David Beeson Weatherall Institute of Molecular Medicine Oxford Pathogenic mechanisms underlying synaptic dysfunction in congenital myasthenic syndromes
Jan 21, 2016
David BeesonWeatherall Institute of Molecular Medicine
Oxford
Pathogenic mechanisms underlying synaptic dysfunction
in congenital myasthenic syndromes
Endplate region
Congenital myasthenic syndromes
• Genetic
• Fatiguable muscle weakness
• Heterogeneous
Clustered AChRAgrin
MuSKRapsyn
CMS-associated genes
AChE
COLQ
CHRNACHRNBCHRNDCHRNECHRNG
RAPSNMUSKDOK-7
CHAT
SCN4A
LRP4
Congenital myasthenic syndromes
Syndrome Kinships
AChR deficiency CHRNE 112
AChR deficiency – (RAPSN) 51
CMS with proximal weakness (DOK7) 55
Slow channel (CHRNA/B/D/E) 22
Fast channel (CHRNA/D/E) 12
AChE deficiency (COLQ) 15
Presynaptic (CHAT) 8
Additional referrals no mutations found
(Studied in Oxford)
Neuromuscular synapse(a complex structure)
Dok-7
Clustered AChR
NERVE
Agrin
MuSKRapsyn
Postsynaptic specialisation
LRP4
Clinical features of Dok-7 CMS
Inheritance - recessive
Onset - 1.5 – 4 years, sometimes respiratory problems at birth
Symptoms - limb girdle pattern of weakness, ptosis, but eye muscles unaffected
Responsive - ephedrine salbutamol
Unresponsive - pyridostigmine
C-terminalPH PTB
1263insC
1339_1342dupCTGG
1143insC
548_551delTCCT
1124_1127dupTGCC
601C>T 1508insC
1378insC
1357_1370del14
Dok-7 mutations
Common mutation
IVS1+14del15
IVS2-1G>T
1504_1505insTA437delC
481G>A
539G>C
496G>A
596delT
230C>T
473G>A
1185C>G
415G>C967C>T
101_141del 1487G>T
325G>T
Differentiate
+ Agrin
Myoblasts
Transfect with mutant cDNA
Myotubes AChR
AChR clusters
In vitro clustering assay
C2C12RAPSN -/-
MUSK -/-
Myotubes
Dok-7 induced AChR clusters in C2C12 cellsMyotubes – no Dok-7
Dok-7 mutant
Dok-7 WT
(Fewer and smaller clusters)
0
50
100
150
200
250
/10
field
s
Mock
Wild
type
Num
ber
of c
lust
ers
548d
elTCCT
1143insC
1124
dupT
GCC
AChR clusters in C2C12 myotubesinduced by truncated Dok-7
Type of AChR clusters formed following expression of Dok-7 in C2C12 cells
BranchedC-shapedPerforated Endplate
c-shaped and perforated
pBABEW
TT77
M
G109C
R158Q
G161R
G180A
1127
insT
GCC
0123456789
1011
per
cen
t o
f cl
ust
ers
Average length of AChR Clusters
pBA
BE
Dok W
T
T77M
G10
9C
R15
8Q
G16
1R
G18
0A
1277in
s
0
5
10
15
20
25
30
35
clust
er len
gth (
m
)
pBA
BE
Dok
-7 W
TT7
7MG
109C
R15
8QG
161R
G18
0A11
27in
sTG
CC
0
100
200
300N
o A
ChR
clu
ster
s per
mm
2
Number of clusters
** * * *
* * * *
1 way ANOVA Bonferroni’s multiple comparison
1 way ANOVA Tukey's Multiple Comparison Test
branched
pBABEW
TT77
M
G109C
R158Q
G161R
G180A
1127
insT
GCC
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
per
cen
t o
f cl
ust
ers
all significant
all significant
AChR clusters in cultured human myotubes
Agrin-induced clusters on myotubes derived from a Dok-7 patient
Can patients with DOK7 mutations be treated?
• Unresponsive to cholinesterase medication
• Some show some benefit fro 3,4-DAP
• Remarkable response to ephedrine salbutamol
Wheelchair/scoliosis to running and jumping
Wheelchair to running 200 metres
Legs raised
Arms raised
Time on treatmentT
ime
Tim
e
Baseline 1st dose 2 months 6-8 months0
3
6
9
12
15
18
21
24
*
QM
G (
max
39)
Time on treatment
Disability score
QM
GDok-7 CMS patients respond to
treatment with ephedrine
Effect of ephedrine on AChR clusters in human myotubes from a Dok-7 patient
homozygous for 1124_1127dupTGCC
0
50
100
150
200
250
300
350
400 Clusters per fieldCluster length
- -+ +Ephedrine Ephedrine
40% 3.4-foldincrease increase
Truncated Dok-7
Clustered AChRAgrin
MuSK
Rapsyn
Impaired kinase signalling
Retrograde signalling
NERVE
reduced MuSK-P*
C-terminal domainPH PTB
1124_1127dupTGCCPTB motif NPXY
LRP4
Ephedrine
2AR
LR
P4
Maintaining synaptic structure
Dr Palace + CliniciansYuji YamanashiAngela Vincent + team
Collaborators:
Extracellular ring
M2
Intermediate ringCytoplasmic ring
K
K
M2
Q
Q
Conductance of ion channel largely governed by three rings of charged amino acids
Fetal Adult
• Larger conductance
• Shorter openings
Imoto et al.
Case Study
• Clinical features:
– 47 yr woman
– Onset at birth: generalised weakness and ptosis
– Progressive course:Fatigable limb weakness
Severely restricted eye movements
Responsive to pyridostigmine (high dose)
– Respiratory arrest aged 45, hypoxic brain injury
DNA screening revealed two mutations
• Epsilon subunit– P282R missense– F266 in frame deletion
NH2
COOH
F266
P282R
0
20
40
60
80
100
120
-B
uTX
surf
ace
bind
ing
WT
P28
2R
F2
66
0
20
40
60
80
100
120
-B
uTX
surf
ace
bind
ing
cont
rol
Surface expression
•Cell-attached patch recordings
•Transfected HEK 293 cells
•Constant low concentration of acetylcholine
Electrophysiological methodology
Closed
Open
Log10 duration (ms)
N (
sqrt
)
Burst length
Hundreds/thousands of bursts are measured
1: 0.11+/- 0.032: 1.19 +/- 0.223: 4.68 +/- 0.74
n=5
Wildtype AChR recordings
Size of F266 opening are reduced
Pipette potential
Slope conductance reduced
Wildtype AChR
F266 AChR
Ohm’s Law:
V = I ×R
R = V ÷ I
Conductance= 1/R
Excess Na+,Ca 2+ Insufficient Na+
PROLONGEDACTIVATION
SHORTENED ACTIVATION
REDUCEDCONDUCTANCE
Insufficient Na+
Kinetic abnormalities of the AChR
A B
Figure 3: Time (seconds) for individual patients in A: Arms raised 90 degrees and B: Legs raised 45 degrees (data points are mean of right side and left side scores).
• 47 year old female with symptoms weakness since birth
• Progrssive bulbar, repiratory and limb muscle involvement
• Positive response to pyridostigmine, but no improvement with 3,4-DAP
• At 45 suffered respiratory arrest with resultant hypoxic brain damage
• No family history
Novel AChR abnormality
Heteroallelic for mutations in CHRNE ( subunit)
0
20
40
60
80
100
120
-B
uT
Xsu
rfac
e bi
ndin
g
WT
P28
2R
F2
66
0
20
40
60
80
100
120
-B
uT
Xsu
rfac
e bi
ndin
g
WT
P28
2R
F2
66
P282R
F266
Agrin-induced clusters on myotubes derived from a Dok-7 patient
Normal Mutant
20 m
AChR deficiency
Inverted screen test
Myotubes – no Dok-7
Dok-7 common mutation
Dok-7 WT
(Fewer and smaller clusters)
Dok-7 induced AChR clusters
Comparison of AChR deficiency phenotypes with early onset
presentationClinical feature Early Onset
rapsyn mutations
AChR deficiency -subunit mutations
Arthrogryposis Common Absent
Episodic crises Common Rare
Ophthalmoplegia
Absent Common
Spontaneous improvement
Common Rare
Clustered AChRAgrin
MuSKRapsyn
CMS-associated genes
AChE
COLQ
CHRNACHRNBCHRNDCHRNECHRNG
RAPSNMUSKDOK-7
CHAT
SCN4A
Mild arthrogryposis
RAPSN mutation
Muscle AChR
Adult Fetal
NH2
COOH
Transgenic slow channel mouse with AChR-EGFP
NH2
COOH
GFP
L221F
NFP /synaptophysinL221F-EGFP merge
S low channel
Normal S low channel
S low channelS low channel
Normal S low channel
Dok-7 induced AChR clusters in C2C12 cellsMyotubes – no Dok-7
Dok-7 mutant
Dok-7 WT
(Fewer and smaller clusters)