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Muscle microRNA signatures as biomarkers of disease progression in amyotrophic lateral sclerosis
Accepted Manuscript Muscle microRNA signatures as biomarkers of disease progression in amyotrophic lateral sclerosis
Ying Si, Xianqin Cui, David K. Crossman, Jiaying Hao, Mohamed Kazamel, Yuri Kwon, Peter H. King PII: DOI: Reference:
S0969-9961(18)30041-X doi:10.1016/j.nbd.2018.02.009 YNBDI 4116
To appear in:
Neurobiology of Disease
Received date: Revised date: Accepted date:
24 November 2017 28 January 2018 21 February 2018
Please cite this article as: Ying Si, Xianqin Cui, David K. Crossman, Jiaying Hao, Mohamed Kazamel, Yuri Kwon, Peter H. King , Muscle microRNA signatures as biomarkers of disease progression in amyotrophic lateral sclerosis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ynbdi(2017), doi:10.1016/j.nbd.2018.02.009
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ACCEPTED MANUSCRIPT King, P.H. 1
Muscle MicroRNA Signatures as Biomarkers of Disease Progression in Amyotrophic
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Lateral Sclerosis
Ying Sia,b, Xianqin Cuic,1, David K. Crossmand, Jiaying Haoc , Mohamed Kazamela, Yuri
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Kwona, Peter H. Kinga,b,d,2
Department of Neurology, University of Alabama, Birmingham, AL 35294
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Birmingham Veterans Affairs Medical Center, Birmingham, AL 35295
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Department of Biostatistics, University of Alabama at Birmingham, Birmingham, AL,
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35295
Department of Genetics, University of Alabama, Birmingham, AL, 35294
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Present address: Department of Biostatistics and Bioinformatics, Emory University,
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Atlanta, GA, 30322
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Correspondence to: Dr. P.H. King; UAB Dept. of Neurology, SC 200, 1530 3rd Avenue
South, Birmingham, AL 35294-0017, USA. Tel. (205) 934-2120; Fax (205) 996-7255; email: [email protected]
ACCEPTED MANUSCRIPT King, P.H. 2 ABSTRACT ALS is a fatal neurodegenerative disorder of motor neurons leading to progressive atrophy and weakness of muscles. Some of the earliest pathophysiological changes occur at the level of skeletal muscle and the neuromuscular junction. We previously identified distinct mRNA patterns, including members of the Smad and TGF-β family,
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that emerge in muscle tissue at the earliest (pre-clinical) stages. These patterns track
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disease progression in the mutant SOD1 mouse and are present in human ALS muscle. Because miRNAs play a direct regulatory role in mRNA expression, we hypothesized in
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this study that there would be distinct miRNA patterns in ALS muscle appearing in early stages that could track disease progression. We performed next-generation miRNA
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sequencing on muscle samples from G93A SOD1 mice at early (pre-clinical) and late (symptomatic) stages, and identified distinct miRNA patterns at both stages with some
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overlap. An Ingenuity Pathway Analysis predicted effects on a number of pathways
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relevant to ALS including TGF-β signaling, axon guidance signaling, and mitochondrial function. A subset of miRNAs was validated in the G93A SOD1 mouse at four stages of
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disease, and several appeared to track disease progression, including miR-206. We
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assessed these miRNAs in a large cohort of human ALS and disease control samples and found that some had similar changes but were not specific for ALS. Surprisingly,
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miR-206 levels did not change overall compared to normal controls, but did correlate with changes in strength of the muscle biopsied. In summary, we identified distinct miRNA patterns in ALS muscle that reflected disease stage which could potentially be used as biomarkers of disease activity. Key words: Myomirs, miRNA sequencing, G93A SOD1 mouse, human ALS muscle biopsies, Ingenuity Pathway Analysis
ACCEPTED MANUSCRIPT King, P.H. 3 INTRODUCTION
ALS is a heterogeneous disease with regard to etiology, location of onset (arm, leg, bulbar), involvement of upper and lower motor neurons, and pace of progression (AlChalabi and Hardiman, 2013). This heterogeneity poses diagnostic challenges to the
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clinician and contributes to the lengthy delay in diagnosis which often takes 9-15 months (Hardiman et al., 2011). One of the earliest sites of pathophysiology in ALS is skeletal
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muscle, the “end-organ”, where neuromuscular junction loss and mitochondrial
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dysfunction appear before evidence of motor neuron damage (Dupuis and Loeffler, 2009; Musaro, 2010). Prior reports have suggested that mitochondrial defects in muscle
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may actually trigger breakdown of the neuromuscular junction and initiate motor neuron degeneration (Dupuis et al., 2009). This observation has spurred the hypothesis that
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motor neuron degeneration is a dying back phenomenon where disease initiation occurs
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in the distal motor neuron terminals at the neuromuscular junction and tracks back proximally to the cell body (reviewed in (Moloney et al., 2014)). The early involvement of
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muscle in ALS prompted us to investigate muscle-specific mRNA patterns in pre-clinical
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disease. We hypothesized that the transcriptome would reflect these early pathophysiological changes, possibly provide insight in disease mechanisms, and serve
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as a potential biomarker of disease initiation or progression. We reported that members of the Smad (1,2,3, 5 and 8) and TGF-β (1,2, and 3) gene families are upregulated in mouse ALS muscle at very early stages of disease, well before any clinical signs of motor dysfunction (Si et al., 2014; Si et al., 2015). These mRNAs and their protein products increase over time in parallel with disease progression. The markers are also upregulated in human ALS muscle, and some correlate with the clinical degree of muscle weakness.
ACCEPTED MANUSCRIPT King, P.H. 4 Micro RNAs (miRNAs) are small molecules ranging from 21-26 nucleotides and play a major role in regulating diverse gene programs through modulation of mRNA stability and translation.(Valencia-Sanchez et al., 2006). The miRNA is initially transcribed as a pri-miRNA in the nucleus, undergoes processing to become a mature miRNA, and then associates with the RNA-induced silencing complex (RISC) to exert its typical
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suppressive effect on targeted mRNAs. With distinct mRNA patterns present in ALS
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muscle beginning at pre-clinical stages, we hypothesized that there would be concomitant miRNA signatures that change with disease progression. The link between
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miRNA maturation and TGF-β signaling through Smads provided further support for this hypothesis (Blahna and Hata, 2012; Davis-Dusenbery and Hata, 2011). Using next
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generation sequencing technology, we identified distinct muscle miRNA patterns in preclinical and symptomatic stages of ALS in the G93A mouse that reflected activation of
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physiologically relevant pathways. Some miRNA targets that emerged were validated in
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human ALS samples and correlated with clinical weakness, making them potential
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candidates for biomarkers of disease progression.
ACCEPTED MANUSCRIPT King, P.H. 5 MATERIALS AND METHODS
Animals
B6.Cg-Tg (SOD1*G93A) 1 Gur/J mice were purchased from The Jackson Laboratory
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(Bar Harbor, ME). Transgenic mice were maintained in the hemizygous state by mating
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G93A males with C57BL/6J females. Nontransgenic littermates were used as controls. All animal procedures were reviewed and approved by the UAB Institutional Animal Care
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and Use Committee in compliance with the National Research Council Guide for the
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Care and Use of Laboratory Animals.
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Tissue collection
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After approval by the UAB Institutional Review Board, muscle biopsy samples of ALS patients were identified as previously described (Si et al., 2014). A cohort of 19 patients
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with clinically probable or definite ALS, based on El Escorial criteria, was used for the
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main analysis. Additional samples were used for analysis of miR-206 as detailed in Supplementary Table 4. Biopsy samples of patients with myopathy, other neurogenic
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disease, or no pathology were used as controls. Muscles sampled from the ALS patients were assessed for power at the bedside using the Medical Research Council (MRC) grading scale. The range for this scale is 0 to 5, with 0 being no detectable movement to 5 where the muscle contracts normally against full resistance. For mouse samples, animals were sacrificed by CO2 inhalation followed by cervical dislocation. Gastrocnemius muscle was dissected at discrete time points between 40 and 150 days postnatal. Samples were briefly rinsed in phosphate buffered saline (PBS), and frozen in liquid nitrogen and stored at -80°C prior to molecular analysis.
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RNA Isolation and qRT-PCR
Total RNA was extracted from frozen tissues with mirVana™ miRNA Isolation Kit (Applied Biosystems, Carlsbad, CA) according to the manufacturer’s instructions. For
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validation studies, relative quantities of mature miRNAs were determined using Applied
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Biosystems TaqMan® microRNA Assays (Applied Biosystems, Carlsbad, CA). Ten nanograms of total RNA were reverse transcribed with TaqMan® MicroRNA Reverse
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Transcription Kit according to the manufacturer’s specifications. Individual assays were performed using On-Demand primers and probes from Applied Biosystems. The
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endogenous controls were snoRNA202 for mouse and RNU48 for human tissues. A standard thermal cycle protocol was used. Data were analyzed with the ViiA 7 Software
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version v1.2 (Applied Biosystems). The baseline was auto set, and threshold values
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were adjusted from 0.1 to 0.2 based on the amplification curve of different targets. Quantification of target miRNAs was done by the ΔΔCT method using snoRNA 202 or
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RNU48 as the internal control.
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MicroRNA profiling
MicroRNA profiling was conducted using the Illumina HiSeq 2500. Briefly, RNA quality was assessed on the Agilent BioAnalyzer and the presence of a strong 5S peak was used an indicator of a retained miRNA fraction. Next-Generation sequencing libraries were produced using the TruSeq small RNA library prep kit from Illumina. The microRNA fraction was used as a substrate for adaptor ligation at the 3’ end through an RNA ligase directed mechanism. The 3’ ligation was followed by addition of a 5’ adaptor and reverse transcription to generate first strand cDNA. An initial PCR step was done to
ACCEPTED MANUSCRIPT King, P.H. 7 produce the 2nd strand and to introduce unique indexes to each sample. Finally, the libraries were purified with magnetic beads and quantitated using the Kapa Biosystems qPCR quantitation for Illumina libraries. The resulting libraries were standardized for concentration and sequenced on the HiSeq2500 using 50bp single end reads.
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Data assessment
miRNAs species with average sequence counts less than 10 were removed from
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downstream analyses. The count data were normalized with the trimmed mean of the log expression ratios method (TMM) (Robinson and Oshlack, 2010). Likelihood ratio test
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was used for pair-wise comparisons. Analyses were conducted in R (Version 3.3.3) with
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Ingenuity Pathway Analysis
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Clustvis (Metsalu and Vilo, 2015).
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package “edgeR” (Robinson et al., 2010). Heatmaps were generated using the web tool,
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Differentially expressed miRNAs that had an average read count value greater than 10 were uploaded into Ingenuity Pathway Analysis (IPA) software
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(http://www.ingenuity.com). The miRNAs were then filtered on those that had a log2 fold change 1 and a p-value < 0.05. The target genes of these significant differentially expressed miRNAs were predicted using the IPA miRNA Target Filter tool. The miRNA gene targets were filtered based on those experimentally observed or predicted with high confidence. Functional analysis of these filtered miRNA gene targets was performed using IPA Core Analysis tool, which identifies statistically significant overrepresentation of these predicted gene targets in a given biological process or toxicological function.
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Statistics
A two-tailed Student’s t test was used for miRNA expression in mouse samples. For human samples, one-way analysis of variance (ANOVA) was used followed by a
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Dunnett’s multiple comparisons test. Pearson correlation coefficients were calculated
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using Graphpad software (San Diego, CA).
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RESULTS
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miRNAs are differentially expressed in ALS muscle and change over time
We performed Next-generation miRNA sequencing on muscle samples from G93A
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SOD1 mice and littermate controls to look for unique miRNA signatures in ALS muscle.
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Gastrocnemius samples were obtained at an early, pre-clinical stage (60 d) and later during overt clinical disease (125 d). These time points were used in our previous study
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of Smad and TGF-β biomarkers and based on published literature (Si et al., 2014; Si et al., 2015). After excluding miRNAs with less than 10 reads on average, there were
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approximately 280 targets identified in both age groups (Fig. 1). There was differential expression of 54 miRNAs at 60 d (P < 0.05) consistent with prior observations of molecular changes in muscle at early, preclinical stages of the disease (Capitanio et al., 2012; Hayworth and Gonzalez-Lima, 2009; Si et al., 2014; Si et al., 2015). At 125 d, this number increased by ~ two-fold with more miRNAs showing increased expression. Differential expression was present when comparing 60 d and 125 d ALS stages, suggesting a dynamic miRNA profile related to disease progression. Differential
ACCEPTED MANUSCRIPT King, P.H. 9 expression was also observed with WT at 60 and 125 d, although with a disproportionately higher number of miRNAs decreasing compared to the ALS mice (70% versus 46%). This finding, however, suggests an aging effect on miRNA expression even within the relatively short interval of 65 days. To gain insight into potential downstream pathways that might be affected by these miRNAs changes, we
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performed an Ingenuity Pathway Analysis on miRNAs that increased or decreased by
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more than two-fold (p < 0.05) in the ALS mouse relative to littermate controls (Fig. 2 and Supplementary Table 1). This analysis can identify cellular/molecular functions or
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signaling pathways significantly linked to the pattern of differential miRNA expression based on the targeted mRNAs. At 60 d, miRNAs associated with axonal guidance
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signaling (e.g. ephrin receptor and RhoA signaling) and changes in mitochondrial transmembrane potential had highly significant enrichment. Other pathways identified
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included p53, TGF-β, hypoxia-inducible factor, and retinoic acid receptor (RAR)
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activation. At 125 d, similar pathways were identified with the addition of NF-kB
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signaling. Apoptotic signaling was enriched at both ages.
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Identification of miRNAs that potentially track disease progression
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To identify potential miRNAs that could track disease progression, we focused on those with p- and q-values less than 0.05 and at least a 2-fold difference. Heat maps were generated for both age groups (Fig. 3). In the younger cohort, 7 of the 9 miRNAs meeting these criteria also appeared in the older cohort raising the possibility that they track disease progression. The most significant differentially expressed miRNA in both cohorts was miR-206 (fold-change, p- and q-values are shown in Supplementary Tables 2 and 3). To identify other miRNAs that potentially track disease progression, we compared the younger and older ALS groups. To exclude miRNAs that change with
ACCEPTED MANUSCRIPT King, P.H. 10 normal aging, we first compared the two littermate control groups and identified a number of targets that changed (Table 1). Interestingly, 17 of the 23 miRNAs that were differentially expressed were identified in prior reports looking at miRNA changes in aging myoblasts and/or aging muscle samples from the hindlimb (Kim et al., 2014; Lee et al., 2015). After removing these targets, we identified 38 miRNAs that were
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differentially expressed in older (symptomatic) versus younger (asymptomatic) ALS mice
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(Table 2). With miRNA-206, there was nearly a 2-fold increase from 60 to 125 d in the ALS group suggesting that its changes parallel disease progression. This was supported
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by the 12.9-fold increase in the ALS group at 125 d (versus control) compared to a 4.9fold increase in ALS mice over controls at 60 d. The vast majority of the miRNAs,
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however, did not reach significance in the younger cohort or were only modestly changed compared to WT, indicating that the miRNA transcriptome qualitatively changes
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with disease stage. Two targets (miR-194-5p and miR-210-3p) showed opposing
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changes in the two age groups. Taken together, these analyses identified several miRNAs, including miR-206, which showed promise in detecting pre-symptomatic
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disease as early as day 60. The majority of miRNAs changed after this time point.
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Validation of a subset of miRNAs in the ALS mouse
We focused on a subset of miRNAs to validate the sequencing results and to characterize changes over the course of the disease (Fig. 4). We chose two additional ages, 105 d and 150 d. The former represents late pre-symptomatic, as defined by maximal weight and rotarod performance, and the latter represents end-stage (Si et al., 2014). The marker which best tracked disease progression was miR-206, increasing at each time interval to end stage. On the other hand, miR-133a and miR-133b (which differ by two nucleotides at the 3’ terminus) decreased with disease progression,
ACCEPTED MANUSCRIPT King, P.H. 11 particularly when compared to age-matched control levels which trended upward in older mice. miR-1, which did not appear in the list of miRNAs, was assessed because it is transcribed bicistronically with miR-133a (Horak et al., 2016). We observed a similar decrease in expression (with a concomitant trend upward in WT). This is in contrast to miR-133b and miR-206 which are also bicistronic but changed in opposite directions. For
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each of these miRNAs, there was a significant change in expression level in the ALS
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mouse between 60 d and later stages, suggesting an association with disease progression. Other miRNAs, including miR-34c, 10b-5p, 99a-5p, 100-5p, and 127-3p
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became significantly altered at all stages but without any stage-dependent trends. miR127-3p showed a gradual reduction in wild-type controls at older ages which provides
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validation for observations in Table 1. Thus, some of the miRNAs that change with age can still be sensors of disease activity. To determine how early the miRNAs change, we
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examined 40 d old mice, and did not find differential expression (Supplementary Fig. 1).
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The 40 - 60 d time frame for onset of miRNA changes is consistent with what we observed for Smads and TGF-βs (Si et al., 2014; Si et al., 2015). Taken together, these
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findings provided validation of our miRNA sequencing data.
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Validation of targets in human ALS muscle
Targets that were validated in mouse were assessed in biopsy samples from human ALS patients (Table 3) and compared to normal or disease controls (Table 4). The mean age for the ALS patients was 60 years which matches the overall mean age of diagnosis at our institution (Kazamel et al., 2013). Biopsies were obtained approximately 1 year after the onset of symptoms. The controls consisted of myopathy and neuropathy similar to our previous publications (Si et al., 2014; Si et al., 2015). Most of the miRNAs that were attenuated in mouse muscle were also attenuated in human ALS samples,
ACCEPTED MANUSCRIPT King, P.H. 12 including miR-1, 10b, 99a-5p, 100-5p, 133a-3p and 133b-3p (Fig. 5). Disease controls, however, were similarly affected. Two miRNAs, 34c and 206, which were elevated in mouse samples were not significantly changed. Although miR-206 showed a trend upward, this finding was in contrast to the ALS mouse where the miRNA increased more than 10-fold in later stages of the disease (Fig. 4). The standard error for miR-206,
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however, was larger than other miRNAs indicating variability among samples. Because
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this miRNA varies in the mouse based on muscle type, showing higher expression in slow twitch-dominant muscles (McCarthy and Esser, 2007; Williams et al., 2009b), we
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assessed it in a broader number of normal human samples. We focused on two commonly biopsied muscles: deltoid and vastus lateralis (Supplementary Table 4). With
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the mean age of each group being similar (62 years for deltoid and 60 years for vastus lateralis), there was more than a two-fold higher level (P < 0.001) in vastus lateralis
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samples (Fig. 6A). We then assessed miRNA-206 in an expanded number of ALS
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biopsy samples (Supplementary Table 4), and compared the relative quantities to the same muscle type from the normal control population (Fig. 6B). A non-significant trend
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toward higher levels in deltoid samples was observed, but no change with vastus
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lateralis. Again, the error bars were increased. To determine whether this variability reflected the degree of disease involvement, we performed a correlative analysis
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between miRNA level and the Medical Research Council (MRC) muscle grade in the original cohort (Fig. 7). For miR-206, there was a significant negative correlation between MRC grade and expression (r = -0.45, P = 0.03). We extended this analysis to the other miRNAs and found positive correlations with miR-133a (r = 0.52, p = 0.03) and miR-1 (r = 0.49, p = 0.04). miR-10b-5p, 133b-3p, and 127-3p, showed trends toward correlation but did not reach significance. miR-34c, 99a and 100-5p showed no correlation. In summary, some miRNAs identified in mouse ALS muscle had similar
ACCEPTED MANUSCRIPT King, P.H. 13 changes in human muscle and showed correlation with clinical assessment of muscle strength.
DISCUSSION
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In this report we show that there are distinct miRNA signatures in muscle samples from
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pre-clinical ALS mice, reflecting early physiological changes, which shift in a later stage of the disease when motor deficits are present. Some of the miRNAs that were
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differentially expressed at both stages paralleled disease progression and were similarly altered in human ALS muscle. Furthermore, a subset of these miRNAs in human
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samples correlated with muscle strength, suggesting that they may reflect disease activity and serve as biomarkers for ALS.
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Skeletal muscle is the “end organ” of ALS, reflecting the ravages of motor neuron
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degeneration as the muscle becomes atrophic, spastic or both. Our previous work showed the emergence of distinct muscle mRNA patterns, including members of the
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TGF-β and Smad families, at very early and pre-clinical stages of disease in the ALS
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mouse (Si et al., 2014; Si et al., 2015). Consistent with this observation, TGF-β signaling ranked near the top in both age groups for pathways predicted to be altered (Fig. 2). A
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recent report suggests that this signaling is linked to increased muscle fibrosis in the symptomatic stages of disease (Gonzalez et al., 2017). miRNAs associated with alterations in mitochondrial membrane potential also ranked high and meshes with prior studies showing that membrane depolarization of muscle mitochondria and other signs of mitochondrial damage occur at the earliest pre-clinical stages of disease (Dupuis et al., 2003; Luo et al., 2013). Likewise, Ephrin/RhoA signaling is increased in muscle of pre-symptomatic ALS and is negatively associated with muscle/NMJ reinnervation (Moloney et al., 2014). P53 signaling appeared in all subsets, ranking at the top of
ACCEPTED MANUSCRIPT King, P.H. 14 significance for enrichment in the 125 d cohort. A recent meta-analysis of transcriptomic experiments identified p53 deregulation as a characteristic feature of ALS muscle, significantly correlating with severity of atrophy (von Grabowiecki et al., 2016). miRNAs associated with RAR signaling were observed in both age groups. This pathway has not been previously identified in ALS muscle, but is triggered early on in injured muscles and
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promotes muscle repair (Di Rocco et al., 2015). Interestingly there were a substantial
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number of miRNAs that changed (mostly in the negative direction) between the two control groups indicating an age effect. A majority of these miRNAs (Table 1) were
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reported to change in the same direction in aging myoblasts and/or muscle isolated from hindlimbs, although the age differences were substantially larger (18-24 months) (Kim et
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al., 2014; Lee et al., 2015). The comparatively young age and short time interval (~ 2 months) in our study suggests that the changes begin very early and relatively fast.
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In the validated subset of miRNAs, many showed differences starting in the 60
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day old group, but not earlier. miRNA 206 was elevated by ~2-fold in contrast to a prior study of the low-copy G93A SOD1 mouse model where induction occurred only after
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disease initiation (as defined by maximal weight) (Williams et al., 2009b). In our study,
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this miRNA gradually increased with age in the ALS mouse suggesting that it could be a marker of disease progression. This miRNA supports neuromuscular junction
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reinnervation through suppression of HDAC4 (Williams et al., 2009b). No differences in miR-206, however, were discerned in human ALS samples compared to controls. Interestingly this miRNA was two-fold higher in vastus lateralis compared to deltoid in our control population. In mouse, slow-twitch fiber predominant muscles such as soleus are reported to have significantly higher miR-206 levels than fast-twitch predominant muscles like plantaris (McCarthy, 2008; Williams et al., 2009b). In humans, however, deltoid and vastus lateralis are “mixed” muscles with variable but overlapping proportions of slow-twitch fibers (Johnson et al., 1973; Saltin and Gollnick, 2010). Our
ACCEPTED MANUSCRIPT King, P.H. 15 findings suggest that other factors besides fiber type proportion are at play for determining miR-206 expression. Even when factoring the variable expression of miR206, there were no differences with control muscle (Fig. 6). The significant inverse correlation with muscle power of the biopsied muscle, however, suggests that it could be a marker of disease activity. This finding also reflects the heterogeneity of disease
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activity in human ALS muscle (versus the relative homogeneity of muscle involvement in
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the ALS mouse) and emphasizes the need to associate miR-206 levels (and other miRNAs) with muscle-specific clinical assessment rather than overall clinical status. This
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heterogeneity, coupled with the differences in baseline miR-206 expression in different muscles, could explain inconsistencies in previous reports. One study, for example,
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observed an increase in miR-206 over normal controls in early stages of disease but no difference in later stages, whereas two others found upregulation in both phases
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(Bruneteau et al., 2013; Di Pietro et al., 2017; Pegoraro et al., 2017). Interestingly, miR-
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133b, which is bicistronic with miR-206 (Horak et al., 2016), gradually decreased in mouse ALS muscle with disease progression and was attenuated in human ALS
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samples. This dichotomy is consistent with a prior report indicating that transcription of
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the two miRNAs, even though located in tandem, is uncoupled (Cesana et al., 2011). Assessment of pri-miRNA levels would be necessary to confirm this possibility. On the
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other hand, miR-1 and miR-133a, which are also bicistronic and transcribed together (Chen et al., 2006), progressively decreased in the ALS mouse in parallel and correlated with muscle weakness in human ALS samples. As with miR-206, changes in these miRNAs vary in prior reports, with the mouse study by Williams et al. being consistent with our findings (in both mouse and humans) versus other reports showing variable changes depending on disease stage or pace of progression (Di Pietro et al., 2017; Pegoraro et al., 2017; Williams et al., 2009b). In one study, patients with slow progression or early stage disease showed increases in miR-133a, 133b and 1 whereas
ACCEPTED MANUSCRIPT King, P.H. 16 rapid progression or late disease was associated with no change or a decrease compared to control samples (Di Pietro et al., 2017). Several miRNAs that were validated in the mouse, including miR-34c-5p and miR-127-3p, were not altered in human ALS samples and/or did not correlate with muscle weakness. It is possible that these miRNAs are unique to mutant SOD1-
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associated ALS as our human patients were predominantly sporadic. All miRNAs that
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were validated in human samples were similarly altered in myopathy and other neurogenic processes, suggesting that they are general responders to muscle tissue
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injury. The trigger for this response and the impact on the underlying disease process is unclear given the pleiotropic effects of the muscle-specific miRNAs on myogenesis,
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muscle repair and reinnervation (Horak et al., 2016; Wang, 2013; Williams et al., 2009a). Myofiber loss cannot explain the selective attenuation of muscle-specific miRNAs since
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many other miRNAs increased with disease progression or did not change compared to
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normal controls (Greco et al., 2009). It is possible that changes in the microenvironment of injured muscle may influence miRNA patterns. A prior study of patients with
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inflammatory myopathy (which was the predominant diagnosis in our myopathy cohort)
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described a similar suppression of miR-1, 133a and 133b and linked it to TNF-α (Georgantas et al., 2014). The inflammatory cytokine TWEAK, which promotes muscle
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wasting in the setting of denervation and other pathological states also suppresses these miRNAs (Bhatnagar and Kumar, 2012; Panguluri et al., 2010). The potential relevance is underscored by a report that this signaling pathway is increased in ALS muscle and that knockout of TWEAK lessens muscle atrophy (Bowerman et al., 2015). Finally, it is important to note that changes observed in some of the miRNAs could be attributable to other cell types in the muscle sample such as macrophages or other immune cells, fibroblasts, endothelial cells and motor neuron termini.
ACCEPTED MANUSCRIPT King, P.H. 17 In conclusion, we have identified distinct muscle miRNA patterns that change with ALS progression and reflect the underlying pathophysiology of the disease at the level of the muscle. We have identified a subset of miRNAs that can track disease progression in the ALS mouse and are also affected in human ALS muscle. These miRNAs would be best suited as markers of disease progression, although the very
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early preclinical appearance of changes suggests they could track disease onset. The
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utility of these miRNAs as markers in the clinic is unclear. It would depend on ease of methodology for measuring them. Several reports have detected significant, albeit non-
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specific, changes in serum levels of miR-1, 133a, 133b and 206 (Toivonen et al., 2014; Waller et al., 2017). Alternatively, as nanoparticle technology advances, in vivo imaging
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of miRNAs by magnetic resonance imaging may be possible down the road (Hernandez
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et al., 2013).
ACCEPTED MANUSCRIPT King, P.H. 18 ACKNOWLEDGEMENTS
This work was supported by NINDS R01NS092651 (PHK) and a Merit Review BX001148 from the Department of Veterans Affairs (PHK).The authors wish to thank the UAB Genomics Core, funded through the UAB Comprehensive Cancer Center
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(CA13148) and CFAR (AI027767).
ACCEPTED MANUSCRIPT King, P.H. 19 REFERENCES
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Dupuis, L., et al., 2009. Muscle mitochondrial uncoupling dismantles neuromuscular
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Dupuis, L., Loeffler, J. P., 2009. Neuromuscular junction destruction during amyotrophic lateral sclerosis: insights from transgenic models. Curr Opin Pharmacol. 9, 341-6.
Georgantas, R. W., et al., 2014. Inhibition of Myogenic MicroRNAs 1, 133, and 206 by Inflammatory Cytokines Links Inflammation and Muscle Degeneration in Adult Inflammatory Myopathies. Arthritis & Rheumatology. 66, 1022-1033.
ACCEPTED MANUSCRIPT King, P.H. 21 Gonzalez, D., et al., 2017. ALS skeletal muscle shows enhanced TGF-β signaling, fibrosis and induction of fibro/adipogenic progenitor markers. PLOS ONE. 12, e0177649.
Greco, S., et al., 2009. Common micro-RNA signature in skeletal muscle damage and
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Johnson, M. A., et al., 1973. Data on the distribution of fibre types in thirty-six human muscles: An autopsy study. Journal of the Neurological Sciences. 18, 111-129.
ACCEPTED MANUSCRIPT King, P.H. 22 Kazamel, M., et al., 2013. Epidemiological features of amyotrophic lateral sclerosis in a large clinic-based African American population. Amyotroph Lateral Scler Frontotemporal Degener. 14, 334-7.
Kim, J. Y., et al., 2014. Genome-wide profiling of the microRNA-mRNA regulatory
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Metsalu, T., Vilo, J., 2015. ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic Acids Res. 43, W566-70.
ACCEPTED MANUSCRIPT King, P.H. 23 Moloney, E. B., et al., 2014. ALS as a distal axonopathy: molecular mechanisms affecting neuromuscular junction stability in the presymptomatic stages of the disease. Front Neurosci. 8, 252.
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Si, Y., et al., 2014. Smads as muscle biomarkers in amyotrophic lateral sclerosis. Ann Clin Transl Neurol. 1, 778-87.
ACCEPTED MANUSCRIPT King, P.H. 24 Si, Y., et al., 2015. Transforming Growth Factor Beta (TGF-beta) Is a Muscle Biomarker of Disease Progression in ALS and Correlates with Smad Expression. PLoS One. 10, e0138425.
Toivonen, J. M., et al., 2014. MicroRNA-206: A Potential Circulating Biomarker
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Candidate for Amyotrophic Lateral Sclerosis. PLoS ONE. 9, e89065.
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von Grabowiecki, Y., et al., 2016. Transcriptional activator TAp63 is upregulated in muscular atrophy during ALS and induces the pro-atrophic ubiquitin ligase
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Trim63. eLife. 5, e10528.
Waller, R., et al., 2017. Serum miRNAs miR-206, 143-3p and 374b-5p as potential
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biomarkers for amyotrophic lateral sclerosis (ALS). Neurobiol Aging. 55, 123-131.
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Wang, X. H., 2013. MicroRNA in myogenesis and muscle atrophy. Curr Opin Clin Nutr
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Metab Care. 16, 258-66.
Williams, A. H., et al., 2009a. MicroRNA control of muscle development and disease. Curr Opin Cell Biol. 21, 461-9.
Williams, A. H., et al., 2009b. MicroRNA-206 Delays ALS Progression and Promotes Regeneration of Neuromuscular Synapses in Mice. Science. 326, 1549-1554.
ACCEPTED MANUSCRIPT King, P.H. 25 Table 1 Differentially expressed miRNAs
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*miRNAs altered in aging myoblasts and/or aging muscle, based on prior reports (Kim
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10.6 2.9 -2.5 -3.0 -3.3 -3.4 -3.8 -4.0 -4.0 -4.1 -4.4 -4.5 -4.7 -4.9 -4.9 -5.6 -5.7 -5.8 -5.8 -5.8 -6.5 -8.3 -9.9
P-value 3.3E-04 1.4E-04 2.9E-03 9.0E-06 1.7E-03 3.6E-04 1.0E-08 3.1E-04 7.9E-04 9.6E-06 3.1E-03 1.0E-04 1.4E-03 1.5E-07 2.4E-07 2.3E-03 7.3E-09 3.3E-05 2.5E-04 8.4E-07 1.3E-04 2.6E-05 3.3E-08
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miRNA* 6244 451a 19b-3p 127-3p* 485-5p* 154-5p 434-3p* 431-5p* 673-5p* 434-5p* 377-5p* 136-5p* 673-3p* 541-5p* 411-5p* 543-3p* 299a-5p 455-5p 300-3p* 127-5p* 1193-3p* 136-3p* 337-5p*
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in WT mice at age 125 d versus 60 d.
et al., 2014; Lee et al., 2015).
ACCEPTED MANUSCRIPT King, P.H. 26 Table 2. Differentially expressed miRNAs (fold-change) in ALS mice at age 125 d versus 60 d (p- and q-values < 0.05). Fold-change for these miRNAs in the ALS v. WT 60 d and 125 d groups are shown for comparison.
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ALS v. WT (125 d) 4.4 15.5 12.2 3.7 4.0 6.3 2.5 2.5 2.5 1.7 2.5 2.3 2.9 2.2 3.3 -0.5 2.3 1.9 1.8 ns 1.7 2.2 12.9 ns 2.2 1.8 -2.6 -2.2 -4.7 -4.0 -6.9 -7.3 -3.9 -3.2 -9.0 -4.3
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ALS v. WT (60 d)1 nd nd nd -2.9 nd nd nd nd nd nd nd nd nd nd nd nd nd nd
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ALS (125 d v. 60 d) 6.5 4.8 4.3 3.7 3.5 3.3 3.0 2.8 2.5 2.5 2.4 2.4 2.3 2.3 2.2 2.2 2.2 2.2 2.2 2.1 2.0 1.9 1.9 1.9 1.8 1.8 -2.1 -2.9 -3.4 -3.6 -4.1 -5.3 -5.7 -5.8 -6.7 -10.5
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mmu-miR 21a-3p 18a-5p 142-3p 194-5p 21a-5p 19a-3p 301a-3p 322-5p 340-5p 223-3p 3535 106b-5p 24-3p 101a-3p 17-5p 98-5p 93-5p 16-5p 210-3p 30a-5p 25-3p 140-3p 206-3p 30e-5p 27b-3p 27a-3p 125a-5p 362-5p 99a-5p 99b-5p 100-5p 1943-5p 501-3p 540-5p 381-5p 329-3p
-1.8 -1.7 nd nd 4.9 -1.9 nd nd nd nd nd nd nd nd nd nd nd nd
ACCEPTED MANUSCRIPT King, P.H. 27 539-5p 409-5p
-6.9 -18.2
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nd, no difference from wild-type control.
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nd nd
-10.8 -47.2
ACCEPTED MANUSCRIPT King, P.H. 28
Table 3: ALS patients (n = 19) 34-78
Mean age (years)
60 ± 13
Gender
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Duration (months)
14 ± 13
Muscle (n)
TA (6); VL (1)
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Age range (years)
15 (4 muscles not tested)
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EMG denervation
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BI (6), DT (6)
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9F
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tibialis anterior; VL, vastus lateralis
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BI, biceps; DT, deltoid; EMG, electromyography; TA,
ACCEPTED MANUSCRIPT King, P.H. 29
Table 4: Control patients Myopathy (n = 10)
Neuropathy (n =6)
Age Range (y)
24-67
38-74
39-88
Mean Age (y)
53 ± 11
58 ± 9
TA (1), VL (4),
TA (1), VL (6),
BI (3), DT (1)
BI (2), DT (1)
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Non-specific (1)
Inflammatory (1)
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BI (1) Diabetic (2)
BI, biceps; DT, deltoid; TA, tibialis anterior; VL, vastus lateralis
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TA (3), VL (2),
Inflammatory (9)
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Diagnosis
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59 ± 21
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Muscle (all)
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Normal (n=9)
Multifocal motor (1) Non-specific (2)
ACCEPTED MANUSCRIPT King, P.H. 30 FIGURE LEGENDS
Figure 1. Differential expression of miRNAs in gastrocnemius samples of G93A SOD1 (ALS) and wild-type (WT) mice at 60 and 125 days of age. miRNA transcriptomes were determined by Next-generation sequencing and compared among four groups: ALS and
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WT mice at 60 and125 d (n = 3 per group). Differential expression was considered
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present when the P value was < 0.05 for miRNAs with greater than 10 reads,
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independent of fold-change.
Figure 2. Ingenuity Pathway Analysis of differentially expressed miRNAs in G93A SOD1
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gastrocnemius muscle, compared to wild-type controls, at 60 and 125 days of age. Only miRNAs with ≥ 2-fold change and P < 0.05 were used in this analysis. Biological
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functions and signaling pathways predicted to be affected by a downregulation (Down)
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or upregulation (Up) of miRNAs in the ALS group are shown. P values are indicated on the x axis. The number of altered miRNAs and downstream genes predicted to be
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affected are shown in parentheses (miRNAs/genes). See Supplementary Table 1 for
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additional information.
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Figure 3. Unsupervised hierarchical clustering of miRNAs differentially expressed in ALS mice compared to controls at 60 and 125 d. Only miRNAs with p- and q-values < 0.05 and a 2-fold or greater difference are shown (9 miRNAs for 60 d and 73 miRNAs for 125 d). See Supplementary Tables 2 and 3 for additional information.
Figure 4. Validation of a subset of differentially expressed miRNAs identified by sequencing. Expression levels of miRNAs in muscle samples from G93A SOD1 or WT mice were assessed by qRT-PCR. Ages ranged from early, pre-clinical (60 d), late pre-
ACCEPTED MANUSCRIPT King, P.H. 31 clinical (105 d), clinically symptomatic (125 d), and end-stage (150 d). Clinical onset was determined by maximal weight and rotarod function as previously reported (Si et al., 2014). Each data point represents the mean ± SEM of 3-6 mice. All values were expressed as a fold-change of WT at age 60 d (set at 1.0). * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 comparing G93A to WT mice at each age. # P < 0.05 comparing
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G93A SOD1 mice at the indicated age to G93A SOD1 mice at 60 d. RQ, relative
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quantity.
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Figure 5. Validation of miRNAs in human ALS muscle samples. Expression levels of miRNAs in muscle samples from human biopsy samples were assessed by qRT-PCR.
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Clinical data are shown in Tables 3 and 4. Data points represent the mean ± SEM of 19 ALS, 9 normal controls, 10 myopathies (Myo) and 6 neuropathies (Neuro). *P < 0.05, **P
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< 0.01, ***P < 0.001, ****P < 0.0001.
Figure 6. miRNA 206 is differentially expressed in normal human muscle. A) Normal
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muscle biopsy samples were assessed for miR-206-3p expression by qRT-PCR. The
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average age for deltoid (DT) and vastus lateralis (VL) samples was 62 ± 12 and 60 ± 7 years. B) miRNA-206 expression was assessed in ALS and normal patients using equal
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numbers of DT and VL samples. RQ, relative quantity.
Figure 7. Correlation of miRNA expression level and strength of muscle biopsied. Muscle strength was determined clinically at the bedside by manual motor testing using the Medical Research Council (MRC) scale (see methods). Total number of samples assessed was 17 (MRC scores were not available for 2 of the 19 muscle samples shown in Fig. 5). For miR-206, there were 24 samples. *P < 0.05.
ACCEPTED MANUSCRIPT King, P.H. 32 Highlights
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• miRNA transcriptomes were assessed in muscle of G93A-SOD1 mice. •Distinct and overlapping miRNA patterns appeared in 60 and 125 d old cohorts. •miRNAs affecting mitochondria, axonal guidance, p53 and TGFβ pathways were enriched. •A subset of miRNAs tracked disease progression in the mouse. •Some miRNAs were validated in human ALS muscle and correlated with motor strength.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
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Ying Si, Xianqin Cui, David K. Crossman, Jiaying Hao, Mohamed Kazamel, Yuri Kwon, Peter H. King PII: DOI: Reference:
S0969-9961(18)30041-X doi:10.1016/j.nbd.2018.02.009 YNBDI 4116
To appear in:
Neurobiology of Disease
Received date: Revised date: Accepted date:
24 November 2017 28 January 2018 21 February 2018
Please cite this article as: Ying Si, Xianqin Cui, David K. Crossman, Jiaying Hao, Mohamed Kazamel, Yuri Kwon, Peter H. King , Muscle microRNA signatures as biomarkers of disease progression in amyotrophic lateral sclerosis. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ynbdi(2017), doi:10.1016/j.nbd.2018.02.009
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ACCEPTED MANUSCRIPT King, P.H. 1
Muscle MicroRNA Signatures as Biomarkers of Disease Progression in Amyotrophic
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Lateral Sclerosis
Ying Sia,b, Xianqin Cuic,1, David K. Crossmand, Jiaying Haoc , Mohamed Kazamela, Yuri
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Kwona, Peter H. Kinga,b,d,2
Department of Neurology, University of Alabama, Birmingham, AL 35294
b
Birmingham Veterans Affairs Medical Center, Birmingham, AL 35295
c
Department of Biostatistics, University of Alabama at Birmingham, Birmingham, AL,
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Department of Genetics, University of Alabama, Birmingham, AL, 35294
1
Present address: Department of Biostatistics and Bioinformatics, Emory University,
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Atlanta, GA, 30322
2
Correspondence to: Dr. P.H. King; UAB Dept. of Neurology, SC 200, 1530 3rd Avenue
South, Birmingham, AL 35294-0017, USA. Tel. (205) 934-2120; Fax (205) 996-7255; email: [email protected]
ACCEPTED MANUSCRIPT King, P.H. 2 ABSTRACT ALS is a fatal neurodegenerative disorder of motor neurons leading to progressive atrophy and weakness of muscles. Some of the earliest pathophysiological changes occur at the level of skeletal muscle and the neuromuscular junction. We previously identified distinct mRNA patterns, including members of the Smad and TGF-β family,
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that emerge in muscle tissue at the earliest (pre-clinical) stages. These patterns track
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disease progression in the mutant SOD1 mouse and are present in human ALS muscle. Because miRNAs play a direct regulatory role in mRNA expression, we hypothesized in
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this study that there would be distinct miRNA patterns in ALS muscle appearing in early stages that could track disease progression. We performed next-generation miRNA
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sequencing on muscle samples from G93A SOD1 mice at early (pre-clinical) and late (symptomatic) stages, and identified distinct miRNA patterns at both stages with some
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overlap. An Ingenuity Pathway Analysis predicted effects on a number of pathways
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relevant to ALS including TGF-β signaling, axon guidance signaling, and mitochondrial function. A subset of miRNAs was validated in the G93A SOD1 mouse at four stages of
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disease, and several appeared to track disease progression, including miR-206. We
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assessed these miRNAs in a large cohort of human ALS and disease control samples and found that some had similar changes but were not specific for ALS. Surprisingly,
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miR-206 levels did not change overall compared to normal controls, but did correlate with changes in strength of the muscle biopsied. In summary, we identified distinct miRNA patterns in ALS muscle that reflected disease stage which could potentially be used as biomarkers of disease activity. Key words: Myomirs, miRNA sequencing, G93A SOD1 mouse, human ALS muscle biopsies, Ingenuity Pathway Analysis
ACCEPTED MANUSCRIPT King, P.H. 3 INTRODUCTION
ALS is a heterogeneous disease with regard to etiology, location of onset (arm, leg, bulbar), involvement of upper and lower motor neurons, and pace of progression (AlChalabi and Hardiman, 2013). This heterogeneity poses diagnostic challenges to the
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clinician and contributes to the lengthy delay in diagnosis which often takes 9-15 months (Hardiman et al., 2011). One of the earliest sites of pathophysiology in ALS is skeletal
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muscle, the “end-organ”, where neuromuscular junction loss and mitochondrial
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dysfunction appear before evidence of motor neuron damage (Dupuis and Loeffler, 2009; Musaro, 2010). Prior reports have suggested that mitochondrial defects in muscle
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may actually trigger breakdown of the neuromuscular junction and initiate motor neuron degeneration (Dupuis et al., 2009). This observation has spurred the hypothesis that
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motor neuron degeneration is a dying back phenomenon where disease initiation occurs
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in the distal motor neuron terminals at the neuromuscular junction and tracks back proximally to the cell body (reviewed in (Moloney et al., 2014)). The early involvement of
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muscle in ALS prompted us to investigate muscle-specific mRNA patterns in pre-clinical
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disease. We hypothesized that the transcriptome would reflect these early pathophysiological changes, possibly provide insight in disease mechanisms, and serve
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as a potential biomarker of disease initiation or progression. We reported that members of the Smad (1,2,3, 5 and 8) and TGF-β (1,2, and 3) gene families are upregulated in mouse ALS muscle at very early stages of disease, well before any clinical signs of motor dysfunction (Si et al., 2014; Si et al., 2015). These mRNAs and their protein products increase over time in parallel with disease progression. The markers are also upregulated in human ALS muscle, and some correlate with the clinical degree of muscle weakness.
ACCEPTED MANUSCRIPT King, P.H. 4 Micro RNAs (miRNAs) are small molecules ranging from 21-26 nucleotides and play a major role in regulating diverse gene programs through modulation of mRNA stability and translation.(Valencia-Sanchez et al., 2006). The miRNA is initially transcribed as a pri-miRNA in the nucleus, undergoes processing to become a mature miRNA, and then associates with the RNA-induced silencing complex (RISC) to exert its typical
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suppressive effect on targeted mRNAs. With distinct mRNA patterns present in ALS
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muscle beginning at pre-clinical stages, we hypothesized that there would be concomitant miRNA signatures that change with disease progression. The link between
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miRNA maturation and TGF-β signaling through Smads provided further support for this hypothesis (Blahna and Hata, 2012; Davis-Dusenbery and Hata, 2011). Using next
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generation sequencing technology, we identified distinct muscle miRNA patterns in preclinical and symptomatic stages of ALS in the G93A mouse that reflected activation of
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physiologically relevant pathways. Some miRNA targets that emerged were validated in
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human ALS samples and correlated with clinical weakness, making them potential
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candidates for biomarkers of disease progression.
ACCEPTED MANUSCRIPT King, P.H. 5 MATERIALS AND METHODS
Animals
B6.Cg-Tg (SOD1*G93A) 1 Gur/J mice were purchased from The Jackson Laboratory
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(Bar Harbor, ME). Transgenic mice were maintained in the hemizygous state by mating
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G93A males with C57BL/6J females. Nontransgenic littermates were used as controls. All animal procedures were reviewed and approved by the UAB Institutional Animal Care
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and Use Committee in compliance with the National Research Council Guide for the
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Care and Use of Laboratory Animals.
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Tissue collection
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After approval by the UAB Institutional Review Board, muscle biopsy samples of ALS patients were identified as previously described (Si et al., 2014). A cohort of 19 patients
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with clinically probable or definite ALS, based on El Escorial criteria, was used for the
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main analysis. Additional samples were used for analysis of miR-206 as detailed in Supplementary Table 4. Biopsy samples of patients with myopathy, other neurogenic
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disease, or no pathology were used as controls. Muscles sampled from the ALS patients were assessed for power at the bedside using the Medical Research Council (MRC) grading scale. The range for this scale is 0 to 5, with 0 being no detectable movement to 5 where the muscle contracts normally against full resistance. For mouse samples, animals were sacrificed by CO2 inhalation followed by cervical dislocation. Gastrocnemius muscle was dissected at discrete time points between 40 and 150 days postnatal. Samples were briefly rinsed in phosphate buffered saline (PBS), and frozen in liquid nitrogen and stored at -80°C prior to molecular analysis.
ACCEPTED MANUSCRIPT King, P.H. 6
RNA Isolation and qRT-PCR
Total RNA was extracted from frozen tissues with mirVana™ miRNA Isolation Kit (Applied Biosystems, Carlsbad, CA) according to the manufacturer’s instructions. For
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validation studies, relative quantities of mature miRNAs were determined using Applied
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Biosystems TaqMan® microRNA Assays (Applied Biosystems, Carlsbad, CA). Ten nanograms of total RNA were reverse transcribed with TaqMan® MicroRNA Reverse
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Transcription Kit according to the manufacturer’s specifications. Individual assays were performed using On-Demand primers and probes from Applied Biosystems. The
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endogenous controls were snoRNA202 for mouse and RNU48 for human tissues. A standard thermal cycle protocol was used. Data were analyzed with the ViiA 7 Software
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version v1.2 (Applied Biosystems). The baseline was auto set, and threshold values
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were adjusted from 0.1 to 0.2 based on the amplification curve of different targets. Quantification of target miRNAs was done by the ΔΔCT method using snoRNA 202 or
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RNU48 as the internal control.
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MicroRNA profiling
MicroRNA profiling was conducted using the Illumina HiSeq 2500. Briefly, RNA quality was assessed on the Agilent BioAnalyzer and the presence of a strong 5S peak was used an indicator of a retained miRNA fraction. Next-Generation sequencing libraries were produced using the TruSeq small RNA library prep kit from Illumina. The microRNA fraction was used as a substrate for adaptor ligation at the 3’ end through an RNA ligase directed mechanism. The 3’ ligation was followed by addition of a 5’ adaptor and reverse transcription to generate first strand cDNA. An initial PCR step was done to
ACCEPTED MANUSCRIPT King, P.H. 7 produce the 2nd strand and to introduce unique indexes to each sample. Finally, the libraries were purified with magnetic beads and quantitated using the Kapa Biosystems qPCR quantitation for Illumina libraries. The resulting libraries were standardized for concentration and sequenced on the HiSeq2500 using 50bp single end reads.
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Data assessment
miRNAs species with average sequence counts less than 10 were removed from
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downstream analyses. The count data were normalized with the trimmed mean of the log expression ratios method (TMM) (Robinson and Oshlack, 2010). Likelihood ratio test
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was used for pair-wise comparisons. Analyses were conducted in R (Version 3.3.3) with
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Ingenuity Pathway Analysis
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Clustvis (Metsalu and Vilo, 2015).
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package “edgeR” (Robinson et al., 2010). Heatmaps were generated using the web tool,
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Differentially expressed miRNAs that had an average read count value greater than 10 were uploaded into Ingenuity Pathway Analysis (IPA) software
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(http://www.ingenuity.com). The miRNAs were then filtered on those that had a log2 fold change 1 and a p-value < 0.05. The target genes of these significant differentially expressed miRNAs were predicted using the IPA miRNA Target Filter tool. The miRNA gene targets were filtered based on those experimentally observed or predicted with high confidence. Functional analysis of these filtered miRNA gene targets was performed using IPA Core Analysis tool, which identifies statistically significant overrepresentation of these predicted gene targets in a given biological process or toxicological function.
ACCEPTED MANUSCRIPT King, P.H. 8
Statistics
A two-tailed Student’s t test was used for miRNA expression in mouse samples. For human samples, one-way analysis of variance (ANOVA) was used followed by a
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Dunnett’s multiple comparisons test. Pearson correlation coefficients were calculated
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using Graphpad software (San Diego, CA).
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RESULTS
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miRNAs are differentially expressed in ALS muscle and change over time
We performed Next-generation miRNA sequencing on muscle samples from G93A
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SOD1 mice and littermate controls to look for unique miRNA signatures in ALS muscle.
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Gastrocnemius samples were obtained at an early, pre-clinical stage (60 d) and later during overt clinical disease (125 d). These time points were used in our previous study
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of Smad and TGF-β biomarkers and based on published literature (Si et al., 2014; Si et al., 2015). After excluding miRNAs with less than 10 reads on average, there were
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approximately 280 targets identified in both age groups (Fig. 1). There was differential expression of 54 miRNAs at 60 d (P < 0.05) consistent with prior observations of molecular changes in muscle at early, preclinical stages of the disease (Capitanio et al., 2012; Hayworth and Gonzalez-Lima, 2009; Si et al., 2014; Si et al., 2015). At 125 d, this number increased by ~ two-fold with more miRNAs showing increased expression. Differential expression was present when comparing 60 d and 125 d ALS stages, suggesting a dynamic miRNA profile related to disease progression. Differential
ACCEPTED MANUSCRIPT King, P.H. 9 expression was also observed with WT at 60 and 125 d, although with a disproportionately higher number of miRNAs decreasing compared to the ALS mice (70% versus 46%). This finding, however, suggests an aging effect on miRNA expression even within the relatively short interval of 65 days. To gain insight into potential downstream pathways that might be affected by these miRNAs changes, we
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performed an Ingenuity Pathway Analysis on miRNAs that increased or decreased by
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more than two-fold (p < 0.05) in the ALS mouse relative to littermate controls (Fig. 2 and Supplementary Table 1). This analysis can identify cellular/molecular functions or
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signaling pathways significantly linked to the pattern of differential miRNA expression based on the targeted mRNAs. At 60 d, miRNAs associated with axonal guidance
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signaling (e.g. ephrin receptor and RhoA signaling) and changes in mitochondrial transmembrane potential had highly significant enrichment. Other pathways identified
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included p53, TGF-β, hypoxia-inducible factor, and retinoic acid receptor (RAR)
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activation. At 125 d, similar pathways were identified with the addition of NF-kB
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signaling. Apoptotic signaling was enriched at both ages.
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Identification of miRNAs that potentially track disease progression
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To identify potential miRNAs that could track disease progression, we focused on those with p- and q-values less than 0.05 and at least a 2-fold difference. Heat maps were generated for both age groups (Fig. 3). In the younger cohort, 7 of the 9 miRNAs meeting these criteria also appeared in the older cohort raising the possibility that they track disease progression. The most significant differentially expressed miRNA in both cohorts was miR-206 (fold-change, p- and q-values are shown in Supplementary Tables 2 and 3). To identify other miRNAs that potentially track disease progression, we compared the younger and older ALS groups. To exclude miRNAs that change with
ACCEPTED MANUSCRIPT King, P.H. 10 normal aging, we first compared the two littermate control groups and identified a number of targets that changed (Table 1). Interestingly, 17 of the 23 miRNAs that were differentially expressed were identified in prior reports looking at miRNA changes in aging myoblasts and/or aging muscle samples from the hindlimb (Kim et al., 2014; Lee et al., 2015). After removing these targets, we identified 38 miRNAs that were
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differentially expressed in older (symptomatic) versus younger (asymptomatic) ALS mice
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(Table 2). With miRNA-206, there was nearly a 2-fold increase from 60 to 125 d in the ALS group suggesting that its changes parallel disease progression. This was supported
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by the 12.9-fold increase in the ALS group at 125 d (versus control) compared to a 4.9fold increase in ALS mice over controls at 60 d. The vast majority of the miRNAs,
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however, did not reach significance in the younger cohort or were only modestly changed compared to WT, indicating that the miRNA transcriptome qualitatively changes
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with disease stage. Two targets (miR-194-5p and miR-210-3p) showed opposing
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changes in the two age groups. Taken together, these analyses identified several miRNAs, including miR-206, which showed promise in detecting pre-symptomatic
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disease as early as day 60. The majority of miRNAs changed after this time point.
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Validation of a subset of miRNAs in the ALS mouse
We focused on a subset of miRNAs to validate the sequencing results and to characterize changes over the course of the disease (Fig. 4). We chose two additional ages, 105 d and 150 d. The former represents late pre-symptomatic, as defined by maximal weight and rotarod performance, and the latter represents end-stage (Si et al., 2014). The marker which best tracked disease progression was miR-206, increasing at each time interval to end stage. On the other hand, miR-133a and miR-133b (which differ by two nucleotides at the 3’ terminus) decreased with disease progression,
ACCEPTED MANUSCRIPT King, P.H. 11 particularly when compared to age-matched control levels which trended upward in older mice. miR-1, which did not appear in the list of miRNAs, was assessed because it is transcribed bicistronically with miR-133a (Horak et al., 2016). We observed a similar decrease in expression (with a concomitant trend upward in WT). This is in contrast to miR-133b and miR-206 which are also bicistronic but changed in opposite directions. For
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each of these miRNAs, there was a significant change in expression level in the ALS
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mouse between 60 d and later stages, suggesting an association with disease progression. Other miRNAs, including miR-34c, 10b-5p, 99a-5p, 100-5p, and 127-3p
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became significantly altered at all stages but without any stage-dependent trends. miR127-3p showed a gradual reduction in wild-type controls at older ages which provides
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validation for observations in Table 1. Thus, some of the miRNAs that change with age can still be sensors of disease activity. To determine how early the miRNAs change, we
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examined 40 d old mice, and did not find differential expression (Supplementary Fig. 1).
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The 40 - 60 d time frame for onset of miRNA changes is consistent with what we observed for Smads and TGF-βs (Si et al., 2014; Si et al., 2015). Taken together, these
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findings provided validation of our miRNA sequencing data.
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Validation of targets in human ALS muscle
Targets that were validated in mouse were assessed in biopsy samples from human ALS patients (Table 3) and compared to normal or disease controls (Table 4). The mean age for the ALS patients was 60 years which matches the overall mean age of diagnosis at our institution (Kazamel et al., 2013). Biopsies were obtained approximately 1 year after the onset of symptoms. The controls consisted of myopathy and neuropathy similar to our previous publications (Si et al., 2014; Si et al., 2015). Most of the miRNAs that were attenuated in mouse muscle were also attenuated in human ALS samples,
ACCEPTED MANUSCRIPT King, P.H. 12 including miR-1, 10b, 99a-5p, 100-5p, 133a-3p and 133b-3p (Fig. 5). Disease controls, however, were similarly affected. Two miRNAs, 34c and 206, which were elevated in mouse samples were not significantly changed. Although miR-206 showed a trend upward, this finding was in contrast to the ALS mouse where the miRNA increased more than 10-fold in later stages of the disease (Fig. 4). The standard error for miR-206,
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however, was larger than other miRNAs indicating variability among samples. Because
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this miRNA varies in the mouse based on muscle type, showing higher expression in slow twitch-dominant muscles (McCarthy and Esser, 2007; Williams et al., 2009b), we
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assessed it in a broader number of normal human samples. We focused on two commonly biopsied muscles: deltoid and vastus lateralis (Supplementary Table 4). With
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the mean age of each group being similar (62 years for deltoid and 60 years for vastus lateralis), there was more than a two-fold higher level (P < 0.001) in vastus lateralis
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samples (Fig. 6A). We then assessed miRNA-206 in an expanded number of ALS
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biopsy samples (Supplementary Table 4), and compared the relative quantities to the same muscle type from the normal control population (Fig. 6B). A non-significant trend
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toward higher levels in deltoid samples was observed, but no change with vastus
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lateralis. Again, the error bars were increased. To determine whether this variability reflected the degree of disease involvement, we performed a correlative analysis
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between miRNA level and the Medical Research Council (MRC) muscle grade in the original cohort (Fig. 7). For miR-206, there was a significant negative correlation between MRC grade and expression (r = -0.45, P = 0.03). We extended this analysis to the other miRNAs and found positive correlations with miR-133a (r = 0.52, p = 0.03) and miR-1 (r = 0.49, p = 0.04). miR-10b-5p, 133b-3p, and 127-3p, showed trends toward correlation but did not reach significance. miR-34c, 99a and 100-5p showed no correlation. In summary, some miRNAs identified in mouse ALS muscle had similar
ACCEPTED MANUSCRIPT King, P.H. 13 changes in human muscle and showed correlation with clinical assessment of muscle strength.
DISCUSSION
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In this report we show that there are distinct miRNA signatures in muscle samples from
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pre-clinical ALS mice, reflecting early physiological changes, which shift in a later stage of the disease when motor deficits are present. Some of the miRNAs that were
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differentially expressed at both stages paralleled disease progression and were similarly altered in human ALS muscle. Furthermore, a subset of these miRNAs in human
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samples correlated with muscle strength, suggesting that they may reflect disease activity and serve as biomarkers for ALS.
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Skeletal muscle is the “end organ” of ALS, reflecting the ravages of motor neuron
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degeneration as the muscle becomes atrophic, spastic or both. Our previous work showed the emergence of distinct muscle mRNA patterns, including members of the
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TGF-β and Smad families, at very early and pre-clinical stages of disease in the ALS
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mouse (Si et al., 2014; Si et al., 2015). Consistent with this observation, TGF-β signaling ranked near the top in both age groups for pathways predicted to be altered (Fig. 2). A
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recent report suggests that this signaling is linked to increased muscle fibrosis in the symptomatic stages of disease (Gonzalez et al., 2017). miRNAs associated with alterations in mitochondrial membrane potential also ranked high and meshes with prior studies showing that membrane depolarization of muscle mitochondria and other signs of mitochondrial damage occur at the earliest pre-clinical stages of disease (Dupuis et al., 2003; Luo et al., 2013). Likewise, Ephrin/RhoA signaling is increased in muscle of pre-symptomatic ALS and is negatively associated with muscle/NMJ reinnervation (Moloney et al., 2014). P53 signaling appeared in all subsets, ranking at the top of
ACCEPTED MANUSCRIPT King, P.H. 14 significance for enrichment in the 125 d cohort. A recent meta-analysis of transcriptomic experiments identified p53 deregulation as a characteristic feature of ALS muscle, significantly correlating with severity of atrophy (von Grabowiecki et al., 2016). miRNAs associated with RAR signaling were observed in both age groups. This pathway has not been previously identified in ALS muscle, but is triggered early on in injured muscles and
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promotes muscle repair (Di Rocco et al., 2015). Interestingly there were a substantial
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number of miRNAs that changed (mostly in the negative direction) between the two control groups indicating an age effect. A majority of these miRNAs (Table 1) were
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reported to change in the same direction in aging myoblasts and/or muscle isolated from hindlimbs, although the age differences were substantially larger (18-24 months) (Kim et
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al., 2014; Lee et al., 2015). The comparatively young age and short time interval (~ 2 months) in our study suggests that the changes begin very early and relatively fast.
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In the validated subset of miRNAs, many showed differences starting in the 60
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day old group, but not earlier. miRNA 206 was elevated by ~2-fold in contrast to a prior study of the low-copy G93A SOD1 mouse model where induction occurred only after
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disease initiation (as defined by maximal weight) (Williams et al., 2009b). In our study,
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this miRNA gradually increased with age in the ALS mouse suggesting that it could be a marker of disease progression. This miRNA supports neuromuscular junction
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reinnervation through suppression of HDAC4 (Williams et al., 2009b). No differences in miR-206, however, were discerned in human ALS samples compared to controls. Interestingly this miRNA was two-fold higher in vastus lateralis compared to deltoid in our control population. In mouse, slow-twitch fiber predominant muscles such as soleus are reported to have significantly higher miR-206 levels than fast-twitch predominant muscles like plantaris (McCarthy, 2008; Williams et al., 2009b). In humans, however, deltoid and vastus lateralis are “mixed” muscles with variable but overlapping proportions of slow-twitch fibers (Johnson et al., 1973; Saltin and Gollnick, 2010). Our
ACCEPTED MANUSCRIPT King, P.H. 15 findings suggest that other factors besides fiber type proportion are at play for determining miR-206 expression. Even when factoring the variable expression of miR206, there were no differences with control muscle (Fig. 6). The significant inverse correlation with muscle power of the biopsied muscle, however, suggests that it could be a marker of disease activity. This finding also reflects the heterogeneity of disease
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activity in human ALS muscle (versus the relative homogeneity of muscle involvement in
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the ALS mouse) and emphasizes the need to associate miR-206 levels (and other miRNAs) with muscle-specific clinical assessment rather than overall clinical status. This
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heterogeneity, coupled with the differences in baseline miR-206 expression in different muscles, could explain inconsistencies in previous reports. One study, for example,
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observed an increase in miR-206 over normal controls in early stages of disease but no difference in later stages, whereas two others found upregulation in both phases
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(Bruneteau et al., 2013; Di Pietro et al., 2017; Pegoraro et al., 2017). Interestingly, miR-
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133b, which is bicistronic with miR-206 (Horak et al., 2016), gradually decreased in mouse ALS muscle with disease progression and was attenuated in human ALS
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samples. This dichotomy is consistent with a prior report indicating that transcription of
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the two miRNAs, even though located in tandem, is uncoupled (Cesana et al., 2011). Assessment of pri-miRNA levels would be necessary to confirm this possibility. On the
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other hand, miR-1 and miR-133a, which are also bicistronic and transcribed together (Chen et al., 2006), progressively decreased in the ALS mouse in parallel and correlated with muscle weakness in human ALS samples. As with miR-206, changes in these miRNAs vary in prior reports, with the mouse study by Williams et al. being consistent with our findings (in both mouse and humans) versus other reports showing variable changes depending on disease stage or pace of progression (Di Pietro et al., 2017; Pegoraro et al., 2017; Williams et al., 2009b). In one study, patients with slow progression or early stage disease showed increases in miR-133a, 133b and 1 whereas
ACCEPTED MANUSCRIPT King, P.H. 16 rapid progression or late disease was associated with no change or a decrease compared to control samples (Di Pietro et al., 2017). Several miRNAs that were validated in the mouse, including miR-34c-5p and miR-127-3p, were not altered in human ALS samples and/or did not correlate with muscle weakness. It is possible that these miRNAs are unique to mutant SOD1-
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associated ALS as our human patients were predominantly sporadic. All miRNAs that
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were validated in human samples were similarly altered in myopathy and other neurogenic processes, suggesting that they are general responders to muscle tissue
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injury. The trigger for this response and the impact on the underlying disease process is unclear given the pleiotropic effects of the muscle-specific miRNAs on myogenesis,
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muscle repair and reinnervation (Horak et al., 2016; Wang, 2013; Williams et al., 2009a). Myofiber loss cannot explain the selective attenuation of muscle-specific miRNAs since
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many other miRNAs increased with disease progression or did not change compared to
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normal controls (Greco et al., 2009). It is possible that changes in the microenvironment of injured muscle may influence miRNA patterns. A prior study of patients with
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inflammatory myopathy (which was the predominant diagnosis in our myopathy cohort)
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described a similar suppression of miR-1, 133a and 133b and linked it to TNF-α (Georgantas et al., 2014). The inflammatory cytokine TWEAK, which promotes muscle
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wasting in the setting of denervation and other pathological states also suppresses these miRNAs (Bhatnagar and Kumar, 2012; Panguluri et al., 2010). The potential relevance is underscored by a report that this signaling pathway is increased in ALS muscle and that knockout of TWEAK lessens muscle atrophy (Bowerman et al., 2015). Finally, it is important to note that changes observed in some of the miRNAs could be attributable to other cell types in the muscle sample such as macrophages or other immune cells, fibroblasts, endothelial cells and motor neuron termini.
ACCEPTED MANUSCRIPT King, P.H. 17 In conclusion, we have identified distinct muscle miRNA patterns that change with ALS progression and reflect the underlying pathophysiology of the disease at the level of the muscle. We have identified a subset of miRNAs that can track disease progression in the ALS mouse and are also affected in human ALS muscle. These miRNAs would be best suited as markers of disease progression, although the very
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early preclinical appearance of changes suggests they could track disease onset. The
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utility of these miRNAs as markers in the clinic is unclear. It would depend on ease of methodology for measuring them. Several reports have detected significant, albeit non-
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specific, changes in serum levels of miR-1, 133a, 133b and 206 (Toivonen et al., 2014; Waller et al., 2017). Alternatively, as nanoparticle technology advances, in vivo imaging
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of miRNAs by magnetic resonance imaging may be possible down the road (Hernandez
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et al., 2013).
ACCEPTED MANUSCRIPT King, P.H. 18 ACKNOWLEDGEMENTS
This work was supported by NINDS R01NS092651 (PHK) and a Merit Review BX001148 from the Department of Veterans Affairs (PHK).The authors wish to thank the UAB Genomics Core, funded through the UAB Comprehensive Cancer Center
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(CA13148) and CFAR (AI027767).
ACCEPTED MANUSCRIPT King, P.H. 19 REFERENCES
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Bhatnagar, S., Kumar, A., 2012. The TWEAK-Fn14 System: Breaking the Silence of Cytokine-Induced Skeletal Muscle Wasting. Current Molecular Medicine. 12, 3-
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ACCEPTED MANUSCRIPT King, P.H. 20 Chen, J. F., et al., 2006. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet. 38, 228-33.
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Di Rocco, A., et al., 2015. Selective Retinoic Acid Receptor gamma Agonists Promote
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ACCEPTED MANUSCRIPT King, P.H. 21 Gonzalez, D., et al., 2017. ALS skeletal muscle shows enhanced TGF-β signaling, fibrosis and induction of fibro/adipogenic progenitor markers. PLOS ONE. 12, e0177649.
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ACCEPTED MANUSCRIPT King, P.H. 24 Si, Y., et al., 2015. Transforming Growth Factor Beta (TGF-beta) Is a Muscle Biomarker of Disease Progression in ALS and Correlates with Smad Expression. PLoS One. 10, e0138425.
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von Grabowiecki, Y., et al., 2016. Transcriptional activator TAp63 is upregulated in muscular atrophy during ALS and induces the pro-atrophic ubiquitin ligase
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Wang, X. H., 2013. MicroRNA in myogenesis and muscle atrophy. Curr Opin Clin Nutr
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ACCEPTED MANUSCRIPT King, P.H. 25 Table 1 Differentially expressed miRNAs
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*miRNAs altered in aging myoblasts and/or aging muscle, based on prior reports (Kim
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10.6 2.9 -2.5 -3.0 -3.3 -3.4 -3.8 -4.0 -4.0 -4.1 -4.4 -4.5 -4.7 -4.9 -4.9 -5.6 -5.7 -5.8 -5.8 -5.8 -6.5 -8.3 -9.9
P-value 3.3E-04 1.4E-04 2.9E-03 9.0E-06 1.7E-03 3.6E-04 1.0E-08 3.1E-04 7.9E-04 9.6E-06 3.1E-03 1.0E-04 1.4E-03 1.5E-07 2.4E-07 2.3E-03 7.3E-09 3.3E-05 2.5E-04 8.4E-07 1.3E-04 2.6E-05 3.3E-08
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miRNA* 6244 451a 19b-3p 127-3p* 485-5p* 154-5p 434-3p* 431-5p* 673-5p* 434-5p* 377-5p* 136-5p* 673-3p* 541-5p* 411-5p* 543-3p* 299a-5p 455-5p 300-3p* 127-5p* 1193-3p* 136-3p* 337-5p*
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in WT mice at age 125 d versus 60 d.
et al., 2014; Lee et al., 2015).
ACCEPTED MANUSCRIPT King, P.H. 26 Table 2. Differentially expressed miRNAs (fold-change) in ALS mice at age 125 d versus 60 d (p- and q-values < 0.05). Fold-change for these miRNAs in the ALS v. WT 60 d and 125 d groups are shown for comparison.
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ALS v. WT (125 d) 4.4 15.5 12.2 3.7 4.0 6.3 2.5 2.5 2.5 1.7 2.5 2.3 2.9 2.2 3.3 -0.5 2.3 1.9 1.8 ns 1.7 2.2 12.9 ns 2.2 1.8 -2.6 -2.2 -4.7 -4.0 -6.9 -7.3 -3.9 -3.2 -9.0 -4.3
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ALS v. WT (60 d)1 nd nd nd -2.9 nd nd nd nd nd nd nd nd nd nd nd nd nd nd
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ALS (125 d v. 60 d) 6.5 4.8 4.3 3.7 3.5 3.3 3.0 2.8 2.5 2.5 2.4 2.4 2.3 2.3 2.2 2.2 2.2 2.2 2.2 2.1 2.0 1.9 1.9 1.9 1.8 1.8 -2.1 -2.9 -3.4 -3.6 -4.1 -5.3 -5.7 -5.8 -6.7 -10.5
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mmu-miR 21a-3p 18a-5p 142-3p 194-5p 21a-5p 19a-3p 301a-3p 322-5p 340-5p 223-3p 3535 106b-5p 24-3p 101a-3p 17-5p 98-5p 93-5p 16-5p 210-3p 30a-5p 25-3p 140-3p 206-3p 30e-5p 27b-3p 27a-3p 125a-5p 362-5p 99a-5p 99b-5p 100-5p 1943-5p 501-3p 540-5p 381-5p 329-3p
-1.8 -1.7 nd nd 4.9 -1.9 nd nd nd nd nd nd nd nd nd nd nd nd
ACCEPTED MANUSCRIPT King, P.H. 27 539-5p 409-5p
-6.9 -18.2
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nd, no difference from wild-type control.
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nd nd
-10.8 -47.2
ACCEPTED MANUSCRIPT King, P.H. 28
Table 3: ALS patients (n = 19) 34-78
Mean age (years)
60 ± 13
Gender
9M
Duration (months)
14 ± 13
Muscle (n)
TA (6); VL (1)
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Age range (years)
15 (4 muscles not tested)
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EMG denervation
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BI (6), DT (6)
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9F
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tibialis anterior; VL, vastus lateralis
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BI, biceps; DT, deltoid; EMG, electromyography; TA,
ACCEPTED MANUSCRIPT King, P.H. 29
Table 4: Control patients Myopathy (n = 10)
Neuropathy (n =6)
Age Range (y)
24-67
38-74
39-88
Mean Age (y)
53 ± 11
58 ± 9
TA (1), VL (4),
TA (1), VL (6),
BI (3), DT (1)
BI (2), DT (1)
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Non-specific (1)
Inflammatory (1)
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BI (1) Diabetic (2)
BI, biceps; DT, deltoid; TA, tibialis anterior; VL, vastus lateralis
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TA (3), VL (2),
Inflammatory (9)
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Diagnosis
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59 ± 21
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Muscle (all)
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Normal (n=9)
Multifocal motor (1) Non-specific (2)
ACCEPTED MANUSCRIPT King, P.H. 30 FIGURE LEGENDS
Figure 1. Differential expression of miRNAs in gastrocnemius samples of G93A SOD1 (ALS) and wild-type (WT) mice at 60 and 125 days of age. miRNA transcriptomes were determined by Next-generation sequencing and compared among four groups: ALS and
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WT mice at 60 and125 d (n = 3 per group). Differential expression was considered
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present when the P value was < 0.05 for miRNAs with greater than 10 reads,
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independent of fold-change.
Figure 2. Ingenuity Pathway Analysis of differentially expressed miRNAs in G93A SOD1
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gastrocnemius muscle, compared to wild-type controls, at 60 and 125 days of age. Only miRNAs with ≥ 2-fold change and P < 0.05 were used in this analysis. Biological
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functions and signaling pathways predicted to be affected by a downregulation (Down)
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or upregulation (Up) of miRNAs in the ALS group are shown. P values are indicated on the x axis. The number of altered miRNAs and downstream genes predicted to be
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affected are shown in parentheses (miRNAs/genes). See Supplementary Table 1 for
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Figure 3. Unsupervised hierarchical clustering of miRNAs differentially expressed in ALS mice compared to controls at 60 and 125 d. Only miRNAs with p- and q-values < 0.05 and a 2-fold or greater difference are shown (9 miRNAs for 60 d and 73 miRNAs for 125 d). See Supplementary Tables 2 and 3 for additional information.
Figure 4. Validation of a subset of differentially expressed miRNAs identified by sequencing. Expression levels of miRNAs in muscle samples from G93A SOD1 or WT mice were assessed by qRT-PCR. Ages ranged from early, pre-clinical (60 d), late pre-
ACCEPTED MANUSCRIPT King, P.H. 31 clinical (105 d), clinically symptomatic (125 d), and end-stage (150 d). Clinical onset was determined by maximal weight and rotarod function as previously reported (Si et al., 2014). Each data point represents the mean ± SEM of 3-6 mice. All values were expressed as a fold-change of WT at age 60 d (set at 1.0). * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 comparing G93A to WT mice at each age. # P < 0.05 comparing
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G93A SOD1 mice at the indicated age to G93A SOD1 mice at 60 d. RQ, relative
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Figure 5. Validation of miRNAs in human ALS muscle samples. Expression levels of miRNAs in muscle samples from human biopsy samples were assessed by qRT-PCR.
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Clinical data are shown in Tables 3 and 4. Data points represent the mean ± SEM of 19 ALS, 9 normal controls, 10 myopathies (Myo) and 6 neuropathies (Neuro). *P < 0.05, **P
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Figure 6. miRNA 206 is differentially expressed in normal human muscle. A) Normal
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muscle biopsy samples were assessed for miR-206-3p expression by qRT-PCR. The
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average age for deltoid (DT) and vastus lateralis (VL) samples was 62 ± 12 and 60 ± 7 years. B) miRNA-206 expression was assessed in ALS and normal patients using equal
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numbers of DT and VL samples. RQ, relative quantity.
Figure 7. Correlation of miRNA expression level and strength of muscle biopsied. Muscle strength was determined clinically at the bedside by manual motor testing using the Medical Research Council (MRC) scale (see methods). Total number of samples assessed was 17 (MRC scores were not available for 2 of the 19 muscle samples shown in Fig. 5). For miR-206, there were 24 samples. *P < 0.05.
ACCEPTED MANUSCRIPT King, P.H. 32 Highlights
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• miRNA transcriptomes were assessed in muscle of G93A-SOD1 mice. •Distinct and overlapping miRNA patterns appeared in 60 and 125 d old cohorts. •miRNAs affecting mitochondria, axonal guidance, p53 and TGFβ pathways were enriched. •A subset of miRNAs tracked disease progression in the mouse. •Some miRNAs were validated in human ALS muscle and correlated with motor strength.
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