1. The document summarizes research on genetic causes and pathogenic mechanisms underlying various forms of Charcot-Marie-Tooth (CMT) disease and distal hereditary motor neuropathies (dHMN). It describes mutations found in genes involved in protein folding, axonal transport, cytoskeletal stability and other pathways.
2. Research shows that mutations in small heat shock proteins HSPB1 and HSPB8, which are implicated in CMT and dHMN, cause motor neuron dysfunction and protein aggregation. Studies using cell and animal models demonstrate disease-relevant gain of toxic function from these mutations.
3. The small heat shock proteins HSPB1 and HSPB8 are normally involved in
Is preeclampsia and spontaneous preterm delivery associate with vascular and ...
Vincent Timmerman - 'Neuropatías periféricas hereditarias'
1. Pathogenic mechanisms of CMT neuropathies
Redrawing of original figures by (A) Tooth, (B,C) Dejerine
& Sottas. By J. Berciano, MUSCLE & NERVE (2003)
2. Exploring the genetic causes of PNS degeneration
Timmerman, Strickland, Züchner GENES (2014) + updates
AIFM1, COX6A1, VCP, …
4. Overlapping molecular pathological themes link CMT and
hereditary spastic paraplegias
Timmerman, Clowes and Reid, Experimental Neurology (2013) + updates
ATL3
VCP
5. Exome sequencing links corticospinal motor neuron disease
to common neurodegenerative disorders
Novarino et al. Science (2014)
6. HMSN
hereditary motor and
sensory neuropathy
= ‘CMT’
Motor Sensory & Autonomic
HSAN
hereditary sensory &
autonomic
neuropathy
HMN
hereditary motor
neuropathy
Harding & Thomas, J. Med. Genet (1980), J. Neurol. Sci (1980) and Brain (1980)
Harding, A.E. (1993) In: Dyck, Thomas, Griffin, Low and Poduslo (Eds) In: Peripheral Neuropathy
Dyck, P.J. (2005) In: Peripheral Neuropathy
Various kinds of inherited peripheral neuropathies
7. SUBTYPE INH GENE PHENOTYPE MUTATIONS
dHMN1 AD 7q34-36 typical HMN (juvenile onset) -
dHMN2A AD HSPB8 typical HMN (adult onset) 4
dHMN2B AD/AR HSPB1 typical HMN (juvenile and adult onset) 18
dHMN2C AD HSPB3 typical HMN 1
dHMN2D AD FBXO38 distal SMA with predominance weakness of the calf muscles 1
DSMA4 AR PLEKHG5 proximal and distal muscle atrophy, scoliosis, respiratory failure 3
dHMN AD SETX HMN with pyramidal signs (juvenile ALS4) 4
DSMA5 AR DNAJB2 typical HMN 1
dHMN5A AD GARS predominant weakness in hands 10
dHMN5B AD REEP1 predominant weakness in hands, pyramidal signs 1
dHMN5C AD BSCL2 predominant weakness in hand, Silver syndrome, rarely sensory symptoms (CMT2D) 2
dHMN6 AR IGHMBP2 juvenile onset; SMARD, diaphragmatic SMA 55
dHMN7A AD SLC5A7 typical HMN; vocal cord paralysis 1
dHMN7B AD DCTN1 bulbar and facial weakness 1
dHMNJ AR 9p21.1-12 juvenile onset; pyramidal signs -
SMARD2 X-linked LAS1L distal muscle weakness, respiratory failure, diaphragm paralysis 1
SMAX3 X-linked ATP7A typical HMN (X-linked distal SMA) 2
SMALED AD BICD2 congenital AD-SMA 4
DYNC1H1 congenital; contractures; predominant in lower limbs; pyramidal signs; learning
difficulties; cortical migration defects
6
PNMHH AD MYH14 typical HMN; distal myopathy; hoarseness; hearing loss 1
SPSMA AD TRPV4 scapuloperoneal SMA with vocal cord paralysis 6
dHMN AD AARS typical HMN 1
ARAN-NM AR HINT1 distal muscle weakness; neuromyotonia involving the hands 8
134 mutations in 21 genes (updated July 2014)
8. Muscle
Motor neuron
High metabolism and
precise connectivity
Protein synthesis / folding
GARS, BSCL2
DNA/RNA processing
SETX, IGHMBP2
Axonal transport
MFN2, DCTN1, DYNC1H1, BICD2
Neurite outgrowth
Axonal guidance
Cytoprotecion
Cytoskeletal stability
GARS, PLEKHG5, FBXO38
Proposed functions for genes in axonal CMT and distal HMN
HSPB1, HSPB8, HSPB3
Cation-channel dysfunction
ATP7A, TRPV4
L. Van Den Bosch and V. Timmerman (updated)
Current Neurology and Neuroscience Reports (2006)
Pareyson, Saveri and Piscosquito, Curr Mol Med (2014)
9. Mutations in small heat shock proteins associated with
peripheral neuropathies
Irobi et al. Nature Genetics (2004); Evgrafov et al. Nature Genetics (2004)
Kolb et al., Neurology (2010); Nakhro et al., Neuromuscul Dis (2013)
Rossor et al. JPNS (2012); Houlden et al. Neurology (2008)
Holmgren, Bouhy, Timmerman (2012), eLS, John Wiley & Sons
G
34RP39L
E41K
G
84R
L99M
R127WS135F
R
136W
/L
R140G
K141Q
T151IS158fs
E175XT180I
R
188W
P182L/S
88 168
P
1 2055 1915
PP
78 82 178 193
66 1431 150
R7S
93 170 196
K141E/N
24 87
IXI/VWDPF
117 151107 160
N-terminal domain α-Crystallin domain C-terminal end
ACD
ACD
ACDβ4 β8
HSPB1
HSPB3
HSPB8
1
P
P
K141T
Q
175X
10. Small HSPs are more than chaperones
Almeida-Souza, Timmerman, Janssens (2011) BioArchitecture
Holmgren, Bouhy, Timmerman (2012) eLS, John Wiley & Sons
Protein folding Apoptosis
Neuronal survival
Cancer
Alzheimer CMT
small HSPs
Translational controlCytoskeleton dynamics
ALS
Splicing regulation
11. HSPB1, HSPB5 and HSPB4 in human cancers:
potent oncogenic role of some of their client proteins
Arrigo & Gibert, Cancers (2014)
Membrane
signalling
proteins
Cytoskeleton, cell adhesion, tissue integrity
Epithelial to mesenchimal transmission
Gene expression
Control of protein
degradation
Cell death, apoptosis, redox rate, aging
Kinases, phosphatases,
signal transduction
Growth factors, receptors
12. HSPB8 (Hsp22) is
involved in many
different processes
…
regulating the
proteolytic
degradation of
unfolded proteins
(Huntington, AD, desmin-related
cardiomyopathy)
Holmgren, Bouhy, Timmerman (2012) eLS, John Wiley & Sons
Role in CMT ?
G93A-SOD1 expression
induces a robust autophagic
response specifically in muscle
Crippa et al. (2013) Frontrier in
Cellular Neuroscience
13. M. Coleman, Nature Review Neuroscience (2005)
Mutant HSPB8 (K141E)
Irobi et al. Human Molecular Genetics (2010)
Mutant HSPB8 causes motor neuron-specific
neurite degeneration
14. Mutant HSPB8 causes motor neuron specific neurite
degeneration in axonal CMT
0
10
20
30
40
50
60
70
80
90
100
0 500 1000 1500 2000 2500
percentageofcells
neuritelenght (µm)
Control GFP
HSPB8 K141N
K141E
Irobi et al. Human Molecular Genetics (2010)
15. Mutant HSPB8 causes protein aggregates in
dermal fibroblasts
Irobi et al. Neuromuscular Disorders (2012)
HSPB8-K141N
scale bar = 20 µm
16. Mutant HSPB8 protein
aggregates recrute the
cellular machinery to
eliminate refolded or
degraded misfolded HSPB8
proteins
Scale bar = 10 µm
Patient and control fibroblast cells
were immunostained with
antibodies against HSP70 (green) or
Ubiquitin (green) and HSPB8 (red).
HSPB8-K141N
17. Mutant HSPB8 causes reduced mitochondrial membrane
potential in dermal fibroblasts
Loss of mitochondrial membrane potential in patient’s
fibroblasts.
∆Ψm was measured in cultured patients and controls
fibroblasts.
A: Valinomycin treated cells were used as a positive control
for loss of ∆Ψm.
B and C: fluorescent lipophilic cationic dye (TMRM) histogram
analysis of ∆Ψm.
D: Statistical analysis (∆Ψm geometric mean and SD) was
performed on 3 different experiments.
Irobi et al. Neuromuscular Disorders (2012)
18. HSPB8 patient fibroblast cells do not elicit marked apoptosis
Quantification of DNA fragmentation in
patient and control fibroblasts
Cell death assay of distal HMN patients and
controls fibroblast cells
Irobi et al. Neuromuscular Disorders (2012)
Caspase-Glo 3/7 assay (Promega)
DNA fragmentation assay (BioVision)
19. Carra et al. JBC (2010)
gmr-GAL4-UAS-SCA3(78Q)/+
gmr-GAL4-UAS-SCA3(78Q)/UAS-HSPB8#2
Wt
K141E
K141N
K141N
Quantification of eye degeneration in flies
overexpressing either SCA3(78Q) alone or in
combination with wild-type or mutated, K141E or
K141N, HSPB8
Total number of eyes scored: 200-300
* = p< 0,05 as compared to SCA3; average values ±
s.e.m. of n = 3 independent experiments.
The K141E mutation significantly affects
HSPB8 protective effect on SCA3-
induced eye degeneration
Drosophila ortholog of HSPB8:
Implication of HSPB8 loss-of-function in
protein folding disease
The small HSP chaperones can impede protein aggregation
in poly-Q disease
21. HSPB1 (Hsp27) is
involved in many
different processes …
interferes with the cell
death signalling
pathways at various
levels
Holmgren, Bouhy, Timmerman (2012) eLS, John Wiley & Sons
Role in CMT ?
24. HSPB1 mutants present higher chaperone activity
0
0 .1
0 .2
0 .3
0 .4
0 .5
0 .6
W T R1 2 7 W S 1 3 5 F R1 3 6 W T 1 5 1 I S 1 5 6 Y P 1 8 2 L E G F P
0.1
0.2
0.3
0.4
0.5
0.6
**
*
*
**
*
**
*
**
*
HS
HR
Relativechaperoneactivity
WT R127W S135F T151I S156Y P182L EGFPR136W
Almeida-Souza et al. JBC (2010)
SH-SY5Y cells stably
expressing HSPB1
and luciferase
No
Heat Shock
Heat Shock
30min/44°C
Heat Shock
30min/44°C
Recovery: 3hs
Luciferase activity
Cycloheximide NO Recovery
25. Search for interacting partners to small HSP genes
Cells expressing wild-type gene
Cells expressing mutated gene
prCMV TAPtag pA
prCMV TAPtag pAWT CMT gene
Mutant CMT gene
Complex purification
(TAP)
“Wild-type complex”
“Mutant complex”
prCMV TAPtag pA
prCMV TAPtag pAWT CMT gene
Mutant CMT gene
WT MUT
m/z
MS
Differential interacting partner identification
26. Overactive HSPB1 mutants present a higher affinity
to their client proteins
Almeida-Souza et al. JBC (2010)
Size Exclusion ChromatographyDifferential HSPB1 protein interactions
28. Hyperactive HSPB1 mutants bind to tubulin and
microtubules
Almeida-Souza et al. Journal of Neuroscience (2011)
IP from HEK293 cells
IP from HEK293 protein extracts Mouse model
29. Co-localization of mutant HSPB1 to microtubules
Almeida-Souza et al. Journal of Neuroscience (2011)
Cos1 cells
Normalized
fluorescence intensity profiles
Mutant HSPB1 disturb MT dynamics
EB1-GFP tracking confirms slower MT polymerization
Mutant HSPB1 stabilize MTs in vitro
30. Looking at the HSPB1 effect on individual MTs
TUBB3-GFP EB1-GFP Microtubule plus ends
Overactive HSPB1 disturb MT dynamics
EB1-GFP tracking confirms slower MT polymerization
Overactive HSPB1 stabilize MTs in vitro
Hela
Almeida-Souza et al. Journal of Neuroscience (2011)
31. HSPB1 facilitates the formation of non-centrosomal
microtubules
Almeida-Souza et al. PlosOne (2013)
HSPB1 facilitates the formation of MTs in vitro
and binds to MTs at early stages of their
polymerization
34. Almeida-Souza et al. Journal of Neuroscience (2011)
MT stability is confirmed in HSPB1 transgenic mice
HSPB1 mouse
Disturbed axonal transport defects in
symptomatic HSPB1 mutant mouse
neurons
35. Decreased acetylated tubulin levels in symptomatic
HSPB1 mutant mouse
d’Ydewalle et al. Nature Medicine (2011)
***p<0.0001
36. HDAC6 is a microtubule-associated deacetylase
HDAC6 : internal duplication of two class II catalytic domains + Ub binding domain
Hubbert et al. Nature (2002)
•Acetylated tubulin is most abundant in stable microtubules
but is absent from dynamic cellular structures such as
neuronal growth cones.
•Decrease in HDAC6 increases tubulin acetylation stable
microtubules
•Inhibition of HDAC6 improves the dynamics of tubulin
improve axonal transport
37. Treatment significantly
increased motor performance of
HSPB1_S135F mice
HDAC6 inhibitors rescue axonal transport defects and
restore the distal HMN/CMT2F phenotype
Treatment increased electrophysiological
parameters such as CMAP and SNAP
d’Ydewalle et al. Nature Medicine (2011)
***p<0.0001
38. HDAC6 inhibitors reverse the axonal loss in
mutant HSPB1 mice
d’Ydewalle et al. Nature Medicine (2011)
Treatment leads to muscle re-innervation and rescues axonal
transport
*** p<0.0001
39. Microtubule dynamics in the PNS
Almeida-Souza, Timmerman & Janssens, Bio-Architecture (2012)
41. HspB1 deficient mice display impaired wound healing
• HspB1 regulates inflammatory gene expression and drives
cell proliferation.
• The expression of HspB1 protein and mRNA is controlled
by the cell cycle.
• HspB1-deficient mice (genOway, LoxP flaking exon 1-3).
• HspB1-deficient fibroblasts have:
– increased expression of the pro-inflammatory
cytokine, IL-6, and reduced proliferation
– reduced entry into S phase and increased expression
of Cdk inhibitors p27(kip1) and p21(waf1)
• There was a significant impairment in the rate of healing
of wounds in hspB1-deficient mice:
– reduced re-epithelialisation and collagen deposition
– increased inflammation
• HspB1 deficiency augments neutrophil infiltration in
wounds, driven by increased chemokine (C-X-C motif)
ligand 1 expression.
Crowe et al. PlosOne (2013)
43. P182L_HSPB1 disrupts neurofilament assembly and the
axonal transport of specific cellular cargoes
• P182L but not wt HSPB1 leads to formation of insoluble intracellular
aggregates and to sequestration in the cytoplasm of selective cellular
components, including NF-M and p150 dynactin
Ackerley et al. HMG 2006;15:347-354
HSPB1wt HSPB1 (P182L)
HSPB1-HA
P182L-HA
Anti-HA GFP
44. NFs are bounded to and
transported by the motor
proteins kinesin and dynein
NFs detached from a motor protein, but
can easily re-associate.
NF phosphorylation state determines
association with kinesin or dynein.
NFs dissociated from the motors and
restricted from re-association,
represented as NF bundles
Neurofilaments can exist in three distinct pools
Holmgren, Bouhy, Timmerman JPNS (2012)
45. Proline-directed kinases and phosphatases in
neurofilament phosphorylation
Holmgren, Bouhy, Timmerman JPNS (2012)
46. HSPB1 mutations increase Cdk5 mediated phosphorylation‑
of neurofilaments
Holmgren et al. Acta Neuropathologica (2013)
Proximal Distal
Wild type HSPB1 Mutant HSPB1
neurofilaments
phosphorylated neurofilament
kinesin
dynein-dynactin complex
microtubules
Proximal Distal
Silencing Cdk5 restores NF-kinesin
interaction and NF solubility in
HSPB1 mutant
47. Complex pathomechanisms of small HSP mutations
stabilization of MT
autophagy deficit
sHSP properties
axonal degeneration
acetylation of MT
sHSP functions
axonal transport defect
mitochondrial depletion
protein aggregation
redox
iron metabolism
ER stress
chaperoning
binding affinity
oligomerization
phosphorylation
cytoskeleton
apoptosis
oxidative stress
48. HMSN
hereditary motor and
sensory neuropathy
= ‘CMT’
Motor Sensory & Autonomic
HSAN
hereditary sensory &
autonomic
neuropathy
HMN
hereditary motor
neuropathy
Harding & Thomas, J. Med. Genet (1980), J. Neurol. Sci (1980) and Brain (1980)
Harding, A.E. (1993) In: Dyck, Thomas, Griffin, Low and Poduslo (Eds) In: Peripheral Neuropathy
Dyck, P.J. (2005) In: Peripheral Neuropathy
Various kinds of inherited peripheral neuropathies
50. Overview of AD-HSAN subtypes
Rotthier, Baets, Timmerman & Janssens, Nature Reviews Neurology (2012)
Updated with: Leipold et al. AJHG (2013), Zhang et al. AJHG (2013), Kornak et al. Brain (2014)
* Online Mendelian Inheritance in Man (OMIM) reference number
‡ Congenital onset in one patient with hypotonia, cataracts, microcephaly and vocal cord paralysis
§ Childhood onset in one patient
HSAN SCN11A loss of pain perception (2 de novo), familial episodic
pain (2 missense)
Childhood 604385
HSAN ATL3 Loss of pain perception and destruction of pedal
skeleton (1 missense in 2 families)
Adolescence
to adulthood
609369
51. HSAN I (CMT2B) caused by mutations in RAB7A
Rotthier, Baets, Timmerman & Janssens, Nature Reviews Neurology (2012)
Updated with: Cogli et al. Acta Neuropathologica (2013), …
Decreased nucleotide affinity and deregulation of nucleotide exchange
Subtle increase in duration of RAB7 association with target membranes
Peripherin
Altering neurofilament
dynamics
52. Comprehensive map of the effectors of Rab GTPases
Gillingham et al. Developmental Cell (2014)
53. Human L129F Rab7 mutation in Drosophila
dWT Drosophila WT RAB7
hWT Human WT RAB7
hL129F Human mutant RAB7
0N3R tau TAU mutant
painless Painless mutant
TrpA1[1] TRPA1 mutant
D42-Gal4 Motor neuron driver
P0163-Gal4 Sensory neuron driver
221-Gal4 UAS-GFP
Rab7 is a highly conserved protein;
the human form shares 76%
identity and 95% similarity with its
single Drosophila orthologue
We used double transgenic lines expressing
Rab7 alleles from both chromosome 2 and 3
54. Mutant Rab7 larvae show decreased motor performance
Distance travelled in two minutes by larvae
Janssens et al. Neurobiology of Disease (2014)
55. Sensory performance is affected in mutant Rab7 larvae
Thermotaxis assay
Nociception assay
„warm side (30°C)‟
„cold side (22°C = preferred )‟
soldering iron with a chisel-shaped tip at 43°C
Janssens et al. Neurobiology of Disease (2014)
56. Transport of mutant Rab7+
vesicles is altered in sensory axons of
3rd
instar larvae and in neurites of SH-SY5Y cells
No changes in neuronal
morphology
Rab7+
vesicles in long axons of sensory ddaE and
ddaD neurons of dissected larvae:
No difference between the two genotypes
with regard to speed and directionality of
vesicle movement
However; L129F Rab7 vesicles pause
less than their wt counterparts
ventral multi-dendritic sensory vpda
neuron of hemisegment 6
Janssens et al. Neurobiology of Disease (2014)
57. CMT2B mutations in rab7 cause dosage-dependent
neurodegeneration due to partial loss of function
Cherry et al. eLIFE (2013)
58. Defective axonal transport of Rab7 results in
dysregulated trophic signaling
Zhang et al. J Neurosci. (2013)
Rab7 mutants
dysregulate axonal
transport and
diminish retrograde
signaling of nerve
growth factor (NGF)
and its TrkA
receptor.
Rab7 mutants induce
premature
degradation of
retrograde NGF-TrkA
trophic signaling.
59. Stuck in traffic: an emerging theme
in diseases of the nervous system
Neefjes & van der Kant, Trends in Neuroscience (2014)