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)