1. Molecular neurology Parkinson's disease
for image
Matthias Georg Ziller
R4 neurology
January 30th 2008

2. Parkinson's disease Clinical picture first described in 1817
Motor features
Resting tremor
Bradykinesia
Rigidity
Postural instability
Non motor features
Autonomic
Cognitive
psychiatric

3. Burden of Parkinson's disease
~ patients in US
1 % of > 50 yo.
2nd most common MD
Lifetime risk
2 % for men, 1.3 % for women
Projected increase
Patient numbers will double to 8-9 million worldwide in 2030
Dorsey et al., NEUROLOGY 2007;68: Projected number
of people with Parkinson disease in the most populous nations, 2005 through 2030

4. PD: A history of understanding
For a long time assumed to be a non hereditary disorder … but :
140 years ago – description of early familial cases
90 years ago – link between parkinsonism and cases of encephalitis and formation of inclusion bodies was exlored
45 years ago – demonstration of dopamine deficiency, discovery of nigro-striatal pathway and first symptomatic use of dopamine
25 years ago – observation of MPTP-induced toxicology
10 years ago – The molecular genetic revolution … monogenic forms
… a "Nature versus nurture" debate

5. Pathophysiology of motor manifestationsD1
Result of the direct circuit is activation of cortex and facilitation of movement

6. Pathophysiology of motor manifestationsD2
Result of the indirect circuit is inhibition of thalamus, decreased activation of cortex
and inhibition of movement – Action of SNpc is antagonistic to this

7. Pathophysiology of motor manifestations
Loss of dopaminergic stimulation of striatum results in greater inhibition of thalamus
and decreased cortical activation  bradykinesia

8. Neuropathology
Loss of dopaminergic pigmented neurons in substantia nigra pars compacta

9. Neuropathology Cell loss in other pigmented nuclei
Ventral tegmental area
Locus coeruleus
Raphe nucleus
Intraneuronal eosinophilic inclusion bodies (Lewy)
Brainstem
Diffuse distribution in cortex
Staining + for alpha-synuclein
Reactive gliosis

10. Lewy body and Lewy neurites
-SYNUCLEINE +

11. Staging of PD pathology
2003 Braak et al.:
Staging of PD pathology
Allows to understand presymptomatic disease and non motor manifestations
Inclusion bodies ascend in the brainstem
Stage 2: coeruleus
Sleep, mood
At stage 3 reach SNpc
Motor manifestations
> stage 4 cortex involved
Dementia
Behavioural symptoms

12. Progression of a-synuclein immunopositive
labelling from stages 3 to 6, Braak 2006
Figure 3. a-d: Stages 3 to 6 of pathological changes associated with sporadic Parkinson's disease (PD) in 100 m sections immunostained for -synuclein (syn-1, Transduction Laboratories). The darkened tissue areas indicate the presence of PD-related lesions that are already recognizable with the naked eye from stage 3 onward in hemisphere sections. a: Hemisphere section of a case at stage 3. The arrow points to a single spot showing the central subnucleus of the amygdala. Allocortical, mesocortical, and neocortical areas are uninvolved at this stage. A close-up of the affected region appears in Figure 2e. b: Hemisphere section of a stage 4 case. More severe involvement of the amygdala (arrow) is accompanied by beginning affection of the anteromedial temporal mesocortex (arrowhead). Note that neocortical areas are not immunolabeled. c: Hemisphere section from a stage 5 case. A thick network of LNs emerges in the superficial layers of the anteromedial temporal mesocortex, and projection neurons located in the deep layers contain LBs (arrowhead). The disease process encroaches upon the related insular and cingulate mesocortex (asterisks). From the mesocortex, the pathology progresses into the high order association fields of the neocortex. The immunoreactivity gradually tapers off the closer it gets to the secondary and primary fields of the temporal neocortex (arrow). Note that the gyrus of Heschl along the superior temporal convolution is unaffected. d: Cortical involvement gains additional momentum in stage 6. Areas of the insular, cingulate (asterisks), and temporal mesocortex (arrowhead) continue to show strong immunolabeling. The cortical changes increase both in severity and extent. The disease process reaches even the secondary and, in advanced stage 6 cases, primary neocortical fields, as seen here from the mild affection of the primary auditory field in Heschl's gyrus (arrow).

13. "Nurture": environmental contribution
Factors that increase the risk
• Influenza epidemic - van Economo’s disease
• Being raised on a farm with well water
• Pesticide / herbicide / manganese exposure
• MPTP toxin exposure
Factors that modify (maybe decrease) the risk
• Regular tobacco use and recent smoking
• Caffeinated beverages (but not EtOH)
• Physical exercise
• Some NSAIDs (Ibuprofen, but not ASA)
• Increased risk with high dairy intake in men
• Higher levels of uric acid in peripheral blood

14. "Nature": Genetic forms Discovered during past 10 years
Monogenic forms may be clinically indistinguishable from "sporadic PD"
2-3 % of sporadic cases probably monogenic
May lead to understanding of pathophysiology

15. Genetic forms

16. Familial forms - Summary
PARK 1-13
5 autosomal dominant:
PARK 1 (=4), 4 is the triplication form of PARK1
3, 5, 8, 13
4 recessive:
PARK 2,6,7,9
1 X-linked:
PARK 12
2 with unknown mode of transmission: 10,11
additional mutations in small families without assigned PARK-numbers

17. Alpha-synuclein (PARK1+4)
First described + associated with familial parkinsonism 1997
SNCA gene, chrom 4q
3 known point mutations
single-allele duplication or triplication in some families
initially named PARK4
Penetrance low, 33 %
Severity depends on gene dosage,
patients with duplication resemble “idiopathic” PD more than triplication cases
Mutations and multiplications are rare
Increased expression of wild type SNCA copies may play a role in "idiopathic disease"
First described + associated with familial parkinsonism 1997
SNCA gene, chrom 4q
3 known point mutations
single-allele duplication or triplication in some families
initially named PARK4
Penetrance low, 33 %
Severity depends on gene dosage,
patients with duplication resemble “idiopathic” PD more than triplication cases
Broad spectrum of phenotypes
Mutations and multiplications are rare
Increased expression of wild type SNCA copies may play a role in "idiopathic disease"

18. What is SNCA ? 140-residue cytosolic and lipid-binding protein
Natively unfolded, presynaptic protein
synaptic vesicle recycling, storage of transmitters
negative coregulator of transmitter release
Identical with Non-amyloid β component precursor
Oligomer-forming SNCA in LBs
truncated, oxidized and phosphorylated variants
Mutants have greater nuclear targeting in cell culture
What does it do ?
has propensity to aggregate due to hydrophobic middle region, presence of fibrillar snca in LB,
truncated forms mediate neurodegeneration in vivo in flies, mice
aggregation is promoted by phosphorylation at Serin 129 and Serin-129-P is major component of LB
role of G-protein coupled receptor kinase 5 is promoting aggregation
Sept4 insuffiency (presynaptic scaffold protein) enhances Ser-129-P SNCA aggregation
Unclear if protein accumulation is toxic or protective
Pharmacologic agents promoting inclusions formation seem to protect against SNCA toxicity
Early events after SNCA accumulation: blockade of ER to Golgi vesicular trafficking
Mice expressing human mutant SNCA develop mitochondrial pathology and SN neurons are vulnerable to MPTP
SNCA may modulate oxidative damage
(mice lacking SNCA are resistant to mitochondrial toxins)
biochemical abnormalities in SNCA activate stress-signaling kinases, affect age-related decrease in neurogenesis, inhibit histone acetylation in nucleus to promote toxicity
role for beta-SNC: blocks aggregation of aSNC and promotes cell survival

19. Speculative Model of the Interactions among Proteins Implicated in Parkinson's Disease
Figure. Speculative Model of the Interactions among Proteins Implicated in Parkinson's Disease. The binding of {alpha}-synuclein to lipid membranes protects it from misfolding and aggregation. Mutations in glucocerebrosidase (GBA) alter lipid composition to favor the accumulation of cytosolic {alpha}-synuclein that can then misfold and aggregate into Lewy bodies. Mutations in DJ-1 may enhance the misfolding and aggregation of {alpha}-synuclein and other substrates. Mutations in parkin and UCH-L1 impair the ability of the cell to clear abnormal proteins. The misfolding and aggregation of {alpha}-synuclein and possibly other proteins trigger oxidative stress and energy depletion. PINK1 mutations compromise mitochondrial function, and the response to oxidative damage relies on normal DJ-1 function.
A-synuclein: Unclear if protein accumulation is toxic or protective
Pharmacologic agents promoting inclusions formation seem to protect against SNCA toxicity
Early events after SNCA accumulation: blockade of ER to Golgi vesicular trafficking
Mice expressing human mutant SNCA develop mitochondrial pathology and SN neurons are vulnerable to MPTP
SNCA may modulate oxidative damage
(mice lacking SNCA are resistant to mitochondrial toxins)
biochemical abnormalities in SNCA activate stress-signaling kinases, affect age-related decrease in neurogenesis, inhibit histone acetylation in nucleus to promote toxicity
Feany M. N Engl J Med 2004;351:

20. LRRK-2 (PARK8)
Leucin-rich repeat kinase 2 gene, identified by 2 groups in 2004
Mutations often associated with late-onset disease
contrary to previous beliefs
Large gene, 51 exons
encoding AA- protein with several functional domains
Certain genotypes may be susceptibility factors
16 sequence changes clearly pathogenic, in only 10 of 51 exons
clustering in C-terminal region of protein
pathology linked to kinase function but unclear*
most frequent mutation is c.6055G
 A, 1.5 % of index cases with late-onset “idiopathic” PD
same mutation found in 40% of Arab PD patients and 20 % of Ashkenazi Jewish PD  likely founder effect
homozygous forms of this mutation exist, but are not more severe than heterozygous
Leucin-rich repeat kinase 2 gene, identified by 2 groups in 2004
Mutations often associated with late-onset disease
contrary to previous beliefs
Large gene, 51 exons encoding AA- protein with several functional domains, more than 50 known variants
Certain genotypes may be susceptibility factors rather than pathogenic
16 sequence changes at least are clearly pathogenic, in only 10 of 51 exons
clustering in C-terminal region of protein
most frequent mutation is c.6055G  A, 1.5 % of index cases with lat-onset
“idiopathic” PD
same mutation found in 40% of Arab PD patients and 20 % of Ashkenazi Jewish PD  likely founder effect
homozygous forms of this mutation exist, but are not more severe than heterozygous
*Greggio et al., Neurobiol Dis 2006,
Kinase activity is required for the toxic effects of mutant LRRK2/dardarin

21. Parkin (PARK2)
Identified 1998, earlier age at onset, slower progression
most common factor (10-20%) for early onset disease, all ethnic groups
large number of known mutations
12 exons, chrom 6
Gene product Parkin: 465 aa protein,
ubiquitin ligase, activity disrupted in mutants
Mediates ubiquitilation of target proteins
loss of ligase activity leads to pathology
May lead to accumulation of substrates
Parkin essential in formation of Lewy bodies ?
Possible role in mitochondrial integrity
Effector mechanisms
Rescues mitochondrial dysfunction in flies after inactivation of PINK
SNCA induced dysfunction is enhanced due to lack of Parkin activity
Posttranslational modification of Parkin due to oxidative or nitrosative stress compromises ligase activity
Parkin (PARK2)
1998, earlier age at onset, slower progression, better response to L-Dopa
2/6 autopsy brains had Lewy inclusions, not other 4
most common factor (10-20%) for early onset parkinsonism, all ethnic groups
large number of known mutations, including small mutations and exon rearrangements
12 exons, chrom 6
gene product Parkin: 465 aa protein, ubiquitin ligase, activity disrupted in mutants
mediates ubiquitilation of target proteins: targeting misfolded proteins to ubiquitin-proteasome pathway for degradation, loss of ligase activity leads to pathology
? Parkin essential in formation of Lewy bodies
possible role in mitochondrial integrity
Effector mechanisms
Activation of IkappaB/NF-kB pathway by Parkin
Rescues mitochondrial dysfunction in flies after inactivation of PINK
SNCA induced dysfunction is enhanced due to lack of Parkin activity
Posttranslational modification of Parkin due to oxidative or nitrosative stress compromises ligase activity
The Parkin gene is expressed in presynaptic and postsynaptic processes and cell bodies of many neurons.19 The Parkin protein is presumed to function as an E3-type, E2-enzyme-dependent ubiquitin ligase that is involved in the proteasomal degradation of target proteins.20 The available E3 activity is disrupted by mutations associated with PD, thereby supporting the predominant loss-of-function theory.21 A growing number of putative ('to-be-ubiquitinated') Parkin substrates have been identified, and accumulation of these proteins is proposed to cause the selective death of neurons in the substantia nigra and locus coeruleus in humans.22 Although these putative substrates await convincing validation in animal models with disrupted parkin alleles, Drosophila melanogaster and mice that are deficient of wild-type parkin revealed unequivocal, systemic signs of increased oxidative stress and mitochondrial dysfunction.23, 24, 25 It is currently unclear if these biochemical changes can be attributed solely to the E3-ligase activity of the protein (or lack thereof) or to an—as yet unknown—function of the parkin homologs. An equally contentious issue is whether Parkin expression is essential for formation of neuronal inclusions in vivo, and if so, whether its ubiquitin ligase activity is responsible for the formation of Lewy bodies, as suggested (but not proven) by the abundance of ubiquitinated proteins in Lewy inclusions isolated from human brain.19 Disappointingly, the pathognomonic loss of human substantia nigra neurons has not been replicated in any of the published mouse models of three monogenic PD variants ( -synuclein [SNCA]-transgenic mice, parkin-null mice and dj1-null mice). By contrast, the fruit fly (D. melanogaster) appears more susceptible to PD-type pathology.26
Recently, two biochemical modifications of Parkin (S-nitrosylation and dopamine quinone-adduct formation) were identified in cellular studies and human brain specimens.27 These data indicated that reduced E3-ligase activity of the wild-type Parkin protein (rather than an autosomal recessive mutation in the two Parkin alleles) could also occur as a result of the principal pathogenetic process that is responsible for the development of sporadic PD.

22. Speculative Model of the Interactions among Proteins Implicated in Parkinson's Disease
Figure. Speculative Model of the Interactions among Proteins Implicated in Parkinson's Disease. The binding of {alpha}-synuclein to lipid membranes protects it from misfolding and aggregation. Mutations in glucocerebrosidase (GBA) alter lipid composition to favor the accumulation of cytosolic {alpha}-synuclein that can then misfold and aggregate into Lewy bodies. Mutations in DJ-1 may enhance the misfolding and aggregation of {alpha}-synuclein and other substrates. Mutations in parkin and UCH-L1 impair the ability of the cell to clear abnormal proteins. The misfolding and aggregation of {alpha}-synuclein and possibly other proteins trigger oxidative stress and energy depletion. PINK1 mutations compromise mitochondrial function, and the response to oxidative damage relies on normal DJ-1 function.
A-synuclein: Unclear if protein accumulation is toxic or protective
Pharmacologic agents promoting inclusions formation seem to protect against SNCA toxicity
Early events after SNCA accumulation: blockade of ER to Golgi vesicular trafficking
Mice expressing human mutant SNCA develop mitochondrial pathology and SN neurons are vulnerable to MPTP
SNCA may modulate oxidative damage
(mice lacking SNCA are resistant to mitochondrial toxins)
biochemical abnormalities in SNCA activate stress-signaling kinases, affect age-related decrease in neurogenesis, inhibit histone acetylation in nucleus to promote toxicity
Feany M. N Engl J Med 2004;351:

23. Ubiquitin Proteasome System

24. Ubiquitine-Proteasome System in PD
Parkin, E3-UB ligase
Loss of function in mutants
Mediates ubiquitylation of synphilin, SNCA-interacting protein
UCHL-1 (PARK5), deubiquitylating enzyme
Impaired UB-hydrolysis
Alpha-synuclein inhibits proteasome
But no generalized dysfunction in brain regions with LBs
Precise mechanism of UPS dysfunction in PD unclear
Decreased function with toxin exposure ?
MPTP inhibits proteasome
Changes linked to mitochondrial function, aging ?

25. PINK1(PARK6) =PTEN-induced kinase 1, mutations found in 2004
3 families with consanguinity and autosomal recessive parkinsonism
1-8 % of populations, considerable variation between ethnic groups
Mutations near kinase domain
 loss of function in vivo
581 aa-protein, mitochondrial localization
Highly conserved kinase domain, mitochondrial targeting motif
no known substrate for kinase activity
Mutations lead to different phosphorylation patterns
Proteasomal stress enables altered cleavage of PINK and possible accumulation in LBs
Interaction with DJ-1: recruited to pathway after oxidative damage due to PINK dysfunction

26. DJ-1 (PARK7) Described 2003, chrom 1, 1-2 % of early onset cases
Localization:
ubiquitous expression
cytosolic, may translocate to mitochondria with pH changes
Function:
chaperone-like activity,
intracellular sensor of oxidative stress
regulates D2 receptor signaling
Direct action as antioxidant ?
DJ-1 mutants
show increased vulnerability to energy metabolism changes
Overexpression of DJ-1 protects against mitochondrial complex 1 inhibitors
effect is abrogated by mutants

27. Speculative Model of the Interactions among Proteins Implicated in Parkinson's Disease
Figure. Speculative Model of the Interactions among Proteins Implicated in Parkinson's Disease. The binding of {alpha}-synuclein to lipid membranes protects it from misfolding and aggregation. Mutations in glucocerebrosidase (GBA) alter lipid composition to favor the accumulation of cytosolic {alpha}-synuclein that can then misfold and aggregate into Lewy bodies. Mutations in DJ-1 may enhance the misfolding and aggregation of {alpha}-synuclein and other substrates. Mutations in parkin and UCH-L1 impair the ability of the cell to clear abnormal proteins. The misfolding and aggregation of {alpha}-synuclein and possibly other proteins trigger oxidative stress and energy depletion. PINK1 mutations compromise mitochondrial function, and the response to oxidative damage relies on normal DJ-1 function.
A-synuclein: Unclear if protein accumulation is toxic or protective
Pharmacologic agents promoting inclusions formation seem to protect against SNCA toxicity
Early events after SNCA accumulation: blockade of ER to Golgi vesicular trafficking
Mice expressing human mutant SNCA develop mitochondrial pathology and SN neurons are vulnerable to MPTP
SNCA may modulate oxidative damage
(mice lacking SNCA are resistant to mitochondrial toxins)
biochemical abnormalities in SNCA activate stress-signaling kinases, affect age-related decrease in neurogenesis, inhibit histone acetylation in nucleus to promote toxicity
Feany M. N Engl J Med 2004;351:

28. Oxidative stress and mitochondrial dysfunction
Mitochondrial complex 1 inhibitors MPTP
rotenone, paraquat
Reproduce parkinsonism with dopaminergic neuron loss
Absence of LB in these cases
Chronic infusion reproduces all features, including LBs
Supports theory of environmental toxin interaction by inhibition respiratory chain
Inhibition of complex I:
Depletion of ATP, impairment of dependent processes
Generation of free radicals causing oxidative stress
Post-mortem evidence of ox stress in PD brains
Elevated lipid peroxidation and nitration levels in SN and LBs
Reduced levels of glutathione and oxidized glutathione (antioxidants)
Reduced complex 1 activity in muscle, brain, platelets of PD patients

29. More questions: Role of susceptibility genes
Exact role is controversial:
genes connected to monogenic forms may also act as susceptibility genes
Single mutations in “recessive” genes are common (Parkin, DJ-1, PINK1)
2. Heterozygous mutations
- may be associated with PET, MRI, ultrasound changes
- Subtle motor signs in “asymptomatic” carriers
- Unclear if these are developmental changes, early disease
markers or adaptive
3. Are modifications of wild-type forms linked to parkinsonism ? ubiquitylation studies of wild-type Parkin linked to sporadic PD …
4. Genetic polymorphisms may be associated with disease
- pG2385 increases risk in Chinese population

31. A model of known pathogenetic events in PD shows a principal imbalance between factors that promote PD (e.g. increased total metal content in the substantia nigra, altered steady-state levels of -synuclein proteins, including its phosphorylation, rise in dopamine-metabolism-related stress, and exposure to neurotoxins) and factors that prevent PD (e.g. cigarette smoking, caffeine consumption, expression of wild-type Parkin, DJ1, and PINK1, and normal levels of glutathione). LRRK2, leucine-rich repeat kinase 2; mt, mutant; phosphor., phosphorylation of -synuclein at residue Ser129; PINK1, phosphatase and tensin homolog (PTEN)-induced putative kinase 1; Ser18, serine at residue 18; Tyr, tyrosine at residue 18; UCHL1, ubiquitin carboxyl-terminal esterase L1; wt, wild-type.

32. References
Continuum 2004, No.3 Movement Disorders, Suchowersky and Furtado
Schlossmacher & Klein, Neurology, 2007, 69, 2093, PD – 10 years after its genetic revolution
Lim et al, BMC Biochemistry 2007, 8, S1, Role of ubiquitin proteasome system in Parkinson's disease
Thomas & Flint Beal, Parkinson's disease, Human Molecular Genetics 2007, Vol 16, Review Issue 2,
Gandhi & Wood, Molecular pathogenesis of PD, Human Molecular Genetics 2005, 14, 18,
Braak et al., Stanley Fahn Lecture: The staging procedure for Inclusion Body Pathology, Mov Disorders 21, no.12, 2006,
Feany M. N Engl J Med 2004;351:
16th Annual National Residents’ Seminar on Movement Disorders Course Notes, Ottawa 2008