protect Dorsal Root Ganglia from Obesity-Induced Endoplasmic Reticulum Stress and Mechanical Allodynia, Cell Rep 25(2) (2018) 271-277 e4. [83] H.C. Hsu, C.Y. Chen, B.C. Lee, M.F. Chen, High-fat diet induces cardiomyocyte apoptosis via the inhibition of autophagy, Eur J Nutr 55(7) (2016) 2245-54. [84] X. Wang, Z.F. Zhang, G.H. Zheng, A.M. Wang, C.H. Sun, S.P. Qin, J. Zhuang, J. Lu, D.F. Ma, Y.L. Zheng, The Inhibitory Effects of Purple Sweet Potato Color on Hepatic Inflammation Is Associated with Restoration of NAD(+) Levels and Attenuation of NLRP3 Inflammasome Activation in High-Fat-Diet-Treated Mice, Molecules 22(8) (2017). [85] Y. Hussien, J.R. Podojil, A.P. Robinson, A.S. Lee, S.D. Miller, B. Popko, ER Chaperone BiP/GRP78 Is Required for Myelinating Cell Survival and Provides Protection during Experimental Autoimmune Encephalomyelitis, J Neurosci 35(48) (2015) 15921-33. [86] M.S. Elitt, H.E. Shick, M. Madhavan, K.C. Allan, B.L.L. Clayton, C. Weng, T.E. Miller, D.C. Factor, L. Barbar, B.S. Nawash, Z.S. Nevin, A.M. Lager, Y. Li, F. Jin, D.J. Adams, P.J. Tesar, Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease, Stem Cell Reports 11(3) (2018) 711-726. [87] P. Acs, S. Komoly, Selective ultrastructural vulnerability in the cuprizone-induced experimental demyelination, Ideggyogy Sz 65(7-8) (2012) 266-70. [88] M. Faizi, A. Salimi, E. Seydi, P. Naserzadeh, M. Kouhnavard, A. Rahimi, J. Pourahmad, Toxicity of cuprizone a Cu(2+) chelating agent on isolated mouse brain mitochondria: a justification for demyelination and subsequent behavioral dysfunction, Toxicol Mech Methods 26(4) (2016) 276-83. [89] F. Luo, K. Herrup, X. Qi, Y. Yang, Inhibition of Drp1 hyper-activation is protective in animal models of experimental multiple sclerosis, Exp Neurol 292 (2017) 21-34. [90] R.J. Youle, M. Karbowski, Mitochondrial fission in apoptosis, Nat Rev Mol Cell Biol 6(8) (2005) 657-63. [91] H.Y. Lin, S.W. Weng, Y.H. Chang, Y.J. Su, C.M. Chang, C.J. Tsai, F.C. Shen, J.H. Chuang, T.K. Lin, C.W. Liou, C.Y. Lin, P.W. Wang, The Causal Role of Mitochondrial Dynamics in Regulating Insulin Resistance in Diabetes: Link through Mitochondrial Reactive Oxygen Species, Oxid Med Cell Longev 2018 (2018) 7514383. [92] L. Haider, M.T. Fischer, J.M. Frischer, J. Bauer, R. Hoftberger, G. Botond, H. Esterbauer, C.J. Binder, J.L. Witztum, H. Lassmann, Oxidative damage in multiple sclerosis lesions, Brain 134(Pt 7) (2011) 1914-24.
Journal Pre-proof
66
Jo
ur
na
lP
re
-p
ro
of
[93] M.E. Witte, D.J. Mahad, H. Lassmann, J. van Horssen, Mitochondrial dysfunction contributes to neurodegeneration in multiple sclerosis, Trends Mol Med 20(3) (2014) 179-87. [94] C.M. Rice, M. Sun, K. Kemp, E. Gray, A. Wilkins, N.J. Scolding, Mitochondrial sirtuins--a new therapeutic target for repair and protection in multiple sclerosis, Eur J Neurosci 35(12) (2012) 1887-93. [95] G. Vakilzadeh, F. Khodagholi, T. Ghadiri, A. Ghaemi, F. Noorbakhsh, M. Sharifzadeh, A. Gorji, The Effect of Melatonin on Behavioral, Molecular, and Histopathological Changes in Cuprizone Model of Demyelination, Mol Neurobiol 53(7) (2016) 4675-84. [96] N. Sanadgol, F. Golab, H. Askari, F. Moradi, M. Ajdary, M. Mehdizadeh, Alphalipoic acid mitigates toxic-induced demyelination in the corpus callosum by lessening of oxidative stress and stimulation of polydendrocytes proliferation, Metab Brain Dis 33(1) (2018) 27-37. [97] U. Funfschilling, L.M. Supplie, D. Mahad, S. Boretius, A.S. Saab, J. Edgar, B.G. Brinkmann, C.M. Kassmann, I.D. Tzvetanova, W. Mobius, F. Diaz, D. Meijer, U. Suter, B. Hamprecht, M.W. Sereda, C.T. Moraes, J. Frahm, S. Goebbels, K.A. Nave, Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity, Nature 485(7399) (2012) 517-21. [98] D. Mehta, C. Miller, D.L. Arnold, E. Bame, A. Bar-Or, R. Gold, J. Hanna, L. Kappos, S. Liu, A. Matta, J.T. Phillips, D. Robertson, C.A. von Hehn, J. Campbell, K. Spach, L. Yang, R.J. Fox, Effect of dimethyl fumarate on lymphocytes in RRMS: Implications for clinical practice, Neurology 92(15) (2019) e1724-e1738. [99] Q. Weng, J. Wang, J. Wang, B. Tan, J. Wang, H. Wang, T. Zheng, Q.R. Lu, B. Yang, Q. He, Folate Metabolism Regulates Oligodendrocyte Survival and Differentiation by Modulating AMPKalpha Activity, Sci Rep 7(1) (2017) 1705. [100] R. Schoenfeld, A. Wong, J. Silva, M. Li, A. Itoh, M. Horiuchi, T. Itoh, D. Pleasure, G. Cortopassi, Oligodendroglial differentiation induces mitochondrial genes and inhibition of mitochondrial function represses oligodendroglial differentiation, Mitochondrion 10(2) (2010) 143-50. [101] J. Wang, C. Zhao, P. Kong, H. Sun, Z. Sun, G. Bian, Y. Sun, L. Guo, Treatment with NAD(+) inhibited experimental autoimmune encephalomyelitis by activating AMPK/SIRT1 signaling pathway and modulating Th1/Th17 immune responses in mice, Int Immunopharmacol 39 (2016) 287-94. [102] I. Ziabreva, G. Campbell, J. Rist, J. Zambonin, J. Rorbach, M.M. Wydro, H. Lassmann, R.J. Franklin, D. Mahad, Injury and differentiation following inhibition of mitochondrial respiratory chain complex IV in rat oligodendrocytes, Glia 58(15) (2010) 1827-37. [103] L.R. Stein, S. Imai, Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging, EMBO J 33(12) (2014) 132140. [104] B. Jablonska, M. Gierdalski, L.J. Chew, T. Hawley, M. Catron, A. Lichauco, J. Cabrera-Luque, T. Yuen, D. Rowitch, V. Gallo, Sirt1 regulates glial progenitor proliferation and regeneration in white matter after neonatal brain injury, Nat Commun 7 (2016) 13866.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
67
Journal Pre-proof
ro
of
Graphical abstract
-p
Highlights
Chronic high fat diet impairs TCA cycle function in the mouse spinal cord
Mitochondria dysfunction occurs in the spinal cord after high fat consumption
Excess fat promotes mitochondrial dysfunction in purified oligodendrocytes
Chronic high fat diet causes oxidative stress in the brain and spinal cord
Chronic high fat diet results in oligodendrocyte loss in the brain and spinal cord
Jo
ur
na
lP
re
68
Monica R. Langley, Hyesook Yoon, Ha-Neui Kim, Chan-Il Choi, Whitney Simon, Laurel Kleppe, Ian R. Lanza, Nathan K. LeBrasseur, Aleksey Matveyenko, Isobel A. Scarisbrick PII:
S0925-4439(19)30358-8
DOI:
https://doi.org/10.1016/j.bbadis.2019.165630
Reference:
BBADIS 165630
To appear in:
BBA - Molecular Basis of Disease
Received date:
20 September 2019
Revised date:
14 November 2019
Accepted date:
2 December 2019
Please cite this article as: M.R. Langley, H. Yoon, H.-N. Kim, et al., High fat diet consumption results in mitochondrial dysfunction, oxidative stress, and Oligodendrocyte loss in the central nervous system, BBA - Molecular Basis of Disease(2019), https://doi.org/10.1016/j.bbadis.2019.165630
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof
1
High Fat Diet Consumption Results in Mitochondrial Dysfunction, Oxidative Stress, and Oligodendrocyte Loss in the Central Nervous System Short running title: High fat diet causes oligodendrocyte loss Authors Monica R. Langleya, Hyesook Yoona,b, Ha-Neui Kima, Chan-Il Choia, Whitney Simona, Laurel Kleppea, Ian R. Lanzab,c, Nathan K. LeBrasseura,b, Aleksey Matveyenkob,c, Isobel
ro
of
A. Scarisbricka,b,*.
Affiliations
Department of Physical Medicine & Rehabilitation, Rehabilitation Medicine Research
-p
a
Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN
lP
b
re
Center, Mayo Clinic, Rochester, MN 55905, USA
Department of Endocrinology, Mayo Clinic, Rochester, MN 55905, USA
Jo
ur
c
na
55905, USA
* Correspondence to: Isobel A. Scarisbrick, Ph.D., Neurobiology of Disease Program, Physical Medicine a& Rehabilitation, Rehabilitation Medicine Research Center, 642B Guggenheim Building, Mayo Clinic, Rochester, 200 First St SW, Rochester, MN 55905, Tel: 507-284-0124, Fax:507-266-4716, Email: [email protected]
Journal Pre-proof
2
Abstract Metabolic syndrome is a key risk factor and co-morbidity in multiple sclerosis (MS) and other neurological conditions, such that a better understanding of how a high fat diet contributes to oligodendrocyte loss and the capacity for myelin regeneration has the potential to highlight new treatment targets. Results demonstrate that modeling metabolic dysfunction in mice with chronic high fat diet (HFD) consumption promotes
of
loss of oligodendrocyte progenitors across the brain and spinal cord. A number of
ro
transcriptomic and metabolomic changes in ER stress, mitochondrial dysfunction, and
-p
oxidative stress pathways in HFD-fed mouse spinal cords were also identified.
re
Moreover, deficits in TCA cycle intermediates and mitochondrial respiration were observed in the chronic HFD spinal cord tissue. Oligodendrocytes are known to be
lP
particularly vulnerable to oxidative damage, and we observed increased markers of
na
oxidative stress in both the brain and spinal cord of HFD-fed mice. We additionally identified that increased apoptotic cell death signaling is underway in oligodendrocytes
ur
from mice chronically fed a HFD. When cultured under high saturated fat conditions,
Jo
oligodendrocytes decreased both mitochondrial function and differentiation. Overall, our findings show that HFD-related changes in metabolic regulators, decreased mitochondrial function, and oxidative stress contribute to a loss of myelinating cells. These studies identify HFD consumption as a key modifiable lifestyle factor for improved myelin integrity in the adult central nervous system and in addition new tractable metabolic targets for myelin protection and repair strategies.
Keywords high fat diet, myelin, oligodendrocyte, mitochondria, apoptosis
Journal Pre-proof Abbreviations 4-HNE= 4-hydroxynonenal aNSCs= adult neural stem cells CLAMS= Comprehensive Lab Animal Monitoring System EDSS= expanded disability status score EAE= experimental autoimmune encephalitis
of
HFD= high fat diet
ro
MetSyn = metabolic syndrome MS= multiple sclerosis
-p
MBP= myelin basic protein (MBP
re
OLC= oligodendrocyte lineage cell
lP
OPCs= Oligodendrocyte progenitor cells
Olig2= oligodendrocyte lineage transcription factor 2
na
PLP= proteolipid protein RER= respiratory exchange rate
ur
RRMS= relapsing remitting MS
Jo
SPMS= secondary progressive MS TCA= tricarboxylic acid
VWMD= vanishing white matter disease
3
Journal Pre-proof
4
1. INTRODUCTION White matter pathology including myelin sheath abnormalities, demyelination, loss of oligodendrocytes, and impaired myelin repair processes are significant pathophysiological characteristics of diverse neurological conditions, including multiple sclerosis (MS), Alzheimer’s disease, neuropsychiatric disorders, and traumatic brain and spinal cord injury [1-6]. Oligodendrocytes not only serve as insulation by
of
ensheathing axons and allowing for increased speed and temporal control of action
ro
potential propagation, but also provide key metabolic and trophic support for neurons [1,
-p
7, 8]. Development of new strategies to prevent oligodendrocyte loss and guide efficient
re
oligodendrogenesis and myelination has the potential to therapeutically benefit individuals affected by white matter-related neurological injury and disease. Since
lP
oligodendrocyte function is intimately linked to neighboring glial cells and neuronal
na
activity, in addition to microenvironmental factors, the most promising interventions will
ur
likely involve a combinatorial approach [1, 9].
Jo
Obesity during childhood or adolescence is associated with an increased risk of developing MS [10-12]. Recent studies support a causal role for elevated body mass index (BMI) in MS etiology which is further increased when interacting with other known genetic and environmental risk factors such as HLA polymorphisms or Epstein Barr virus positivity [10, 13, 14]. Interestingly, metabolic dysfunction resulting from excessive dietary fat intake may also influence the initiation, progression, or disability burden of MS [13, 15, 16]. In terms of conversion from relapsing remitting (RRMS) to secondary progressive (SPMS), elevated BMI is associated with an increased risk of SPMS in smokers, but not non-smokers [15]. There is also a significant positive association
Journal Pre-proof
5
between higher BMI or adverse lipid profile and higher Expanded Disability Status Score (EDSS) [16]. Since obesity and metabolic syndrome are common co-morbidities which impact quality of life for individuals with MS, recent studies have evaluated the utility of drugs and interventions to overcome metabolic syndrome as a strategy to improve patients’ physical and mental health or radiological outcomes [16-18].
of
Disruptions in brain lipid metabolism, serum lipid profiles, and cholesterol levels
ro
have all been reported during active disease in MS [16, 19]. How to manage dietary fat consumption for optimal CNS health is still controversial since accurate myelin
-p
assembly requires large amounts of lipids and fatty acid synthesis, yet overconsumption
re
of saturated fats is detrimental for overall brain function [19, 20]. HFD consumption
lP
exacerbates both autoreactive immune responses and neuroinflammation in experimental autoimmune encephalitis (EAE) [21, 22]. Metabolic derangement resulting
na
from obesogenic diets or excessive saturated fatty acids can lead to oxidative and ER
ur
stress responses and ultimate death of vulnerable cell types [23-25]. Elevated levels of oxidative stress and ER stress-related proteins have been observed in MS lesions [26,
Jo
27]. Oligodendrocytes are particularly sensitive and susceptible to oxidative stress having extremely high energetic and ER demands in order to support myelin membranes [28]. Moreover, our recent findings show that chronic consumption of a Western style diet, containing high fat and high sucrose, causes significant loss of oligodendrocytes and their progenitors in the intact adult murine spinal cord [29]. Therefore, to elucidate mechanisms underlying oligodendrocyte loss observed in the context of a Western diet, here we specifically address whether similar effects occur with consumption of a diet high in saturated fat alone. The brain and spinal cord of mice
Journal Pre-proof
6
consuming a regular or a high fat diet for 4 to 12 wk were analyzed using transcriptomic, metabolomic, functional, and immunohistochemical outcome measures to reveal potential mechanisms of oligodendrocyte loss. These approaches identified perturbations in mitochondrial function among the key mechanisms by which HFDconsumption is likely to impair myelinating cell function in vivo. Parallel disruptions in mitochondrial function and differentiation were also identified when HFD was modeled
of
in cultures of purified oligodendrocytes grown under high saturated fat conditions.
ro
Altogether these findings highlight new pathogenic mechanisms by which excess
-p
dietary fat contributes to myelin injury in the adult brain and spinal cord and uncover
lP
2. MATERIALS AND METHODS
re
new targets for therapy.
2.1 Experimental Method Details
na
2.1.1 Animal husbandry and diet
ur
All diets, procedures, and experiments were approved by The Mayo Clinic
Jo
Institutional Animal Care and Use Committee (IACUC) and performed in accordance with appropriate institutional and regulatory guidelines. Male C57BL6/J mice were obtained from Jackson Labs (Bar Harbor, ME) at 8 wk of age and were randomly assigned to feeding on either a regular diet (RD, 10 % kcal fat, D12450K, Research Diets, New Brunswick, NJ) or a high fat diet (HFD, 60% kcal fat, D12492, Research Diets, New Brunswick, NJ) beginning at 10 wk of age. Saturated fat comprised 22.7 and 32% of the total fat content within the diets, respectively. Carbohydrate comprised 70% kcal for RD and 20% HFD, while both diets had 20% kcal protein. Mice were
Journal Pre-proof
7
provided ad libitum access to the diets along with tap water and marked by tail tattoo for individual identification. Food intake and body weights were monitored weekly. 2.1.2 Glucose tolerance test Following a 6 h fast, baseline glucose levels were measured by needle puncture of the tail of mice consuming RD or HFD for 3 wk (Bayer Breeze2, Bayer, Mishawaka, IN). Next, an intraperitoneal injection of glucose (1g/kg) was administered and blood
of
glucose levels measured again at each time point (0, 15, 30, 60, 90, 120 min) as
ro
previously described [30].
-p
2.1.3 Behavioral Assessment
re
Behavioral analysis was performed at baseline and following 4 or 12 wk of RD or HFD consumption. To examine spontaneous movement, mice were placed in a
lP
chamber (27 x 27 x 20.3 cm, Med Associates Inc., St. Albans, VT) equipped with
na
infrared beams to record horizontal and vertical movements over a 30 min interval during the light phase. Mice were allowed to acclimate to the room conditions for 1 h
ur
prior to recording at 50 ms resolution in Activity Monitor Software. Equipment was
Jo
cleaned with 70% ethanol between mice. The same testing chamber and period was also used to evaluate anxiety-like behavior by quantifying the amount of time a mouse spent in the center zone (4.5, 4.5 to 12, 12 cm) versus the residual area. 2.1.4 Immunohistochemistry 4% Paraformaldehyde-fixed, paraffin-embedded brain and lumbosacral spinal cord tissues were cut to 6 µm and utilized for all immunostaining procedures. Immunoperoxidase and immunofluorescence was performed as previously described [29]. Briefly, sections were washed, steamed in antigen retrieval solution (sodium
Journal Pre-proof
8
citrate,10 mM, pH 6.0), blocked (20 % normal goat serum with 0.25 % Triton-x), then incubated in primary antibodies for Olig2 (Millipore, MABN50, 1:200), CC-1 (Millipore, OP80, 1:50), Nkx2.2 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa city, IA), NG2 (Millipore, ab5320), 4-HNE (Abcam, ab46545, 1:500), Bax (Invitrogen, MA514003,1:200 ), Bcl-2 (Abcam, AB59348, 1:500), cleaved caspase-3 (Cell Signaling, 9664, 1:1000), GFAP (Abcam, ab4647, 1:2000) for 18 h. For
of
immunoperoxidase staining, subsequent incubation with an appropriate biotinylated
ro
secondary antibody (Jackson, 1:200) followed by incubation with an avidin peroxidase
-p
solution (Vector) was used to yield a brown reaction product after incubation with 3,3-
re
diaminobenzidine (DAB, Sigma D5637). Immunostained sections were counter stained with methyl green (Sigma, 67060), dehydrated, cover slipped using DPX Mountant
lP
(Sigma), and imaged using an Olympus BX51 microscope (Olympus, Center Valley,
na
PA).
Immunofluorescence staining was achieved by incubating with appropriate
ur
fluorochrome-conjugated secondary antibodies (Jackson, 1:200) and counterstaining
Jo
with 4’,6’-diamindino-2-phenylindole (DAPI). Cell counting and thresholding of images from the dorsal column of the spinal cord and corpus callosum of the brain was performed in ImageJ (NIH) without knowledge of treatment groups using cropped regions of interest averaged from 2-3 sections per animal with at least n= 4 per group. 2.1.5 Western blotting One half of the spinal cord (cervical and thoracic) was homogenized in radioimmunoprecipitation buffer and 35 µg protein per sample was resolved by SDS-PAGE (Bio-Rad Laboratories, Hercules, CA) and electroblotted as described previously [29].
Journal Pre-proof
9
Membranes were probed for AMPK (Cell Signaling, 2532S), pAMPK (Cell Signaling, 2531S), SIRT1 (Abcam, Ab121193), PGC1α (Abcam, Ab54481), and β-actin (Novus Biological, NB600-501). Detection was accomplished using appropriate HRPconjugated secondary antibodies (GE Healthcare), Pierce chemiluminescent developer (ThermoFisher), and ImageLab 2.0 Software (Bio-Rad). Relative optical density (ROD) for each protein was normalized to the actin of the same membrane.
of
2.1.6 RNA isolation, qPCR, and sequencing
ro
Total RNA was isolated from half of the spinal cords or primary murine cells
-p
using RNA Stat60 (Tel-Test, Friendswood, TX) per manufacturer’s instructions and
re
used for qPCR using BioRad iTaq Universal Probes (ThermoFisher) or primers (Integrated DNA Technologies) and SYBR Green One-step kits. RNA expression of
lP
each gene was normalized to Rn18s.
na
RNA libraries were prepared using 200 ng of total RNA according to the manufacturer’s instructions for the TruSeq RNA Sample Prep Kit v2 (Illumina, San
ur
Diego, CA). The concentration and size distribution of the completed libraries was
Jo
determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). Libraries were sequenced at 100 million reads per sample following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 X 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.20 collection software. Base-calling was performed using Illumina’s RTA version 2.5.2. One regular diet-fed mouse was excluded because of poor sequencing.
Journal Pre-proof
10
mRNA-seq data was processed by the Mayo Bioinformatics Core Facility to identify genes with differential expression among groups. MAP-RSeq [31] workflow (v1.2.1.3) was used to process mRNA-seq data, including read alignment, quality control, gene expression quantification and finally summarizing the data across samples. Paired-end reads were aligned by TopHat (v2.0.12) against the mouse genome build (mm10) using the bowtie1aligner [32], and the gene counts were
of
generated using Subread package [33] (v1.4.4). The workflow also provides detailed
ro
quality control metrics across genes using the RSeQC [34] (v2.3.2).
-p
Differential expression analysis was performed with edgeR [35] package (v2.6.2)
re
to identify genes with altered expression. A cutoff for false discovery rate-adjusted pvalue was set at < 0.05 to determine the genes with significant expression changes
lP
between conditions. The differential genes were further submitted to Ingenuity Pathway
na
Analysis (IPA®) for pathway enrichment analysis and causal network analysis. For causal network analysis (also described as “master regulator analysis”), only directed
ur
and experimentally observed interactions with a false discovery rate of less than 0.1
Jo
were used. The overall heatmap represents RPKM values representing the entire gene profile differences between groups irrespective of FDR organized by hierarchical clustering with the row distance of one minus Pearson correlation, while the scatterplot depicts only significantly differentially expressed genes based on a FDR of less than 0.15. Heatmaps derived from select PantherGo Pathways as well as the scatterplots from 500 gene sets for each cell type [36] were represented regardless of FDR. 2.1.7 Qualitative large-scale metabolite profiling and data analysis
Journal Pre-proof
11
Untargeted metabolomics analysis was performed by the Metabolomics Resource Core Facility at Mayo Clinic in spinal cord tissues of RD or HFD-fed animals by adapting previously described methods [37]. At study endpoints, spinal cords were quickly removed by flushing PBS through the vertebral column, split in half vertically and immediately snap frozen on dry ice for subsequent metabolic and transcriptomic analyses. Briefly, spinal cord samples that were stored at -80 C were pulverized in liquid
of
nitrogen using a custom mortar and pestle into a fine powder. Accurate weight was
ro
obtained using an analytical balance making sure the pulverized sample remained
-p
frozen. Pulverized samples were kept at -80 0C. Before metabolite extraction, samples
re
were thawed on ice for 15 min and 1X PBS was added to make a 100 mg/ml homogenate. An aliquot of 50 µl (~ 5 mg tissue) was used for metabolite extraction. 13
lP
C6-phenylalanine (2 µl at 250 ng/µl) was added as internal standard (IS) to all samples
na
and tubes were sonicated in the ice bath for 2 min (30 sec x 4 pulses). Samples were then deproteinated with cold acetonitrile: methanol (1:6 ratio), kept on ice with
ur
intermittent vortexing and centrifuged at 18000xg for 30 min at 4 C. The supernatant
Jo
was divided into 2 aliquots and dried down using a stream of nitrogen gas for analysis on a Quadruple Time-of-Flight Mass Spectrometer (Agilent Technologies 6550 Q-TOF) coupled with an Ultra High Pressure Liquid Chromatograph (1290 Infinity UHPLC Agilent Technologies). Profiling data was acquired under both positive and negative electrospray ionization conditions over a mass range m/z of 100 - 1700 at a resolution of 10,000 (separate runs) in scan mode. Metabolite separation was achieved using two columns of differing polarity, a hydrophilic interaction column (HILIC, ethylene-bridged hybrid 2.1
Journal Pre-proof
12
x 150 mm, 1.7 mm; Waters) and a reversed-phase C18 column (high-strength silica 2.1 x 150 mm, 1.8 mm; Waters) with gradient described previously [37]. Run time was 18 min for the HILIC and 27 min for the C18 column using a flow rate of 400 µl/min. A total of four runs per sample were performed to give maximum coverage of metabolites. Samples were injected in duplicate, wherever necessary, and a pooled quality control (QC) sample, made up of all of the samples from each study was injected several times
of
during a run. A separate plasma quality control (QC) sample was analyzed with pooled
ro
QC to account for analytical and instrumental variability. Dried samples were stored at -
-p
20 C until analysis. Samples were reconstituted in running buffer and analyzed within
re
48 h of reconstitution. Auto-MS/MS data was also acquired with pooled QC sample to aid in unknown metabolite identification using fragmentation pattern. The tissue pellet
lP
from pulverized samples remaining after the extraction was stored at -80 C.
na
Total protein content from the tissue pellet was determined using Pierce BCA protein assay kit using a Spectramax Plus microplate reader. Briefly, the tissue pellet,
ur
after metabolite extraction, was solubilized in 0.3 M sodium hydroxide and 10 µl lysate
Jo
was used to determine total protein concentration. Protein determination was performed in triplicate and bovine serum albumin (BSA) was used to generate the standard curve. Data alignment, filtering, univariate, multivariate statistical and differential analysis was performed using Mass Profiler Professional (Agilent Inc, USA). Metabolites detected in at least ≥ 80% of one of two groups were selected for differential expression analyses. Metabolite peak intensities and differential regulation of metabolites between groups were determined as described previously [37]. Each
Journal Pre-proof
13
sample was normalized to the protein content and log2 transformed. Unpaired t-test with multiple testing correction p < 0.05 was used to find the differentially expressed metabolites between two groups. Missing values are excluded from the analysis for the calculation of fold change and p-values. Default settings were used with the exception of signal-to-noise ratio threshold (3), mass limit (0.0025 units), and time limit (9 s). Putative identification of each metabolite was done based on accurate mass (m/z)
of
against METLIN database using a detection window of ≤ 7 ppm. The putatively
ro
identified metabolites were annotated as Chemical Abstracts Service (CAS), Kyoto
-p
Encyclopedia of Genes and Genomes (KEGG), Human Metabolome Project (HMP) database, and LIPID MAPS identifiers. The differentially expressed metabolites are
re
analyzed for pathway enrichment using MetaCore (Genego, St. Joseph, MI) – this
lP
identifies pathways using 3 metabolite identifiers – that is, KEGG, CAS and HMDB.
na
2.1.8 Targeted TCA cycle metabolomics
Spinal cord levels of tricarboxylic acid (TCA) cycle metabolites were measured
ur
using the same samples prepared for untargeted metabolomics after the derivatization
Jo
with N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide + 1% tbutyldimethylchlorosilane by gas chromatography/mass spectrometry under electron impact and selected ion monitoring conditions as previously described [37]. All measurements were performed against 12-point calibration curves that underwent the same derivatization with internal standard. 2.1.9 Mitochondrial functional and structural analyses Oroboros oxygraph O2k was used for high resolution respirometry readings from isolated spinal cord mitochondria and Seahorse extracellular flux assay (Agilent) was
Journal Pre-proof
14
used to assess mitochondrial function in cell cultures as described in more detail in our previous publications [38]. Oxygen consumption rates (OCR) were normalized to BCA protein estimation. In brief, fresh whole spinal cord tissue was placed in mitochondria isolation buffer (225 mM Mannitol, 75 mM sucrose, 20 mM MOPS, 1 mM EGTA, 0.5 mM DTT) containing 0.05% Nargase (w/v)/1g tissue and homogenized for 10 min. Next, differential centrifugation was used to isolate mitochondria from other fractions, the
of
mitochondria pellet was resuspended in mitochondria isolation buffer with 0.02%
ro
Digitonin for 10 min, then centrifuged and resuspended again in fresh buffer (125 µl/100
-p
mg tissue) for running the assay. 50 µl of isolated mitochondria suspension was run for
re
each of two technical duplicates. The oxygraph chamber was pre-washed and filled with respiration buffer (120 mM mannitol, 40 mM MOPS, 5mM KH 2PO4, 0.1mM EGTA,
lP
60mM KCl, 5 mM MgCl2) to equilibrate and oxygenate for at least 30 minutes. Oxygen
na
calibrations for ambient air and zero were acquired for each chamber during each run. Oxygen consumption rates were measured in DatLab (Oroboros) after adding the
ur
mitochondria suspension (state I), glutamate (10 mM)/Malate (2 mM)/succinate (10 mM)
Jo
mixture (state II), ADP (14.25 mM), oligomycin (2 µg/ml), FCCP (0.005 mM) titrations, and antimycin A (2.5 mM) using Hamilton syringes. State 3 mitochondrial respiration was assessed following the addition of glutamate, malate, and succinate and ADP to the chamber, giving the basal respiration rate for complexes I and II. Oligomycin injection then inhibited ATP synthase to induce state 4 respiration, attributable to proton leak across the inner mitochondrial membrane. FCCP addition results in an uncoupled state, allowing for determination of maximum capacity, while non-mitochondrial respiration was assessed after antimycin inhibits complex III. State III respiration measurements
Journal Pre-proof
15
were taken from a stable segment following the addition of ADP, while State IV measurements followed oligomycin injection. Maximum respiration was recorded during the highest segment of uncoupled respiration following the FCCP titrations, and nonmitochondrial respiration was measured after anitmycin A addition. All measurements were normalized to protein content of the mitochondria fraction. The Seahorse XF24 (Agilent) extracellular flux assay was used to monitor
of
oxygen consumption rates at various stages of respiration in primary murine
ro
oligodendrocytes as previously described [39]. Following cartridge equilibration with
-p
injection ports filled with oligomycin (1 μg/ml), FCCP (1 μM), and antimycin A (10 μM), a
re
plate pre-treated in serum-free media was placed into the Seahorse analyzer. OCR readouts at each stage were measured in pmol/min and normalized to protein.
lP
For structural analysis of mitochondria, 0.09 x 106 cells primary oligodendrocytes
na
were grown per well on poly-L-lysine (PLL, 10 μg/ml) coated glass coverslips in 24-well plates, treated with 0 or 100 μM palmitate (PA) for 24 h, and then washed twice with
0
ur
fresh media. CMXRos MitoTracker Red (ThermoFisher, 100 nM) was incubated at 37
Jo
C for 30 min. Immunocytochemistry procedures and DAPI counterstaining were then
performed. ImageJ was used to quantify mitochondrial parameters as described previously [39, 40]. Averaged values are reported from analyzed cells within five separate images per coverslip. 2.1.10 Primary glia isolation, OLC culture Mixed glial preparations from postnatal day 0-3 murine cerebral cortex were cultured in DMEM (Fisher 11960-044) with 1 mM sodium pyruvate (Corning 11360070), 20 mM HEPES (Gibco 15630-080), 100 U/ml PenStrep (Life Technologies 15140122),
Journal Pre-proof
16
and 10% fetal bovine serum (FBS, Atlanta Biologicals S11150) for 10-12 d. Oligodendrocytes, astrocytes, and microglia cultures were separated by differential adhesion/shaking overnight at 225 rpm on an Innova 2000 orbital shaker as previously described [41]. For oligodendrocyte treatments and differentiation, 0.09 x 10 6 cells were plated on each well of PLL-coated coverslips or 24 well plates and grown in Neurobasal A
of
media (Life Technologies, 10888022) with N2 (17502048), B27 (17504044), PenStrep,
ro
sodium pyruvate, 2 mM glutamax (35050-061), and 5% BSA (Sigma A4503) for two d
-p
before treating with palmitate (PA, 100 µM, 24 h). This dose was lower than toxic
re
amounts previously reported in other cells types at the same time point [42, 43] and did not result in significant lipotoxicity to oligodendrocytes or aNSCs. RNA levels for MBP,
lP
PLP, and Olig2 were determined in technical duplicates using and an iCycler iQ5
na
system (BioRad) with primers identified within the Key Resources Table. Relative starting quantities were normalized to Rn18S and reported as percent of control. PLP
ur
protein levels were determined based on threshold of immunoreactivity from five fields
Jo
per coverslip quantified in ImageJ. 2.1.11 Adult neural stem cell culture Adult neural stem cells were isolated from the subventricular zone (SVZ) of 8 wk old C57Bl6/J mice as previously described [44]. Briefly, mice were anesthetized with sodium pentobarbital and decapitated to collect coronal brain sections and further dissect stem cells from within the SVZ. Collected cells were dissociated with Accutase and grown in DMEM/F12 with B27, antibiotic-antimycotic, insulin (20 µg/mL), epidermal
Journal Pre-proof
17
growth factor (EGF, 20 ng/mL) and basic fibroblast growth factor (bFGF, 20 ng/mL, PeproTech, Rocky Hill, NJ) in T75 tissue culture treated flasks as neurospheres. For myelin RNA and protein expression experiments, cells were dissociated and plated onto poly-L-ornithine (20 µg/ml) and laminin (10 µg/ml) coated 6 well plates and glass coverslips, respectively. To induce differentiation toward oligodendrocyte lineage, cells were switched to media containing Neurobasal A, antibiotic-antimycotic, B27,
of
insulin (20 µg/mL), T3 (60 ng/ml), glutamax (2 mM) and cultured for an additional 72 h.
ro
To identify effects of PA (100µM) on differentiation to oligodendrocyte lineage, we
-p
compared protein and RNA marker expression to controls by immunofluorescence
re
staining and qRT-PCR. 2.1.12 Statistical analysis
lP
Data were tested for normality using the Shapiro-Wilk test, graphed, and analyzed by
na
Student’s t-test unless noted otherwise in GraphPad Prism 7.0 software. Mann-Whitney nonparametric test was used for histological analysis of 4-HNE immunoreactivity in
ur
brain and spinal cord tissue, while body weight, food intake, and glucose tolerance test
Jo
in mice were analyzed by two-way ANOVA with Bonferroni post-test. p-values ≤ 0.05 were considered significant.
Journal Pre-proof 2.2 KEY RESOURCES TABLE
Abcam
ab28486; AB_776593
AB12193; AB_298923 74.5A5; AB_531794
ab5320; AB_11213678
lP
na
Jo
rabbit anti-4hydroxynonenol rabbit anti-cleaved caspase-3 rabbit anti-Bcl-2 rabbit anti-Neurofilament biotinylated goat anti-rat secondary biotinylated donkey antirabbit secondary biotinylated donkey antimouse secondary goat-anti-rabbit AF488 secondary goat anti-rat Cy3
2531S; AB_330330
Millipore
MAB386; AB_94975
Abcam Novus Biological Abcam
Ab54481; AB_881987 NB600-501; AB_10077656
Cell Signaling Technology Abcam Sigma Aldrich Jackson
9664; AB_10694088
Jackson
711-065-152; AB_2340593
Jackson
715-066-151; AB_2340788
Jackson
111-545-047; AB_2338051
Jackson
112-166-072; AB_2338257
ur
anti-Neural glial antigen2 (NG2) rabbit anti-Proteolipid protein (PLP) rat anti-Myelin basic protein (MBP) rabbit anti-PGC1-α mouse anti-ᵝ-actin
MABN50; AB_10807410 MA514003; AB_10979735 Ab9610; AB_10141047 Ab16794; AB_443473 2532S; AB_330331
re
mouse anti-SIRT1 anti-Nkx2.2
Millipore Invitrogen Millipore Abcam Cell Signaling Technology Cell Signaling Technology Abcam Development al Studies Hybridoma Bank, University of Iowa, Iowa city, IA Millipore
of
rabbit anti-pAMPK
Identifier
ro
Antibodies mouse anti-Olig2 rabbit anti-BAX rabbit anti-Olig2 mouse anti-CC-1 rabbit anti-AMPK
Source
-p
Reagent or Resource
ab46545; AB_722490
AB59348; AB_2064155 N4142; AB_477272 112-066-072; AB_2338185
18
Journal Pre-proof
FIS1
PINK1
SOD2 GFAP
SOD2 Rn18s
Primary isolation (Yoon 2015) Primary isolation (Choi 2017)
N/A
The Jackson Laboratory
N/A
of
N/A
NM_016967; Mm.PT.58.42319010
lP
Applied Biosystems Integrated DNA Technologies Integrated DNA Technologies Applied Biosystems Integrated DNA Technologies Integrated DNA Technologies
ur Jo
PGC1-α
711-166-152; AB_2313568
na
MBP
PLP
Jackson
re
oligonucleotides, (primers/probes) OLIG2
111-545-041; AB_2341130
ro
Experimental models: cell lines Mouse:C57BL/6 oligodendrocyte lineage cells (OLCs) Mouse:C57BL/6 adult neural stem cells (aNSCs) Experimental models: organisms/strains Mouse: C57BL/6J
Jackson
-p
secondary goat anti-rabbit AF488 secondary donkey anti-rabbit Cy3 secondary
19
Integrated DNA Technologies Applied Biosystems Applied Biosystems
NM_001025251; CCAGTAGTCCATTTCTTCAAGAACAT/GC CGATTTATAGTCGGAAGCTC NM_011123.2; TCTTTGGCGACTACAAGACCAC/CACAA ACTTGTCGGGATGTCCTA Mm01208835_m1 Mm.PT.56a.21878911
Mm.PT.58.23711353
Mm01313000_m1 NM_010277.2; GCAGATGAAGCCACCCTGG/ GAGGTCTGGCTTGGCCAC NM_013671.3; Mm01313000_m1 NR_003278.3; Mm03928990_g1
Journal Pre-proof
Chemicals, Peptides, and recombinant proteins palmitic acid dapi Mitotracker Red
20
Sigma Aldrich Cat#P0500 ThermoFisher Cat#D1306; AB_2629482 ThermoFisher Cat#m7512
Commercial kits Seahorse XF Cell MitoStress Test Pierce BCA protein estimation
Agilent
Cat#103015-100
Other Regular diet (10 kcal % fat) High fat diet (60 kcal % fat)
Research Diets Research Diets
https://www.graphpad.com/scientificsoftwar e/prism https://imagej.net/Fiji
D12450K D12492
Jo
ur
na
lP
GraphPad Prism 7
https://www.med-associates.com/
-p
Fuji ImageJ
Med Associates Inc GraphPad Software NIH
re
Software and Algorithms Activity Monitor Software
ro
of
ThermoFisher Cat#23225
2.3 CONTACT FOR REAGENT AND RESOURCE SHARING Additional information and requests for resources can be directed to the corresponding author/Lead Contact, Isobel Scarisbrick ([email protected]).
Journal Pre-proof
21
3. RESULTS 3.1 Chronic high fat diet consumption impairs metabolic parameters and diminishes the number of oligodendrocytes and oligodendrocyte progenitors in the central nervous system Since our prior studies revealed that a diet high in fat and sucrose results in a loss of myelinating cells and their progenitors in the spinal cord [29], we chose to
of
investigate the differential impact of high fat alone and any temporal effects by feeding
ro
10 wk old male C57Bl/6 mice a regular diet (RD, 10% of kcal from fat) or high fat diet
-p
(HFD, 60% kcal from fat) for either 4 or 12 wk (Figure 1A). As expected, the HFD mice appeared noticeably larger (Figure 1B) compared to the RD-fed controls and consumed
re
more kcal of food per day (Figure 1C). Beginning at 3 wk through 12 wk, mice also
lP
gained a significant amount of weight (Figure 1D). After 3 wk, glucose sensitivity was
na
assessed using a glucose tolerance test. As expected, HFD-fed mice demonstrated significantly higher fasting blood glucose levels and impaired glucose tolerance (Figure
ur
1E, F) [30]. A set of RD and HFD mice were monitored in a Comprehensive Lab Animal
Jo
Monitoring System (CLAMS) to reveal significant changes in several metabolic parameters including percent fat/lean mass, VO2, VCO2, respiratory exchange rate (RER), and metabolic rate (Supplemental Figure 1). In a separate cohort, a number of movement measurements were also affected by HFD consumption including anxietylike behavior and decreased vertical motion at 4 wk, and also involving horizontal activity by 12 wk (Supplemental Figure 2). After establishing the systemic and behavioral effects of HFD consumption in mice, we next tested the direct consequences on oligodendrocyte lineage cells (OLCs).
Journal Pre-proof
22
At study endpoints, spinal cord tissues were separated into cervical-thoracic and lumbosacral segments for analysis as depicted in (Figure 1G). The impact of consuming a HFD on myelin producing cells was determined by counting OLC markers in the postfixed lumbosacral spinal cord of each group. Oligodendrocyte progenitor cells (OPCs) were identified using antibodies recognizing NG2, Nkx2.2, and Olig2 (oligodendrocyte lineage transcription factor 2, marks progenitors and early mature cells), while mature
of
oligodendrocytes were considered CC-1+. After 4 wk of HFD, no change in the number
ro
of Olig2+ cells was observed in the spinal cord dorsal column (Figure 1H). RNA
-p
expression of oligodendrocyte markers Olig2 (Figure 1I), myelin basic protein (MBP,
re
Figure 1J), and proteolipid protein (PLP, Figure 1K) remained unchanged after 12 wk of HFD-consumption. By contrast counts of oligodendrocyte progenitors immunoreactive
lP
for Nkx2.2 (Figure 1L, 40x, and Supplemental Figure 3A , 20x), NG2 (Figure 1 M, 40x,
na
and Supplemental Figure 3B, 20x) and Olig2 (Figure 1N, 40x, and Supplemental Figure 3C, 20x) were all reduced at 12 wk. Furthermore, counts of CC-1+ mature
ur
oligodendrocytes (Figure 1O, 40x, and Supplemental Figure 3D, 20x) demonstrated a
Jo
significant depletion. We did not observe a significant change in Olig2 (Supplemental Figure 4A, B), in MBP isoforms (Supplemental Figure 1A, C and Supplemental Figure 5 E-H) or PLP (Supplemental Figure 5 A,D) protein expression by Western blot. Considering we observed a loss of oligodendrocytes and their progenitors in the spinal cord at 12 wk of HFD consumption, we next examined any regional specificity by parallel quantification in the brain. The corpus callosum (Figure 1P) from mice fed a HFD had fewer Olig2+ cells (Figure 1Q) at 12 wk, but staining for MBP (Supplemental Figure 3E), a marker of myelin structural integrity, and mature oligodendrocytes (CC-1+,
Journal Pre-proof
23
Supplemental Figure 3F) were unaffected. Taken together, these findings suggest that oligodendrocyte progenitors in both the brain and spinal cord are vulnerable to death in response to chronic HFD consumption; however, only spinal cord mature oligodendrocytes appeared susceptible to HFD-induced toxicity at the timepoint examined. Interestingly, remaining oligodendrocytes within both central nervous system
Jo
ur
na
lP
re
-p
ro
of
(CNS) regions maintained normal PLP and MBP levels at all time points examined.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
24
Journal Pre-proof
25
Fig 1. Chronic high fat diet consumption impairs metabolic parameters and diminishes the number of oligodendrocytes and progenitors in the central nervous system. A, Schematic depicting experimental design in which 10 wk old male mice were fed a regular diet (RD) or a high fat diet (HFD) for 4 or 12 wk. Representative images show mice consuming a HFD gained weight (B) after consuming more kcal food per day (C, n=3 cages of 5 mice per group). HFD mice gained weight (D, n=10 per
of
group. RD 25.84 g and HFD 24.97 g at study start; RD 27.53 g and HFD 43.02 g at
ro
study end) and had impaired glucose tolerance after 3 wk (E, F; AUC=area under the
-p
curve, n=8 mice per group). Graphical results represented as the mean ± SEM. G, Schematic shows spinal cord regions used in analyses. H, Representative images from
re
immunostained sections of spinal cord dorsal column and counts of the number of
lP
Olig2+ cells at 4 wk (n=5 mice per group). RNA expression of Olig2 (I), MBP (J), and
na
PLP (K) at 12 wk (n=4 mice per group). Representative images of dorsal column and corresponding counts of Nkx2.2+ (L), NG2+ (M), Olig2+ (N), and CC-1+ (O) cells after
ur
12 wk (n=7 mice per group). P, Schematic depicting region of corpus callosum
Jo
examined for immunostaining. Representative images from immunostained sections of the corpus callosum (Q) and counts of Olig2+ cells at 12 wk (n=5 mice per group). Bar graphs shown represent the mean number of cells per area of spinal cord ± SEM and are expressed as percent of RD-fed control mice. (*, p<0.05; **, p<0.01; and ***, p<0.001). Scale bar = 20 μm (L-O) and 50 μm (Q).
Journal Pre-proof
26
3.2 Chronic high fat diet consumption differentially regulates the transcriptional profile of the adult mouse spinal cord To begin to address the mechanisms driving oligodendrocyte depletion in the face of HFD-consumption, we performed RNA sequencing from half of each spinal cord as demonstrated in Figure 2A. An overall heatmap (Figure 2B and Supplemental Figure 4) depicts differential gene expression in the spinal cords of mice consuming a RD (n=3)
of
and HFD (n=4) for 12 wk, where blue is the relative row minimum and red is the relative
ro
row maximum. Using a false discovery rate (FDR) of P < 0.15, a scatter plot of
-p
differentially expressed genes (DEGs, Figure 2C) showed 75 to be transcriptionally
re
distinct between diet groups (32 up and 43 down). Among these, we observed an upregulation of Hba-a2, Sdf2L1, Hmgcs2, Creld2, Cryab, Hspa5/Grp78, Grn, Kdr,
lP
Ddit4l, Fos, Xbp1, Paqr6, Fn14, Gstp1, Ccdc42ep2, and Litaf. Significantly
na
downregulated genes included Lcn2, Ch25h, Sgk1, Slc17a7, Pdyn, Socs3, Plaur, Dbp, Acer2, Pla2g3, Kirrel2, Cp, Fn1, and Klf9.
ur
A number of ER stress induced genes including Creld2, Xbp1, and Hspa5 were
Jo
upregulated in HFD spinal cord. Not surprisingly, ingenuity canonical pathway analysis revealed ER Stress and the Unfolded Protein Response pathways as top pathways affected in spinal cord tissue by HFD consumption (Table 1). Next, to identify transcription factors which may act as master regulators for the observed transcriptional changes, we performed a causal network analysis with only directed and experimentally observed interactions with a FDR less than 0.1 (Table 2). PTK6 (Figure 2D), predicted to be activated, and NFKBIA (Figure 2E), predicted as an inhibited upstream transcriptional regulator, influence expression of Xbp1 and HSPA5, respectively.
Journal Pre-proof
27
Moreover, PDGFRA is a crucial mediator of oligodendrogenesis [45] and was predicted to be inactivated. Loss of Mecp2 in oligodendrocytes down regulates myelin gene expression and was predicted to have lower activation in the HFD mice [46]. We next focused attention on expression of myelin and cholesterol related genes using PantherGO Pathways (Figure 3A), where there was surprisingly a trend for increases in a number of genes, which may reflect a compensatory response in
of
remaining oligodendrocytes and explain why frank loss of myelin was not observed at
ro
the 12 wk endpoint despite the loss of mature myelinating cells and progenitors. Having
-p
characterized overall transcriptomic differences between the spinal cord of mice
re
consuming a RD or HFD, we next used the gene sets described by Zhang et al [36] to profile transcriptional changes enriched at distinct stages of oligodendrocyte lineage
lP
progression (Figure 3B-E), including oligodendrocyte precursors (OPCs, Figure 3C),
na
new OLCs (Figure 3D), and myelinating OLCs (Figure 3E)[36]. Of the OPC-enriched gene set, Slc6a3 (p=0.27), Lox (p=0.18) , Cdc25c (p=0.16), Snora17 (p=0.015), Bglap2
ur
(p=0.79), Gsx1 (p=0.07), Ccnb1 (p=0.18), Alas2 (p=0.10) , Raet1d, and Troap (p=0.19)
Jo
were all increased above 1.5-fold, while Ddx43 (p=0.002) and Cpz were below 0.5-fold in the spinal cord of mice consuming HFD relative to RD (Figure 3C, all genes included regardless of FDR). New OL gene changes included increased Krt28 (p=0.10), S100a14 (p=0.19), Snora26 (p=0.25), 1700001P01Rik (p=0.10), and Sis (p=0.08) and decreased Erp27 (p=0.18), Corin (p=0.09), and Chrng (Figure 3D). In myelinating OLs, Hist1h2bg (p=0.40), 1700001P01Rik (p=0.10), A930003A15Rik (p=0.09), Hist1h2ae (p=0.43), Hist1h4i (p=0.04), and Hist1h2ac (p=0.34) were increased, while Hist1h2bn (p=0.006), Erp27 (p=0.18), and Mpl (p=0.04) were decreased (Figure 3E).
Journal Pre-proof Together, these results suggest that HFD consumption promotes complex transcriptional changes in the spinal cord and in oligodendrocyte lineage cells that include pathways essential for maintaining homoeostasis and highlight several
Jo
ur
na
lP
re
-p
ro
of
important new targets for further investigation.
28
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
29
Journal Pre-proof
30
Fig 2. Transcriptional changes in the spinal cord of adult high fat-fed mice. A, One half of the spinal cord from regular diet (RD, n=3) and high fat diet (HFD, n=4) fed mice were processed whole genome RNA sequencing. B, Heat map of transcriptional changes observed in the spinal cord of male mice fed RD or HFD for 12 wk representing the entire gene profile differences between groups irrespective of FDR, organized by hierarchical clustering with the row distance of one minus Pearson
of
correlation where blue is relative row minimum and red is the row maximum. C, Scatter
ro
plot of differentially expressed genes (DEGs, red dots; 32 up, 43 down with a FDR <
-p
0.15) where the x-axis is false discovery rate (FDR) and y-axis is log fold change (log FC), with annotated gene names for select up (red) and down (blue) regulated genes.
Jo
ur
na
lP
upstream transcriptional regulator.
re
PTK6 (D) was predicted to be activated while NFKBIA (E) was predicted as an inhibited
2.67
2.55E-06
MYOCD
Transcription regulator
3
2.12
31
Target molecules in dataset
Network biascorrected p-value
Predicting activation Z-score
3
0.013
CDC42EP2, CRYAB, GNA12, GSTP1,HBA1/HBA2,H MGCS2,KDR, PDIA6,XBP1 ARL4D, COL8A2,CP, FBN1,FN1, GRN, LCN2,LFNG,
-p
ro
of
Molecule Type Kinase
Exp Log Ratio
PTK6
Master Regulator
Depth
P-value of overlap
Journal Pre-proof
0.012
2
5.69E-04
0.015
HSPA5,PDIA6,SDF2L1, FN1
3
2
1.76E-04
0.022
HSPA5,KDR,SGK1, FN1
2
1.89
3.92E-05
0.026
ARC, GSTP1,KDR,LCN2, COL8A2,FBN1,FN1
transcription regulator
2
1.73
1.06E-03
0.042
KDR,SGK1, FN1
0
transcription regulator
3
1.5
7.52E-05
0.033
ARC, CRYAB, HBA1/HBA2,HMGCS2, HSPA5,KDR, SERPINB1,TNFRSF12 A,XBP1, CH25H,COL8A2, DBP,FBN1,GRN, LAMA4,LRG1
0.04
transcription
1
1.41
6.47E-05
0.0032
HSPA5,XBP1
0.38
Transcription regulator
TAF12
0.04
transcription regulator
SRF
0.11
transcription regulator
NFYC
-0.03
CCNE1
CREB3
1
Jo
ur
na
XBP1
lP
re
8.00E-06
ARC,GSTP1,KDR, XBP1, COL8A2,FBN1,FN1, LCN2,
Journal Pre-proof
32
regulator -0.07
transcription regulator
3
1.41
6.39E-06
0.0094
ARC,CDC42EP2,GSTP 1,KDR,LCN2, COL8A2,FBN1,FN1
ATF6B
0.10
transcription regulator
1
1
6.30E-03
0.0318
XBP1
NFYC
-0.03
transcription regulator
1
1
1.67E-02
0.0449
SGK1
PIK3CA
-0.06
kinase
3
1
1.32E-05
0.049
ARC, CDC42EP2, CRYAB, GNA12,GSTP1,HMGCS 2,HSPA5, PDIA6, XBP1, ARL4D, CIART,CP, DBP, LCN2,LFNG, SGK1
DDIT3
0.13
transcription regulator
1
0.58
PRKAB1
0.00 5
kinase
PRKAG1
0.00 2
RPS6KA 4
-0.04
PRKACA
0.07
2
0.45
1.11E-04
0.0435
HMGCS2, DBP,FN1, LCN2,SGK1
kinase
2
0.45
1.11E-04
0.0435
HMGCS2, DBP,FN1, LCN2,SGK1
kinase
2
0.33
6.49E-06
0.0083
ARC,GSTP1,HSPA5, ,CIART,COL8A2,FBN1, FN1, LCN2,SGK1
chemical endogenous mammalian
3
0.33
3.15E-05
0.0162
ARC,GSTP1,HSPA5, PDIA6, XBP1, CIART,FN1, LCN2, SGK1
kinase
3
0.23
7.34E-06
0.032
ARC, CRYAB,GSTP1,HMGC S2,HSPA5,KDR, SERPINB1, ARL4D,CH25H,CIART, COL8A2,CP,
ur
na
lP
re
heme
0.008
Jo
-p
ro
of
MKL1
1.60E-04
HSPA5, XBP1, LCN2
Journal Pre-proof
33 DBP,FBN1,FN1,GRN, LCN2,LFNG,Podxl
CLOCK
0.02
ARNTL
0.28
PPRC1
-0.05
SMARCA 2
-0.02
kinase
3
0
3.62E-07
0.0008
1
0
3.33E-04
0.0049
HMGCS2, DBP
1
0
0.0184
HMGCS2, DBP
4.05E-03
0.0319
GNG11,LCN2
2.20E-03
0.0344
KDR, CP
0.0153
CDC42EP2, CRYAB, GNA12, HBA1/HBA2,HSPA5,K DR, PDIA6,SERPINB1, XBP1, CH25H,COL8A2,CP, FBN1,FN1, GRN, LCN2,LFNG, SGK1
0.0126
ARC, CDC42EP2, CRYAB,GNA12, GSTP1,HBA1/HBA2,H MGCS2,HSPA5,KDR, PDIA6, SDF2L1,SERPINB1, XBP1, ARL4D, CH25H,CIART,CP, DBP,FN1, GRN, LAMA4,LFNG, PLA2G3, SGK1
re 0
lP 1
na
-0.12
1
kinase
3
2.07E-03
0
-0.23
1.19E-06
Jo
ur
PDGFRA
transcription regulator transcription regulator transcription regulator transcription regulator
ro
of
-0.07
-p
MAP3K3
ARC,CDC42EP2, ,CRYAB,GNA12,HSPA 5,SDF2L1,SERPINB1,T NFRSF12A, ARL4D, ,CH25H,CIART,COL8A 2,CP, ,DBP,FBN1,FN1,GRN, LCN2,LFNG,LRG1,PLA 2G3, SGK1
HTT
-0.04
transcription regulator
3
-0.41
9.99E-07
Journal Pre-proof
MECP2
-0.11
transcription regulator
1
-0.58
1.25E-04
0.0021
HBA1/HBA2,SGK1,SL C17A7
CREB1
0.05
transcription regulator
1
-0.82
2.90E-04
0.0314
ARC,HSPA5, CIART, FN1, LCN2,SGK1
1
-1
8.39E-03
0.0162
SLC17A7
-0.19
transcription regulator transcription regulator transcription regulator
1
-1
1.46E-02
0.0341
DBP
1
-1
1.67E-02
0.0408
LFNG
of
34
ARC, HSPA5, CIART, FN1, GRN, LCN2, SGK1
FEZF2 PER2 MSGN1
0.05
kinase
2
-1.13
1.43E-04
NFKBIA
-0.37
transcription regulator
1
-1.41
liganddependent nuclear receptor
1
-1.73
-p
transcription regulator
re
0.07
CRYAB ,HSPA5, CH25H, CP, FN1,GRN,LCN2, SGK1
2.27E-03
0.0453
GSTP1,HMGCS2,LCN2
3
-2
1.76E-04
0.0222
HSPA5,KDR, FN1, SGK1
6.19E-07
na
TAF11
0.0025
lP
NR1I2
0.0373
ro
PASK
ur
Table 1. Summary of ingenuity canonical pathways significantly affected in spinal cord
Jo
tissue by HFD consumption. Bold indicates increased.
Journal Pre-proof
35
Ingenuity Canonical Pathways
-log(pvalue)
log(B -H pvalue )
Endoplasmic Reticulum Stress Pathway
3.45
1.56
0.0004
0.028
2
XBP1,HSPA5
Aldosterone Signaling in Epithelial Cells
2.86
1.29
0.0014
0.051
3
CRYAB,HSPA5,SGK1
Unfolded protein response
2.63
1.29
0.002
0.051
Glutamate Receptor Signaling
2.58
1.29
0.003
Huntington's Disease Signaling
2.4
1.21
0.004
Ceramide Degradation
2.1
Sphingosine and Sphingosine-1phosphate Metabolism
1.97
#D EG s
Molecules
2
GNG11,SLC17A7
0.062
3
GNG11,HSPA5,SGK1
0.008
0.104
1
ACER2
0.94
0.011
0.115
1
ACER2
1.89
0.94
0.013
0.115
2
GNG11,PLA2G3
0.051
-p
re
lP
ur
0.99
ro
XBP1,HSPA5
na
of
FDR
2
Jo
CCR3 Signaling in Eosinophils
P-value
Ketogenesis
1.88
0.94
0.013
0.115
1
HMGCS2
Mevalonate Pathway I
1.76
0.87
0.017
0.134
1
HMGCS2
Table 2. Master regulator analysis shows significantly affected transcriptional regulators in HFD-fed mouse spinal cord. Bold indicates increased.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
36
Journal Pre-proof
37
Fig 3. Myelin-related transcriptional changes in the spinal cord of adult high fatfed mice. A, Heat map of myelin-related genes in the spinal cord of male mice fed RD or HFD for 12 wk where blue indicates relative minimum value and red is the row maximum value. Genes within groups are sorted by highest to lowest average HFD relative values compared to RD, with red asterisk indicating genes with p<0.05 Schematic depicting differentiation of oligodendrocyte lineage cells (B) and scatterplots
of
of RNA expression of genes enriched in oligodendrocyte progenitors (OPCs, C), new
ro
oligodendrocytes (OL, D) or myelinating OLs (E) in HFD mice normalized relative to RD
Jo
ur
na
lP
re
-p
controls.
Journal Pre-proof
38
3.3 Functional and metabolic changes in spinal cord mitochondria following HFD consumption are time-dependent Prior to initiation of cell death, ER stress pathway activation can have a number of effects on metabolic and bioenergetic processes [28, 47, 48]. Therefore, since the ER Stress Pathway was the top ingenuity canonical pathway identified from transcriptomic profiling of spinal cord tissue, we further explored potential mechanisms for
of
oligodendrocyte loss in this model by comparing metabolite profiles from the spinal
ro
cords of mice fed a RD or HFD using untargeted UPLC-ToF-MS-based metabolomics
-p
as described previously in detail [37].
HFD consumption significantly altered 238 metabolites analyzed using an
re
unpaired t-test with Benjamini-Hochberg multiple testing correction and identified using
lP
the METLIN metabolite database. To determine which pathways were most affected by
na
the metabolites altered due to chronic HFD-consumption, we performed metabolite sets enrichment analysis (MSEA) with MetabaCore depicted with most significant p-values in
ur
red and least significant in white (Figure 4A). These results suggest that protein
Jo
biosynthesis, TCA cycle, glutathione metabolism, and the mitochondrial electron transport chain were among the significantly enriched pathways. Accordingly, we sought to validate these findings by quantifying TCA cycle metabolites at 4 and 12 wk using a targeted GC-MS-based approach [37]. No significant differences were observed at 4 wk (Figure 4B), but intermediates from the TCA cycle including oxaloacetate (p=0.04) , citric acid (p=0.003), and α-ketoglutarate (p=0.03) were strongly depleted in the 12 wk HFD spinal cord when compared to RD controls (Figure 4B, C and Supplemental Figure 7).
Journal Pre-proof
39
A separate cohort of mice was fed a RD or HFD for 4 or 12 wk to assess the functional impact on spinal cord mitochondria using Oroboros oxygraph [38]. Unexpectedly, we observed a significant increase in State 3 Respiration at 4 wk on HFD (Figure 4D), while 12 wk HFD spinal cord mitochondria had diminished State 3 oxygen consumption compared to RD (Figure 4D). Even State 4 respiration was increased at 4 wk, but declined at 12 wk of HFD consumption (Figure 4E). Maximum (Figure 4F) and
of
non-mitochondrial respiration (Figure 4G) were not altered by HFD consumption at 4 or
ro
12 wk. Taken together, these data provide evidence for temporally distinct changes in
-p
TCA cycle metabolites and mitochondrial function, where at 12 wk TCA cycle
Jo
ur
na
lP
re
metabolites are depleted and mitochondrial function is impaired.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
40
Journal Pre-proof
41
Fig 4. Functional and metabolic changes in spinal cord mitochondria following HFD consumption are time-dependent. Metabolite Sets Enrichment Overview (A) summarizing significantly affected pathways in HFD-fed mice as revealed by untargeted LC-MS-MS profiling of spinal cord tissue. Heat map (B) summarizes targeted LC-MSMS profiling of tricarboxylic acid (TCA) cycle metabolites at 4 and 12 wk of RD or HFD consumption with metabolites depleted at 12 wk identified within the schematic by blue
of
arrows (C). Gray indicates metabolite was not detected in samples, red indicated
ro
highest fold expression and blue lowest fold abundance. High resolution respirometry by
-p
Oroboros oxygraph of isolated spinal cord mitochondria indicates oxygen consumption is increased in 4 wk HFD mice (D, E), but decreased by 12 wk (D, E). Injections of
re
glutamate/malate/succinate (GMS), ADP, oligomycin (O), and trifluoromethoxy
lP
carbonylcyanide phenylhydrazon (FCCP) allow for oxygen consumption rates (OCRs) to
na
be determined during various stages of mitochondrial respiration. State 3 mitochondrial respiration was assessed following the addition of glutamate, malate, and succinate to
ur
the chamber, giving the basal respiration rate for complexes I and II. Oligomycin
Jo
injection then inhibited ATP synthase to induce state 4 respiration, attributable to proton leak across the inner mitochondrial membrane. FCCP addition results in an uncoupled state, allowing for determination of maximum capacity, while non-mitochondrial respiration was assessed after antimycin inhibits complex III. OCRs during state 3 (D), state 4 (E), Maximum (F), and non-mitochondrial respiration (G). Graphical results represented as the mean ± SEM (*, p<0.05; **, p<0.01; and ***, p<0.001; n=4-6/group).
Journal Pre-proof
42
3.4 Altered mitochondrial quality control processes, oxidative stress, and apoptosis in the spinal cord following 12 wk of high fat diet consumption. HFD consumption was previously shown to alter the metabolic regulators AMPK and SIRT1 in peripheral tissues and to affect mitochondrial function and quality control processes [49, 50]. Therefore, we next explored this signaling pathway (Figure 5A) in the spinal cord of RD and HFD-fed mice. With 12 wk of HFD, immunoblotting
of
demonstrated reductions in AMPK phosphorylation (Figure 5B, C) and in total AMPK
ro
(Figure 5B, C). SIRT1 protein levels (Figure 5B, C) were also significantly reduced in
-p
the spinal cord of HFD mice compared to controls. PGC1α, a direct target of AMPK and
re
SIRT1 and crucial regulator of mitochondrial biogenesis , showed diminished protein levels (Figure 5B, C) and RNA expression (Figure 5D) in the HFD-fed mice compared to
lP
RD-fed controls. There was also an upregulation of the fission gene, Fis1 (Figure 5D),
na
and a reduction in the mitophagy-related gene PINK1 (Figure 5D), which could indicate an overall accumulation of fragmented mitochondria in the spinal cord of HFD-fed mice.
ur
Oligodendrocytes are particularly vulnerable to cell death mediated by oxidative
Jo
damage due to their relatively low antioxidant levels and high amounts of iron storage [51, 52]. Notably, gene expression of the oxidative defense enzyme SOD2 (Figure 5D) was significantly lower in HFD spinal cords compared to RD. Next, in a heatmap of mitochondrial-related genes (Figure 5E), we noticed a trend for increased Bax gene expression (p=0.03, FDR=0.63), while anti-apoptosis Bcl-2 appeared less (p=0.69, FDR=0.99) in the spinal cords of HFD mice versus RD controls. Free radical production results in oxidative modification of lipids which can damage biological membranes including the myelin sheath. 4-hydroxynonenal (4-HNE) lipid peroxidation products are
Journal Pre-proof
43
found in demyelinating MS lesions and are highly toxic to OLCs [47, 53, 54]. White matter tracts within both the spinal cord dorsal column (Figure 5F) and corpus callosum (Figure 5G) of mice consuming HFD had significantly greater immunoreactivity for 4HNE compared to RD controls. Furthermore, immunofluorescence staining demonstrated increased levels of pro-apoptotic protein Bax (Figure 5H) and decreased levels of anti-apoptotic Bcl-2 (Figure 5I) in the dorsal column of HFD-fed mice.
of
Quantification of the Bax/Bcl-2 ratio (Figure 5H) and double positive Olig2/cleaved-
ro
caspase-3 cells (Figure 5I) suggest apoptotic cell death signaling is underway in
-p
oligodendrocytes chronically fed a HFD. These results point to HFD-related changes in
re
key metabolic regulators such as AMPK and SIRT1, decreased mitochondrial biogenesis and an impaired potential for antioxidant responses as contributing factors to
lP
the increases in oxidative stress markers such as 4-HNE and loss of myelinating cells
Jo
ur
na
observed in mice consuming HFD.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
44
Journal Pre-proof
45
Fig 5. Altered Mitochondrial Quality Control Processes, Oxidative Stress, and Apoptosis in the spinal cord following 12 wk of high fat diet consumption. Schematic (A) and Western blots (B) demonstrate significant reductions in AMPK phosphorylation, SIRT1, and PGC1α. C, Bar graphs show quantification of relative optical density (ROD) highlighting reductions in AMPK phosphorylation and total AMPK, SIRT1, and PGC1α protein expression in the spinal cord of high fat diet (HFD)-
of
consuming mice compared to those fed a regular diet (RD). The first HFD mouse
ro
samples protein level changes were less affected than others due to animal variability
-p
despite consistent conditions between mice. D, Quantitative PCR demonstrated differences in RNA expression of PGC1α, Fis1, PINK1, and SOD2. E, Heat map
re
summarizes RNAseq data for mitochondria-related transcriptional changes.
lP
Representative 4-hydroxynonenol (4-HNE) immunostained sections and corresponding
na
quantification of immunoreactivity normalized to RD control revealing increased lipid peroxidation in the spinal cord (SC) (F) and corpus callosum (G) of HFD-fed mice by 12
ur
wk. Immunofluorescence images and associated bar graphs show increased Bax/Olig2
Jo
double positive cells (H) and an increased Bax/Bcl2 ratio as well as more cleaved caspase-3/Olig2 double positive cells (I) in the white matter of spinal cord tissue from HFD-fed mice versus RD controls, indicating oligodendrocyte apoptosis. Graphical results represented as the mean ± SEM (*, p<0.05, **, p<0.01, and ***, p<0.001 (n=5-7 mice per group)). Scale bar= 50 µm (F), 100 µm (G), and 20 µm (H, I).
Journal Pre-proof
46
3.5 Mitochondrial changes and differentiation in oligodendrocytes treated with saturated fat. To model HFD consumption in vitro and study potential direct effects towards oligodendrocytes, we cultured primary murine oligodendrocytes in media containing the saturated fatty acid palmitate (PA, 100 µM) for 24 h. Seahorse extracellular flux analysis (Figure 6A) was performed to evaluate mitochondrial bioenergetics. PA significantly
of
decreased basal, ATP-linked, and maximal oxygen consumption rates in
ro
oligodendrocytes, but had no effect on proton leak. Since we observed transcriptional
-p
changes in fission and mitophagy genes in the spinal cord of mice consuming a HFD, we also visualized the integrity and structure of oligodendrocyte mitochondria using
re
MitoTracker. Healthy control oligodendrocytes have a long, filamentous network of
lP
mitochondria (Figure 6B), while PA-treated oligodendroglia have mitochondria that are
na
smaller, more compact, and circular. Overall, mitochondrial function and structure are impaired in oligodendrocytes cultured with high levels of saturated fat, in a manner
ur
recapitulating the changes observed in the spinal cord of HFD-fed mice (Figure 4).
Jo
To determine the influence of PA on oligodendrocyte differentiation, PLPimmunoreactivity and RNA transcripts for PLP, MBP and Olig2 were quantified. Addition of PA (100 µM, 24 h) to differentiating primary oligodendrocyte cultures reduced PLP protein (Figure 6C) to half that of controls. Supporting this, PLP, MBP and Olig2 RNA expression were also depleted (Figure 6D). In addition to oligodendrocyte progenitors, neural stem cells also contribute to replacement of oligodendrocytes following injury [44, 55]. Therefore, we next determined if the oligotoxic effects of PA extend to monolayer cultures of neural stem cells (aNSCs) derived from the adult subventricular zone (SVZ).
Journal Pre-proof
47
Supporting deleterious effects of PA across the oligodendrocytes and neural stem cells, the abundance of PLP protein (Figure 6E) and MBP RNA expression (Figure 6F) were significantly less in aNSCs cultured in the presence of PA. Taken together, these findings suggest excess PA impairs the ability of OLCs or aNSCs to differentiate into
Jo
ur
na
lP
re
-p
ro
of
myelinating oligodendrocytes.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
48
Journal Pre-proof
49
Fig 6. Differentiation and mitochondrial changes in oligodendrocyte progenitors cultured in the presence of high saturated fatty acid. High fat consumption was modeled in primary murine oligodendrocyte lineage cell (OLC) cultures by including palmitate in the media (PA, 100µM, 24 h). Bioenergetics characterization (A) of PA in OLCs was measured using a Seahorse XF24 analyzer. Basal, ATP-linked, and maximum capacity oxygen consumption rates were decreased in OLCs treated with PA.
of
Significant changes in proton leak respiration were not observed. MitoTracker staining
ro
(B) showed increased mitochondrial fragmentation in PA-treated OLCs as determined
-p
by mitochondrial parameters including significantly increased solidity and circularity and a smaller average size. Proteolipid (PLP) immunoreactivity of OLCs (C) and qPCR
re
quantification of RNA (D) for PLP, myelin basic protein (MBP) and Olig2 illustrate
lP
impaired differentiation toward a mature myelinating oligodendrocyte when treated with
na
PA. Similarly PA-treated adult SVZ-derived neural stem cell (aNSC) cultures (M) also express less PLP protein (E) and MBP RNA (F). Graphical results represented as the
Jo
ur
mean ± SEM (n=3-6/group; *, p<0.05, **, p<0.01, and ***, p<0.001). Scale bar= 50 µm.
Journal Pre-proof
50
4. Discussion Diet-induced obesity increases the risk for a number of diseases including diabetes, cardiovascular disease, cancer, and cognitive dysfunction and has been steadily growing in prevalence in recent decades [20, 56-59]. The role of oligodendrocytes and myelin plasticity is likewise implicated in a variety of neuropsychiatric and cognitive conditions [1, 2, 6]. Notably, white matter integrity is
of
negatively correlated with BMI and is decreased in the corpus callosum of obese
ro
individuals [4, 14, 60]. Elevated BMI is also linked to an increased incidence and
-p
disease burden in MS, especially in combination with other risk factors [10, 13, 61, 62].
re
Here we explored the potential link between a HFD, myelin integrity and metabolic dysfunction in the CNS. Our findings demonstrate for the first time that a HFD alone
lP
depletes oligodendrocyte progenitors in the brain and spinal cord and high saturated fat
na
impedes oligodendrocyte differentiation in vitro. Overall, we observed changes in genes and pathways involving oligodendrogenesis and differentiation, iron homeostasis,
ur
oxidative stress, ER stress, insulin signaling, and inflammation all of which may
Jo
contribute mechanistically alone or collectively to the loss of myelinating cells in the CNS of mice chronically consuming HFD. In the spinal cords of HFD fed mice, RNA sequencing and untargeted metabolomics point towards ER stress and mitochondrial dysfunction as key mechanisms contributing to oligodendrogliopathy. These findings were validated by results showing depletion of TCA cycle intermediates and reduced oxygen consumption rates in the spinal cords of HFD-consuming mice and in isolated oligodendrocytes. Moreover, deficits in AMPK-SIRT1-PGC1α signaling were present in HFD spinal cords, whereas the oxidative stress marker 4HNE was increased. Overall,
Journal Pre-proof
51
the vast influence of metabolic dysfunction on oligodendrocytes observed here is of high relevance to demyelinating, neuropsychiatric and cognitive disorders and to considerations for strategies to manage myelin protection and repair. The results of the current study suggest that consumption of excess high fat alone is sufficient to trigger oligodendrogliopathy and impairments in oligodendrocyte differentiation across the brain and spinal cord and links these to several disturbances
of
in metabolic status. Oligodendrocytes play critical roles in modulating motor [63, 64] and
ro
psychiatric [5, 65] function by improving the speed and integrity of impulse conduction,
-p
providing neuronal metabolic support, and regulating synapses [8]. In a recent study, we
re
discovered that a diet high in saturated fat (41%) and sucrose (34%), the so-called Western diet, depletes oligodendrocytes and their precursors after 7 wk of consumption
lP
in the adult (4 month) spinal cord [29]. A later study, reported neuroinflammation,
na
cerebrovascular decline, and white matter damage with an increase in Olig2+ progenitors, but a decrease in MBP in the frontal parietal cortex/corpus callosum
ur
following long term (10 month) consumption of a “Western-style” diet (16.4% calories
Jo
from fat with added high-fructose corn syrup) [4]. Importantly, in both prior studies examining the effects of a high fat high sugar diet on myelin, exercise prevented the deleterious effects. The findings of the current study suggest that consumption of high fat alone is sufficient to trigger demyelinating effects in the brain and spinal cord of adult mice. Additional study will be needed to determine if the demyelinating effects of HFD can likewise be prevented by exercise interventions. Many of the transcriptional changes we observed are increased in the spinal cord of mice consuming a HFD are already established to be differentially expressed in
Journal Pre-proof
52
individuals with MS and its experimental models. Fos, Grn, Sdf2l1, Fn14, and Paqr6 RNA accumulation are each separately found in glia of white matter tissue of MS patients, and Grn polymorphisms may influence MS disease course and relapse recovery [66-72]. Down regulated genes in the spinal cord of HFD mice were also affected in MS animal models, include Slc17a7 and Pdyn [73]. Although Cryab was shown to inhibit inflammation and disease in the EAE model, recent studies in the
of
Cuprizone model suggest that Cryab activates astrocytes and exacerbates
ro
demyelination [74]. Moreover, activation of the Sgk1 pathway following oxidative stress
-p
exacerbates EAE [75]. Among many genes identified by Moyon et. al in a study of
re
transcriptional profiling of Cuprizone-“activated” oligodendrocytes, higher expression of Socs3, Ddit4, and lower expression of Kdr and Cdc42ep2 genes may be required to
lP
activate OPCs enabling remyelination [76]. Kruppel like factor 9 (Klf9), down regulated
na
by HFD, is a transcription factor downstream of T3 that regulates oligodendrocyte differentiation and myelin regeneration in the Cuprizone model [77, 78]. Ddit3 is
ur
expressed by activated astrocytes following Cuprizone exposure, while knockout mice
Jo
had less oligodendrocyte apoptosis, demyelination, and microglial and astrocyte activation [79]. HFD also exacerbates disease symptoms and neuroinflammation in EAE models [21, 22]. Notably, the genes identified as having higher expression in HFD-EAE versus RD-EAE did not overlap with those we observed in the spinal cord of adult mice consuming HFD, indicating unique mechanisms contribute to HFD-triggered pathology depending on the disease context [22]. ER stress and inflammation closely follow lipid dysregulation in diabetic neuropathy and obesity-associated allodynia models and cooperate in a feed forward
Journal Pre-proof
53
manner [80-82]. In line with studies showing ER stress in liver, heart, muscle, hypothalamus, and hippocampus in diet-induced obesity models [7, 48, 83, 84], RNA sequencing results in the current study uncovered ER Stress and the Unfolded Protein Response as prominently altered pathways in the adult spinal cord after 12 wk of HFD consumption. Since myelinating cells produce large amounts of plasma membrane, they are particularly susceptible to disruptions in secretory pathways including ER stress and
of
eventual apoptosis if left unchecked [3, 26, 85]. Thus, controlling ER overload may be
ro
beneficial to restoring oligodendrocytes and myelin in various white matter diseases [3,
-p
26, 85, 86]. Xbp1, involved in ER stress responses, is reported to increase in MS
re
lesions as well as in vanishing white matter disease (VWMD) [3, 26]. ER chaperones help control cellular responses to ER stress and some were shown to be required for
lP
oligodendrocyte survival during EAE [85]. ER and mitochondria interact both physically
MS pathology [27].
na
and functionally, and alterations in these connections have recently been described in
ur
Alterations in ER stress are connected to mitochondrial deficits in MS [27].
Jo
Mitochondrial morphology and impaired mitochondrial function and antioxidant responses are also reported in experimental models of demyelinating disease [87-89]. Here we document fragmented mitochondria in cultures of oligodendrocytes grown under high fat conditions. Fis1 is involved in mitochondrial fission and in early events in cell-death signaling, and expression was also upregulated in the spinal cord of mice consuming high fat. Similar changes are likewise engaged in OLCs in the context of oxidative stress or inflammation [40, 89-91]. Moreover, inhibition of fission is protective against demyelination and neuroinflammation, although has little impact on OLC
Journal Pre-proof
54
differentiation in EAE and Cuprizone MS models [89, 92]. Impairments in mitochondria also lead to increased reactive oxygen species and downstream effects on proteins, lipids, and nucleic acids [92-94]. Notably, in both the corpus callosum and spinal cord white matter regions where OLC death occurred following chronic HFD consumption, we observed increased lipid peroxidation. Increased lipid peroxidation was also observed in the spinal cord with consumption of high fat and sucrose [29]. In MS
of
pathogenesis and its animal models, oxidative stress mediates apoptosis of
ro
oligodendrocytes and is commonly documented by an elevated Bax/Bcl2 ratio and
-p
caspase-3 cleavage [95, 96]. Our in vivo data suggest that mitochondrial-dependent
re
apoptosis also occurs in oligodendrocytes in relation to HFD-induced mitochondrial dysfunction and increased oxidative damage.
lP
In the current study, we discovered substantial deficits in TCA cycle
na
intermediates and oxygen consumption rates in the spinal cords of HFD mice. Key substrates at all stages of the TCA cycle, from citric acid to oxaloacetate were reduced
ur
in the spinal cord of mice consuming HF. In addition, these effects were paralleled by
Jo
diminished mitochondrial oxidative respiration. Oligodendrocytes have extremely high energy demands and die hours faster than neurons following nutrient deprivation by arterial occlusion [28, 97]. Impaired energy metabolism has already been proposed as a contributing factor to oligodendrogliopathy in pattern III MS lesions and is described in animal models of the disease [87, 89, 93]. Normally, metabolism of pyruvate and other fuel sources through the mitochondrial TCA cycle leads to ATP synthesis via the electron transport chain. Our findings that the dysmyelinating effects of HFD were associated with significant impairments in TCA cycle function, are of particular interest
Journal Pre-proof
55
given the efficacy of dimethyl fumarate, itself a TCA cycle metabolite, as a first line treatment for relapsing remitting MS [98]. AMPK, SIRT1 and PGC1α were all substantially reduced in the spinal cord of mice consuming HF. AMPK and SIRT1 are crucial metabolic sensors that impact PCG1α and thus mitochondria biogenesis and function. In addition to promoting cell survival, AMPK-SIRT1 activation and mitochondrial function regulate oligodendrocyte
of
differentiation [50, 99-102]. Recently, folate was determined to regulate oligodendrocyte
ro
survival and differentiation through AMPK activity [99]. SIRT1 and SIRT2 activation
-p
redundantly mediate differentiation of neural stem or progenitor cells towards the oligodendrocyte lineage, likely by upregulation of PDGFRα, Sox10, Nkx2.2 and
re
downregulation of p21 [9, 103, 104]. Moreover, SIRT1 activation through
lP
overexpression or agonists is neuroprotective in MS models, while expansion of
na
oligodendrocyte progenitors occurs following SIRT1 inactivation [101, 104]. Exercise and other activators of the AMPK-SIRT1-PGC1α system may be beneficial in reducing
ur
deleterious effects of obesogenic diets on oligodendrocyte lineage cells and myelin
Jo
integrity by promoting mitochondrial homeostasis [4, 29, 49, 101]. Interestingly, we observed a predicted decrease in PDGFRα signaling in the transcriptomic data along with decreased AMPK-SIRT1 signaling, leading to a loss of both progenitors and mature oligodendrocytes in the spinal cord following long term HFD. Whether or not the disruptions we observe in TCA cycle metabolite depletion, impaired mitochondrial function and oligodendrocyte loss observed in HFD-fed spinal cords are a direct consequence of the altered AMPK-SIRT signaling, as specified in other tissues and cell types, should be more directly addressed in future studies.
Journal Pre-proof
56
Mature CC-1+ oligodendrocytes along with Olig2+ progenitors are lost in the spinal cord white matter with HFD consumption, but only the later are impacted in the corpus callosum at the same 12 wk time point. Given their increased energy demands for proliferation and myelin membrane production it is possible that oligodendrocyte progenitors and young oligodendrocytes as labeled by Olig2 are more vulnerable to the stresses associated with HF consumption compared to mature oligodendrocytes. We
of
cannot exclude that mature oligodendroglia in the corpus callosum would be lost with
ro
even more chronic HFD feeding. Region specific and possible temporal differences in
-p
HF-elicited oligotoxicity may also relate to heterogeneity in the local oligodendrocyte
re
populations, region specific effects on neurons, or differential effects across the astrocyte and microglial compartments that so significantly contribute to the
lP
microenvironment [41]. Our studies also demonstrate an anxiety-like phenotype and
na
overall activity level changes in the HFD-fed mice that mirror a number of previous reports [20, 58], however future studies will be required to determine if HFD-induced
ur
behavioral changes are in fact dependent on the oligodendrocyte loss documented.
Jo
Although we show that impaired differentiation and mitochondrial fragmentation occurring with HFD are recapitulated across regions and in cultures of oligodendrocytes, a Ribotag approach or single cell sequencing will be beneficial for the identification of cell-specific transcriptional changes in each tissue, and this is another goal for future experiments.
Journal Pre-proof
57
5. Conclusions The findings of the current study demonstrate significant perturbations in CNS function occur in response to long term consumption of a diet laden with excessive fat, including direct impairment of oligodendrocyte mitochondrial function that ultimately impairs oligodendrocyte survival and differentiation. Previous reports in Western diet-fed mice suggest exercise may mitigate deleterious effects on myelin integrity and
of
behavioral outcomes, and our new data in mice fed a high fat diet suggest that
ro
modulation of mitochondrial function, ER and oxidative stress pathways, in addition to
-p
the TCA cycle may also be important areas to explore as targets to improve myelin
re
protection and regeneration. Accordingly, this work should inspire new research to evaluate ways to mitigate the negative consequences of a chronic high fat diet on
6. Author Contributions
na
lP
oligodendrocytes alone and in the context of white matter injury.
ur
IAS, MRL and AM conceived and designed the study. MRL, HY and AM performed all
Jo
in vivo experiments, necropsies, histological staining and analysis. WS and HNK assisted with necropsies and LK performed some of the histological staining. CC generated heat maps of RNA sequencing data and contributed to neural stem cell cultures. MRL performed and analyzed oxygraph and in vitro experiments. MRL and IAS interpreted the data and wrote the manuscript. All authors reviewed and edited the manuscript.
Journal Pre-proof
58
7. Acknowledgements The work was supported by Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology (CMSAN), the Eugene and Marcia Applebaum Foundation, the Mayo Clinic Center for Biomedical Discovery (CBD) and the Mayo Clinic Metabolomics Core (U24DK100469 and UL1TR000135). Portions of this work were also supported by R01NS052741, RG4958 from the National Multiple Sclerosis Society, a grant from the
of
Craig H. Neilsen Foundation, and the Minnesota State Spinal Cord Injury and Traumatic
ro
Brain Injury Research Program.
-p
We would like to thank Thomas White for his assistance in analyzing the glucose
re
tolerance data and Katherine Klaus for her help the Oroboros oxygraph system. We also thank Xuewei Wang and Tumpa Dutta for their initial assistance with Bioinformatics
lP
for RNAseq and LC-MS-MS measurements, respectively. The diagrams within figures
ur
Declaration of Interests
na
were created using elements from the Biomedical-PPT-Toolkit-Suite (Motifolio).
Jo
The authors have no declarations of interests to disclose.
Journal Pre-proof
59
References
Jo
ur
na
lP
re
-p
ro
of
[1] R. Ohtomo, A. Iwata, K. Arai, Molecular Mechanisms of Oligodendrocyte Regeneration in White Matter-Related Diseases, Int J Mol Sci 19(6) (2018). [2] R.C. Armstrong, A.J. Mierzwa, G.M. Sullivan, M.A. Sanchez, Myelin and oligodendrocyte lineage cells in white matter pathology and plasticity after traumatic brain injury, Neuropharmacology 110(Pt B) (2016) 654-659. [3] B. van Kollenburg, J. van Dijk, J. Garbern, A.A. Thomas, G.C. Scheper, J.M. Powers, M.S. van der Knaap, Glia-specific activation of all pathways of the unfolded protein response in vanishing white matter disease, J Neuropathol Exp Neurol 65(7) (2006) 707-15. [4] L.C. Graham, W.A. Grabowska, Y. Chun, S.L. Risacher, V.M. Philip, A.J. Saykin, I. Alzheimer's Disease Neuroimaging, S.J. Sukoff Rizzo, G.R. Howell, Exercise prevents obesity-induced cognitive decline and white matter damage in mice, Neurobiol Aging 80 (2019) 154-172. [5] K. Roy, J.C. Murtie, B.F. El-Khodor, N. Edgar, S.P. Sardi, B.M. Hooks, M. BenoitMarand, C. Chen, H. Moore, P. O'Donnell, D. Brunner, G. Corfas, Loss of erbB signaling in oligodendrocytes alters myelin and dopaminergic function, a potential mechanism for neuropsychiatric disorders, Proc Natl Acad Sci U S A 104(19) (2007) 8131-6. [6] V. Haroutunian, P. Katsel, P. Roussos, K.L. Davis, L.L. Altshuler, G. Bartzokis, Myelination, oligodendrocytes, and serious mental illness, Glia 62(11) (2014) 1856-77. [7] M. Cai, H. Wang, J.J. Li, Y.L. Zhang, L. Xin, F. Li, S.J. Lou, The signaling mechanisms of hippocampal endoplasmic reticulum stress affecting neuronal plasticity-related protein levels in high fat diet-induced obese rats and the regulation of aerobic exercise, Brain Behav Immun 57 (2016) 347-359. [8] R.E. Pepper, K.A. Pitman, C.L. Cullen, K.M. Young, How Do Cells of the Oligodendrocyte Lineage Affect Neuronal Circuits to Influence Motor Function, Memory and Mood?, Front Cell Neurosci 12 (2018) 399. [9] A. Alizadeh, S. Karimi-Abdolrezaee, Microenvironmental regulation of oligodendrocyte replacement and remyelination in spinal cord injury, J Physiol 594(13) (2016) 3539-52. [10] M.A. Gianfrancesco, B. Acuna, L. Shen, F.B. Briggs, H. Quach, K.H. Bellesis, A. Bernstein, A.K. Hedstrom, I. Kockum, L. Alfredsson, T. Olsson, C. Schaefer, L.F. Barcellos, Obesity during childhood and adolescence increases susceptibility to multiple sclerosis after accounting for established genetic and environmental risk factors, Obes Res Clin Pract 8(5) (2014) e435-47. [11] A. Langer-Gould, S.M. Brara, B.E. Beaber, C. Koebnick, Childhood obesity and risk of pediatric multiple sclerosis and clinically isolated syndrome, Neurology 80(6) (2013) 548-52. [12] K.L. Munger, T. Chitnis, A. Ascherio, Body size and risk of MS in two cohorts of US women, Neurology 73(19) (2009) 1543-50. [13] T. Olsson, L.F. Barcellos, L. Alfredsson, Interactions between genetic, lifestyle and environmental risk factors for multiple sclerosis, Nat Rev Neurol 13(1) (2017) 2536.
Journal Pre-proof
60
Jo
ur
na
lP
re
-p
ro
of
[14] T.M. Wassenaar, K. Yaffe, Y.D. van der Werf, C.E. Sexton, Associations between modifiable risk factors and white matter of the aging brain: insights from diffusion tensor imaging studies, Neurobiol Aging 80 (2019) 56-70. [15] A. Manouchehrinia, A.K. Hedstrom, L. Alfredsson, T. Olsson, J. Hillert, R. Ramanujam, Association of Pre-Disease Body Mass Index With Multiple Sclerosis Prognosis, Front Neurol 9 (2018) 232. [16] P. Tettey, S. Simpson, Jr., B. Taylor, L. Blizzard, A.L. Ponsonby, T. Dwyer, K. Kostner, I. van der Mei, An adverse lipid profile is associated with disability and progression in disability, in people with MS, Mult Scler 20(13) (2014) 1737-44. [17] L. Negrotto, M.F. Farez, J. Correale, Immunologic Effects of Metformin and Pioglitazone Treatment on Metabolic Syndrome and Multiple Sclerosis, JAMA Neurol 73(5) (2016) 520-8. [18] A. Sicras-Mainar, E. Ruiz-Beato, R. Navarro-Artieda, J. Maurino, Comorbidity and metabolic syndrome in patients with multiple sclerosis from Asturias and Catalonia, Spain, BMC Neurol 17(1) (2017) 134. [19] P. Dimas, L. Montani, J.A. Pereira, D. Moreno, M. Trotzmuller, J. Gerber, C.F. Semenkovich, H.C. Kofeler, U. Suter, CNS myelination and remyelination depend on fatty acid synthesis by oligodendrocytes, Elife 8 (2019). [20] M. Ogrodnik, Y. Zhu, L.G.P. Langhi, T. Tchkonia, P. Kruger, E. Fielder, S. Victorelli, R.A. Ruswhandi, N. Giorgadze, T. Pirtskhalava, O. Podgorni, G. Enikolopov, K.O. Johnson, M. Xu, C. Inman, A.K. Palmer, M. Schafer, M. Weigl, Y. Ikeno, T.C. Burns, J.F. Passos, T. von Zglinicki, J.L. Kirkland, D. Jurk, Obesity-Induced Cellular Senescence Drives Anxiety and Impairs Neurogenesis, Cell Metab (2019). [21] S. Timmermans, J.F. Bogie, T. Vanmierlo, D. Lutjohann, P. Stinissen, N. Hellings, J.J. Hendriks, High fat diet exacerbates neuroinflammation in an animal model of multiple sclerosis by activation of the Renin Angiotensin system, J Neuroimmune Pharmacol 9(2) (2014) 209-17. [22] M. Hasan, J.E. Seo, K.A. Rahaman, H. Min, K.H. Kim, J.H. Park, C. Sung, J. Son, M.J. Kang, B.H. Jung, W.S. Park, O.S. Kwon, Novel genes in brain tissues of EAE-induced normal and obese mice: Upregulation of metal ion-binding protein genes in obese-EAE mice, Neuroscience 343 (2017) 322-336. [23] R. Ghemrawi, S.F. Battaglia-Hsu, C. Arnold, Endoplasmic Reticulum Stress in Metabolic Disorders, Cells 7(6) (2018). [24] C.M. Mayer, D.D. Belsham, Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation, Endocrinology 151(2) (2010) 576-85. [25] K.S. Gwiazda, T.L. Yang, Y. Lin, J.D. Johnson, Effects of palmitate on ER and cytosolic Ca2+ homeostasis in beta-cells, Am J Physiol Endocrinol Metab 296(4) (2009) E690-701. [26] A.N. Mhaille, S. McQuaid, A. Windebank, P. Cunnea, J. McMahon, A. Samali, U. FitzGerald, Increased expression of endoplasmic reticulum stress-related signaling pathway molecules in multiple sclerosis lesions, J Neuropathol Exp Neurol 67(3) (2008) 200-11.
Journal Pre-proof
61
Jo
ur
na
lP
re
-p
ro
of
[27] Y. Haile, X. Deng, C. Ortiz-Sandoval, N. Tahbaz, A. Janowicz, J.Q. Lu, B.J. Kerr, N.J. Gutowski, J.E. Holley, P. Eggleton, F. Giuliani, T. Simmen, Rab32 connects ER stress to mitochondrial defects in multiple sclerosis, J Neuroinflammation 14(1) (2017) 19. [28] L. Rosko, V.N. Smith, R. Yamazaki, J.K. Huang, Oligodendrocyte Bioenergetics in Health and Disease, Neuroscientist (2018) 1073858418793077. [29] H. Yoon, A. Kleven, A. Paulsen, L. Kleppe, J. Wu, Z. Ying, F. Gomez-Pinilla, I.A. Scarisbrick, Interplay between exercise and dietary fat modulates myelinogenesis in the central nervous system, Biochim Biophys Acta 1862(4) (2016) 545-55. [30] M.E. McGee-Lawrence, T.A. White, N.K. LeBrasseur, J.J. Westendorf, Conditional deletion of Hdac3 in osteoprogenitor cells attenuates diet-induced systemic metabolic dysfunction, Mol Cell Endocrinol 410 (2015) 42-51. [31] K.R. Kalari, A.A. Nair, J.D. Bhavsar, D.R. O'Brien, J.I. Davila, M.A. Bockol, J. Nie, X. Tang, S. Baheti, J.B. Doughty, S. Middha, H. Sicotte, A.E. Thompson, Y.W. Asmann, J.P. Kocher, MAP-RSeq: Mayo Analysis Pipeline for RNA sequencing, BMC Bioinformatics 15 (2014) 224. [32] B. Langmead, C. Trapnell, M. Pop, S.L. Salzberg, Ultrafast and memory-efficient alignment of short DNA sequences to the human genome, Genome Biol 10(3) (2009) R25. [33] S. Anders, P.T. Pyl, W. Huber, HTSeq--a Python framework to work with highthroughput sequencing data, Bioinformatics 31(2) (2015) 166-9. [34] L. Wang, S. Wang, W. Li, RSeQC: quality control of RNA-seq experiments, Bioinformatics 28(16) (2012) 2184-5. [35] M.D. Robinson, D.J. McCarthy, G.K. Smyth, edgeR: a Bioconductor package for differential expression analysis of digital gene expression data, Bioinformatics 26(1) (2010) 139-40. [36] Y. Zhang, K. Chen, S.A. Sloan, M.L. Bennett, A.R. Scholze, S. O'Keeffe, H.P. Phatnani, P. Guarnieri, C. Caneda, N. Ruderisch, S. Deng, S.A. Liddelow, C. Zhang, R. Daneman, T. Maniatis, B.A. Barres, J.Q. Wu, An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex, J Neurosci 34(36) (2014) 11929-47. [37] T. Dutta, Y.C. Kudva, X.M. Persson, L.A. Schenck, G.C. Ford, R.J. Singh, R. Carter, K.S. Nair, Impact of Long-Term Poor and Good Glycemic Control on Metabolomics Alterations in Type 1 Diabetic People, J Clin Endocrinol Metab 101(3) (2016) 1023-33. [38] G.N. Ruegsegger, S. Manjunatha, P. Summer, S. Gopala, P. Zabeilski, S. Dasari, P.M. Vanderboom, I.R. Lanza, K.A. Klaus, K.S. Nair, Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes, FASEB J 33(3) (2019) 4458-4472. [39] M. Langley, A. Ghosh, A. Charli, S. Sarkar, M. Ay, J. Luo, J. Zielonka, T. Brenza, B. Bennett, H. Jin, S. Ghaisas, B. Schlichtmann, D. Kim, V. Anantharam, A. Kanthasamy, B. Narasimhan, B. Kalyanaraman, A.G. Kanthasamy, MitoApocynin Prevents Mitochondrial Dysfunction, Microglial Activation, Oxidative Damage, and Progressive Neurodegeneration in MitoPark Transgenic Mice, Antioxid Redox Signal 27(14) (2017) 1048-1066.
Journal Pre-proof
62
Jo
ur
na
lP
re
-p
ro
of
[40] R.K. Dagda, S.J. Cherra, 3rd, S.M. Kulich, A. Tandon, D. Park, C.T. Chu, Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission, J Biol Chem 284(20) (2009) 13843-55. [41] H. Yoon, G. Walters, A.R. Paulsen, I.A. Scarisbrick, Astrocyte heterogeneity across the brain and spinal cord occurs developmentally, in adulthood and in response to demyelination, PLoS One 12(7) (2017) e0180697. [42] Y.W. Ng, Y.H. Say, Palmitic acid induces neurotoxicity and gliatoxicity in SH-SY5Y human neuroblastoma and T98G human glioblastoma cells, PeerJ 6 (2018) e4696. [43] S. Kim, C. Kim, S. Park, Mdivi-1 Protects Adult Rat Hippocampal Neural Stem Cells against Palmitate-Induced Oxidative Stress and Apoptosis, Int J Mol Sci 18(9) (2017). [44] C.I. Choi, H. Yoon, K.L. Drucker, M.R. Langley, L. Kleppe, I.A. Scarisbrick, The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation, Sci Rep 8(1) (2018) 9360. [45] A. Hall, N.A. Giese, W.D. Richardson, Spinal cord oligodendrocytes develop from ventrally derived progenitor cells that express PDGF alpha-receptors, Development 122(12) (1996) 4085-94. [46] X.R. Jin, X.S. Chen, L. Xiao, MeCP2 Deficiency in Neuroglia: New Progress in the Pathogenesis of Rett Syndrome, Front Mol Neurosci 10 (2017) 316. [47] A.D. Roth, M.T. Nunez, Oligodendrocytes: Functioning in a Delicate Balance Between High Metabolic Requirements and Oxidative Damage, Adv Exp Med Biol 949 (2016) 167-181. [48] K.R. Bohnert, J.D. McMillan, A. Kumar, Emerging roles of ER stress and unfolded protein response pathways in skeletal muscle health and disease, J Cell Physiol 233(1) (2018) 67-78. [49] N.L. Price, A.P. Gomes, A.J. Ling, F.V. Duarte, A. Martin-Montalvo, B.J. North, B. Agarwal, L. Ye, G. Ramadori, J.S. Teodoro, B.P. Hubbard, A.T. Varela, J.G. Davis, B. Varamini, A. Hafner, R. Moaddel, A.P. Rolo, R. Coppari, C.M. Palmeira, R. de Cabo, J.A. Baur, D.A. Sinclair, SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function, Cell Metab 15(5) (2012) 675-90. [50] M. Bonora, E. De Marchi, S. Patergnani, J.M. Suski, F. Celsi, A. Bononi, C. Giorgi, S. Marchi, A. Rimessi, J. Duszynski, T. Pozzan, M.R. Wieckowski, P. Pinton, Tumor necrosis factor-alpha impairs oligodendroglial differentiation through a mitochondria-dependent process, Cell Death Differ 21(8) (2014) 1198-208. [51] O. Baud, R.F. Haynes, H. Wang, R.D. Folkerth, J. Li, J.J. Volpe, P.A. Rosenberg, Developmental up-regulation of MnSOD in rat oligodendrocytes confers protection against oxidative injury, Eur J Neurosci 20(1) (2004) 29-40. [52] H. Lassmann, J. van Horssen, Oxidative stress and its impact on neurons and glia in multiple sclerosis lesions, Biochim Biophys Acta 1862(3) (2016) 506-10. [53] E. McCracken, V. Valeriani, C. Simpson, T. Jover, J. McCulloch, D. Dewar, The lipid peroxidation by-product 4-hydroxynonenal is toxic to axons and oligodendrocytes, J Cereb Blood Flow Metab 20(11) (2000) 1529-36. [54] G.G. Ortiz, F.P. Pacheco-Moises, O.K. Bitzer-Quintero, A.C. Ramirez-Anguiano, L.J. Flores-Alvarado, V. Ramirez-Ramirez, M.A. Macias-Islas, E.D. Torres-
Journal Pre-proof
63
Jo
ur
na
lP
re
-p
ro
of
Sanchez, Immunology and oxidative stress in multiple sclerosis: clinical and basic approach, Clin Dev Immunol 2013 (2013) 708659. [55] M.E. Silva, S. Lange, B. Hinrichsen, A.R. Philp, C.R. Reyes, D. Halabi, J.B. Mansilla, P. Rotheneichner, A. Guzman de la Fuente, S. Couillard-Despres, L.F. Batiz, R.J.M. Franklin, L. Aigner, F.J. Rivera, Pericytes Favor Oligodendrocyte Fate Choice in Adult Neural Stem Cells, Front Cell Neurosci 13 (2019) 85. [56] C.S. Fox, S.H. Golden, C. Anderson, G.A. Bray, L.E. Burke, I.H. de Boer, P. Deedwania, R.H. Eckel, A.G. Ershow, J. Fradkin, S.E. Inzucchi, M. Kosiborod, R.G. Nelson, M.J. Patel, M. Pignone, L. Quinn, P.R. Schauer, E. Selvin, D.K. Vafiadis, L. American Heart Association Diabetes Committee of the Council on, H. Cardiometabolic, C.o.C. Council on Clinical Cardiology, C.o.C.S. Stroke Nursing, C.o.Q.o.C. Anesthesia, R. Outcomes, A. American Diabetes, Update on Prevention of Cardiovascular Disease in Adults With Type 2 Diabetes Mellitus in Light of Recent Evidence: A Scientific Statement From the American Heart Association and the American Diabetes Association, Diabetes Care 38(9) (2015) 1777-803. [57] R.J. Koene, A.E. Prizment, A. Blaes, S.H. Konety, Shared Risk Factors in Cardiovascular Disease and Cancer, Circulation 133(11) (2016) 1104-14. [58] W. Mizunoya, K. Ohnuki, K. Baba, H. Miyahara, N. Shimizu, K. Tabata, T. Kino, Y. Sato, R. Tatsumi, Y. Ikeuchi, Effect of dietary fat type on anxiety-like and depression-like behavior in mice, Springerplus 2(1) (2013) 165. [59] D. Frasca, B.B. Blomberg, R. Paganelli, Aging, Obesity, and Inflammatory AgeRelated Diseases, Front Immunol 8 (2017) 1745. [60] J. Xu, Y. Li, H. Lin, R. Sinha, M.N. Potenza, Body mass index correlates negatively with white matter integrity in the fornix and corpus callosum: a diffusion tensor imaging study, Hum Brain Mapp 34(5) (2013) 1044-52. [61] L.E. Mokry, S. Ross, N.J. Timpson, S. Sawcer, G. Davey Smith, J.B. Richards, Obesity and Multiple Sclerosis: A Mendelian Randomization Study, PLoS Med 13(6) (2016) e1002053. [62] J.J. Guerrero-Garcia, L. Carrera-Quintanar, R.I. Lopez-Roa, A.L. Marquez-Aguirre, A.E. Rojas-Mayorquin, D. Ortuno-Sahagun, Multiple Sclerosis and Obesity: Possible Roles of Adipokines, Mediators Inflamm 2016 (2016) 4036232. [63] I.A. McKenzie, D. Ohayon, H. Li, J.P. de Faria, B. Emery, K. Tohyama, W.D. Richardson, Motor skill learning requires active central myelination, Science 346(6207) (2014) 318-22. [64] S. Schneider, A. Gruart, S. Grade, Y. Zhang, S. Kroger, F. Kirchhoff, G. Eichele, J.M. Delgado Garcia, L. Dimou, Decrease in newly generated oligodendrocytes leads to motor dysfunctions and changed myelin structures that can be rescued by transplanted cells, Glia 64(12) (2016) 2201-2218. [65] X. Chen, W. Zhang, T. Li, Y. Guo, Y. Tian, F. Wang, S. Liu, H.Y. Shen, Y. Feng, L. Xiao, Impairment of Oligodendroglia Maturation Leads to Aberrantly Increased Cortical Glutamate and Anxiety-Like Behaviors in Juvenile Mice, Front Cell Neurosci 9 (2015) 467. [66] M. Luan, Z. Shang, Y. Teng, X. Chen, M. Zhang, H. Lv, R. Zhang, The shared and specific mechanism of four autoimmune diseases, Oncotarget 8(65) (2017) 108355-108374.
Journal Pre-proof
64
Jo
ur
na
lP
re
-p
ro
of
[67] J.S. Yu, T. Hayashi, E. Seboun, R.M. Sklar, T.H. Doolittle, S.L. Hauser, Fos RNA accumulation in multiple sclerosis white matter tissue, J Neurol Sci 103(2) (1991) 209-15. [68] M. Vercellino, C. Fenoglio, D. Galimberti, A. Mattioda, C. Chiavazza, E. Binello, L. Pinessi, D. Giobbe, E. Scarpini, P. Cavalla, Progranulin genetic polymorphisms influence progression of disability and relapse recovery in multiple sclerosis, Mult Scler 22(8) (2016) 1007-12. [69] A. Groves, Y. Kihara, D. Jonnalagadda, R. Rivera, G. Kennedy, M. Mayford, J. Chun, A Functionally Defined In Vivo Astrocyte Population Identified by c-Fos Activation in a Mouse Model of Multiple Sclerosis Modulated by S1P Signaling: Immediate-Early Astrocytes (ieAstrocytes), eNeuro 5(5) (2018). [70] R. Waller, M.N. Woodroofe, S.B. Wharton, P.G. Ince, S. Francese, P.R. Heath, A. Cudzich-Madry, R.H. Thomas, N. Rounding, B. Sharrack, J.E. Simpson, Gene expression profiling of the astrocyte transcriptome in multiple sclerosis normal appearing white matter reveals a neuroprotective role, J Neuroimmunol 299 (2016) 139-146. [71] B. Serafini, R. Magliozzi, B. Rosicarelli, R. Reynolds, T.S. Zheng, F. Aloisi, Expression of TWEAK and its receptor Fn14 in the multiple sclerosis brain: implications for inflammatory tissue injury, J Neuropathol Exp Neurol 67(12) (2008) 1137-48. [72] S. Desplat-Jego, L. Feuillet, R. Creidy, I. Malikova, R. Rance, M. Khrestchatisky, K. Hahm, L.C. Burkly, J. Pelletier, J. Boucraut, TWEAK is expressed at the cell surface of monocytes during multiple sclerosis, J Leukoc Biol 85(1) (2009) 132-5. [73] I. Sevastou, G. Pryce, D. Baker, D.L. Selwood, Characterisation of Transcriptional Changes in the Spinal Cord of the Progressive Experimental Autoimmune Encephalomyelitis Biozzi ABH Mouse Model by RNA Sequencing, PLoS One 11(6) (2016) e0157754. [74] H.F. Kuipers, J. Yoon, J. van Horssen, M.H. Han, P.L. Bollyky, T.D. Palmer, L. Steinman, Phosphorylation of alphaB-crystallin supports reactive astrogliosis in demyelination, Proc Natl Acad Sci U S A 114(9) (2017) E1745-E1754. [75] L. Wang, B. Li, M.Y. Quan, L. Li, Y. Chen, G.J. Tan, J. Zhang, X.P. Liu, L. Guo, Mechanism of oxidative stress p38MAPK-SGK1 signaling axis in experimental autoimmune encephalomyelitis (EAE), Oncotarget 8(26) (2017) 42808-42816. [76] S. Moyon, A.L. Dubessy, M.S. Aigrot, M. Trotter, J.K. Huang, L. Dauphinot, M.C. Potier, C. Kerninon, S. Melik Parsadaniantz, R.J. Franklin, C. Lubetzki, Demyelination causes adult CNS progenitors to revert to an immature state and express immune cues that support their migration, J Neurosci 35(1) (2015) 4-20. [77] M.M. Herrmann, S. Barth, B. Greve, K.M. Schumann, A. Bartels, R. Weissert, Identification of gene expression patterns crucially involved in experimental autoimmune encephalomyelitis and multiple sclerosis, Dis Model Mech 9(10) (2016) 1211-1220. [78] J.C. Dugas, A. Ibrahim, B.A. Barres, The T3-induced gene KLF9 regulates oligodendrocyte differentiation and myelin regeneration, Mol Cell Neurosci 50(1) (2012) 45-57. [79] F. Fischbach, J. Nedelcu, P. Leopold, J. Zhan, T. Clarner, L. Nellessen, C. Beissel, Y. van Heuvel, A. Goswami, J. Weis, B. Denecke, C. Schmitz, T. Hochstrasser,
Journal Pre-proof
65
Jo
ur
na
lP
re
-p
ro
of
S. Nyamoya, M. Victor, C. Beyer, M. Kipp, Cuprizone-induced graded oligodendrocyte vulnerability is regulated by the transcription factor DNA damage-inducible transcript 3, Glia 67(2) (2019) 263-276. [80] K.K. Ryan, S.C. Woods, R.J. Seeley, Central nervous system mechanisms linking the consumption of palatable high-fat diets to the defense of greater adiposity, Cell Metab 15(2) (2012) 137-49. [81] S. Lupachyk, P. Watcho, A.A. Obrosov, R. Stavniichuk, I.G. Obrosova, Endoplasmic reticulum stress contributes to prediabetic peripheral neuropathy, Exp Neurol 247 (2013) 342-8. [82] C.K. Gavini, A.L. Bookout, R. Bonomo, L. Gautron, S. Lee, V. Mansuy-Aubert, Liver X Receptors Protect Dorsal Root Ganglia from Obesity-Induced Endoplasmic Reticulum Stress and Mechanical Allodynia, Cell Rep 25(2) (2018) 271-277 e4. [83] H.C. Hsu, C.Y. Chen, B.C. Lee, M.F. Chen, High-fat diet induces cardiomyocyte apoptosis via the inhibition of autophagy, Eur J Nutr 55(7) (2016) 2245-54. [84] X. Wang, Z.F. Zhang, G.H. Zheng, A.M. Wang, C.H. Sun, S.P. Qin, J. Zhuang, J. Lu, D.F. Ma, Y.L. Zheng, The Inhibitory Effects of Purple Sweet Potato Color on Hepatic Inflammation Is Associated with Restoration of NAD(+) Levels and Attenuation of NLRP3 Inflammasome Activation in High-Fat-Diet-Treated Mice, Molecules 22(8) (2017). [85] Y. Hussien, J.R. Podojil, A.P. Robinson, A.S. Lee, S.D. Miller, B. Popko, ER Chaperone BiP/GRP78 Is Required for Myelinating Cell Survival and Provides Protection during Experimental Autoimmune Encephalomyelitis, J Neurosci 35(48) (2015) 15921-33. [86] M.S. Elitt, H.E. Shick, M. Madhavan, K.C. Allan, B.L.L. Clayton, C. Weng, T.E. Miller, D.C. Factor, L. Barbar, B.S. Nawash, Z.S. Nevin, A.M. Lager, Y. Li, F. Jin, D.J. Adams, P.J. Tesar, Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease, Stem Cell Reports 11(3) (2018) 711-726. [87] P. Acs, S. Komoly, Selective ultrastructural vulnerability in the cuprizone-induced experimental demyelination, Ideggyogy Sz 65(7-8) (2012) 266-70. [88] M. Faizi, A. Salimi, E. Seydi, P. Naserzadeh, M. Kouhnavard, A. Rahimi, J. Pourahmad, Toxicity of cuprizone a Cu(2+) chelating agent on isolated mouse brain mitochondria: a justification for demyelination and subsequent behavioral dysfunction, Toxicol Mech Methods 26(4) (2016) 276-83. [89] F. Luo, K. Herrup, X. Qi, Y. Yang, Inhibition of Drp1 hyper-activation is protective in animal models of experimental multiple sclerosis, Exp Neurol 292 (2017) 21-34. [90] R.J. Youle, M. Karbowski, Mitochondrial fission in apoptosis, Nat Rev Mol Cell Biol 6(8) (2005) 657-63. [91] H.Y. Lin, S.W. Weng, Y.H. Chang, Y.J. Su, C.M. Chang, C.J. Tsai, F.C. Shen, J.H. Chuang, T.K. Lin, C.W. Liou, C.Y. Lin, P.W. Wang, The Causal Role of Mitochondrial Dynamics in Regulating Insulin Resistance in Diabetes: Link through Mitochondrial Reactive Oxygen Species, Oxid Med Cell Longev 2018 (2018) 7514383. [92] L. Haider, M.T. Fischer, J.M. Frischer, J. Bauer, R. Hoftberger, G. Botond, H. Esterbauer, C.J. Binder, J.L. Witztum, H. Lassmann, Oxidative damage in multiple sclerosis lesions, Brain 134(Pt 7) (2011) 1914-24.
Journal Pre-proof
66
Jo
ur
na
lP
re
-p
ro
of
[93] M.E. Witte, D.J. Mahad, H. Lassmann, J. van Horssen, Mitochondrial dysfunction contributes to neurodegeneration in multiple sclerosis, Trends Mol Med 20(3) (2014) 179-87. [94] C.M. Rice, M. Sun, K. Kemp, E. Gray, A. Wilkins, N.J. Scolding, Mitochondrial sirtuins--a new therapeutic target for repair and protection in multiple sclerosis, Eur J Neurosci 35(12) (2012) 1887-93. [95] G. Vakilzadeh, F. Khodagholi, T. Ghadiri, A. Ghaemi, F. Noorbakhsh, M. Sharifzadeh, A. Gorji, The Effect of Melatonin on Behavioral, Molecular, and Histopathological Changes in Cuprizone Model of Demyelination, Mol Neurobiol 53(7) (2016) 4675-84. [96] N. Sanadgol, F. Golab, H. Askari, F. Moradi, M. Ajdary, M. Mehdizadeh, Alphalipoic acid mitigates toxic-induced demyelination in the corpus callosum by lessening of oxidative stress and stimulation of polydendrocytes proliferation, Metab Brain Dis 33(1) (2018) 27-37. [97] U. Funfschilling, L.M. Supplie, D. Mahad, S. Boretius, A.S. Saab, J. Edgar, B.G. Brinkmann, C.M. Kassmann, I.D. Tzvetanova, W. Mobius, F. Diaz, D. Meijer, U. Suter, B. Hamprecht, M.W. Sereda, C.T. Moraes, J. Frahm, S. Goebbels, K.A. Nave, Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity, Nature 485(7399) (2012) 517-21. [98] D. Mehta, C. Miller, D.L. Arnold, E. Bame, A. Bar-Or, R. Gold, J. Hanna, L. Kappos, S. Liu, A. Matta, J.T. Phillips, D. Robertson, C.A. von Hehn, J. Campbell, K. Spach, L. Yang, R.J. Fox, Effect of dimethyl fumarate on lymphocytes in RRMS: Implications for clinical practice, Neurology 92(15) (2019) e1724-e1738. [99] Q. Weng, J. Wang, J. Wang, B. Tan, J. Wang, H. Wang, T. Zheng, Q.R. Lu, B. Yang, Q. He, Folate Metabolism Regulates Oligodendrocyte Survival and Differentiation by Modulating AMPKalpha Activity, Sci Rep 7(1) (2017) 1705. [100] R. Schoenfeld, A. Wong, J. Silva, M. Li, A. Itoh, M. Horiuchi, T. Itoh, D. Pleasure, G. Cortopassi, Oligodendroglial differentiation induces mitochondrial genes and inhibition of mitochondrial function represses oligodendroglial differentiation, Mitochondrion 10(2) (2010) 143-50. [101] J. Wang, C. Zhao, P. Kong, H. Sun, Z. Sun, G. Bian, Y. Sun, L. Guo, Treatment with NAD(+) inhibited experimental autoimmune encephalomyelitis by activating AMPK/SIRT1 signaling pathway and modulating Th1/Th17 immune responses in mice, Int Immunopharmacol 39 (2016) 287-94. [102] I. Ziabreva, G. Campbell, J. Rist, J. Zambonin, J. Rorbach, M.M. Wydro, H. Lassmann, R.J. Franklin, D. Mahad, Injury and differentiation following inhibition of mitochondrial respiratory chain complex IV in rat oligodendrocytes, Glia 58(15) (2010) 1827-37. [103] L.R. Stein, S. Imai, Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging, EMBO J 33(12) (2014) 132140. [104] B. Jablonska, M. Gierdalski, L.J. Chew, T. Hawley, M. Catron, A. Lichauco, J. Cabrera-Luque, T. Yuen, D. Rowitch, V. Gallo, Sirt1 regulates glial progenitor proliferation and regeneration in white matter after neonatal brain injury, Nat Commun 7 (2016) 13866.
Jo
ur
na
lP
re
-p
ro
of
Journal Pre-proof
67
Journal Pre-proof
ro
of
Graphical abstract
-p
Highlights
Chronic high fat diet impairs TCA cycle function in the mouse spinal cord
Mitochondria dysfunction occurs in the spinal cord after high fat consumption
Excess fat promotes mitochondrial dysfunction in purified oligodendrocytes
Chronic high fat diet causes oxidative stress in the brain and spinal cord
Chronic high fat diet results in oligodendrocyte loss in the brain and spinal cord
Jo
ur
na
lP
re
68