Amyotrophic Lateral Sclerosis: Perspectives from Genetics · Amyotrophic Lateral Sclerosis:...
Transcript of Amyotrophic Lateral Sclerosis: Perspectives from Genetics · Amyotrophic Lateral Sclerosis:...
Amyotrophic Lateral Sclerosis: Perspectives from Genetics
Robert H. Brown, Jr., D.Phil., M.D.
University of Massachusetts Medical Center
for Shirley H. Wray Collection
NOVEL Library
August 7, 2008 Boston, MA
Department of Neurology
Massachusetts General Hospital Harvard Medical School
Mutations Mendelian and Complex Genetics Mechanisms Recent lessons in pathophysiology Medicines Therapeutic advances
ALS is a devastating orphan disease
Incidence Total 1-2/100,000 3,000-6,000 US 70,000 World Prevalence 5/100,000 ~20,000 US 500,000 World
Mean age of onset = 55 years
….. uniformly fatal, in 4-5 years.
The hallmark of ALS is muscle denervation and atrophy due to loss of spinal motor neurons; this begins focally.
Degeneration of brain motor neurons causes spasticity.
Ross 1881
Cytoskeletal pathology and ubiquitinated protein aggregates are seen in motor neurons.
axonal spheroid
ubquitinated proteins
Ubiquitinated proteins in most ALS motor neurons include TDP-43.
Neumann M, Trojanowski J, Lee V et al. , Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314:130-133, 2006.
nuclear protein transcription repressor part of splicing apparatus in early ALS and FTD
cleaved hyperphosphorylated ubiquitinated
There is neuroinflamation in ALS spinal cord.
ALS Control
McGeer PL, McGeer PG. Muscle and Nerve 26: 459–470, 2002
Activated microglia in anterior gray matter of ALS spinal cord.
The development of ALS probably reflects an interplay: environment, genetic factors, and chance, aging.
The development of ALS probably reflects an interplay: environment, genetic factors, and chance, aging.
Factor Odds Ratio Toxins Agricultural Toxins -- herbicides 3.0 -- fertilizers 2.3 Metals -- lead exposure 1.6 Respiratory -- smoking 3.3 Infectious Agents -- retroviruses -- Lyme -- other Infectious Toxins Diet -- high fat 3.8 -- high glutamate 3.2 -- high fiber 0.3
The development of ALS probably reflects an interplay: environment, genetic factors, and chance, aging.
10% of ALS: autosomal dominant
Amyotrophic Lateral Sclerosis: 13 Mendelian loci, 7 genes
ALS1 AD 21q SOD1ALS2 AR 2q alsinALS3 AD 18q ?ALS4 AD 9q senataxinALS5 AD 15q ?ALS6 AD 16q ?ALS7 AD 20p ?ALS8 AD 20q VAPBALS AD 2p dynactinALS AD 1p TDP-43ALS AR 19p NTE
ALS-FTD AD 9q ?ALS-X AD X cen ?
Amyotrophic Lateral Sclerosis: 13 Mendelian loci, 7 genes
5 have ALS or ALS-like phenotype
*
* *
ALS1 AD 21q SOD1ALS2 AR 2q alsinALS3 AD 18q ?ALS4 AD 9q senataxinALS5 AD 15q ?ALS6 AD 16q ?ALS7 AD 20p ?ALS8 AD 20q VAPBALS AD 2p dynactinALS AD 1p TDP-43ALS AR 19p NTE
ALS-FTD AD 9q ?ALS-X AD X cen ?
*
*
Mutations Mendelian Genetics - FALS Mechanisms Recent lessons in pathophysiology Medicines Therapeutic advances
William Osler 1890 First description of familial ALS
1849-1919
10% of ALS cases are inherited as a dominant, highly penetrant (>90%) trait.
25% of familial ALS (3% of all ALS) arises from mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1).
135 mutations (124 missense, 11 truncation)
SOD1A4V cases are common in North America but rare in Europe;
extremely rapidly progressive MND.
A4V Farr Family
In northern Sweden, SOD1D90A/+ are asymptomatic; SODD90A/D90A slowly progressive MND.
A4V
The A4V variant of SOD1 arose in Asia and was carried across the Bering Strait about 10,000 years ago.
Amyotrophic Lateral Sclerosis: Mendelian loci
ALS1 AD 21q SOD1ALS2 AR 2q alsinALS3 AD 18q ?ALS4 AD 9q senataxinALS5 AD 15q ?ALS6 AD 16q ?ALS7 AD 20p ?ALS8 AD 20q VAPBALS AD 2p dynactinALS AD 1p TDP-43ALS AR 19p NTE
ALS-FTD AD 9q ?ALS-X AD X cen ?
Tunisian family Kuwaiti family
• corticospinal, corticobulbar signs • lower motor neuron signs
• exclusively corticospinal, corticobulbar signs • no lower motor neuron findings
Ben Hamida, M., et al., Brain 113, 347-363 (1990).
Lerman-Sagie, T, Filiano, J., Smith, D.W. & Korson, M. J. Child Neurol. 11, 54-57 (1996).
ALSIN deficiency in juvenile ALS causes defective microtubule assembly, motility and trafficking of cellular
constituents.
Rho-GEF
Nuclear trafficking,
spindle formaion, microtubule assembly
Signaling cell growth, motility
Protein, vesicle,
endocytitc trafficking
Ran-GEF Rab5-GEF
• 184 kd • Prominently expressed in large neurons • Implicated in vesicle trafficking
Yang, Y., et al , Nat Genet, 2001. 29(2): p. 160-5. Hadano, S., et al., Nat Genet, 2001. 29(2): p. 166-73.
Amyotrophic Lateral Sclerosis: Mendelian loci
ALS1 AD 21q SOD1ALS2 AR 2q alsinALS3 AD 18q ?ALS4 AD 9q senataxinALS5 AD 15q ?ALS6 AD 16q ?ALS7 AD 20p ?ALS8 AD 20q VAPBALS AD 2p dynactinALS AD 1p TDP-43ALS AR 19p NTE
ALS-FTD AD 9q ?ALS-X AD X cen ?
Dynactin subunit mutations are implicated in MND.
• Onset average 34 (23-44) • Dominant inheritance • Predominantly lower MNs • Early stridor and shortness of breath
– vocal fold paralysis • Weakness in hands > arms and legs • Thenar weakness early • Steppage gait late • Chronic reinnervation by EMG
Puls I, Oh S, Sumner CJ, Wallace KE, Floeter MK, Mann EA, Kennedy WR, Wendelschafer-Crabb G, Vortmeyer A, Powers R, Finnegan K, Holzbaur ELF, Fischbeck KH, Ludlow CL. Distal Spinal and Bulbar Muscular Atrophy
Caused by Dynactin Mutation. Ann Neurol 2005;57:687–694
Release
KINESIN
DYNEIN (dynactin)
Anterograde transport
Retrograde transport
Microtubules
Anterograde cargo
Neurotrophic factors
Mitochondrion
Retrograde cargo
Dynein-Dynactin complex (retrograde motor) Kinesin (anterograde motor)
Golgi - ER
Synthesis and trafficking of vesicles and proteins
Nucleus
Uptake
Neurofilaments
Amyotrophic Lateral Sclerosis: Mendelian loci
ALS1 AD 21q SOD1ALS2 AR 2q alsinALS3 AD 18q ?ALS4 AD 9q senataxinALS5 AD 15q ?ALS6 AD 16q ?ALS7 AD 20p ?ALS8 AD 20q VAPBALS AD 2p dynactinALS AD 1p TDP-43ALS AR 19p NTE
ALS-FTD AD 9q ?ALS-X AD X cen ?
AJHG 82:780, 2008
- Juvenile onset - Corticospinal and lower motor neurons involved - No known exposure to organophosphates
Novel Motor Neuron Disease Phenotype Madras Motor Neuron Disease:
Juvenile onset of motor weakness and deafness Rapidly progressive, severely disabling
Drs. Gourie-Devi, Nalini, Nithin NIMHANS, Bangalore
Dr. C. Thangaraj CCMB, Hyderabad
Overview: More than 60 gene defects
cause human motor neuron diseases (ALS, HSP, SMA, X-SBMA, PN)
These gene defects implicate common elements
in the pathogenesis of these diseases:
Axonal motors and filaments (7) vesicle trafficking (4) DNA/RNA metabolism (6) Cell signaling (5) Heat shock proteins (3)
Mutations Complex Genetics - SALS (1) Candidate genes in SALS Mechanisms Recent lessons in pathophysiology Medicines Therapeutic advances
Candidate genes in sporadic ALS* • VEGF
• Angiogenin
• Survival Motor Neuron
• Neurofilaments
• 35 other MN disease genes
* implicated in some but not all populations studied
Angiogenin
• 14 kda • Multifunctional
– Promotes angiogenesis – Ribonuclease – Permissive for VEGF
• 7 variants implicated in SALS. – Scotland, Ireland – Not US, UK
Greenway MJ, Hardiman O et al. Nat Genet 2006:38:411-413.
• Paraoxonases PON1, PON2, and PON3 • Gene cluster, chr 7
• Hydrolases • PON1 and PON3
– expressed in the liver – associated with HDL
• PON2
– intracellular, also associated with a plasma membrane – ubiquitous
• Functions: – antioxidative: attenuate lipid peroxidation by LDL anti-atherogenic – cytoprotective: inactivate wide range of toxins
Paraoxonases (PON) may be risk factors for SALS
Properties of PON1
• 41 kd - monomer • conserved in evolution • central hydrophobic channel • 6 bladed propeller
c
Harel et al. Nat Str Biol 11(5):412, 2004
• Ca in active site • 3 helices closed channel • Insert into HDL particle membranes • Highly polymorphic • Multiple substrates
Slowik A, Figlewicz DA et al. Paraoxonase gene polymorphisms and sporadic ALS. Neurology, epub July, 2006 PON1
Saeed M, Siddique T et al. Paraoxonase cluster polymorphisms are associated with sporadic ALS. Neurology, epub July, 2006. PON2 Morahan JM, Yu B, Trent RM, Pamphlett R. A gene environment study of the paroxonase 1 gene and pesticides in amyotrophic lateral sclerosis. Neurotoxicology, 2006. PON1.
Cronin S, Greenway MJ, Prehn JHM, Hardiman O. Paraoxonase promoter and intronic variants modify risk of sporadic amyotrophic lateral sclerosis. J Neurol Neurosurg Psychiatry 2007;78:984–986. PON1
Paraoxonases (PON) may be risk factors for SALS
HYPOTHESIS – diminished toxin degradation or anti-oxidant capacity predisposes to ALS.
Mutations Complex Genetics (2) SNP-based genome analyses in SALS Mechanisms Recent lessons in pathophysiology Medicines Therapeutic advances
• ITPR2 – highly expressed in motor neurons • Controls release of calcium from ER – calibrating intracellular
calcium levels in motor neurons • Release is mediated by inositol tri-phosphate • Excessive Ca2+ is potentially toxic via multiple mechanisms
Mutations Mendelian and Complex Genetics Mechanisms Recent lessons in pathophysiology Medicines Therapeutic advances
Over-expression of mutant SOD1 generates animal models of ALS.
G93A mouse (Day Lab)
H46R rat (Day Lab)
A4V fly (Mel Feaney, HMS)
ALS worm (Rick Morimoto)
ALS zebrafish
Mouse experiments reveal that mutant SOD1 protein is cytotoxic.
Mouse SOD1 Human SOD1 (transgene) Mouse Type Phenotype
SOD1 KO
Normal
Tg–WT SOD1 Normal
Mouse level WT human level Mutant human level
Naive
Normal
ALS Tg–Mut SOD1
3% of ALS arises from mutations in the gene encoding cytosolic Cu/Zn superoxide dismutase (SOD1).
SOD1 catalyzes the conversion of superoxide anion to oxygen and hydrogen peroxide.
Transgenic expression of mutant SOD1 apoenzyme, devoid of Cu, mouse ALS.
G93A mouse (Day Lab)
X X
X X X
Hypothesis: the critical factor in SOD1mutant ALS is conformational instability of this abundant protein.
SOD1 dimer
Unstable monomer
Pore-like proto-aggregate Large aggregate
Mutant
Wildtype
Toxicity
Instability of the mutant SOD1 protein contributes to its toxicity
Excessive bursting Abnormal axonal transport
Early axonal
retraction
Early events in ALS are intrinsic to the motor neurons.
Binds mSOD1 Decreased ATP, Ca++
apoptosis
Squid Loligo pealii Drs. Scott Brady, Gerardo Morfini, University of Illinois, Wood Hole Hannah Brown, Harvard Medical School, Woods Hole
• isolated axon
• not influenced by - cell body - gene transcription
• direct impact of SOD1 on transport motor apparatus
Rat
e of
tran
spor
t
Cell body NM Junction
Anterograde
Retrograde
Normal SOD1 has no effect on transport.
Anterograde
Retrograde
SOD1 mutants slow anterograde transport.
SOD1 mutants impair the function of axonal kinesin, probably by altering phosphorylation.
Retrograde
Anterograde
Urushitani M, Sik A, Sakurai T, Nukina N, Takahashi R, Julien J-P. Chromogranin-mediated secretion of mutant superoxide dismutase proteins linked to amyotrophic
lateral sclerosis. Nat Neurosci 9:108-118, 2006.
CgA CgA
Microglia Astrocytes
Inactive
Active
Mutant SOD1 binds chromogranin and is secreted.
Activated astrocyte
AstrocytesmutSOD1 adversely affect motor neurons in vitro.
Co-culture of stem-cell derived MNs and astrocytes
Surv
ivng
Mot
or N
euro
ns
WT MNs + WT Glia
+ Glia mut SOD1
DiGeorgio, Carrasco, Siao, Maniatis, Eggan Nature Neuroscience 19(5):608-614, 2007
Reduction of oxidative bursting by microglia (NADPH / Nox2 knock-out)
enhances survival in SOD1G93A mice.
Marden JJ, Harraz MM, Williams AJ, Nelson K, Luo M, Paulsen H, Englehardt JF. Redox modifier genes in amyotrophic lateral sclerosis. J Clin Inv 119(10):2913, 2007
Does neuroinflamation alter disease course?
Time (Months)
Aspirin use __ __ __ No aspirin use _______
0.0
0.2
0.4
0.6
0.8
1.0
0 10 20 30 40 50 60
Aspirin use __ __ __ No aspirin use _______
Time (Months)
Surv
ival
Fr
actio
n
Merit Cudkowicz (MGH)
Retrospective analysis of 4 studies, n=596: topiramate placebo, celebrex, creatine, MGH longitudinal 2/15 meds affected survival: ASA, NSAIDs
Survival
10 months
Does this relate to sporadic ALS?
Does this relate to sporadic ALS?
WT SOD1 is neurotoxic when oxidized.
Ezzi SA, Urushitani M, Julien J-P. Wild-type superoxide dismutase acquires binding and toxic properties of ALS-linked mutant forms through oxidation. epub 2007
M
otor
Neu
ron
Surv
ival
Protein
SOD1 G93A
WT SOD1 OXID
WT SOD1
Motor Neurons
TNFα
Sec
retio
n
SOD
1 G
93A
WT
SOD
1
Con
trol
WT
SOD
1 O
XID
Microglia
Mab C4F6 recognizes a toxic form of mutant SOD1 protein.
MAB C4F6
Mutant SOD1
Urushitani M, Ezzi SA, Julien J-P. PNAS 104(7): 2495-2500, 2007
G93A G37R G85R
Mab C4F6 reacts with a sub-fraction of WT-SOD1 in SALS spinal cords.
Julien J-P personnal communication
Spinal cord CSF G37R
ControlSALS SALS
SALS
Sporadic ALS
Spinal cord
CSF
Hypothesis: Some cases of sporadic ALS are mediated by modified WT
SOD1, with toxicity similar to that of mutant SOD1.
MAB C4F6
Mutant SOD1
WT-SOD1 Post-translational
modifications
Mutations Mendelian and Complex Genetics Mechanisms Recent lessons in pathophysiology Medicines Therapeutic advances
….the ALS pipeline has many drugs in trials
• Minocycline • IGF-1 (#3) • Ceftriaxone • Sodium Phenylbutrate • Coenzyme Q10 • Thalidomide • Ritonavir/hydroxyurea • Valproic acid • Copaxone • Memantine • Celebrex/Creatine vs
Minocycline/Creatine
• Arimoclomol (phase IIB) • Talampanal • Trophos • R+ Pramipexole
Current Planned
• Stem Cells • Drug discovery • Gene therapy • Protein delivery • Allele inactivation • Protein inactivation
Understanding molecular pathology in ALS has aided therapy development
• Stem Cells • Drug discovery • Gene therapy • Protein delivery • Allele inactivation • Protein inactivation
Understanding molecular pathology in ALS has aided therapy development
Microwell screening can be done with high throughput: >200,000 drugs tested/day
400 cases $10 M / 3 years
40 mice $50 K / 6 mo’s
2 wells $5 / 24 hr
2 x 106 less expensive
103 faster
Instability of the mutant SOD1 protein contributes to its toxicity.
Stabilization of the dimer may be beneficial.
SOD1 dimer
Unstable monomer Pore-like proto-aggregate Large non-toxic aggregate
Mutant
Wildtype
Toxicity
1.5 million compounds
3000 molecules are predicted to stabilize SOD1
Parallel processing on Beowulf cluster speed up computation.
Computational Docking: A fast and effective way to screen big libraries of compounds
S. Ray
1.5 million compounds
3000 molecules predicted to bind SOD1
Parallel processing on Beowulf cluster speed up computation.
Computational Docking: A fast and effective way to screen big libraries of compounds
S. Ray
SO
N
NHN
O
(1)
NH
O NH
O
NH
Cl
(2)
NH
NHN
O
S
Cl
O
(3)
NH
NHN
S
S
CH3
O
(4)
NH
NHN
O
S
F
O
(7)
NH2
CH3
NH2
CH3(6)
NOH
Br
F(5)
NHNH
NH2O
F
(8)
OH
NH
NH2
O
(9) NH2
NH
N
OH
O
(10)
NH
N NH
NH2O
(12)
NH
NH2
O
OH(13)
F
OH
O
OH
OH
CH3OH
O
CH3
(14)
N
N
NNH
2
N
O
O
P
OH
OO
H
OH
OH
(15)
S
N
NH2
O
(11)
Multiple molecules identified by cyber-pharmacology do stabilize SOD1.
Ray SS, Nowak RJ, Brown, RH Jr., Lansbury PT. Inhibition of aggregation of familial amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutants by drug-like structural stabilizers PNAS,102(10),3639-3644, 2005
• Stem Cells • Drug discovery • Gene therapy • Protein delivery • Allele inactivation • Protein inactivation
Understanding molecular pathology in ALS has aided therapy development
Viral vectors may allow delivery of genes to the nervous system – e.g. by retrograde transport from muscles.
Kaspar BK, Llado J, Sherkat N, Rothstein JD, Gage FH. Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model. Science. 301:839-842, 2003.
AAV2 – IGF1
AAV2-IGF1 gene therapy potently increases ALS survival.
AAV-IGF1 delivered- 60 days old
IGF-1 GDNF
GFP
Survival
AAV2-IGF1 gene therapy potently increases ALS survival.
AAV-IGF1 delivered- 60 days old
IGF-1 GDNF
GFP
Survival
40 days
• Stem Cells • Drug discovery • Gene therapy • Protein delivery • Allele inactivation • Protein inactivation
Understanding molecular pathology in ALS has aided therapy development
• Stem Cells • Drug discovery • Gene therapy • Protein delivery • Allele inactivation • Protein inactivation
Understanding molecular pathology in ALS has aided therapy development
Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model.
Ralph GS, Azzouz M et al.. Nat Medicine 11(4):424-433, 2005
Rabies-G-pseudotyped EIAV lentivirus Many muscle injections at age 7 days (face, tongue, diaphragm, intercostal muscles, hindlimb) At end-stage: more MN persisted in treated than in untreated mice
Ralph GS, Radcliffe PA, Day DM, Carthy JM, Leroux MA, Lee DCP, Wong L-F, Bilsland LG, Greensmith L, Kingsman SM, Mitrophanous KA, Mazarakis ND, Azzouz M. Silencing mutant SOD1
using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat Med 11(4):424-433, 2005.
Ralph GS, Radcliffe PA, Day DM, Carthy JM, Leroux MA, Lee DCP, Wong L-F, Bilsland LG, Greensmith L, Kingsman SM, Mitrophanous KA, Mazarakis ND, Azzouz M. Silencing mutant SOD1
using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat Med 11(4):424-433, 2005.
100 days
Trials of allele inactivation therapy for SOD1-mediated ALS are planned (Isis)
Intraventricular infusion of anti-SOD1 anti-sense oligonucleotides prolongs survival in transgenic SOD1G93A rats.
Smith RA, Mller T, Bennett CF, Cleveland DW et al. J. Clin. Inv. 116(8): 2290-2296, 2006.
∆ 2 weeks
• Stem Cells • Drug discovery • Gene therapy • Protein delivery • Allele inactivation • Protein inactivation
Understanding molecular pathology in ALS has aided therapy development
Will vaccination therapy inactivate toxic proteins in ALS?
Vaccination with SOD1 prolongs survival in ALS mice and reduces titer of toxic form of SOD1.
C4F
6 (+
) mut
ant S
OD
1
Sur
viva
l Fra
ctio
n
∆ 25 days
Urushitani M, Ezzi SA, Julien J-P. Therapeutic effects of immunization with mutant superoxide dismutase in mice models of amyotrophic lateral sclerosis. PNAS 104(7): 2495-2500, 2007
Conclusions
• Four gene defects cause familial ALS; PON gene variants may be robustly associated with SALS.
• In SOD1mutant ALS, pathological processes
– multi-factorial and begin early. – biphasic
• initially intrinsic to motor neurons • then involve non-neural cells (neuroinflamation).
• WT SOD1 may be implicated in some cases of SALS.
• These molecular insights are yielding new therapeutic strategies in ALS.