論文の公開元へA mutation in DOK7 in congenital myasthenic syndrome forms aggresome in cultured cells, and reduces DOK7 expression and MuSK phosphorylation in patient-derived iPS cells
概要
主論文の要旨
A mutation in DOK7 in congenital myasthenic syndrome
forms aggresome in cultured cells, and reduces DOK7
expression and MuSK phosphorylation in
patient-derived iPS cells
先天性筋無力症症候群におけるDOK7の変異は、培養細胞で
アグリソーム形成を誘導し、患者由来iPS細胞で
DOK7の発現とMuSKリン酸化を減少させる
名古屋大学大学院医学系研究科
先端応用医学講座
総合医学専攻
神経遺伝情報学分野
（指導：大野 欽司
张 少川
教授）
【Introduction】
Congenital myasthenic syndromes (CMS) are a heterogeneous group of rare inherited
diseases characterized by muscle weakness and fatigue resulting from compromised
signal transduction at the neuromuscular junction (NMJ). Mutations in a total of 34
genes have been reported to cause CMS. Within these gene molecules, the phosphorylation
of MuSK plays a central role in the clustering of acetylcholine receptors (AChRs) at the
motor endplate. Both defective and excessive phosphorylation of MuSK reduces AChR
clusters.
DOK7 enhances MuSK phosphorylation and mediate AChRs clustering. DOK7
mutations account for 10-15% of CMS, and are the most common cause of limb-girdle
myasthenia. DOK7 is comprised of a pleckstrin homology (PH) domain, a phosphotyrosinebinding (PTB) domain, and a long unstructured C-terminal region containing a nuclear
exporting signal (NES) and two tyrosine residues that can be phosphorylated. More than
70 missense, truncation, and splicing mutations have been reported in DOK7 in CMS.
Two reports showed that twelve missense mutations in DOK7 (p.E3K, p.P31T, p.A33V,
p.S45L, p.T77M, p.G109C, p.V139L, p.R158Q, p.G161R, p.G166R, p.G171D, and
p.G180A) reduced the activity of DOK7 on the MuSK phosphorylation. However, none
reduced its expression and aggregate formations of mutant DOK7 has not been
investigated to date.
【Materials and Methods】
We utilized patient-derived induced pluripotent stem cells (CMS-iPSCs) combined
with a gene-editing tool, CRISPR/Cas9, to characterize two mutations, c.653-1G>C and
c.190G>A (Figure 1). We also evaluate the effects of these mutations on protein
expression, MuSK phosphorylation, and AChR clustering in DOK7 transfected cells.
【Results】
Activation of a cryptic 3’ splice site in DOK7 exon 6 due to c.653-1G>C at the 3’
end of intron 5
To examine the effects of c.653-1G>C on splicing, we analyzed DOK7 transcripts in CMSiPSCs (Figure 1). RT-PCR spanning DOK7 exon 6 and sequencing of cloned fragments
showed two aberrantly spliced transcripts in CMS-iPSCs (Figure 2A). One was due to
skipping of exon 6, and the other was due to an activation of a cryptic splice site deleting
seven nucleotides at the 5’ end of exon 6 (Figure 2BC). The in-frame deletion lacking
codons 218-257 (p.D218_G257del) was similarly expressed compared to wild-type
(WT)-DOK7 in COS7 cells. The 7-nt-deleted transcript (c.653_659delACCCAAG)
predicting p.D218Afs*34 showed markedly reduced expression in COS7 cells (Figure
2DE).
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Effects of p.G64R on protein expression, AChR clustering, and MuSK interaction
We next evaluated the effects of p.G64R on protein expression and AChR clustering
in transfected C2C12 myoblasts. Western blotting showed that p.G64R significantly
reduced its expression (Figure 3A). In differentiated C2C12 myotubes (Figure 3BC),
WT-DOK7 induced AChR clustering but p.G64R-DOK7 had no activity on the clustering.
As C2C12 myotubes express DOK7 endogenously, we used COS7 cells that express
negligible amounts of DOK7 and MuSK. In co-immunoprecipitation assay with FLAGtagged MuSK along with WT-DOK7 or p.G64R-DOK7 in COS7 cells (Figure 3D), G64R
had no effect on the total amount of MuSK, but reduced the DOK7-associated MuSK.
Similarly, tyrosine phosphorylation of MuSK was markedly reduced in p.G64R-DOK7transfected COS7 cells.
Ubiquitin inhibitor MG132 accelerates the reduction of soluble p.G64R-DOK7
through forming insoluble aggregates
The ubiquitin-proteasome system (UPS) is the major system for protein degradation.
We next examined whether the UPS accelerated the clearance of p.G64R-DOK7 on
mRNA or protein level. The proteasome inhibitor (MG132) had no effects on
transcription but decreased the expression of p.G64R-DOK7 in transfected COS7 cells
(Figure 4AB). This indicated that an accelerated UPS-mediated degradation of DOK7
was not the cause of the reduced expression of p.G64R-DOK7.
MG132 usually enhances protein degradation through autophagy activation, which is
an alternative pathway to clear the irregularly folded or unfolded proteins. These
improperly folded proteins tend to form insoluble aggregates. We thus examined
whether p.G64R-DOK7 form a detergent-insoluble fraction. WT-DOK7 was mostly
present in the detergent-soluble fraction, whereas p.G64R-DOK7 was mostly located in
the detergent-insoluble fraction (Figure 4C). p.G64R also decreased the sum of the
soluble and insoluble fractions of DOK7 (Figure 4D). MG132 treatment increased the
amount of p.G64R-DOK7 in the insoluble fraction in dose- and time-dependent manners
(Figure 4EF). Immunostaining of DOK7 in the COS7 cells also showed that p.G64RDOK7 was prone to form aggregated puncta at the juxtanuclear region, whereas WTDOK7 was diffusely dispersed in the cytoplasm (Figure 4GH). Nocodazole, an inhibitor
of microtubule formation, markedly reduced the aggresome formations of p.G64RDOK7 in COS7 cells (Figure 5A). p.G64R-DOK7 aggregates were recruited at the
microtubule-organizing center (MTOC, Figure 5B) and colocalized with the aggresome
markers (ubiquitin, parkin, P62, and HSP70, Figure5CDE). These results suggested that
p.G64R caused abnormal folding of DOK7, which made DOK7 ubiquitinated, transported
to MTOC, and formed aggresomes at the juxtanuclear region.
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Comparison of CMS-iPSCs with p.G64R and isogenic CMS-iPSCs Cas9 without
p.G64R
To dissect the effect of p.G64R-DOK7 in patient-derived iPSCs, we established an
isogenic cell line, CMS-iPSCs Cas9 , from CMS-iPSCs by correcting c.190A>G (p.G64R),
while retaining c.653-1G>C on another allele (Figure 6ABC). First, myogenic marker
genes (MYH3 and MYOD1) and NMJ-associated genes (LRP4, DOK7, MUSK, and
CHRNG) were induced in myogenic differentiation of both CMS-iPSCs and CMSiPSCs Cas9 (Figure 6D). Next, we observed that, in contrast to CMS-iPSCs Cas9 , endogenous
DOK7 was undetectable even after immunoprecipitation (Figure 7A), and the
phosphorylation of endogenous MuSK was markedly low in myogenically differentiated
CMS-iPSCs (Figure 7B). In myogenically differentiated CMS-iPSCs, DOK7 made
aggregates at the juxtanuclear region (Figure 7CD). In contrast to COS7 cells, however,
HSP70 was not colocalized with DOK7. Taken together, p.G64R reduced the expression
of DOK7 and MuSK phosphorylation, and formed aggregates at the juxtanuclear region
in myogenically differentiated CMS-iPSCs.
【Discussion】
We identified compound heterozygous mutations (c.653-1G>C and p.G64R) in DOK7 in a
patient with CMS. c.653-1G>C activated a cryptic 3’ splice site in DOK7 exon 6 and
generated two abnormal transcripts. Both lacked the NES domain and the frame-shift
transcript remove two key tyrosine residues. The deletion of the C-terminal region of DOK7
in one or two alleles is observed in most DOK7-CMS patients, which markedly reduces the
expression levels of DOK7. In addition, the disruption of the NES domain and the deletion
of the C-terminal region in DOK7 compromise DOK7-mediated MuSK phosphorylation
and impair AChR clustering in C2C12 myotube. Since the lack of the C-terminal region of
DOK7 cause aberrantly small and simplified neuromuscular synapses in CMS, it largely
compromises the NMJ formation but does not nullify the effect of DOK7.
We observed that p.G64R caused an overload to the UPS, and unprocessed p.G64R-DOK7
made aggresomes at the MTOC. Myogenically differentiated CMS-iPSCs showed that DOK7
made aggregates at the juxtanuclear region without colocalization of HPS70. DOK7
expression and MuSK phosphorylation were markedly reduced in the CMS-iPSCs.
CRISPR/Cas9-medited correction of p.G64R in CMS-iPSCs rescued these phenotypes. Lack
of aggresomes in CMS-iPSCs was likely to be accounted for by high UPS activities in iPSCs.
Alternatively, this was due to a technical constraint that iPSCs could not form mature myotubes
compared to C2C12 myoblast. Although aggresomes were observed in cultured cells and
possibly in CMS-iPSCs, the roles of aggresomes in the patient remain elusive.
【Conclusion】
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In this study, we reveal the pathogenic mechanisms of compound heterozygous
mutations in CMS-patient. c.653-1G>C disrupted the normal splicing and generated two
dysfunctional transcripts. c.190G>A generated residue substitution, p.G64R which
interfered with protein folding. p.G64R-DOK7 makes aggresomes in cultured cells and
is likely to compromise MuSK phosphorylation for AChR clustering in the CMS-patient.
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Figure1. Mutations in DOK7.
(A) Schematic of DOK7 gene (NM_173660.5) showing the location of mutations (red asterisks). The 5’ and 3’
untranslated regions are shown in black. (B) Sequencing chromatograms showing heteroallelic mutations. An invariant
‘ag’ dinucleotide at the 3’ end of intron 5 is indicated by a box.
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Figure 2. Aberrantly spliced transcripts due to c.653-1G>C.
(A) RT-PCR spanning DOK7 exon 6 of control 454E2-iPSCs and patient-derived CMS-iPSCs. The upper band was
comprised of 537-bp and 530-bp fragments. (B) Sequence chromatograms of three RT-PCR products in CMS-iPSCs.
Chromatograms of the two transcripts in the upper band in A are indicated on the right. (C) Schematic showing the
positions of the c.653-1>C mutation and activated cryptic 3’ splice site that is 7 nucleotides downstream of the intron
5/exon 6 junction. (D) Schematic presentation of DOK7 proteins encoded by three transcripts in B. (E) Representative
Western blotting and quantitative analysis of mutant DOK7 arising from an allele with c.653-1G>C in transfected
COS7 cells. Expression levels were normalized to that of GAPDH and to the ratio of wild-type (WT)-DOK7. Mean
and SD (n = 3 experiments) are indicated with individual values in red dots. One-way ANOVA with Dunnett’s post
hoc multiple comparison test was applied (ns, no significance; *p < 0.05).
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Figure 3. Effects of p.G64R-DOK7 on protein expression, AChR clustering, and MuSK interacion.
(A) Representative Western blotting and quantitative analysis of DOK7 in C2C12 cells transfected with wild-type
(WT)-DOK7 or p.G64R-DOK7. Mean and SD (n = 3 experiments) are indicated with individual values in red dots.
Student’s t-test (*P < 0.05 and **P < 0.01). (B) Transfection of wild-type (WT)-DOK7 but not p.G64R-DOK7 induced
AChR clustering visualized by Alexa 594-conjugated α-bungarotoxin (red) in C2C12 cells without agrin. Scale bar =
50 μm. (C) Quantitative analysis of the number, total area, average length, and total signal intensity per visual field
(0.143 mm2) of AChR clusters in C2C12 cells expressing WT-DOK7 or p.G64R-DOK7. p.G64R-DOK7 markedly
reduced AChR clustering. The mean values of 30 visual images are indicated by red dots. One-way ANOVA and
Dunnett’s multiple comparison test (ns, no significance; *p < 0.05, **p < 0.01, and ***p < 0.001). (D) FLAG-MuSK
were co-transfected with wild-type (WT)-DOK7 or p.G64R-DOK7 into COS7 cells. At 24 h after transfection, cell
lysates were immunoprecipitated (IP) by anti-FLAG antibody, and followed by immunoblotting (IB) with the indicated
antibody. PY, anti-phosphotyrosine antibody.
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Figure 4. Effects of p.G64R-DOK7 on mRNA expression, protein solubility, and aggregation after MG132
treatment.
(A) At 18 h after transfection of COS7 cells with WT-DOK7 or p.G64R-DOK7, 10 μM MG132 was added for 3 h. Red
dots indicate the ratio of DOK7 mRNA normalized for GAPDH mRNA (n = 3 independent experiments). P-value was
0.08 by one-way ANOVA, and Dunnett’s multiple comparison test yielded no significance (ns) compared to WTDOK7. (B and D) Representative Western blotting and quantitative analysis of DOK7 and GAPDH without ß-actin
(B) or with ß-actin (D) in detergent-soluble fractions (S) and total protein lysates (T), in transfected COS7 cells. MG132
(20 µM) was added 3 h before harvesting cells (B). Expression levels were normalized to that of GAPDH, and also to
the ratio of wild-type (WT)-DOK7. Mean and SD (n = 3 experiments) are indicated with individual values in red dots.
Multiple Student t-test was applied. (ns, no significance, *p < 0.05, ****p < 0.0001). Triplicated (C) and representative
(E and F) Western blotting of DOK7, ß-actin, and GAPDH in detergent–soluble (S) and insoluble (In) fractions, in
transfected COS7 cells. (E) MG132 at 0-20 µM was added 3 h before harvesting cells. (F) MG132 at 20 µM was added
at indicated time points. (G) Representative images of WT-DOK7 and p.G64R-DOK7 treated with or without 20 µM
MG132 treatment for 3 h in transfected COS7 cells. Green and blue signals represent DOK7 and DAPI, respectively.
Scale bar = 10 μm. (H) The ratio of transfected COS7 cells with aggregates in each experiment is indicated in red dot.
Each experiment is an average of five visual fields and is comprised of at least 100 cells. Mean and SD (n = 3
experiments) are indicated. One-way ANOVA with Dunnett's multiple comparison test (ns, no significance;
****p < 0.0001).
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Figure 5. Characterization of aggregates of p.G64R-DOK7.
(A) Representative immunostaining of p.G64R-DOK7 in transfected COS7 cells with or without 10 µM nocodazole,
an inhibitor of microtubule polymerization. Scale bar = 10 µm. The ratio of aggresome-positive cells after 10 µM
nocodazole treatment for 3 h in five visual fields is plotted in red for each experiment. Mean and SD (n = 3 experiments)
are indicated. *P < 0.05 by Student’s t-test. (B-E) Co-localization of DOK7 and aggresome markers of α-tubulin (B),
ubiquitin (C), parkin (D), P62 (E), and HPS70 (E). COS7 cells transfected with wild-type (WT)-DOK7 or p.G64RDOK7 were immunostained with indicated antibodies at 18 h after transfection. Scale bar = 10 μm.
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Figure 6. Myogenic differentiation of iPSCs.
(A) RT-PCR of pluripotency markers in control 454E2-iPSCs and patient-derived CMS-iPSCs. (B) Schematic
illustration showing the workflow of iPSCs differentiation into myogenic cells. E8: Essential 8 Medium; DF:
DMEM/F12 with 5% KSR; α5: αMEM with 5% KSR; Puro: puromycin; Y: Y27632; SDN: SB431542, dorsomorphin,
and N-acetyl-L-cysteine; C: CHIR99021; R: retinoic acid; Dox: doxycycline; and β-ME: β-mercaptoethanol. (C)
Representative phase-contrast images of undifferentiated and myogenically differentiated iPSCs on day 8. Scale bar =
200 μm.
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Figure 7. The expression level of DOK7, the phosphorylation of MuSK, and the formation of aggregates in
patient-derived CMS-iPSCs and isogenic CMS-iPSCCas9.
(A) Representative Western blotting of DOK7 in myogenically differentiated iPSCs on day 8. Low expression level of
DOK7 necessitated the enrichment of DOK7 by immunoprecipitation (IP) before immunoblotting (IB). Anti-DOK7
antibodies are indicated in parentheses. (B) Phosphorylated MuSK was immunoblotted (IB) by an anti-phosphotyrosine
antibody (PY) after immunoprecipitation (IP) of MuSK. (C) Representative immunostaining of DOK7 and HSP70 in
CMS-iPSC- and CMS-iPSCCas9-derived myogenic cells. Juxtanuclear aggregates are indicated by dotted circles. Scale
bar = 10 µm. (D) The number of juxtanuclear aggregates was blindly counted in five visual fields (~30 nuclei each).
Mean and SD are plotted. ***P = 0.005 by Student’s t-test.
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