Genes and Parkinson’s Disease
Valina L. Dawson, PhD
Institute for Cell Engineering
Departments of Neurology and Neuroscience
Johns Hopkins University School of Medicine
Parkinson’s disease by the Numbers
 Second
most common
neurologic disorder
 4 million people worldwide
 North America: ~500,000 to 1M,
~50,000 diagnosed every year
 An expected doubling by 2040
 50% are diagnosed after age
60; 1/2 are affected before then
 Treatments: Drug therapy and
deep-brain stimulation to
alleviate symptoms
 No treatment for the causes
 ~90% Sporadic, ~10% Familial
Pathologic Hallmarks of PD
PD is characterized pathologically by a
selective loss of dopaminergic neurons in
the substantia nigra par compacta
Cecil’s Textbook of Medicine
UBC Fluorodopa
Normal
PD
Staging
Cell Tissue Res. 2004;318:121
MPTP: Man, Monkey, Marmoset,
Mouse
N=4
MPTP Pathways
Nat Neurosci. 5 Suppl:1058-61, 2002
MPTP Pathways ~ Clincial Trials
MPTP mimics symptoms of
PD but may not mimic how
neurons die in PD?
Poor clincial trial design?
Unreasonable trial end
points?
Start treatment too late?
Nat Neurosci. 5 Suppl:1058-61, 2002
What Causes Parkinson’s
Disease?
Environment
Age
Loss of
dopamine
producing
cells
Heredity
Parkinson’s
The Current Century - “Post-Genomic Era”
Loci and genes linked to familial PD or implicated as genetic causes for PD
Susceptibility genes
GBA
Tau
Moore et al., Annu. Rev. Neurosci. 2005
ATP13A2, AR, lysosomal type 5 P-type ATPase.
POLG, AD catalytic subunit of the mitochondrial DNA (mtDNA)
polymerase (rare)
Modeling Parkinson’s disease
Saccharomyces cerevisiae zebra fish
c. elegans
drosophila
1. Biology
2. Pathobiology of mutated
proteins
3. Validate in human
4. AD - gain of function
5. AR - loss of function
6. Development of
Biomarkers
mouse
Familial Parkinson’s disease
Genes are broadly expressed in many cell types and organs
Suggests that these genes do not serve strictly neuronspecific functions
Why cell type specificity of the disease?
Gene mutations may sensitize cells to intrinsic or extrinsic
toxic insults that are particularly prominent for midbrain
dopamine neurons
Alpha-Synuclein Physiology
Named for its localization to
synapses and nuclei
Functions is not well understood
Knockout mice have synaptic
deficits
May regulate the reserve pool of
synaptic vesicles
Knockout mice do not have PDlike symptoms
Annu. Rev. Biochem. 2005. 74:29–52
Genetics of Parkinson´s disease: a-synuclein
Autosomal dominant PD, point mutations and triplication
repeat
Can self-aggregate/oligomerize and fibrillize, part of the
synuclein protein family (ß- and -synuclein)
May modulate synaptic plasticity and dopaminergic
neurotransmission (vesicle release)
Major component of Lewy bodies and neurites (the
pathological hallmark of PD)
Alpha-Synuclein
Motifs in the α-synuclein protein. The natively unfolded α-synuclein protein is shown
in a linear form. Cookson M, Annu Rev Biochem. 2005;74:29-52
Alpha-Synculein and Parkinson’s
disease
RNA
HuSyn
Syn-1
Percent Survival
100
50
G2-3
H5
N2-5
0
 Sudden Onset associated with slight
0
4
8
12 16 20 24
ataxia, “hunch-back”, & slowness
Age (Months)
 Rapid Progression to significant ataxia,
slowness, freezing, dystonia,and slight
muscle atrophy
Lee et al., Proc Natl Acad Sci U S A. 99:8968-73
 Within 14-21 days following initial clinical (2002).
symptoms, the affected mice are
completely catatonic and moribund.
Giasson et al., Neuron. 2002 34(4):521-33
Pathology of Alpha-Synuclein
• Mutations promote the formation of
oligomers, fibrils
• Metals, pesticides, and oxidizing conditions
all promote aggregation
• phosphorylated forms are found in Lewy
bodies, effect on aggregation is unclear
• Tyrosine nitration and methionine oxidation
occur and promote aggregation
• cleavage by calpain I, toxic fragments
• lipids can promote α-synuclein aggregation
Pathology of Alpha-Synuclein
Continuous MPTP administration
induces neuronal inclusions
Proc Natl Acad Sci U S A. 2005 102:3413-8.
Proc Natl Acad Sci U S A. 2005 102:3413-8.
Deletion of alpha-synuclein alleviates neurodegeneration
caused by continuous MPTP infusion
a-Synuclein inclusions



Characteristic for:
 Parkinson´s disease (PD)
 Dementia with Lewy bodies (DLB)
 Lewy body variant of Alzheimer´s disease (LBVAD)
 Multiple system atrophy (MSA) (inclusions in glial cells)
 Neurodegeneration with brain iron accumulation type-1
(Hallervorden-Spatz) (NBIA-1)
Morphological appearence:
 Lewy body (round, filamentous, with core and halo)
 neuroaxonal spheroids
 dystrophic neurites ("Lewy Neurites", LN) (possibly
underestimated so far)
Ultrastructurally:
 bundles of 10-25 nm filaments comprised of polymerized
alpha-synuclein
Genetics of Parkinson´s disease: parkin (PARK2)
 Autosomal Recessive Juvenile (early onset) PD (ARJP)
 Pathology: Loss of SNC DA and LC neurons, absence of Lewy
bodies
 52kD protein
 Oxidized, Nitrated, Nitrosylated and Phosphorylated - Sporadic
PD ~ experimentally decreases enyzymatic activity
UBLs
Ubiquitin
Maturation
C-terminal
hydrolase
a
Mature
UBLs
Ubiquitin Pathway
Activating Conjugating Ligase
Enzyme
Enzyme
E1
E2
E3
ATP
S
S
UBA1
Substrate
S
UBCH 7
UBCH 8
Parkin
CDCrel-1
Parkin
LINKAGES: K29, K48, K63
Function
Proteasome-dependent
proteolysis
Shimura, et al., Nat. Genet. 25: 302-305 (2000)
Zhang et al., Proc. Natl. Acad. Sci. U.S.A. 97: 13,354-59 (2000)
Imai et al., J. Biol Chem 275:35661-64 (2000)
Impaired Ubiquitin Proteasomal Processing of Parkin
Substrates with Familial Associated Mutants
R42P
K161N
R256C
R42P
T240R
R275W
R256C
Q311X
Q311X
R275W
T415N
C431F
G328E
P437L
W453X
G430D
C431F
W453X
Sriram, et al. Human Mol
Gen, 14:2571-86 (2005)
Lack of Enhanced Sensitivity to MPTP in Parkin Knockouts
Loss of TH-IR in the LC, but not SNpc or
striatum of parkin null mice
PNAS 101: 10744-10749, 2004
Ubiquitin Proteosome
System
Parkin
p38/JTV-1
Synphilin-1
a-synuclein
Sept5 (CDCrel-1)
Sept4(CDCrel-2)
Pael-R
Cyclin E
ß-tubulin
Synaptotagmin XI
Cell Tissue Res. 318(1):175-84 (2004)
Parkin/a-Synuclein Double-Mutant mice:
Summary / Conclusions
• The absence of parkin does not alter
– Onset and progression of the lethal motor phenotype induced by
overexpression of human A53T a-synuclein;
– Neuropathologic abnormalities in human A53T a-synuclein
transgenic mice
– Formation of ubiquitin-positive protein aggregates
– Biochemical abnormalities in human A53T a-synuclein transgenic
mice
– Behavioral abnormaties in human A53T a-synuclein transgenic
mice or parkin KO mice.
• Therefore, parkin does not appear to be a critical factor in the
pathogenesis of synucleinopathy.
• Furthermore, these results do not support an essential role of parkin
for the ubiquitination, degradation and/or sequestration of a-synuclein
in vivo.
Accumulation of p38/Jtv-1/AIMP-2 in 18 M Ventral
Midbrain of Parkin Knockout Mice
J. Neurosci., 25:7968-78 (2005)
Accumulation of p38/Jtv-1/AIMP-2 in AR-JP
Parkin interacts with p38/JTV1 in PD/DLBD
•
•
•
•
•
PD and DLBD Patients have
remarkable elavated brain
levels of S-nitrosylation
Parkin is S-nitrosylated both
in vitro and in vivo
S-nitrosylation inhibits
parkin’s ubiquitin E3-ligase
activity and it’s protective
function.
Our results link parkin
function with the more
common sporadic form of
Parkinson’s disease and the
related a-synucleinopathy,
DLBD, through nitrosylative
stress.
Science, 304:1328-31 (2004)
p38/JTV-1/AIMP-2 is an Integral Component of
the Aminoacyl-tRNA Complex
tRNA
ATP
Aminoacyl-tRNA
+AMP
Aminoacyl-AMP
Amino Acid
PPi
ATP
Ap4A + AMP
• Mutation in Glycyl-tRNA causes Charcot-Marie Tooth Disease
(Antonellis, AJHG 72:1293-1299)
• Mutation in Alanyl-tRNA causes neurodegeneration in mice (Lee, et al
Nature, 2006)
• AIMP2 is known to interact with and promotes the ubiquitination and
proteasome-dependent degradation of the far upstream element (FUSE)
binding protein 1 (FBP1)
DJ-1
Early onset PD, relatively benign with long duration
No autopsy material to date
Over expression provides protection against a variety
of toxic insults
Knock-down or knockout sensitizes cells to toxic
insult and sensitizes animals to MPTP injury
DJ-1 Models
Sci Aging Knowledge Environ. 2006 Jan 11;2006(2):pe2
DJ-1 Antibodies are Specific
Human Molecular Genetics (2005) 14:2063–2073
DJ-1 Subcellular Distribution
~75% Cytosolic, ~25% Mitochondria
Human Molecular Genetics (2005) 14:2063–2073
Reduced homo-dimerization of
pathogenic DJ-1 mutants
Human Molecular Genetics (2005) 14:2063–2073
Parkin differentially associates with
pathogenic DJ-1 mutants
Human Molecular Genetics 2005 14(1):71-84
Parkin fails to ubiquitinate DJ-1 but
may stabilize DJ-1 expression
Human Molecular Genetics 2005 14(1):71-84
Increased levels of insoluble DJ-1 in
PD/dementia with Lewy bodies (DLB)
Human Molecular Genetics 2005 14(1):71-84
Reduced levels of insoluble DJ-1
in parkin-linked AR-JP brains
Human Molecular Genetics 2005 14(1):71-84
DJ-1 KO have an increase in H2O2
production & decrease in Aconitase
activity
Andres-Mateos et al., under revision
DJ-1 does not scavenge H2O2 like
peroxidase or catalase but instead is an
atypical peroxidredoxin-like peroxidase
Andres-Mateos et al., under revision
Pink1
PINK1, patients with
mutations have early
to late onset PD
The course is benign
with long disease
duration
Some have dystonia
No autopsy studies
are yet available
Hum Mutat. [Mar 26, Epub ahead of print] 2007
PINK1 KO flies =
Parkin KO flies
KO mice are
uninformative
PINK1 and Parkin
PTEN-induced putative kinase 1 (PINK1)
In a recent issue of Nature, two independent reports by and show that loss of Drosophila
PINK1 leads to defects in mitochondrial function resulting in male sterility, apoptotic muscle
degeneration, and minor loss of dopamine neurons that is rescued by overexpression of the
ubiquitin E3 ligase, parkin. Thus, PINK1 and parkin appear to function in a common
pathway suggesting a convergence of the two genes most commonly associated with
autosomal recessive PD.
Neuron 50(4):527-9, 2006
LRRK2 - leucine-rich repeat kinase 2
Mutations in the LRRK2 gene are the most common cause of late onset
PD with clinical and neurochemical overlap with idiopathic disease
13% (averaged) of familial PD cases are compatible with dominant
inheritance
Sporadic PD: LRRK2 mutations occur in high frequency, from 1%-7% of
PD patients of European origin and 20%-40% of PD in Ashkenazi Jews
and North African Arabs
Kinase: MAPK, MLK, now known to be a serine-threonine kinase ~ MLK
inhibitor - CEP1347 not effective in clinical trials in PD
Lewy body pathology in a LRRK2
G2019S case
locus ceruleus (H&E)
superior temporal
cortex
spinal cord
CA2 hippocampus
olfactory bulb
myenteric plexus
large intestine
Ann Neurol. Vol.59: 388-393
LRRK2 Human Brain
A) visual cortex pyramidal
neurons
C) layer V of the visual cortex.
D) substantia nigra pars compacta
localized to melanin-containing
dopaminergic perikarya (arrows)
and to dendritic and axonal
processes (arrowheads).
E) caudate putamen - mediumsized spiny neurons (arrowheads)
and large interneurons (arrows).
F/G) large branching interneuron
caudate putamen resembling a
cholinergic subtype.
Ann. Neurol. 60(5), in press (2006)
Ann. Neurol. 60(5), in press (2006)
LRRK2 immunogold labeling in rodent brain
A) Golgi transport vesicles (arrowheads) Golgi apparatus B) mitochondria and a lysosomal vesicle C) Axon labeling
microtubules and an associated transport vesicle (arrowhead), mitochondria D) synapses with mitochondria and a
clathrin-coated endosomal vesicle (arrowhead). synaptic vesicles are not labeled for LRRK2. A) and B) rat basal ganglia,
C-E) mouse substantia nigra.
Familial-linked PD mutations increase
kinase activity
Proc. Natl. Acad.
Sci. U.S.A.,
102:16842-16847
(2005)
West et al., HMG 2006
  


Biochemical Characterization of LRRK2
Missense Mutations
Amino Acid
Substi tution
I112 2V
I137 1V
R1441C
R1441G
R1514Q
Y1699C
I201 2T
G2019S
I202 0T
G2385R
Disease
Segregation
Possible
Possible
Yes
Yes
Possible
Yes
Possible
Yes
Yes
Unlikely
Number of
Probands
1
2
Many
Many
2
2
2
Many
2
Many
Domain
LRR
GTPase
GTPase
GTPase
COR
COR
Kinase
Kinase
Kinase
None
GTP-binding
Activity
No change
Increase
Increase
Increase
No change
Increase
No change
No change
No change
No change
Kinase Activity
Increas e
No Cha nge
Increase
Increase
Increase
Increase
Decrease
Increase
Increase
No Cha nge
West et al., Hum Mol Genet. 16(2):223-32, 2007
LRRK2 - cytoplasmic aggregates and
cytotoxicity
100
80
60
40
G2019S
WT LRRK2
20
0
Smith et al. Proc Natl Acad Sci U S A. 102:18676, 2005
Smith et al. Nat Neurosci. 9(10):1231-3, 2006
neurons
Vector
Htt
Relative viability (%)
120
LRRK2 toxicity requires kinase activity
West et al., Hum Mol Genet. 16(2):22332, 2007
LRRK2 Summary
LRRK2 is heterogeneously distributed throughout
the brain including the nigrostriatal pathway
LRRK2 is Localized to Membranous and
Vesicular Structures
Familial Associated Mutations lead to Enhanced
Kinase Activty
Familial Associated Mutations lead to
Neurotoxicity that is Kinase and GTPase
Dependent.
Pathways to Parkinson’s Disease
Sporadic PD
PINK1
DJ-1
Mitochondrial Dysfunction
Loss of oxidative
capacity
DJ-1
Oxidative/Nitrosative
Stress
LRRK2?
Parkin
a-Syn
Formation of inclusions
Accumulation of toxic
substrates (AIMP2, FBP-1)
a-Syn Aggregation
Cell Death
Familial Associated Mutations Enhances
LRRK2’s MLK-like Kinase Activity
LRRK2
Mata et al., Trends Neurosci. 2006
At least 20 LRRK2 mutations have been linked to autosomal dominant
parkinsonism
The frequency of a G2019S mutation
5-6% of autosomal dominant PD patients
39% of familial PD
41% of sporadic cases in North African Arabs
29% of familial Ashkenazi Jewish patients
Genetic Alterations in LRRK2 are most common known cause of PD