Proteomic Profiling of Brain and Testis Reveals the Diverse Changes in Ribosomal Proteins in fmr1 Knockout Mice

Accepted Manuscript Proteomic profiling of brain and testis reveals the diverse changes in ribosomal proteins in fmr1 knockout mice Benhong Xu, Yusheng Zhang, Shaohua Zhan, Xia Wang, Haisong Zhang, Xianbin Meng, Wei Ge PII: DOI: Reference:

S0306-4522(17)30913-2 https://doi.org/10.1016/j.neuroscience.2017.12.023 NSC 18193

To appear in:

Neuroscience

Received Date: Accepted Date:

7 June 2017 18 December 2017

Please cite this article as: B. Xu, Y. Zhang, S. Zhan, X. Wang, H. Zhang, X. Meng, W. Ge, Proteomic profiling of brain and testis reveals the diverse changes in ribosomal proteins in fmr1 knockout mice, Neuroscience (2017), doi: https://doi.org/10.1016/j.neuroscience.2017.12.023

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Proteomic profiling of brain and testis reveals the diverse changes in ribosomal proteins in fmr1 knockout mice Benhong Xu1,2,3*, Yusheng Zhang2*, Shaohua Zhan2, Xia Wang2 , Haisong Zhang1, Xianbin Meng4 and Wei Ge1,2* 1. Affiliated Hospital of Hebei University, No.212, Yu Hua East Rd, Nan Shi District, Baoding, Hebei 071000 China. 2. State Key Laboratory of Medical Molecular Biology & Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, No. 5 Dongdansantiao, Dongcheng District, Beijing, 100005 China. 3. Key Laboratory of Modern Toxicology of Shenzhen, Institute of Toxicology, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China. 4. MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing 100084, China.

* These authors contribute equally to this manuscript.

Address correspondence to: Wei Ge, PhD, Institute of Basic Medical Sciences, School of Basic Medicine, Peking Union Medical College; No. 5 Dongdansantiao, Dongcheng District, Beijing 100005, China. Email: [email protected].

1

Abstract Fragile X syndrome (FXS), the leading cause of inherited forms of mental retardation and autism, is caused by the transcriptional silencing of fmr1 encoding the fragile X mental retardation proteins (FMRP). FMRP is an RNA-binding protein that is a widely expressed, but primarily in the brain and testis, and associated approximately 4% of transcripts. Macro-orchidism is a common symptom associated with FXS both in humans and mice. Thus, we analyze the pooled samples of cerebral cortex, hippocampus and testis from both the fmr1-KO and wild-type mice by a LC-MS/MS proteomic study. Among the identified proteins, most of those showing significant changes in expression were up- or downregulated in the absence of FMRP. Proteins (FMRP, RPS8, RPL23a and ATPIF1, RPL6, GAP43, MTCH2 and MPZ in brain, and FMRP, CAH3, AKR1B7 and C9 in testis) identified by MS/MS were also verified by Western blotting. The Gene Ontology and WikiPathways analysis revealed that the differentially expressed proteins were clustered in the polyribosome and RNA-binding protein categories in both cerebral cortex and hippocampus, but not in testis. Although this study was limited by the little number of samples, our results provide detailed insights into the ribosomal protein profiles of cerebral cortex, hippocampus and testis in the absence of FMRP. Our studies also provide a better understanding of protein profile changes and the underlying dysregulated pathways arising from fmr1 silencing in FXS.

Key words: FMRP, Proteomics, Ribosome, Hippocampus, Testis

Running title: Quantitative protein profiles of brain and testis in fmr1-KO mice

2

Introduction Fragile X syndrome (FXS) is the most well-known inherited of the mental retardation and autism spectrum disorders (ASD) (Hagerman and Hagerman 2016; Rudelli, et al. 1985). In most of cases, FXS is caused by the silencing of fmr1 due to the CGG triplet repeat expansion and high levels of methylation in the 5ʹ-untranslated region resulting the absence of the fragile X mental retardation protein (FMRP) or missense mutations in the protein (Esanov, et al. 2016). FMRP is ubiquitous but mainly impacts the central nervous systems (Ludwig, et al. 2014). Studies of the structural domains and function of FMRP have revealed the presence of four canonical RNA-binding motifs, including three KH domains and an arginine-glycine-rich (RGG) box (Valverde, et al. 2007; Vasilyev, et al. 2015). FMRP is involved in the early translation stage mediated via the tropomyosin-like kinase B (TrkB)-MAP-kinase-interacting kinase (MNK) pathway (Panja, et al. 2014). FMRP plays a vital role in neuronal development by binding to mRNA targets associated with alterations in synaptic plasticity and dendritic spine dynamics (Smith, et al. 2014). One of the vital molecular functions of FMRP is closely related to ribosomal translation and numerous studies have shown that FMRP is associated with the polyribosome in cells (Corbin, et al. 1997; Stefani, et al. 2004). FMRP reversibly stalls ribosomes on the targeting mRNA, resulting in the loss of the translational brake, which may be the cause of FXS (Darnell, et al. 2011). Studies have revealed that FMRP inhibits translation initiation through its interactions with the FMRP binding partner-cytoplasmic FMRP interacting protein (CYFIP1) and brain cytoplasmic RNA1 (BC1)(Napoli, et al. 2008; Zalfa, et al. 2003). In addition, some studies have revealed that binding of FMRP to the ribosome interrupts protein synthesis by preventing binding of tRNA and elongation factors to the ribosome (Chen, et al. 2014b). Most previous studies focused on the mechanism by which the interaction between FMRP and the ribosome influences translation (Darnell, et al. 2011; Smith, et al. 2014). In contrast, few studies have focused on changes in the ribosomal proteins or the whole cell proteome as a result of FMRP-deficiency. 3

Longer and thinner dendritic spines are the most prominent neuroanatomical difference in the brain of FXS patients compared with normal individuals (Irwin, et al. 2000). This phenotype is also observed in the fmr1 knockout (KO) mouse, which also showed dysregulated dendritic spine morphology in the hippocampus (Bostrom, et al. 2016; Desai, et al. 2006; Grossman, et al. 2006). The absence of FMRP also causes a deficit in cortical long-term potentiation in this model as well as an imbalance in brain metabolites (Shi, et al. 2012), which is another feature that resembles FXS in humans. In addition, the absence of FMRP also caused hippocampus-depended learning disrupts in adult mice (Guo, et al. 2011). However, changes in the proteome of both the hippocampus and cortex remain to be fully investigated, especially in adult mice. Macro-orchidism is another characteristic observed in some male FXS patients and fmr1-KO mice,

which

is

caused by increased

Sertoli cell proliferation

(Slegtenhorst-Eegdeman, et al. 1998), although the underlying mechanism is also still largely unknown. A combination of proteomics and genomic ontology analyses is an efficient way to identify changes in the proteomes and related pathways under diverse conditions. Although the manner of synaptic development and protein changes (Klemmer, et al. 2011) have been deciphered in previous proteomics studies, the influence of FMRP-deficiency on different organs or regions of the brain in fmr1-KO mice remain to be elucidated. Here, we used tandem mass tag (TMT™) labeling proteomics strategies to identify the changes on the proteomes of the hippocampus, cortex and testis in fmr1-KO and wild-type mice with the aim of identifying relevant pathways and biological functions that influence the phenotype of fmr1-KO mice.

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Materials and Methods Reagents and antibodies The following reagents and antibodies were used in this study: Urea, iodoacetamide and dithiothreitol (GE Healthcare, LC, UK); proteinase inhibitor cocktail tablet mini (Roche, BS, CH); TMT™ Mass Tagging Kits (Thermo Scientific, NJ, USA); sequencing-grade trypsin/Lys-C mix (Promega, WI, USA); anti-myelin protein zero (MPZ), anti-FMRP and anti-ribosomal protein S8 (RPS8), anti-ATPase inhibitor (ATPIF1), anti-ribosomal protein L6, anti-complement 9 (C9), anti-neuromodulin (GAP43), anti-mitochondrial carrier homolog 2 (MTCH2) (Abcam, MA, USA); anti-ribosome protein L23a, and anti-carbonic anhydrase 3 (CAH3) (Santa Cruz, CA, USA); anti-β-actin (GeneTeX, CA, USA); Enhanced Chemiluminescence (ECL) Kit (Millipore, MA, USA). All other reagents were obtained from Sigma (MO, USA).

Animals FVB.129P-Fmr1tm1Cgr/J (fmr1-KO) mice were a kind gift from Dr. Chen Zhang (Peking University) and were bred in our facility with a 12:12 h dark/light cycle. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Association for Assessment and Accreditation of Laboratory Animal Care.

Hippocampus, cerebral cortex and testis isolation

Adult wild-type and fmr1-KO mice (male, aged 8–10 weeks) were euthanized using 0.1% pentobarbital and perfused with 0.1 M PBS. Four paired WT and fmr1-KO generated from same parental lines were used in proteomic studies. The cerebral cortex, hippocampus and testis were then dissected and stored at -80 °C for later use.

Sample preparation Tissues were prepared as described previously (Xu, et al. 2016). Briefly, each tissue was frozen immediately under liquid nitrogen and homogenized before transfer to 5

ice-cold homogenization buffer (8 M urea in PBS, pH 8.0, 1× cocktail [Roche, IN, USA]). Tissue debris was removed via centrifugation at 4 °C for 10 min at 12,000 rpm. Protein concentration was determined using a Nanodrop 2000 (Thermo Scientific, NJ, USA) according to the manufacturer’s instructions.

TMT labeling Pooled proteins (100 µg) prepared from four individual samples were treated with 10 mM DTT for 1 h at 37°C. The pooled samples were then incubated with 25 mM IAA for 30 min in the dark at room temperature prior to digestion with trypsin/Lys-C mix (1:100 w/w) overnight at 37°C. After acidification with 1% formic acid (FA), the samples were desalted by reversed-phase column chromatography (Oasis HLB; Waters, MC, USA) according to the manufacturer’s instructions, dried under vacuum and dissolved in 100 µl triethylammonium bicarbonate buffer (TEAB, 200 mM, pH 8.5).

Peptides from each group were labeled with TMT reagents according to the manufacturer’s instructions. Briefly, samples were labeled with TMT reagent (0.8 mg TMT dissolved in 40 µl 99.9% acetonitrile) for 1 h at room temperature before the reaction was terminated by the addition of 5 µl 5% hydroxylamine for 5 min. Peptides were labeled with different TMT labels: [TMT-126, hippocampus of fmr1-KO mice; TMT-127, hippocampus of WT mice; TMT-128, cortex of fmr1-KO mice; TMT-129, cortex of WT mice] set 1; [TMT-130, testis of fmr1-KO mice; and TMT-131, testis of WT mice] set 2. All the labeled peptides in each set from the pools were mixed together, desalted, dried and dissolved in 100 µl 0.1% FA (Figure 1).

High performance liquid chromatography (HPLC) separation Fractionation of the labeled peptides was performed as described previously (Gokce, et al. 2011). Briefly, peptides (100 µl in 0.1% FA) were transferred to the MS tube for HPLC (UltiMate 3000 UHPLC, Thermo Scientific) analysis using an Xbridge BEH300 C18 column (4.6 × 250 mm, 2.5 µm; Waters). Column temperature was 6

maintained at 45°C with a flow rate of 1.0 ml/min, and UV absorbance was detected at 214 nm. Fractions were collected every 1.5 min in 47 tubes and then dissolved in 20 µl 0.1% FA for further liquid chromatography (LC)-MS/MS analysis.

Peptide analysis by LC-MS/MS Peptide analysis of the labeled digestion fractions was performed by LC-MS/MS using a the UltiMate 3000 RSLCnano System (Thermo Scientific, NJ, USA) directly interfaced with a Thermo Q Exactive Benchtop mass spectrometer (Thermo Scientific, NJ, USA). Peptides were separated by gradient elution (120 min at 0.30 µl/min) using an analytical capillary column (internal diameter (ID), 75 µl; length, 150 mm; Upchurch, Oak Harbor, WA, USA) packed with C18 silica resin (300 Å, 5 µl; Varian, Lexington, MA, USA).The Q Exactive mass spectrometer was manipulated using Xcalibur 2.1.2 software in a data-dependent acquisition mode. A single full-scan mass spectrum in Orbitrap (400-1, 800 m/z, 60,000 resolution) was followed by 10 data-dependent MS/MS scans at 27% normalized collision energy (higher-energy C-trap dissociation, HCD). The MS/MS spectra from each LC-MS/MS run were compared with the UniProt mouse FASTA database (released on June 19, 2016) using Proteome Discoverer 2.1 software (Thermo Scientific) using the following alterations to the recommended search criteria: full tryptic specificity required; two missed cleavages allowed; static modifications, carbamidomethylation (C, +57.021 Da) and TMT plex (lysine [K] and any N-terminal); dynamic modification, oxidation (methionine, M); precursor ion mass tolerances, 20 ppm for all MS acquired on an Orbitrap mass analyzer; fragment ion mass tolerance, 20 mmu for all MS2 spectra acquired. Relative protein quantification was performed using the reporter ion intensities per peptide according to manufacturer’s instructions. Quantitative precision was expressed as protein ratio variability. The upregulation and downregulation thresholds were set at 1.10 and 0.90, respectively.

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These mass spectrometry proteomics data have been reported to the ProteomeXchange Consortium via the PRIDE partner repository (data identifier PXD005089).

Bioinformatics analysis Gene ontology (GO) enrichment analysis of the altered proteins was performed using FunRich software following the instructions provided (http://www.funrich.org). An online WEB-based GEne SeT AnaLysis Toolkit (WebGestalt; Vanderbilt University, Nashville, TN) was used for the pathway analysis. Pathways were considered to be statistically significant on the basis of a P-value < 0.001 and the presence of at least two target genes in the Wiki pathway. For the protein-protein interaction analysis, we used STRING database version 10.0 (http://string-db.org) with a medium confidence threshold 0.4. The interaction network was mapped by Cytoscape (3.4.0).

Western blot analysis Protein concentrations (determined using a Nanodrop 2000 (Thermo Scientific, NJ, USA) according to the manufacturer’s instructions) were normalized before all Western blot analyses. Equal amounts of protein (20 µg) were loaded onto 12% or 15% polyacrylamide gels for SDS-PAGE (Chen, et al. 2011). Proteins were then electrophoretically transferred to nitrocellulose membrane, before blocking with 5% non-fat dry milk in TBS-T (TBS plus 0.5% Tween) for 30 min. Membranes were then incubated with primary antibodies [anti-RPS8 and anti-RPL23a (1:200), anti-MPZ, anti-RPL6, ATPIF1, AKR1B7, GAP43, MTCH2, C9 and anti-FMRP (1:1,000), and anti-β-actin (1:100,000)] overnight at 4°C, before washing and incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 40 min at room temperature and ECL reagents were added after washing. Images were acquired and analyzed by an imaging system (Tanon 5500, Shanghai, China). The protein expression level relative to the corresponding loading control was calculated according to grayscale values and the significant difference of proteins in KO and WT mice was evaluated using a paired t-test. 8

Results Quantitative MS analysis of protein profiles For more efficient identification of the protein profiles of different tissues after labeling with TMT tags, brain and testis were divided into two different panels for MS analysis and the proteomic studies were repeated twice. In the hippocampus and cerebral cortex, a total of 4,419 proteins were identified with two or more unique peptides with a false discovery rate (FDR) of less than 1%. In the hippocampus of fmr1-KO mice, 59 proteins were upregulated and 58 proteins were downregulated compared with the levels detected in WT mice (Table 1). In the cerebral cortex, 39 proteins were upregulated and 84 proteins were downregulated (Table 2). The differentially expressed proteins in the hippocampus and cerebral cortex exhibited marked changes; only 23 proteins, including plasma membrane calcium-transporting ATPase 4, hippocalcin-like protein 1 and high mobility group protein B1, were downregulated both in hippocampus and cerebral cortex (Table 1 and 2). In the testis, 4,679 proteins were identified, including at least two unique peptides with a FDR of less than 1%. Of these, seven proteins were upregulated and 38 proteins were downregulated (Table 3). The protein profiles showed that FMRP was also downregulated in both brain and testis, which indicated the existence of unstable FMRP peptides in the fmr1-KO mouse, since the proteomic analysis is a peptide-dependent strategy.

Bioinformatics analysis All the differentially expressed proteins were uploaded for analysis using the functional enrichment tool (FunRich). The Venn diagram of the results showed that all the differentially expressed proteins were largely different among the tissues as shown in Figure 3D. Thirty-two proteins were changed both in hippocampus and cerebral cortex, with the exception of FMRP. These included one protein (cytochrome b-c1 complex subunit 6) that was differentially expressed in all the tissue types except for FMRP. For the GO analysis, the top six enriched items were identified based on the 9

P-value. Interestingly, in the GO analysis, the structural constituent of ribosome and poly (A) RNA-binding were the two most significantly changed items in molecular function in both the hippocampus and the cerebral cortex. In testis, the most significantly changed groups were histone binding and protein domain specific binding (Figure 2F). The most differentially expressed proteins in terms of biological process were translation in both the hippocampus and the cerebral cortex, but not in testis (Figure 2). To gain an improved understanding of the signaling pathways affected by the absence of FMRP, we performed bioinformatics analysis using the WEB-based GEne SeT AnaLysis Toolkit (WebGestalt: http://bioinfo.vanderbilt.edu/webgestalt/) and Cytoscape (3.4.0) software. All the differentially expressed proteins identified in the hippocampus enriched in the cytoplasmic ribosomal proteins pathway (P < 0.001). Thus, we mapped all the proteins identified in the hippocampus, cerebral cortex or testis in the cytoplasmic ribosomal proteins pathway in a comprehensive analysis. The proteins were shown in the gene name form in the pathway. Overall, one third of the ribosomal protein subunits in the hippocampus and cerebral cortex were perturbed compared with the WT mice, while there were no changes in testis in the fmr1-KO mice (Figure 3). Only 60S ribosomal protein L23a (RPL23a), 60S ribosomal protein L27a (RPL27a), RPL22, 40S ribosomal protein S23 (RPS23), RPS24 and RPS25 were downregulated in both hippocampus and cerebral cortex. No proteins in ribosome were upregulated in the cerebral cortex of fmr1-KO mice. While, three ribosomal proteins, included 40S ribosomal protein S2 (RPS2), RPS7 and RPS18, were slightly upregulated in hippocampus of fmr1-KO mice. To further elucidate the relationships among all the differentially expressed proteins, we investigated the protein-protein interactions using the STRING software (10.0) to create comprehensive networks of the altered proteins based on the same criteria used in pathway analysis (<0.9-fold or >1.1-fold). The networks were visualized with Cytoscape 3.4.0 to identify the potential relationships among the proteins. In the hippocampus and cerebral cortex, more than half of the differentially expressed proteins showed interactions with at least one protein. However, only 26 10

proteins among those differentially expressed in testis showed simple interactions. Although the differentially expressed proteins identified in the hippocampus and cerebral cortex were largely different, the protein-protein interaction networks were formed mainly by the ribosomal subunit proteins in both tissues (Figure 4).

Verification of protein expression levels by Western blot analysis To further validate our MS/MS results, the individual samples were subjected to Western blot analysis. Based on the results of our proteomics analysis, we selected proteins (FMRP, MPZ, RPS8, RPL23a and GAP43 for hippocampus, FMRP, RPS8, ATPIF1, RPL6, RPL23a and MTCH2 for cerebral cortex; FMRP, CAH3, AKR1B7 and C9 for testis) for validation by Western blotting. β-actin was used as the background reference protein. The Western blot results for these proteins were consistent with the proteomics data. Interestingly, MPZ and RPL23a were significantly downregulated in hippocampus of fmr1-KO mice, but not changed in cerebral cortex (data not shown). RPS8 was downregulated in cerebral cortex of fmr1-KO mice, but was not changed in the hippocampus. As shown in Figure 5C, CAH3 was downregulated and C9 was upregulated in testis of fmr1-KO mice. Taken together, all the results were totally in accordance with those of the proteomics analysis (Figure 5).

Discussion Here, we performed a TMT labeling proteomic study to reveal the diverse expression profiles of the cerebral cortex, hippocampus and testis in both the fmr1-KO and WT mouse. As far as we know, this was one of only a few proteomics studies focusing on the widely used animal models (Kalinowska, et al. 2015; Klemmer, et al. 2011; Liao, et al. 2008; Matic, et al. 2014; Tang, et al. 2015), and is also the first study to report the complete proteomic analysis of the cerebral cortex, hippocampus and testis in fmr1-KO mice. In the central nervous system, the absence of FMRP leading to defects in synaptic plasticity and cognition has been observed in many models of the disease(Gatto and Broadie 2009). This could be accounted for by the regulation of 11

various synaptic plasticity-related proteins by FMRP, which was associated either with polyribosomes in the translational elongation or with repression of translational initiation when binding to mRNPs. One recent proteomic studies based on the neocortical synaptic fractions revealed that the absence of FMRP caused upregulation of hundreds of proteins in young fmr1-KO mice, while the upregulation was largely diminished in adult fmr1-KO mice (Tang, et al. 2015); thus, the regulatory effects of FMRP on neuroplasticity-related proteins appear to be age-dependent in this mouse model. Our proteome profiles in the absence of FMRP, based on the whole tissue lysates of hippocampus, cerebral cortex and testis, will be beneficial for FMRP functional

studies

using

this

mouse

model.

We

also

found

that

the

neuroplasticity-related protein, Ly-6/neurotoxin-like protein 1, was upregulated in cerebral cortex, but not in the hippocampus. Neuromodulin and nestin, which are important for axonal and dendritic filopodia induction, were upregulated in the hippocampus of Fmr1 KO mouse, but were downregulated in cerebral cortex. These findings also suggest that the regulatory function of FMRP is tissue-dependent. In the GO enrichment analysis, we found most of differentially expressed proteins were enriched in the structural constituents of ribosomes and RNA-binding categories in the hippocampus and cerebral cortex of the adult fmr1-KO mice. This is in accordance with the role of FMRP as an RNA-binding protein and may also indicate that FMRP plays an as yet undefined role in translation in addition to stalling the process by blocking binding of the RNA protein and elongation factor to the ribosome (Chen, et al. 2014b; Darnell, et al. 2011). However, the exact mechanisms underlying these predictions require further investigation. Furthermore, in the proteomic studies of testis tissues, only the sequence-specific RNA-binding proteins were enriched, while there were no changes in the ribosomal proteins in the absence of FMRP. In this proteomic study, hippocampus, cerebral cortex and testis tissues were pooled from four paired fmr1-KO and WT littermates. The results may be limited by the number of animals used in the study, and thus we verified the differently expressed proteins by additional samples. In the verification studies, we also found differences in protein expression levels between mice with the same genotypes. 12

However, the protein expression levels of littermates between KO and WT mice detected by Western blotting were consistent with those of the pooled proteomics analysis. In the functional analysis, we defined the protein ratios of 1.1 and 0.9 as the thresholds for up- or downregulated expression, respectively. However, it is difficult to define exactly the thresholds for all the changed proteins in this study. FMRP is widely known as a polyribosome-associated neuronal RNA-binding protein that regulates the expression of various proteins in brain (O'Donnell and Warren 2002). FMRP interacts with the encoding region of transcripts for synaptic proteins and reversibly stall ribosomes on the targeting RNA, which may be responsible for the cognitive and related defects in FXS (Darnell, et al. 2011). In addition, it has recently been shown that FMRP binds directly to L5 subunit of the 80S Drosophila ribosome (Chen, et al. 2014a). Thus, FMRP plays a pivotal role in the translation of various neuronal synaptic proteins and functions in combination with ribosomes. The results of our proteomic analysis revealed downregulation of a set of ribosomal subunit proteins in the hippocampus and cerebral cortex of fmr1-KO mice, but not in the testis. RPL23A was downregulated in both hippocampus and cerebral cortex of fmr1-KO. Previously, it was found that mammalian target of rapamycin complex 2 (mTORC2) could mediate phosphorylation of the nascent Akt polypeptide at Thr450 by stable interaction with RPL23A (Oh, et al. 2010). The depletion of FMRP in the neuron of a conditional KO mice resulted in activated Akt-mTOR pathway signaling in the hippocampus, but without behavioral phenotypes (Amiri, et al. 2014). The downregulation of RPL23A in the hippocampus and cerebral cortex may also show the connections of the FMRP, RPL23A and Akt-mTOR signaling pathway. In addition, there was variation in the expression levels of the ribosomal protein between the hippocampus and cerebral cortex in the absence of FMRP. RPS2, RPS7 and RPS18 were upregulated in the hippocampus of fmr1-KO mice, but were unchanged in the cerebral cortex of fmr1-KO mice. Taken together, these observations indicate that FMRP plays different roles in the cortex and hippocampus and influences the translation of ribosomal proteins themselves. However, the detailed underlying mechanisms and the influence of the dysregulated ribosomal proteins on translation 13

require further investigation. Importantly, we also found some potential dysregulated proteins in our proteomic result which may play pivotal role in the FXS and provide potential guidance for future studies. The transmembrane glycoprotein, MPZ, is a major component of the myelin sheath in the peripheral nervous system (PNS) and is essential for the spiraling, compaction and maintenance of the PNS. MPZ mutations are closely associated with various demyelination and axonal inherited neuropathies (Mandich, et al. 2009; Shy 2006).

Myelin

oligodendrocyte

basic

protein

(MBP),

glycoprotein

proteolipid (MOG)

protein and

(PLP),

myelin

2’,3’-cyclic

nucleotide-3’-phosphohydrolase (CNP) are also components of myelin proteins. FMRP has been shown to bind MBP mRNA both in vivo and in vitro (Wang, et al. 2004). However, the expression of MBP, PLP, CNP and MOG were unchanged in the absence of FMRP in mouse brain (Giampetruzzi, et al. 2013). Our proteomic analysis revealed that these proteins were unchanged in hippocampus and cerebral cortex of fmr1-KO mice. Interestingly, both our proteomic and Western blot analyses showed that MPZ was downregulated in the hippocampus of fmr1-KO mice, but was unchanged in the cerebral cortex. MPZ is the major structural protein of myelin in the PNS, while PLP is the major myelin protein in CNS (Yin, et al. 2006). Although the precise function of MPZ in the CNS is largely unknown, MPZ downregulation in the absence of FMRP may indicate a close functional relationship in the compaction and maintenance of myelination. Methyl-CpG-binding protein 2 (MECP2), a methyl-CpG binding protein, represses transcription from methylated promoters in vitro (Nan, et al. 1997). Various mutations of the MECP2 gene are associated with Rett syndrome (RTT), which is a progressive neurodevelopmental disorder. Both FXS and RTT are known monogenetic syndromic autism spectrum disorders (ASDs) (Couvert, et al. 2001; Wang, et al. 2015), although little is known about the underlying mechanisms and connections of FXS and RTT. FMRP overexpression in fmr1-KO mouse rescues the elevated MECP2 levels in the cortex of fmr1-KO mice, but has no effects in WT mice (Arsenault, et al. 2016). In contrast, we found that MECP2 expression was 14

significantly downregulated in the hippocampus in fmr1-KO mice compared with that in WT mice and our proteomic analysis showed that MECP2 expression in cerebral cortex were slightly downregulated or unchanged in the absence of FMRP. The functions of MECP2 in the hippocampus of fmr1-KO mice as well as the regulatory effects of FMRP on MECP2 in this model remain to be fully elucidated.

Disclosure statement No additional conflicts of interest exist.

Acknowledgments We thank Professor Haiteng Deng and the Protein Chemistry Facility at the Center for Biomedical Analysis of Tsinghua University for the sample analysis.

Grant Support This study was supported by CAMS Innovation Fund for Medical Sciences (2016-I2M-1-003 to W.G), State Key Laboratory Special Fund 2060204 and National Postdoctoral Program for Innovative Talents (BX201700162 to B.H.X), Sanming Project of Medicine in Shenzhen (SZSM201611090), Tsinghua University Initiative Scientific Research (2014z01005).

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Table and Figure legends Tables Table 1 Top 10 up- and down-regulated proteins identified by proteomics in the hippocampus.

Table 2 Top 10 up- and down-regulated proteins identified by proteomics in the cerebral cortex.

Table 3 The upregulated and top 10 downregulated proteins identified by proteomics in the testis.

Figures Figure 1. Strategies for protein profiling of brain and testis of fmr1-KO mouse. Proteins were extracted from four individual samples and pooled (1:1:1:1) for the subsequent proteomic studies. Proteins in each group were digested into peptides, labeled with TMT reagents (hippocampus: 126-WT and 127-KO; cerebral cortex 128-WT and 129-KO, testis 130-WT and 131-KO) and combined into two groups (brain and testis). Fractions were separated by liquid chromatography and then detected by a tandem mass spectrometry. Data were processed using Proteome Discoverer 1.4 software and the information was analyzed using bioinformatics strategies (Gene Ontology, Wiki pathway and STRING).

Figure 2. The enrichment of biological process and molecular function categories of differentially expressed proteins identified by FunRich. Differentially expressed proteins identified in (A, B) hippocampus, (C, D) cerebral cortex and (E, F) testis were analyzed using FunRich software. Top six items of biological process and molecular functions in each group are listed based on P-values. Percentage of differentially expressed proteins against the UniProt database is shown on the y axis. Significance of enrichment is indicated by a hyper geometric P-value (P < 0.05). 19

Figure 3. Venn diagram and Visualization of the differentially expressed proteins in the ribosome pathway. Differentially expressed proteins from (A) hippocampus, (B) cerebral cortex and (C) testis. All the differentially expressed proteins identified in the proteomics were mapped to related Wiki pathways based on the published database. In the pathway, red and green boxes indicate up- or downregulation, respectively. Gray boxes indicate proteins that were not identified in this study, and white boxes indicate proteins with unchanged expression levels between the fmr1-KO mice and wild-type mice. The degree of fold changes is indicated by color intensity. The Venn diagram of differentially expressed proteins is shown in (D).

Figure 4. protein-protein interaction networks of differentially expressed proteins in each group analyzed using STRING. With the exception of FMRP, only seven proteins were differentially expressed in both the hippocampus and cerebral cortex. None of the tested proteins were differentially expressed in all the three tissue types. The interaction networks are based on differentially expressed proteins in (A) hippocampus, (B) cerebral cortex and (C) testis. All the differentially expressed proteins in each group were analyzed using STRING 10.0 software and visualized and mapped using Cytoscape 3.4.0. The unconnected proteins were removed from the protein-protein interaction networks. Proteins in the networks are shown as nodes. Red and green nodes indicate up- or downregulated expression, respectively.

Figure 5. Western blot validation of proteins identified by mass spectrometry. Protein samples from individual mice were validated by Western blot analysis. FMRP, MPZ, RS8, RPL23a and GAP43 expressions were validated in the hippocampus (A), FMRP, RS8 and RPL23a, ATPIF1, RPL6 and MTCH2 in cerebral cortex (B). FMRP, CAH3, AKR1B7 and C9 expression was validated in the testis (C). β-actin was used as the loading control.

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Figure 1

Figure 2

Figure 3

Figure 4

Table 1 Top 10 up- and downregulated proteins identified by proteomics in the hippocampus

34.77 255.93 13.03 21.32

Batch 1 KO/WT 1.33 1.32 1.30 1.28

Batch2 KO/WT 1.31 1.20 1.22 1.13

11.74 80.79 25.07 52.70 66.55 18.25

1.28 1.27 1.27 1.24 1.23 1.22

1.21 1.21 1.19 1.13 1.12 1.21

33.61 29.59 12.61 42.66 19.88 75.65 60.57

0.74 0.72 0.72 0.72 0.71 0.70 0.69

0.69 0.68 0.75 0.77 0.87 0.74 0.59

25.02 54.89

0.66 0.47

0.70 0.41

11.30

0.04

0.09

Accession

Description

Score

O55142 P06837 Q6P5H2 Q8CA95

60S ribosomal protein L35a GN=Rpl35a Neuromodulin GN=Gap43 Nestin GN=Nes cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A GN=Pde10a Somatostatin GN=Sst Hemoglobin subunit epsilon-Y2 GN=Hbb-y Opioid growth factor receptor GN=Ogfr 40S ribosomal protein S7 GN=Rps7 60S ribosomal protein L13 GN=Rpl13 39S ribosomal protein L30, mitochondrial GN=Mrpl30 60S ribosomal protein L18 GN=Rpl18 60S ribosomal protein L24 GN=Rpl24 Protein turtle homolog B GN=Igsf9b 40S ribosomal protein S6 GN=Rps6 Cystatin-B GN=Cstb Methyl-CpG-binding protein 2 GN=Mecp2 Heterochromatin protein 1-binding protein 3 GN=Hp1bp3 60S ribosomal protein L14 GN=Rpl14 Synaptic functional regulator FMR1 GN=Fmr1 Myelin protein P0 GN=Mpz

P60041 P02104 Q99PG2 P62082 P47963 Q9D7N6 P35980 Q8BP67 E9PZ19 P62754 Q62426 Q9Z2D6 Q3TEA8 Q9CR57 P35922 P27573

Table 2 Top 10 up- and down-regulated proteins identified by proteomics in the cerebral cortex

Accession

Description

Score

P11087

Collagen alpha-1(I) chain GN=Col1a1

P50153

Guanine

nucleotide-binding

G(I)/G(S)/G(O) subunit gamma-4 Q9D3P8

Plasminogen receptor (KT)

protein

Batch 1

Batch 2

KO/WT

KO/WT

14.43

1.40

1.31

60.44

1.19

1.13

11.33

1.18

1.14

GN=Gng4

GN=Plgrkt

P15864

Histone H1.2 GN=Hist1h1c

79.44

1.17

1.24

Q8BU31

Ras-related protein Rap-2c

95.23

1.16

1.11

Q66GT5

Phosphatidylglycerophosphatase

and

10.91

1.16

1.12

2

25.96

1.15

1.17

101.96

1.14

1.15

10.43

1.14

1.10

GN=Rap2c

protein-tyrosine phosphatase 1 GN=Ptpmt1 Q9D6K8

FUN14

domain-containing

protein

GN=Fundc2 Q791V5

Mitochondrial carrier homolog 2

GN=Mtch2

Q9JJ59

ATP-binding cassette sub-family B member 9 GN=Abcb9

P10922

Histone H1.0 GN=H1f0

53.77

1.14

1.14

Q9ET01

Glycogen phosphorylase, liver form GN=Pygl

99.23

0.70

0.89

P26350

Prothymosin alpha GN=Ptma

35.60

0.67

0.68

P62852

40S ribosomal protein S25 GN=Rps25

17.12

0.66

0.64

Q6P5H2

Nestin GN=Nes

13.03

0.64

0.87

Q9JJI8

60S ribosomal protein L38

18.72

0.64

0.57

P02104

Hemoglobin subunit epsilon-Y2

GN=Hbb-y

80.79

0.61

0.75

O35143

ATPase inhibitor, mitochondrial

GN=Atpif1

114.57

0.60

0.71

P47911

60S ribosomal protein L6

GN=Rpl38

35.51

0.59

0.59

O88990

Alpha-actinin-3

GN=Actn3

GN=Rpl6

138.41

0.38

0.86

P35922

Synaptic functional regulator FMR1 GN=Fmr1

54.89

0.35

0.30

Table 3 The upregulated and top 10 downregulated proteins identified by proteomics in the testis Batch 1 Batch 2 Accession Description Score KO/WT KO/WT Haptoglobin GN=Hp 20.14 1.82 1.69 Q61646 Ig gamma-2A chain C region, membrane-bound 63.58 1.23 1.29 P01865 form GN=Igh-1a Complement component C9 GN=C9 68.57 1.15 1.13 P06683 NADH dehydrogenase [ubiquinone] 1 alpha 23.44 1.14 1.11 Q9D8B4 subcomplex subunit 11 GN=Ndufa11 D-beta-hydroxybutyrate dehydrogenase, 27.14 1.13 1.12 Q80XN0 mitochondrial GN=Bdh1 Ropporin-1 GN=Ropn1 159.34 1.12 1.11 Q9ESG2 Hydroxyacylglutathione hydrolase-like protein 17.09 1.11 1.11 Q9DB32 GN=Haghl X-linked lymphocyte-regulated protein PM1 10.04 0.80 0.88 P05531 GN=Xlr Ras-related protein Rab-5A GN=Rab5a 43.66 0.75 0.90 Q9CQD1 Collagen alpha-2(I) chain GN=Col1a2 118.52 0.74 0.75 Q01149 Collagen alpha-1(I) chain GN=Col1a1 175.37 0.73 0.81 P11087 protein, adipocyte 73.37 Fatty acid-binding 0.67 0.70 P04117 GN=Fabp4 protein 1 132.38 0.61 Aldose reductase-related 0.41 P21300 GN=Akr1b7 Carbonic anhydrase 3 GN=Ca3 69.06 0.55 0.53 P16015 Creatine kinase M-type GN=Ckm 17.40 0.44 0.40 P07310 Cysteine-rich secretory protein 1 GN=Crisp1 30.10 0.43 0.52 Q03401 Synaptic functional regulator FMR1 76.47 0.34 0.41 P35922 GN=Fmr1

Highlights

1. We performed a TMT labeled proteomic study on brain and testis of fmr1 deficient mouse. 2. Expression of a number of proteins in hippocampus and cerebral cortex were changed in the absence of fmr1. 3. Ribosome proteins were largely disturbed in the absence of fmr1.

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