Skeletal muscle cell protein dysregulation highlights the pathogenesis mechanism of myopathy-associated p97/VCP R155H mutations

p97/VCP, a hexametric member of the AAA-ATPase superfamily, has been associated with a wide range of cellular protein pathways, such as proteasomal degradation, the unfolding of polyubiquitinated proteins, and autophagosome maturation. Autosomal dominant p97/VCP mutations cause a rare hereditary multisystem disorder called IBMPFD/ALS (Inclusion Body Myopathy with Paget’s Disease and Frontotemporal Dementia/Amyotrophic Lateral Sclerosis), characterized by progressive weakness and subsequent atrophy of skeletal muscles, and impacting bones and brains, such as Parkinson’s disease, Lewy body disease, Huntington’s disease, and amyotrophic lateral ALS. Among all disease-causing mutations, Arginine 155 to Histidine (R155H/+) was reported to be the most common one, affecting over 50% of IBMPFD patients, resulting in disabling muscle weakness, which might eventually be life-threatening due to cardiac and respiratory muscle involvement. Induced pluripotent stem cells (iPSCs) offer an unlimited resource of cells to study pathology’s underlying molecular mechanism, perform drug screening, and investigate regeneration. Using R155H/+ patients’ fibroblasts, we generated IPS cells and corrected the mutation (Histidine to Arginine, H155R) to generate isogenic control cells before differentiating them into myotubes. The further proteomic analysis allowed us to identify differentially expressed proteins associated with the R155H mutation. Our results showed that R155H/+ cells were associated with dysregulated expression of several proteins involved in skeletal muscle function, cytoskeleton organization, cell signaling, intracellular organelles organization and function, cell junction, and cell adhesion. Our findings provide molecular evidence of dysfunctional protein expression in R155H/+ myotubes and offer new therapeutic targets for treating IBMPFD/ALS.


Introduction
Inclusion Body Myopathy and Frontotemporal Dementia with early-onset Paget's disease/Amyotrophic Lateral Sclerosis (IBMPFD/ALS) is characterized by progressive muscle weakness, bone deformities, and extensive neurodegeneration that affects muscles and bones, but also the heart and lungs due to atrophy of cardiac and respiratory muscles (1)(2)(3)(4). Three distinct disease pathologies of variable penetrance have been identified: 1) inclusion body myopathy (IBM), an autosomal dominant myopathies with adult-onset resulting in degeneration of pelvic and shoulder girdle muscles (5, 6); 2) Paget's disease of bone (PDB) characterized by excessive osteoblastic and osteoclastic activity, and subsequent bone remodeling with focal areas of increased bone growth, leading to bone deformities and fractures (2,7,8); and 3) frontotemporal dementia (FTD) affecting the frontal and anterior lobes of the brain and leading to impaired language and behavior (3,9). This disease accounts for a substantial portion of primary degenerative dementia that occurs before age 65 (2,7,8). Rimmed vacuoles in IBMPFD muscle and brain tissue samples positive for p97 and ubiquitin staining is a common histological feature of the pathology. Although autosomal dominant VCP/p97 mutations have been associated with IBMPFD (9), other mutations of these genes have been linked in a wide variety of neurodegenerative disorders, including Parkinson's disease, Lewy body disease, in both isolated familial and sporadic ALS (6), and in spinocerebellar ataxia type III (10). Specifically, VCP/p97 pathogenic mutations span the N-terminal half of the protein, which contains domains involved in ubiquitin binding and protein interactions (4). Substitution of arginine residue 155 to histidine (R155H) is the most common VCP mutation linked to IBMPFD, with mutations at this position occurring in more than 50% of IBMPFD patients (2,3). A subset of mutations is also associated with 1-2% of amyotrophic lateral sclerosis (ALS) cases (11).
The mouse model containing VCP/p97 mutations recapitulates the clinical manifestation of the myopathy observed in IBMPFD patients. Treatment with a VCP/p97 inhibitor leads to successful correction of the associated myopathy (1,2).
Although a significant amount of information is available on p97/ VCP mutations and the associated pathology, very little is known about differently expressed genes in this condition and how they impact the biology of the muscle. hiPSCs (human induced Pluripotent Stem Cells) derived from patients exhibiting p97/VCP mutations have widened the range of in vitro experiments enabling further investigation on these pathologies (19)(20)(21)(22). To investigate the impact VCP/P97 mutations have on muscle function, we generated hiPSCs carrying the R155H mutation before differentiating them into skeletal muscle cells using established protocols (19). We then used proteomics to identify molecular mechanisms mediating VCP/P97-associated muscle dysfunction and detected dysregulated expression from several proteins involved in skeletal muscle function, intracellular organelles, and cytoskeleton organization. These findings provide an opportunity to develop new therapeutic approaches to correct the expression of disease-specific proteins.

Human fibroblasts
Fibroblasts (GM22369, GM21752, and GM22600) were purchased from The Coriell Institute (Table 1). All diseased VCP/p97 iPSC were derived from R155H/+ patients' own fibroblast cells and were compared to a related, unaffected control group (GM22246).

Alkaline Phosphatase (AP) staining
Human iPSCs were fixed with 4% paraformaldehyde (PFA) at room temperature (RT) prior to staining using the Alkaline Phosphatase (AP) detection kit (Cell Biolabs Inc. # CBA-300) according to the manufacturer's instructions.

Lentiviruses and packaging plasmids production
The doxycycline (Dox)-inducible PAX7 system consisted of two lentiviral vectors: the rtTA-FUGW lentivirus that carries the reverse tetracycline transactivator and hPAX7-pSAM2 that carries the tetracycline response element (TRE-promoter) to control hPAX7 Frontiers in Neurology 04 frontiersin.org induction and ires-GFP (19). Viruses were generated by cotransfection with packaging plasmids in 293 T cells (24). The virus supernatant was collected at 24-and 48 h post-transfection and concentrated by centrifugation (22,000 g for 2 h). iPSCs were transduced using 13 MOI for pSAM2-PAX7 (calculated on 293 T cells) using spin infection (centrifugation at 2,600 g for 1.5 h and 4 h recovery). A transduction rate greater than 20% GPF-positive cells was used in the downstream experiments.

Statistical analysis
Data were expressed as mean +/− SEM, and statistical significance was measured using the unpaired Student's t-test. The statistical significance was set at p ≤ 0.05.

Western Blot analysis
Protein samples and dual plus molecular weight ladders were separated by SDS-PAGE using Precast Gels with a 4-15% gradient (Bio-Rad #4561083). Proteins were transferred to nitrocellulose membranes (Bio-Rad #170-4,159) using the Bio-Rad Trans-Blot Turbo Transfer System for 7 min. Total proteins on membranes were detected using the Ponceau S staining. Membranes were blocked with 5% non-fat milk in TBS-T and incubated with primary antibodies against human Myf5 (Abcam #125301), and MyoD1 (Abcam #16148) in TBS-T with 2.5% non-fat milk at 4°C overnight. HRP-conjugated secondary antibodies (1:3000), anti-rabbit-HRP (Invitrogen #31460), and anti-mouse HRP (Invitrogen #31430) were used, were incubated with the membrane in TBST with 2.5% non-fat dry milk for 1 h at room temperature. Membranes were exposed to the chemiluminescence reagent (Millipore #WBKLS0500) for 2 min at room temperature and visualized using Chemidoc (Bio-rad).

Mass spectrometry
Myotubes from each Group were lysed using a lysis buffer  Proteomic data processing was performed through Proteome Discoverer 1.4 (Fisher) using the Uniprot human database and the Sequest HT Search Engine. The search allowed for a precursor mass tolerance of 10 ppm, a minimum peptide length of 6, and a minimum peptide sequence number of 1. Upon identification of dysregulated protein expression levels from the control sample and correction for false discovery rate (t-test <0.05), we analyzed interaction protein using the STRING program.

Generation of hiPSCs and isogenic control lines
We generated hiPSCs from three human fibroblasts harboring the p97/VCP R155H +/− mutation and from one unaffected related control. Successfully reprogrammed hiPSCs were validated with Alkaline Phosphatase assay and the SSEA-4, TRA-1-60, and Oct4 markers, confirming their pluripotency ( Figures 1A,B and Supplementary Figures S1A,B). All clones derived from a specific patient were labeled as belonging to the same Group. Selected hiPSC p97/VCP R155H+/− clones were corrected to p97/VCP isoWT using the CRISPR/Cas9 and homology recombination (HR) as previously described (26-28). The mutation R155H/+ is in the exon 5 of the VCP/p97 gene ( Figure 1C). We used a homologous recombination DNA template containing a Guanidine (G) in codon CGT (coding for Arginine) to replace the Adenine (A) in codon CAT. The DNA template also had a missense mutation for Glycine to introduce the Sph1 digestion site and a missense mutation for Valine, V (GTA) to disrupt the PAM sequence ( Figure 1D). To generate p97/VCP R155H from isogenic Control, we prepared a similar DNA template containing the CAT codon to replace histidine in the arginine 155 ( Figure 1E). Successful modifications were verified through sequencing ( Figure 1F).

Differentiation of hiPSCs into myotubes skeletal muscle cells
hiPSC clones from Control and R155H/+ groups (for a total number of 8 lines) were differentiated into skeletal muscle following a multi-step schematic protocol that included transduction of the hiPSCs with an inducible PAX7 expression, Embryoid Bodies (EBs) formation, purification, and expansion of myogenic precursors, and finally the formation of the multinucleated muscle fibers or myotubes (Figure 2A) (20). Diseased and isogenic control iPSCs were successfully differentiated into myoblast expressing PAX7 (GFP+ cells), CD29, and alpha-7 integrin (Figures 2B,C). Further differentiation was performed to generate multinucleated myofibers expressing skeletal muscle marker MHC ( Figure 2D). The same procedure was applied to the cells of all other iPSC groups 2-4 (Supplementary Figures S2A-I).
Next, we measured gene expression of the myoblast's progenitor markers PAX3 and MYF5 and of differentiated myoblasts MYOD1 and MYOGENIN in both Control vs. diseased myoblast. Our results reveal that p97/VCP R155H +/− myoblasts had lower expression of PAX3 and MYF5 than controls and that p97/VCP R155H +/− myotubes had a significative lower expression of MYOD1 ( Figure 2E). We obtained similar results with all 4 groups (Supplementary Figure S2J). Using Western Blot analysis, we assessed MYF5 and MYOD1 protein levels in myoblasts and myotubes in Group 1 cells. We detected higher levels of MYF5 protein in the p97/VCP R155H +/− lines, but it did not reach statistical differences. MYOD remained unchanged ( Figures 2F,G). The proteins MYF5 (28KDa) and MYOD1 (35KDa) are shown in cropped blot images ( Figures 2F,G), and their corresponding full-length blot images are shown as well (Supplementary Figures S5A,B).

Global proteomic analysis in skeletal muscle fibers of R155H/+ and isogenic control
To investigate the global differences in IBMPFD/ALS myotubes, we performed an unbiased proteomic analysis in group 1. Proteomic analysis revealed dysregulated protein (p value <0.05) shown in the Volcano plot. Red dots represent upregulated proteins, and the green dots are down-regulated proteins ( Figure 3A). Similar pattern of dysregulated myotubes is present in groups 1-4 (Supplementary Figure S3A). Pathway analysis showed that many dysregulated proteins are involved in skeletal muscle, intracellular organelles, cytoskeleton organization, cellular communication, and signaling. Each pathway comprises serial sub-pathways that specify the protein functions ( Figure 3B and Supplementary Figure S3B).

R155H/+ impacts skeletal muscle, autophagy, and mitochondrial function
The myopathy described in IBMPFD/ALS leads to muscle weakness (1,2). We found that several constituents of the skeletal muscle architecture, including myosin, troponins, and tropomyosin, as well as several involved in muscle contraction, were downregulated in p97/VCP R155H +/− . Other proteins involved in muscle filament sliding, sarcomere organization, myosin complex, and phosphatase activity were downregulated. In addition, several proteins required in the reuptake of cytosolic calcium into the sarcoplasmic reticulum and in calcium-binding function were downregulated. In contrast, proteins involved in actin stress, a mechanism of myosin and actin contraction in Frontiers in Neurology 06 frontiersin.org Human iPSC (hiPSCs) derived from patients' fibroblast harboring R155H mutation in the p97/VCP gene and the isogenic control cells.

(Continued)
Frontiers in Neurology 08 frontiersin.org non-muscle fibers, were upregulated ( Figure 4A). It was also proposed that the actin stress function as the template for sarcomere formation in cardiac cells, suggesting that diseased cells may not fully differentiate into mature sarcomere (29, 30). A similar protein expression pattern was found in the myotubes of groups 1-4 (Supplementary Figure S4A). p97/VCP is crucial for autophagosome maturation (4, 8), and multiple studies report that VCP/p97 mutants impair autophagy mechanisms (9,16). We found several proteins involved in lysosomal homeostasis and endosome recycling that were downregulated in diseased myotubes. On the contrary, proteins involved in vesicular trafficking and clathrin endocytosis were upregulated ( Figure 4B). Our data show that mutation of VCP/ p97 in IBMPFD disrupts mTOR signaling, a serine/threonine kinase that contributes to myopathy and which has been showing to worsen the severity of the disease (18) ( Figure 4B). Proteins with very similar biological functions were also found in the myotubes groups 1-4 (Supplementary Figure S4B).
Dysfunction in mitochondria, unique organelles essential for various cellular processes such as energy metabolism, calcium homeostasis, lipid biosynthesis, and apoptosis, is a known prevalent feature of many neurodegenerative diseases and motor neuron disorders such as ALS. Disruption of mitochondria structure, dynamics, bioenergetics, and calcium buffering has been extensively reported in ALS patients (31). In diseased myotubes, we found that several proteins involved in the formation of mitochondrial respiratory complexes were downregulated. In contrast, proteins involved in glycolysis and assembly of complex 1 were upregulated, suggesting an enhancement of glycolysis mechanisms as compensation for dysfunctional mitochondria. In addition, proteins relevant for mitochondrial translation, DNA inheritance, and protein import were downregulated, indicating dysfunctional mitochondria ( Figure 4C and Supplementary Figure S4C). Finally, proteomic data of diseased myotubes revealed that proteins involved in cellular stress responses, such as DNA repair, protein degradation, and protein folding, were dysregulated (Supplementary Figure S4D) (12,14,32). p97/VCP also has a chaperone function, and its mutations interfere with cellular methylation, affecting numerous protein features such as turnover, activity, and molecular interactions (13). Some dysregulated proteins promote p53/TP53 degradation and protein degradation (Supplementary Figure S4D).

Discussion
p97/VCP is essential in many cellular functions, including proteasomal degradation and autophagosome maturation. In addition, this protein complex is required to dislocate proteins from the endoplasmic reticulum (ER) to the cytosol during the endoplasmic reticulum-associated degradation (ERAD) (15).
The most common mutation of VCP/p97 in IBMPFD patients is located on the R155H site (3,9). IBMPFD affects the function of muscles, bones, lungs, and the brain. Pathological features in IBMPFD samples include rimmed vacuoles found in p97 and ubiquitin-positive muscle tissues and nuclear inclusions in p97 and ubiquitin-positive neurons in brain tissues (6). IBMPFD mice exhibiting the R155H and A232E mutations showed progressive weakness and atrophy of skeletal muscles (2). To understand the impact of VCP/p97 mutants on myotubes and myoblasts, we generated patients-derived iPSC and differentiated them into myoblasts and myotubes. We found that while diseased and Control cells could generate myotubes, diseased myotubes had a decreased expression of MyoD1. Although MYF5 protein levels were increased in diseased myoblasts, no significant difference in MYOD and MYF5 protein levels were found, probably due to high variation among clones.
Our global protein analysis on myotubes revealed that several proteins involved in key muscle function structure, contraction, and calcium uptake were downregulated, suggesting a dysfunction in muscle contraction ability in p97/VCP R155H +/− cells. We also discovered that diseased muscle had increased protein levels involved in actin stress. These contracting proteins are usually expressed in non-muscle fibers, such as in smooth muscle cells, and function as a template to generate mature sarcomeres (29, 30), suggesting an abnormal contraction mechanism in this pathology. Our findings agree with previously published data providing molecular evidence of myopathy.
p97/VCP mutants inhibit proper autophagy, a degradation system that processes proteins too large for the proteasome. Our proteomic data suggest that proteins involved in protein degradation via proteasome or lysosome are downregulated. MTOR, a key negative regulator of autophagy initiation, is upregulated in our study. It was previously shown that mTOR activity is inhibited in R155H mutant myoblasts, which promotes autophagosome formation, and inhibits autophagosome maturation, thus blocking the mTOR function downstream of the autophagy pathway (18). In addition, an increase in mTOR inhibition was shown to worsen the myopathy associated with the disease, suggesting that the accumulation of autophagosomes that cannot proceed to full maturation is more harmful than impaired autophagy. Therefore, increasing mTOR may counteract the mTOR inhibition (18) by decreasing autophagosomes that cannot progress to maturation due to VCP/p97 mutation (4, 8).
VCP/p97 mutation dysregulates several mitochondrial proteins. Mitochondria dysfunction is a prevalent feature of many neurodegenerative diseases and motor neuron disorders such as ALS. Disruption of mitochondria structure, dynamics, bioenergetics, and calcium buffering has been extensively reported Group 1 myoblasts were previously purified by FACS selection of only GFP-positive (PAX7 + ) cells and then expanded. (C) After 1 week of expansion, the myogenic precursors were stained for two early skeletal muscle markers, Alpha 7 integrin and CD29. The percentage indicates cells staining positive for GFP, Alpha 7 Integrin, and CD29. SSC side scatter. (D) Myogenic progenitors were differentiated into myotubes over 6-8 days, and immunofluorescence determined the myotube formation. Blue nuclei as stained with Hoechst. Red, Myosin Heavy Chain (MHC), myotubes; Scale Barr 400 nm. (E) qRT-PCR of Group 1 myoblast and myotubes, showing expression of PAX3, MYF5 (Marker of myogenic precursors: myoblasts), MYOD1, and MYOGENIN (Late marker of terminally differentiated skeletal muscle cells). Western Blot analysis for the expression analysis and quantification of the proteins Myf5 (28KDa) and MyoD1 (35KDa) in myoblasts (F) and myotubes (G), where are showed the cropped blot images (F,G). Their corresponding uncropped full-length blot images are also represented (Supplementary Figures S5A,B).  in ALS patients (31). Skeletal muscle requires a lot of energy, and abundant mitochondria provide the energy in physiological conditions. Patients harboring VCP/p97 mutation have mitochondria dysfunction, resulting in reduced ATP synthesis and dysregulation in the mitochondria function (33). Our proteomic analysis reveals a decrease in the expression of proteins involved in ATP formation and the import of proteins into the mitochondria, providing molecular targets responsible for mitochondrial dysfunction.

Conclusion
Our data show how VCP/p97 mutations can impair several essential biological processes in skeletal muscles, such as autophagy and mitochondria function, leading to disease progression in IBMPFD/ALS patients. Identifying the protein for which the expression is dysregulated in this disease shines a light on key therapeutic targets for developing a treatment that can reduce the severity of the disease and slow down its progression.

Data availability statement
The original contributions presented in the study are publicly available. This data can be found in PRIDE under the accession number PXD044004: https://www.ebi.ac.uk/pride/archive/projects/ PXD044004.

Funding
This work was supported by funds from the National Institute of Neurological Disorders and Stroke (NINDS), R01NS102279.