Dysregulation of Myosin Complex and Striated Muscle Contraction

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Dysregulation of Myosin Complex and Striated Muscle Contraction Pathway in the Brain of ALS-SOD1 mouse model Benhong Xu, Chengyou Zheng, Xiao Chen, Zaijun Zhang, Jianjun Liu, Peter Spencer, and Xifei Yang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00704 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 20, 2019

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Dysregulation of Myosin Complex and Striated Muscle Contraction Pathway in the Brain of ALS-SOD1 mouse model Benhong Xu1*; Chengyou Zheng2*; Xiao Chen1; Zaijun Zhang3; Jianjun Liu1; Peter Spencer4 and Xifei Yang1#

1. Key Laboratory of Modern Toxicology of Shenzhen, Institute of Toxicology, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China., Shenzhen 518055, China 2. School of Chemical Biology and Biotechnology, Shenzhen Graduate School, Peking University, Shenzhen, China. 3. Institute of New Drug Research and Guangzhou Key Laboratory of Innovative Chemical Drug Research in Cardio-cerebrovascular Diseases, Jinan University College of Pharmacy, Guangzhou, China 4. Department of Neurology, Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR 97201, United States

* These authors contributed equally to this study. Address correspondence to Xifei Yang, PhD. Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention. No. 8 Longyuan Road, Nanshan District, Shenzhen 518055, China. Email: [email protected]

Running tittle: Proteomic profiling of SOD1G93A mouse brain

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Abstract Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease characterized by cortical and spinal motor neuron degeneration, some inherited cases of which are caused by mutations in the gene coding for copper-zinc superoxide dismutase-1 (SOD1). The SOD1G93A mutant mouse model, which expresses large amounts of mutant SOD1, develops adult-onset neurodegeneration of spinal motor neurons and progressive motor deficits leading to paralysis. We used the Tandem Mass Tag technique to investigate the proteome profile of hippocampus, cerebral cortex and medulla oblongata of the SOD1G93A mutant mouse model as compared with that of wild-type (WT) mice. Fifteen proteins were significantly increased or decreased (i.e. changed) in all three tissues. Gene ontology analysis revealed that the changed proteins were mainly enriched on negative regulation of reactive oxygen species, myosin complex and copper ion binding. In the Striated Muscle Contraction Pathway, most of the identified proteins were decreased in the SOD1G93A mice compared with the WT mice. Myosin-1 (MYH1), Fructose-2, 6-bisphosphatase TIGAR (TIGAR) and Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (ATP2A1) were significantly reduced in mutant vs. WT mice, as confirmed by Western blot analysis. Since myosins and tropomyosins are specific for synapse function and drive actin dynamics in the maturation of dendritic spines, changes in these proteins may contribute to perturbations of brain neuronal circuitry in addition to spinal motor neuron disease.

Key words: Amyotrophic lateral sclerosis; Proteomics; Muscle Contraction; SOD1

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Introduction Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons, resulting in progressive muscular weakness and death usually due to respiratory problems 1. Approximately 90% of ALS cases are sporadic (sALS) with causes that presumably include unknown environmental exposures, as is evident in the disappearing Western Pacific form ALS 2. A minority (5-10%) of ALS cases has a familial pattern (fALS), many cases of which arise from mutant genes 3. More than 30 genes have been identified to be associated with familial ALS, including superoxide dismutase 1 (SOD1), C9orf72, TAR DNA binding protein 43 (TDP-43) and Fused in Sarcoma/Translocated in Liposarcoma (FUS) 4-5. Nearly 20% of fALS cases are related with point mutations of SOD1 gene that codes for copper-zinc superoxide dismutase 1, an enzyme responsible for elimination of free superoxide radicals 6. The SOD1G93A transgenic mouse is a well-studied model of ALS that develops adult-onset degeneration of spinal motor neurons and progressive limb weakness and muscle atrophy 7-9.

Several cellular and molecular processes have been implicated in the pathogenesis of ALS-associated motor neuron degeneration, including but not limited to mitochondrial dysfunction, toxic protein aggregation and oxidative stress

10.

Elucidation of the

relationships between SOD1 and other proteins is needed to understand the molecular mechanisms of neuronal degeneration and potential drug targets to block this process.

The progressive motor deficits in ALS are sometimes accompanied by cognitive impairment

11

consistent with frontal-temporal dementia

12.

Cognitive impairment in

ALS has been related with alterations in mitochondria and synaptic loss

7, 13.

mutations have been considered to cause purely motor neuron disease

12,

SOD1

but the

hippocampus and cerebral cortex, which play vital roles in learning and memory

14,

have not been investigated. The present study focuses on proteomic changes in the hippocampus and cerebral cortex of the SOD1G93A mouse model of ALS. The medulla

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oblongata is also examined because of its central role in respiration control, failure of which is a common cause of death in ALS patients 15.

We employ a combination of tandem mass tag (TMT)-labeled proteomics and bioinformatics that has been widely used for the investigation of proteome and pathway changes under certain experimental conditions. We use this strategy to reveal proteome changes of cerebral cortex, hippocampus and medulla oblongata of SOD1G93A mice. Hundreds of proteins and many numerous biological processes were found to be perturbed in this transgenic mouse model of ALS.

Results Initial characterization of 12 week-old SOD1G93A mutant mice Twelve-week-old SOD1G93A but not age-matched WT mice developed characteristic motor system phenotypes. SOD1G93A mice showed decreased locomotor activity compared with that of age- matched WT mice (Figure 1A and 1B). SOD1G93A mice spent more time than WT mice on the pole test and less time on the rotating cylinder (Figure 1C and 1D). SOD1G93A mice showed less grasping force than that of WT mice (Figure 1E). Hundreds of changed proteins were identified by quantitative MS analysis A total of 4171 proteins was identified and quantified by MS (see Supplementary Table S1). Only proteins with high confidence (false discovery rate (FDR) < 1%) were considered. Compared with that of control mice, 25 and 87 proteins were increased and decreased respectively in hippocampus while only 69 proteins were dysregulated in the cerebral cortex of SOD1G93A mice. In the medulla oblongata, 70 and 37 proteins were increased and decreased respectively in transgenic vs. WT mice. Only 15 proteins were changed in all the tested tissues as listed in Table 1. All the unique peptides of proteins listed in table 1 were analyzed and shown in Figure S1. Copper chaperone for superoxide dismutase (CCS), a 54 kDa homodimeric protein required for post4

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translational maturation of SOD1 (Banci et al., 2006), was significantly increased in the cortex, hippocampus and medulla oblongata of the SOD1 mutant mouse model. In addition, copper ion-binding proteins Metallothionein-1, -2 and -3 (MT1, MT2 and MT3), were reduced in the sampled brain regions of SOD1 mutant vs. WT mice. Gene Ontology analysis for the changed proteins Changed proteins included those differentially expressed (increased or decreased) in SOD1G93A vs. WT mouse brain tissues. Changed proteins were uploaded to the ClueGO software and processed for analysis. In the hippocampus, 10 groups of enriched items were found for the changed proteins. The top 3 enriched items were myosin complex, cellular response to metal ion and striated muscle thin filament referred to the enrichment p-value. Other enriched items were: mitochondrial intermembrane space, unfolded protein binding and chloride channel complex, platelet aggregation, negative regulation of reactive oxygen species metabolic process, and regulation of phospholipid. All the changed proteins were shown as nodes with gene names as shown in Figure 3A. In the cerebral cortex, only four items, named copper ion binding, zinc ion homeostasis, myosin complex and negative regulation of reactive oxygen species metabolic process, were enriched for the changed proteins (Figure 3B). In the medulla oblongata, the most enriched items were copper ion binding, myofibril and myosin complex. Other items included: endoplasmic reticulum calcium ion homeostasis, positive regulation of response to endoplasmic reticulum stress, and ADP metabolic process, were other more highly expressed proteins in the medulla oblongata of SOD1G93A vs. WT mouse (Figure 3C). Scaled protein abundance of each protein in each group are also shown in Figure 3D. The changed proteins showed closely protein-protein interaction. STRING (Version: 10.5) software was used to elucidate interactions among changed proteins. In the hippocampus, tropomyosin proteins (Tropomyosin beta chain, Tpm2), troponin proteins (Tnnt3 and Tnni2) and myosin heavy-chain and myosin light-chain proteins displayed close interactions (Figure 4A). In the cerebral cortex, only Myosin5

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1 (Myh1), Myh4, Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (Atp2a1) and Troponin T (Tnnt3) appeared in the protein-protein interaction network. In the medulla oblongata, a more complicated protein interaction network was identified (Figure 4C).

Fructose-2,6-bisphosphatase TIGAR (Tigar) and NAD-dependent protein deacetylase sirtuin-3 (Sir3) were decreased in both the hippocampus and cerebral cortex of SOD1 vs. WT mice

(Figure 4A and B). NAD(P) transhydrogenase (Nnt) was reduced in all

three brain regions studied. Striated Muscle Contraction pathway was enriched for the changed proteins. Signaling pathways affected by the SOD1 mutation were detected with the WEB-based GEne SeT AnaLysis Toolkit (Web Gestalt: http://www.webgestalt.org/option.php) and Cytoscape (3.6.0) software. The Striated Muscle Contraction pathway was enriched in the hippocampus, cortex and medulla of SOD1G93A vs. WT mice. As shown in Figure 5A, hippocampus of SOD1G93A mutant mice had low levels of proteins of titin (Ttn), troponin (Tnnt3, Tnni2, and Tnnc2), tropomyosin (Tpm1, Tpm2 and Tpm3) and myosin (Myl1, Myl9, Myh1, and Myh4). Cerebral cortex of SOD1G93A mutant mice (Figure 5B) had low levels of proteins of titin (Ttn), troponin (Tnnt3) and myosin (Myh1 and Myh4). Medulla oblongata (Figure 5C) of SOD1G93A mutant mice had low levels of α-actinin (Actn2), titin (Ttn), troponin (Tnnt3, Tnni2 and Tnnc2), tropomyosin (Tpm1, Tpm2 and Tpm3) and myosin (Myl1, Myh1, and Myh4) while they had higher level of dystrophin (Dmd) and vimentin (Vim). Taken together, proteins in the striated muscle contraction pathway were fully affected in brain of SOD1G93A mutant mice.

Verification of protein expression levels by Western blot analysis Western blotting was used to validate MS/MS data of regional brain tissues. Actin-beta protein (ACTB) was unchanged in the proteomics study and therefore used as the background reference protein. Five proteins (MYH1, ATP2A1, TIGAR, SOD1 and SOD2) were selected for validation. ATP2A1, sarcoplasmic/endoplasmic reticulum 6

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calcium ATPase 1, in an enzyme that participates in the hydrolysis of ATP and also in muscular excitation and contraction; TIGAR, TP53-inducible glycolysis and apoptosis regulator primarily controls glucose breakdown and also allows DNA repair, organelle degradation and prevention of cell death; SOD2, superoxide dismutase 2, is also known as manganese-dependent superoxide dismutase (MnSOD). MYH1, TIGAR and ATP2A1 were significantly lower in the hippocampus of SOD1G93A vs. WT mice. While TIGAR in the cortex and ATP2A1 in the medulla showed no significant difference between mutant and WT mice, the general trend was consistent with the proteomic results. In addition, the expressions of MYH1 in brain of both SOD1G93A mutant and WT mice were also verified by immunohistochemistry (Figure S2). Taken together, all Western blot analysis results were concordant with the proteomics data (Figure 6). Discussion It has been reported that both ALS patients and SOD1G93A mouse model showed cognitive impairment and anxiety-like behavior11,

16.

Thus, it was essential to

investigate the proteome changes in the brain of SOD1 mutant mouse model. In this study , we performed a TMT-labeled proteomic and Western blotting study of the hippocampus, cerebral cortex and medulla oblongata designed to reveal proteome changes associated with SOD1 mutant mouse model of ALS. The fully mature SOD1 protein exists as a dimer in eukaryotes, with a copper ion and zinc ion in each subunit. SOD1 functions as an efficient enzyme for catalysis of superoxide to oxygen and hydrogen peroxide

17-18.

Demonstration of SOD1 mutations in familial ALS

19

led to

the development of transgenic mice that overexpressed SOD1 protein and which have been widely used for basic research and drug screening. One proposed gain of toxicity was caused by SOD1 misfolding and aggregation from the unstable mutant form

20.

Overexpression of WT human SOD1 (hSOD1) in mouse model could also develop ALS symptom after 370 days later21 and the mice was also widely used as control mice in

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previous studies20, 22. The lack of WT hSOD1 overexpression mice may be one of the limitations for this study.

The copper ion is essential for the catalytic activity of SOD1. Copper chaperone for superoxide dismutase (CCS), a 54 kDa homodimeric protein required for posttranslational maturation of SOD1

23,

was significantly increased in the cortex,

hippocampus and medulla of the SOD1 mutant mouse model. It has been confirmed that CCS overexpression accelerates disease in SOD1G93A mice24. Our proteomic results demonstrated that CCS was increased in brain (including hippocampus, cerebral cortex and medulla oblongata) of SOD1G93A mice. Other copper ion-binding proteins, namely Metallothionein-1, -2 and -3 (MT1, MT2 and MT3), were reduced in the sampled brain regions of SOD1 mutant vs. WT mice. Previous studies have shown that both MT-1/-2 mRNA and proteins are increased in the spinal cord of SOD1 mice, and overexpression of MT-1 attenuates intracellular copper dyshomeostasis and extends the lifespan of the ALS mouse model

25-26.

Perturbation of catalytic copper ion binding affects 40% of

Japanese SOD1-ALS patients 12.

The p53-inducible protein TIGAR (Tp53-induced Glycolysis and Apoptosis Regulator), also described as fructose-2, 6-bisphosphatase TIGAR, functions by reducing cell mitochondrial reactive oxygen species (ROS) and protecting from cell death

27.

Overexpression of TIGAR is critical for neuroprotection in ischemic neuronal injury 28. TIGAR is also responsible for redox homeostasis and meiosis during oocyte maturation 29,

which is required to maintain fertility. Both female SOD1 knock-out mice and

SOD1G93A mutant heterozygous mice have reduced fertility 30, which is consistent with reduced protein expression of TIGAR. Why overexpression of human mutant SOD1 results in reduced TIGAR expression in brain tissue merits investigation.

It was reported that cerebral cortex neurons were changed before motor system phenotype and pathology were present16, 31.

There is no doubt that motor symptoms

and pathology of 16-weeks-old SOD1G93A mice are well underway. A number of 8

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protein networks were changed in the brain of 16-weeks-old SOD1 mutant mice, including cellular response to metal ions, myosin complex and negative regulation of neuron apoptosis process. It is possible that changes in the dysregulated pathways are secondary in the disease course

32.

Myosin proteins (Myh1 and Myh4) and

tropomyosins (Tpm2, Tnnt3, Tnni3 and Tnnc2) were also decreased in the hippocampus, cortex and medulla of SOD1 mice. Myosins and tropomyosins are specific for synapse function and drive actin dynamics in the maturation of dendritic spines

33-35.

Dendritic spines are tiny protrusions from neuronal dendrites that form

excitatory synapses required for neuronal circuitry

36-37.

The actin cytoskeleton plays

an important role in regulating spine morphogenesis and function of synapses

38-39.

Reduced myosin and tropomyosins in SOD1G93A vs. WT mouse suggest that widespread CNS synaptic perturbation occurs in the transgenic model of SOD1-ALS. Conceivably, in human motor neuron disease, synaptic dysfunction in the hippocampus could associate with memory dysfunction while comparable changes in the medulla oblongata might account for respiratory dysfunction. Materials and methods Reagents and antibodies Proteinase inhibitor cocktail was obtained from Roche (IN, USA). The Tandem Mass Tag (TMT) Kit and the Enhanced Chemiluminescence (ECL) Kit were obtained from Thermo Scientific (NJ, USA). Mass spec grade trypsin/Lys-C mix was bought from Promega (WI, USA).

Anti-ATP2A1 (ab2819, also called anti-SERCA1 ATPase1), anti-TIGAR (ab62533), anti-SOD1 (ab13498) and anti-SOD2 (ab13533) were purchased from Abcam (MA, USA). Anti-ACTB was purchased from Santa Cruz (CA, USA). Anti-MYH1 (abs117520) was purchased from Absin (Shanghai, China).

Animals

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B6SJL.SOD1G93A (SOD1G93A) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and breeding was generated by crossing male SOD1G93A hemizygous with wild-type (WT) C57BL/6 females. The mice were bred with a 12-h light-dark cycle. Mouse genotypes were identified by PCR analysis and phenotypes of 12-week-old mice were evaluated by behavior tests. All experiments were approved by the ethics committee of Shenzhen Center for Disease Control and Prevention.

Hippocampus, cerebral cortex and medulla oblongata isolation and preparation Sixteen-week-old SOD1G93A mice and age matched WT mice were euthanized by 0.1% pentobarbital. After perfused with ice cold 0.01 M PBS (pH 7.2), the brain was removed and the hippocampus, cerebral cortex and medulla oblongata were dissected on ice and stored at -80 °C. 8 M urea dissolved in PBS (0.1 M, pH 8.0, 1 × cocktail) was used for proteins homogenization and sonicated for 10 cycles (15 second on and 15 second off). Protein solutions were obtained by centrifugation at 12,000 rpm for 10 min. Nanodrop 2000C (Thermo Scientific, NJ, USA) was used for determination of protein concentration.

TMT labeling In each group (as indicated in Figure 2) six individual samples were pooled together with a total of 100 µg proteins. Then, samples were reduced and methylated with 10 mM Dithiothreitol (DTT) and 25 mM 2-Iodoacetamide (IAA). Protein digestion and labeling were followed as we reported previously40. Samples were labeled by TMT as follow (Figure 2): [TMT-126 and TMT-127 were used for hippocampus of WT mice and SOD1G93A mice, respectively; TMT-128 and TMT-129 were used for cerebral cortex of WT mice and SOD1G93A mice, respectively; TMT-130 and TMT-131 were used for medulla oblongata of WT mice and

SOD1G93A mice, respectively]. The

Pierce High-pH Reversed-Phase Peptide Fractionation Kit™ (Thermo Scientific, NJ, USA) was used for fractionation before analysis by LC-MS/MS according to the manufacturer’s instruction. 10

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LC-MS/MS for peptide analysis Each fraction was separated by a silica capillary column (75 μl internal diameter (ID), 150 mm length; Upchurch, Oak Harbor, WA, USA) packed with C18 resin (300 Å, 5 μl; Varian, Lexington, MA, USA) and then analyzed by Q Exactive mass spectrometer (Thermo Scientific, NJ, USA). The data-dependent acquisition (DDA) mode was used and full-scans in Orbitrap mass analyzer (400-1, 800 m/z, 70,000 resolution) were followed by top 10-20 data-dependent MS/MS scans (100-1,800 m/z).

MS/MS spectra were searched using SEQUEST search algorithms embedded into Proteome Discoverer 2.1 (Thermo Scientific) against the Uniprot_mouse database (download from Uniprot database on March 22th, 2018). The searching parameters were set as default with modifications. Enzyme was set as trypsin and two missed cleavage sites were allowed. Carbamidomethylation (C, +57.021 Da) was set as static modification. TMT6-plex (lysine [K] and any N-terminal of peptides) and the oxidation (methionine, M) were set as dynamic modification as previously reported40. Protein changes were evaluated by the comparison of reporter ions from each group, with 1.25 and 0.8 set as up- and down-regulation thresholds. All the proteomics data were deposited with the ProteomeXchange Consortium via the PRIDE partner repository 41, with the data identifier PXD010553.

Bioinformatics analysis All the dysregulated (both increased and decreased) proteins were analyzed using Clue GO

software

embedded

in

Cytoscape

(3.6.0)

as

described

(http://apps.cytoscape.org/apps/cluego). The online WEB-based GEne SeT AnaLysis Toolkit (http://www.webgestalt.org/option.php) was applied for pathway enrichment. One of the significant enriched pathways (P < 0.001) was drawn on Cytoscape. Proteinprotein interaction analysis was performed using STRING database (10.0, http://stringdb.org).

Western blot analysis 11

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Equal amounts of protein samples were loaded on to 10% or 12% polyacrylamide gels after degeneration by heating. Proteins were transferred to PVDF membrane (0.22 μM) and then the membrane was blocked with 5% nonfat milk powder dissolved in TBS-T (TBS with 0.5% Tween) for 40 min. Primary antibodies (Anti-ATP2A1, anti-TIGAR, anti-SOD1 and anti-SOD2, Anti-ACTB and Anti-MYH1) were incubated overnight at 4°C, then washed with TBS-T for three times and incubated with secondary antibodies conjugated with horseradish peroxidase. ECL reagents were used for membrane exposure, and bands were captured and analyzed with digital imaging system (GE Healthcare, MA, USA). Behavior test Open-field testing was used to assess the locomotor and exploratory activity of animals in a novel environment. Mice were placed in a 50 × 50 × 40 cm opened box. Activity was recorded for 5 min with a computer-linked video camera placed above the testing box. Movement traces and distances moved were analyzed. The Rotarod test was performed to assess motor coordination, strength and balance. Mice were trained daily for 1 week before data acquisition. Animals were placed on the cylinder at a speed of 30 rpm for 3 min and tested three times. The average latency time (fall off from the cylinder) was recorded. Motor function was also assessed with a pole test using a homemade 50-cm stake with a diameter of 1 cm. Mice received training daily for 3 days before recording the data. An average of climbing time from three consecutive tests of each animal was recorded and analyzed. Hind-limb grip strength was measured by a digital force gauge (YLS-13A, Zhenghua Biology, Anhui, China) as reported42. The animal was allowed to grasp the T-bar and the strength of the grip was recorded until both of its hind limbs released the bar. The force measurement was recorded by ten separate trials.

Statistics

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Prism (GraphPad) was used for all statistical analysis. Data were presented as mean± SEM. The significance between two groups was evaluated by a two-tailed unpaired t test (Mann-Whitney). Values is considered significant if p < 0.05.

Acknowledgement This work was supported by the National Natural Science Foundation of China (81801089, 81673134, 81401570), Guangdong Natural Science Foundation (2018A030313242), China Postdoctoral Science Foundation funded project (BX201700162, 2018M630992), Sanming Project of Medicine in Shenzhen (SZSM201611090)

and

Basic

Research

Program

(Grant

NO.

JCYJ20160428142632408).

Author Contributions X. Y., B. X. and C. Z. designed the studies. B. X., X. C. and X. Y. wrote the paper. B. X. and C. Z. performed the experiments. Z. Z., J. L. and P. S. revised the manuscript.

Conflict of Interest Statement The authors declare that there was no potential conflict of interest existed.

Supporting Information:

Table S1. All the proteins of (A) hippocampus, (B) cerebral cortex and (C) medulla oblongata identified by the mass spectrum. Figure S1. The MS/MS spectra of unique peptide for the identification of proteins. Figure S2. Immunohistochemical studies of MYH1 expression in the brain of 19weeks old SOD1G93A mutant and WT mouse.

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Table and Figure legends Table 1 Proteins increased and decreased in hippocampus, cerebral cortex and medulla oblongata Figure 1. Behavior test for the SODG93A mouse model. The representative movement trajectories of WT and SOD1G93A mouse from open filed test (OFT) are shown in (A); total distance travelled in OFT were shown in (B); Climbing time on pole test were shown in (C); Time spent on the rotarod were shown in (D) and Grip strength was shown in (E). Data represent Mean ± S.E.M (n=6), *P