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Neurology
01
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Skeletal muscle cell protein
dysregulation highlights the
pathogenesis mechanism of
myopathy-associated p97/VCP
R155H mutations
Anna Luzzi
1
, Feng Wang
1
, Shan Li
1
, Michelina Iacovino
1
,
2
* and
Tsui-Fen Chou
1
,
3
*
1
The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA,
United States,
2
Department of Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA,
United States,
3
Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena, CA, United States
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.
KEYWORDS
VCP/p97, IBMPFD/ALS, iPSCs, skeletal muscle, myopathy, R155H mutation
OPEN ACCESS
EDITED BY
Benedikt Schoser,
LMU Munich University Hospital, Germany
REVIEWED BY
Chiara F. Valori,
University Hospital of Tübingen, Germany
Elena Maria Pennisi,
Ospedale San Filippo Neri, Italy
*CORRESPONDENCE
Michelina Iacovino
miacovino@lundquist.org
Tsui-Fen Chou
tfchou@caltech.edu
RECEIVED
25 April 2023
ACCEPTED
30 June 2023
PUBLISHED
03 August 2023
CITATION
Luzzi A, Wang F, Li S, Iacovino M and Chou T-F
(2023) Skeletal muscle cell protein
dysregulation highlights the pathogenesis
mechanism of myopathy-associated p97/VCP
R155H mutations.
Front. Neurol.
14:1211635.
doi: 10.3389/fneur.2023.1211635
COPYRIGHT
© 2023 Luzzi, Wang, Li, Iacovino and Chou.
This is an open-access article distributed under
the terms of the
Creative Commons Attribution
License (CC BY)
. The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic practice.
No use, distribution or reproduction is
permitted which does not comply with these
terms.
TYPE
Original Research
PUBLISHED
03 August 2023
DOI
10.3389/fneur.2023.1211635
Luzzi et al.
10.3389/fneur.2023.1211635
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Neurology
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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
–
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
).
p97/VCP is a member of the AAA-ATPase superfamily and has
been associated with a wide range of cellular protein pathways
involved in cellular stress (
12
–
14
), Golgi and endoplasmic reticulum
assembly, proteasomal and ER-associated degradation (ERAD),
apoptosis (
15
), unfolding poly-ubiquitinated proteins (
16
). In
particular, p97/VCP is involved in protein degradation via autophagy
(
16
), a pathway found dysfunctional in many degenerative diseases,
including myopathies (
17
). In IBMPDF-associated VCP/p97
mutations, abnormalities of autophagosome maturation lead to
impaired autophagosome-lysosome fusion and autolysosome
generation (
8
,
16
). In addition, VCP/p97 mutations may disrupt
mTOR signaling, a well-established autophagy regulator, which can
contribute to IBMPFD/ALS disease pathogenesis (
18
).
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
–
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.
Materials and methods
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).
hiPSCs generation
Human iPSCs (hiPSCs) were generated using episomal plasmids
containing the following genes: Oct4 (pCE-oct3/4, Addgene #27076),
Sox2 and Klf4 (pCE-hSK, Addgene #27078), c-myc and LIN28 (pCE-
hUL, Addgene #27080), Dominant-negative p53 (pCE-mp53DD,
Addgene #41856) and EBNA1 (pCXB-EBNA1, Addgene #41857).
Nucleofection was performed using the Amaxa Human Stem Cell
Nucleofector kit (Lonza, VPH 5002). Post-transduction cells were
cultured in TeSR
™
-E7
™
medium (Stem Cells # 5914) for 10
days
before culture in mTESR basal medium (Stem Cells #05850). Colonies
were picked and expanded in mTESR basal medium in matrigel-
coated plates (Corning #354277).
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.
Immunofluorescence (IF) staining of
hiPSCs
Human iPSCs were fixed in 4% PFA at room temperature and then
stained with antibodies against the following makers: Oct3/4 (1:500
Abbreviations: INDEL, Insertion/Deletion; MHC, Myosin Heavy Chain; MYF5,
Myogenic factor 5; MYOD1, Myogenic Differentiation 1; OCT-4, Octamer-binding
transcription factor 4; PAX3, Paired box gene 3; PAX7, Paired box gene 7; SSEA-4,
Stage-specific embryonic antigen-4; TRA-1-60, T cell receptor Alpha locus; VCP,
Valosin Containing Protein.
Luzzi et al.
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dilution Abcam #ab27985) and SSEA-4 (1:100 dilution Abcam#
ab16287) and AlexFluor-488 conjugated antibodies against human
TRA-1-60 (1:100 dilution BD Pharmigen #560173) in 0.1% Triton
(Fisher X-100) and and 1% Fetal Bovine Serum (FBS) in PBS. For
secondary antibodies goat IgG (1:250 in PBS, Invitrogen #A11055) and
mouse IgG (1:250 in PBS, Invitrogen #A11055) were used to detect
Oct3/4 and anti-SSEA-4, respectively. Cell nuclei were counterstained
with Hoechst 33342 (10
ng/mL of Thermo Scientific #H1399).
Gene editing of hiPSCs R155H/+ using
RNA-based methods
gRNA and DNA templates were purchased from Integrated DNA
technology (IDT) as follows: Guide RNA 5
′
-CCACAGCACG
CATCCCACCA-3
′
), H155R-Reverse Complement 5
′
-ATCTGTTT
CCACCACTTT GAACTCCACAGCACGCATGCCACCACGTACA
AGAAAAATGTCTCCTGCGAGAGCAAACAGTA-3
′
), (R155H-
Reverse Complement 5
′
-ATCTGTTTCCA CCACTTTGAACTC
CACAGCACGCATGCCACCA TGTACAAGAAAAATGTCTCC
TGCGAGAGCAAACAGTA-3
′
. Briefly, a pre-annealed mixture of
Atl
®
CRISPR-Cas9 crRNA Guide 1, tracrRNA ATTO
™
550
[(200
μ
M each, IDT #1075928), and Atl
®
S. p. HiFi Cas 9 Nuclease
V3(IDT, cat #1081061] were prepared following manufacturer
instructions. The Ribonucleoprotein (RNP) complex was prepared
by mixing 240
pmol of Atl
′
CRISPR-Cas9 crRNA Guide
1 + CRISPR-Cas9 tracr RNA ATTO
™
550, 208
pmol of Alt-R
®
Cas9 enzyme, and 240
pmol of 100
μ
M Ultramer DNA Oligo
(Integrated DNA Technology). Before nucleofection, 10.8
μ
M of
Alt-R
®
Cas9 Electroporator Enhancer was added to the
nucleofection mixture and integrated into cells. The emission of
red light indicates the introduction of the CRISPR/Cas9 complex
into the cells by the tracrRNA-ATTO and the yield of the
transfection. The cells were let to grow for a few days and then
dissociated into single cells for clonal selection.
Digestion with Sph1 restriction enzyme and
sequencing clones
iPSC DNA (~310
bp) was amplified by PCR (Platinum SuperFi
PCR Master Mix, Invitrogen #12358) using custom Reverse primers
(IDT). Primers (VCP-Ex5-F 5
′
-TGGAGTTGGGGAGAGGTAGGG-3
′
,
and VCP-Ex5-R 5
′
-AAAATCGGATACTGGAATCAGGGAGA-3
′
).
PCR product was digested with Sph1 HF (New England Biolabs
#R3182L), and clones positive for the Sph1 digestion were purified
using agarose gel (Qiagen, #28704). Before sequencing, amplicons
were treated with Exonuclease I (10
U Thermo Fisher Scientific #
EN0581) and FastAP
™
Thermosensitive AP (1
U, Thermo Fisher
Scientific #EF0654). The samples were mixed and incubated at 37
°
C
for 15
min, followed by incubation at 85
°
C for 15
min. Samples were
sent for sequencing and analyzed using the software FinchTV
(Geospiza, Inc., WA, United States) (
23
).
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
TABLE 1
Representation of groups 1–4: each Group represents a specific GM number.
Experimental group
GM
Clones
Gender
Age
Genotype
Notes
Group 1
22,369
Control 1
Male
42 years old
Wild-type
Group 1
22,369
Control 2
Male
42 years old
Wild-type
Group 1
22,369
Control 3
Male
42 years old
Wild-type
Group 1
22,369
R155H/+ Clone 1
Male
42 years old
Heterozygous
Group 1
22,369
R155H/+ Clone 2
Male
42 years old
Heterozygous
Group 1
22,369
R155H/+ Clone 3
Male
42 years old
Heterozygous
Group 2
22,246
Control 4
Male
40 years old
Wild-type
From unaffected
family’s members
Group 2
22,246
Control 5
Male
40 years old
Wild-type
From unaffected
family’s members
Group 2
22,246
Control 6
Male
40 years old
Wild-type
From unaffected
family’s members
Group 2
22,246
R155H/+ Clone 4
Male
40 years old
Heterozygous
Group 3
21,752
Control 7
Male
46 years old
Wild-type
Group 3
21,752
R155H/+ Clone 5
Male
46 years old
Heterozygous
Group 3
21,752
R155H/+ Clone 6
Male
46 years old
Heterozygous
Group 4
22,600
Control 8
Female
36 years old
Wild-type
Group 4
22,600
Control 9
Female
36 years old
Wild-type
The table also shows the number of clones and their genotype; Control is for the isogenic cell line after CRISPR/Cas9 editing and unaffected family members of Group 2. R155H/+ is for
heterozygous clones that have a mutation in one allele.
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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.
Skeletal muscle differentiation
For skeletal muscle differentiation we used previously published
method (
20
). Briefly, embryoid bodies (EBs) were generated in
mTESR and then grown using EB differentiation medium (IMDM
(1X) + GlutaMAX
™
-I, Gibco #31980-030) supplemented with 15%
FBS (Atlanta #S11150), 10% Horse Serum (Gibco #26050-088),
4.5
mM Monothyoglycerol (Alfa Aesar), 25
mg of Ascorbic Acid
(Acros, Organics), 100
mg of human Holo-Transferrin (RD System
#2914-HT), and 1% Penicillin/Streptomycin. To perform myoblasts
differentiation, we induced Pax7 expression using 0.75
mg/mL
Doxycycline for 4
days (Millipore Sigma # D9891) in EB medium.
After 7
days of differentiation, EBs were plated as a monolayer culture,
and we isolated GFP and PAX7-positive myoblasts using FACS
sorting (BD FACS Aria III). Cells were then expanded and terminally
differentiated into myotubes using the myotube differentiation
medium: DMEM low glucose (Gibco #11885–084), supplemented
with 20% of KnockOut SR (Gibco, #10828–028), 10
mM of SB431545
(Cayman Chemical #13031), 10
m of DAPT (Adipogen), and 1%
Penicillin/Streptomycin.
FACS analysis
To evaluate the efficiency of myoblast formation, we stained the
expanded myoblasts with
α
-Alpha 7 integrin-PE (AbLab, Cat
#67–0010-05) and
α
-human CD29-APC (eBioscience #17–029942) as
previously described (
25
).
Immunostaining for myosin heavy chain
(MHC)
Myotubes were fixed in PFA 4%, permeabilized with 0.3% Triton
X-100 in PBS for 20
min at room temperature and stained using a
primary antibody against MF-20 (1:20, Developmental Studies
Hybridoma Bank – DSHB) and an Alexa-Fluor 555-tagged secondary
antibody anti-mouse (1:250, Invitrogen #A28180). Nuclei were stained
with Hoechst, and imaging was performed using Evos FL
Fluorescence microscope.
qRT-PCR analysis of skeletal muscle cells
Total RNA was extracted using the kit Direct-zol
™
RNA
Miniprep Plus (Zymo Research #R2072) following the manufacturer’s
protocol. The cDNA was synthesized using 1
μ
g of the total RNA with
the reverse transcriptase (Bioline, Sensi FAST Kit). Gene expression
levels were measured by RT-PCR using the cDNA with the Sensi-Fast
Hi-Rox Kit (Bioline #Bio-82,020). The target genes’ relative expression
was normalized to that of glyceraldehyde 3-phosphate dehydrogenase
(GAPDH Hs02786624-g1 20x Applied Biosystem). The expression of
the following genes was analyzed PAX3 (Hs00240950), Myf5
(Hs00929416-g1), MyoD1(Hs02330075-g1), and Myogenin
(Hs01072232-m1, Applied Biosystems).
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
(10
M Urea, 40
mM HEPES pH 7.5, 200
mM NaCl) containing a
Protease & Phosphatase Inhibitor Cocktail (Fisher #78440) and
10
mM MG132. Proteins were digested in MS buffer (0.1
M Tris–
HCl, Boston BioProducts #BT-P-920) containing 0.5
M TCEP
(Fisher #20491, prepared in MS buffer), 0.5
M 2-chloroacetamide
(MP Biomedicals #ICN15495580), 0.25
μ
g/
μ
L Lys-C (FujiFilm
Wako Chemicals United States Corporation #125–05061, prepared
in MS grade water), incubated at 37
°
C for 4
h with shaking at
750 rpm; 100 mM CaCl
2
and 0.5
μ
g/
μ
L Trypsin (Fisher #90058)
were added to the samples and incubated at 37
°
C for 20
h with
shaking at 750
rpm. The digested samples were then desalted
using C18 columns (Fisher #89870) and dried using a vacuum
centrifuge. Before running mass spec samples, samples were
dissolved in 0.2% FA solution, and peptide concentration was
tested through Pierce Quantitative Fluorometric Peptide Assay
(Fisher #23290). LC–MS/MS experiments were performed using
an EASY-nLC 1,000 connected to a Q Exactive Orbitrap Mass
Spectrometers (Fisher). 0.25
μ
g sample was loaded onto an Easy
Luzzi et al.
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Spray Column (25
cm x 75
μ
m, 2
μ
m C18, ES802, Fisher) and
separated over 195
min at a flow rate of 0.5
μ
L/min with the
following gradient: 2–35% B (180
min), 35–85% B (5
min), and
85% B (10
min). Solvent A consisted of 99.9% H
2
O and 0.1%
formic acid, and solvent B consisted of 19.9% H2O, 80% ACN, and
0.1% formic acid. A full MS scan was acquired at 70,000
resolutions with a scan range of 350–2000
m/z, the AGC target
was 1 × 106, and the maximum injection time was 100
ms. MS2
scan was acquired at 17,500 resolutions with a scan range of
200–2000
m/z, the AGC target was 5 × 104, the maximum
injection time was 64
ms, and the isolation window was 2.0
m/z.
System control and data collection were performed by
Xcalibur software.
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.
Results
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
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FIGURE 1
Human iPSC (hiPSCs) derived from patients’ fibroblast harboring R155H mutation in the p97/VCP gene and the isogenic control cells.
(A)
hiPSCs colony
for alkaline phosphatase.
(B)
Pluripotency was confirmed via immunofluorescence. Bright-field nuclei stained with Hoechst (Blue), Oct4; SSEA-4 and
TRA-1-60 (green), Scale Barr 400 nm.
(C)
sequence of patient iPSC exon 5 carrying the p97/VCP
R155H/+
.
(D)
sequence of the DNA template used to
generate isotype control and to correct R155H mutation.
(E)
sequence of the DNA template used to generate R155H from control cells. Red codon
R155H, green codon H155R, Cas9 PAM blue. H histidine, R arginine, V valine, G glycine. Histogram of sequenced Exon 5 showing G base at the place of
the A base.
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FIGURE 2
Differentiation of hiPSC into skeletal muscle fibers via expressing PAX7.
(A)
Schematic representation of skeletal muscle differentiation protocol and
terminal differentiation of skeletal muscle cells into myotubes.
(B,C)
Representative FACS profile of PAX7-induced proliferating myogenic progenitors.
(Continued)
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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
).
FIGURE 2 (
Continued
)
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FIGURE 3
Bioinformatics analysis of proteomic results.
(A)
Volcano plot analysis of statistically significant myotubes (
p
-value >0.05). The p-value is represented in
the Y ax, and the fold change in the X ax. The green dots are the down-regulated proteins, and the red dots are the upregulated proteins.
(B)
DE
Pathways analysis of Group 1 myotubes: skeletal muscle, splicing, intracellular organelles, cytoskeleton, signaling, nucleus and apoptosis, enzymes and
cell junction, cell adhesion, and extracellular matrix. DE Pathways analysis of all myotubes: skeletal muscle, splicing, intracellular organelles,
cytoskeleton, signaling/cancer and enzymes, and nucleoside binding.
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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.
FIGURE 4
Dys-regulation of proteins in the Group 1 myotubes: Classification of dysregulated proteins (
p
-value<0.05 cut off) based on fold changes (logFC) in
the Group 1 myotubes. Y ax logFC: negative values, downregulated proteins positive values, upregulated protein. X ax: name of the proteins.
(A)
constituent of the skeletal muscle (red), muscle contraction (green), Calcium ATPase and uptake (purple), adhesion, development, and
differentiation(yellow), and actin stress (blue).
(B)
Dysregulated proteins in the autophagy: lysosomal homeostasis (red), vesicular trafficking (green),
mTOR pathway (purple), and clathrin endocytosis (blue).
(C)
Dysregulated proteins in the mitochondria: ATP generation: glycolysis, Krebs cycle and
respiratory chain (red), mitochondrial ribosome and protein encoded by the mitochondrial DNA, mtDNA (green), voltage and Import proteins
(purple), regulatory functions (blue).