Parkinson’s Disease

Three Main Symptoms

1. akinesia, bradykinesia
2. muscle rigidity
3. resting tremor (can be unilateral)

Additional features
 loss of postural relexes
 Depression

Affects 200-300/100'000 people. Common but not as common as AD.

Early stage Parkinson’s patient

Slight tremor in one hand.
Initial control and then tremor reappears.
Does not swing affected arm as much although she can walk fine.

Late stage Parkinson’s:

Struggling initiating walk. Once he starts, he can walk.
Not a muscular problem per se, but initiation movement problem.
Changing direction in walking direction also very difficult. Cortical control of movement.

Pathophysiology of PD
 Loss of nigrostriatal DA neurons and corresponding loss of DA in caudate/putamen.
 Remaining neurons show Lewy body inclusions (contain amyloid-like fibrils, mainly
aggregated -synuclein)
 Additional cell loss in locus coeruleus and nucleus basalis.

Substantia Nigra Pars compacta has DA neuron body, pars reticulata is a relay.
Green neurons: cholinergic.
Red neurons: GABAergic.

Effect of dopamine on these neurons depends on D1 and D2 presence of receptors.

Division is not absolute though.

 In PD: loss of output to the motor cortex.

Location of SN in brain
Upper midbrain, black staining neurons (neuromelanin, thought to be due to by-product of dopamine)

Fluorodopa PET scan for diagnosis

- Positron emitting Fluorodopa (fluorine)
- DOPA taken up by dopaminergic neurons and emits protons.
- Patient with PD has a smaller positron emission.

Not necessary in many patients, symptoms are obvious.

History of the disease

1. Arvid Calsson first demonstrated dopamine in the striatum
2. Oleh Hornykiewicz discovered the basis for PD was loss of DA from the striatum and first
used L-DOPA therapy

Main treatment for PD is still replacement therapy

Synthesis of DA
DA from amino acid tyrosine (tyrosine hydroxylase, rate-limiting enzyme) into L-DOPA mainly
but not entirely in DA neurons  (L-DOPA decarboxylase, not just in DA neurons) into Dopamine

Modulating tyrosine does not increase the synthesis of dopamine where it is needed in the brain, while
L-DOPA does. Can't give dopamine, one reason is that it doesn't get into the brain (charged)

Genetics of PD
- sporadic PD does not show Mendelian inheritance (very low concordance in twin studies, low
evidence of family history)
- But, > 15 genes responsible for familial forms have been identified (number keeps growing)
Includes: SNCA, PRKN, PINK1, DJ-1, LRKK2, ATP13A2, etc..

Autosomal Recessive
 PARK2 (PRKN, parkin gene).
Juvenile form (early 20s)
But slow progression
Due to mutations in parkin gene on chromosome 6
Identified as E2- ubiquitin protein ligase (action of proteosome, involved in degradation of misfolded
protein).
Normally it ubiquitinates synuclein, and targets it for proteosomal decay (also other things)
 Mutations cause loss of function and failure of this form of proteolysis
Heterozygotes for Parkin mutations: risk factor for late onset Parkinson's Disease

 PARK6
Mutation in gene PINK-1, kinase with a mitochondrial targeting sequence
Cause a recessive early onset Parkinson-like syndrome
PINK-1 binds to and phosphorylates PARKIN

Autosomal dominant
 PARK1
Early adult onset (40s)
Rapid progression
Associated with Lewy bodies in S. Nigra
Missense mutation in -synuclein on chromosome 4
 Aggregation of -synuclein (due to misfolding)

 PARK4
Also -synuclein mutation
Genomic triplication of normal synuclein: 4 functional copies
Leads to early onset PD

 PARK8:
Gene encoding LRKK2 (leucine-rich repeat kinase, with many substrates) also called dantarine
Late onset, most similar to sporadic Parkinson’s disease

G2019S is a common mutation in some populations (37% in N African Arabs, 10% in Ashkenazi
Jews, 1.8% in UK) but does not have complete penetrance (even if have mutation, may not get
disease).
- LRKK2 variants may be responsible for many cases of "sporadic" PD
- No synaptic substrates but many substrates.

Possible Causes of Neurodegeneration

-synuclein: cytoplasmic protein, even under normal conditions is quite prone to aggregation, still
not known what it does. Mutations make it even more likely to aggregate.
- Present in all neurons but quite high in Substantia Nigra, so maybe stress on neurons may tip
-synuclein into aggregating

Unclear whether lewy body is detrimental to the cell itself or if the cell uses it to contain misfolded
proteins and avoiding further damage caused by misfolding.

Other possible events:

- Mitochondrial damage (PARKIN and PINK1 associated with mitochondria, so important)
- Protein dysfunction
- Cell death

Role of Mitophagy
Mutant parkin/mutant PINK1 inhibit mitophagy of damaged mitochondria
Parkin and Pink1 normally promote mitophagy of damaged mitochondria and maintain neuronal
health.
 Hence tip the balance.

Gaucher's disease: PD link

Recessive, caused by mutation in gene GBA encoding Glucocerebrosidase (lysosomal storage
disease)
Heterozygotes have increased risk for PD.
Carrier rate among Ashkenazy Jews is 8.9%
Other mutations that increase susceptibility
- GenomeWideAssociationStudies: found SNPs in a-synuclein and tau associated with sporadic PD
- A SNP in VMAT2 is protective
- A mutation in VPS35, subunit of the Retromer Complex, associated with late-onset PD.
- Also found mutations in sporadic cases.

Aetiology of sporadic PD

 Post-encephalitis
 Drug-induced (MPTP)
 Neurotoxin-induced (paraquat, rotenone)
 CO poisoning
 Traumatic injury
 Stroke induced
 Mutation increase risk

(Oliver Saks, Awakenings - encephalitis had affected striatum, when given L-DOPA were able to
move again, not instantaneous)

Cumulative Damage Model

Affected individuals have an accelerated cell loss.

Many neurons need to be lost before onset of motor symptoms.

Environmental factors and genetic factors are likely to interact

Genetic susceptibility + Environmental factors - may enter through nose or gut.

- Route of nose into brain  early PD patients complain of loss of smell.
- Also GI dysfunctions-constipation, complained about by PD patients.
- There may be loss of enteric dopaminergic neurons.

Free radicals/Reactive O2 species (ROS)/ oxidative stress and PD

- Neuromelanin builds up in S. Nigra, dopamine breakdown product. It leads to the production of
superoxides such as O2-
- Dopamine is substrate of MAO --> H2O2
- H2O2 --> *OH and OH- catalysed by Fr (which is sequestered by neuromelanin)
- H2O2, *OH, O2- and OH- can all lead to lipid peroxidation and destruction of lipid membrane.
Could be one of the reasons why DA neurons are selectively prone to damage.

 Although you can correlate amount of radicals and PD, this is not causation.
- Might be a secondary event.
 No sorts of antioxidant therapy has been effective in slowing PD.
- May be that people are detected quite late so difficult to slow the disease.

 Need animal models to answer question of causation.

Does oxidative stress cause PD?

- Rotenone, paraquat and MPTP inhibit mitochondrial complex I
- A single nucleotide polymorphism (SNP) in NADH dehydrogenase (component of Complex I)
results in a reduced risk of sporadic PD

 Mitochondria seem intimately involved in Parkinson’s disease

Why are S. Nigra neurons particularly susceptible?

Pacemaker activity: DA is fired continuously, needs to be there for the circuit to function properly.
- Rely on L-type calcium channel (low threshold) Cav1.3.
- Continuous Ca2+ entry and may be more susceptible than most neurons to a defect in Ca2+
handling.
 Calcium channel blockers may be protective in PD (1,4-dihydropyridines)
- They produce dopamine and its metabolism may lead to oxidative stress
- Mitochondria dysfunction leads to disrupted Ca2+ sequestration
- Ca2+ continuously going into cell may make them more prone to stress.
 Production of dopamine, particularly susceptible.

Involves excitotoxic activation of NMDA receptors, elevated Ca2+i, free radicals and eventually cell
death.

PD as a neuroinflammatory disease
- Tumour necrosis factor (TNF, a pro-inflammatory cytokine) is elevated in CSF of patients with
PD
- TNF is elevated in animal models of PD
- Activation of microglia has been shown in PD
- Chronic use of non-steroidal anti-inflammatory drugs can lower risks for development of PD in
humans by 46%

“Blocking soluble Tumour Necrosis Factor signalling with dominant-negative tumour necrosis factor
inhibitor attenuates loss of DA neurons in models of PD” McCoy et al 2006

However retrospective studies are not very helpful.

Animal models of PD
1. 6-OH dopamine kills substantia nigra neurons, toxin. Normally injected. test drugs that you think
may be beneficial in Parkinson’s disease
2. MPTP product of narcotic synthesis, parkinsonian-like symptoms in mice and monkeys. No effect
in rats (developed to metabolise drugs that are very harmful)
3. Transgenic over-expression of -syn in mice and drosophila
4. Vesicular monoamine transporter knockout in mice.
Reserpine treatment:
 blocks VMAT, depletes stored DA, NA and is reversed by L-DOPA
 Rats cannot move, comatose.
Older model.

6-OH dopamine (addition of just one hydroxyl group) and MPTP are newer models.
Both taken up into dopaminergic neurons.

6-OH DA
 Unilateral lesion of S.Nigra
 Causes turning behaviour

Reliable measure of denervation and supersentivitiy of DA receptors (too many receptors)

Internal control with contralateral hemisphere
Problem: there are compensatory changes, not a model of a progressive disease.
 it's good for testing potential treatments

MPTP
 Destroys catecholamine containing neurons
Product of illicit narcotic synthesis. Cooking drugs, turned out wrong.
When one died: looked at brain, complete loss of nigrostriatal tract.
 Completely unable to move.

MPTP substrate for MAO  MPP+ pyridinium ion

Dopamine transporter takes up MPP+
MPP+ inhibits complex I in mitochondria.
 Cause oxidative effect

Effectiveness depends on species (macaque monkeys, mice, humans)

MAO in glia also uptakes MPTP into MPP+

MPP+ can also be taken up by vesicles by vesicular transporter and once it is in the vesicles it is
protected.
- If you destroy vesicles, MPP+ becomes even more toxic.
- Paraquat found to have a similar mode of action to MPP+.

How exactly neurons are killed by MPTP is still unclear.

- increase in lipid peroxidation
- decrease in number of S. Nigra neurons
- Decrease in striatal DA
- Responds to drug therapy used in PD
- Useful in elucidating possible mechanisms of PD but MPTP model does not show progression.

Vesicular monoamine transporter 2 model

- Transgenic mouse with 5% loss of VMAT2
- Less DA release, increase DA turnover as more is in cytoplasm. DA not stored in vesicles.
 Movement problems that seem to be age related.

However, mice only live a couple of years, hard to get a progressive model.
Mouse has increased sensitivity to MPTP, cannot take up MPP+ into vesicles, more in cytoplasm and
toxic to mitochondria.
- Does not show progressive DA cell loss but mice is not a good model anyway
Some SNPs in VMAT2 are protective in human PD.
 Study: increase VMAT2 enhances DA release and opposes PD related neurodegeneration in
vivo.

-synuclein model of PD
- 140 aa cytoplasmic protein, normal role not very well known, probably synaptic function.
- Aggregated protein is constituent of lewy bodies
- Mutations cause familial PD

Over-expression of normal -synuclein in transgenic mice models leads to lewy bodies’ formation
and motor impairments (also in drosophila)
-synuclein knockout mice are almost completely fine and are resistant to MPTP toxicity!
 Holds promise for PD research.

Yeast assay for synuclein toxicity: yeast with two copies of synuclein.
Too much synuclein is bad for yeast as well as substantia nigra neurons.

PARKIN model of PD
In Drosophila
- Expression of mutant but not wild type parkin in drosophila causes age-dependent, selective
degeneration of DA neurons accompanied by a progressive motor impairment

Overexpression or knockdown of drosophila Vesicular Monoamine Transporter partially rescues or

exacerbates, respectively, the degenerative phenotypes

Treatment options for PD

L-DOPA:
- L-DOPA first line, co-administration with DDC inhibitors carbidopa or benserazide to reduce
peripheral SE and the dose required.
- Anticholinergics reduce tremor but side effects are prohibited for some people (autonomic,
memory etc)

L-DOPA can be converted in lots of places.

- As disease progresses though the control of dopamine production, storage and release in neurons
decreases and people complain of side effects. This is because of neuronal degeneration.
- You can fractionate the dose or have an L-dopa pump.

Lots of ON/OFF effects:

1. Ameliorates
2. Peak causes dyskinesias and dystonia
3. End of dose deterioration when L-DOPA wears off.
 These effects can become more and more unpredictable.

L-DOPA also induces hallucinations and sleep disorders.

Dopamine Agonists:
 drug companies have developed dopamine agonists
 initially thought that it may cause accelerated cell loss, no clear evidence though but possible that
they might since dopamine itself may be detrimental to cells, produces neuromelanin etc.

Ropinirole, Apomorphine
- Longer lasting than L-DOPA
- Specific for DA receptor subtypes
- No metabolic conversion needed but peripheral DA antagonist require
- May reduce DA turnover (detrimental)
- Less effective than L-DOPA but may be useful in combination with L-DOPA

MAO inhibitors:
Deprenyl (selegiline), MAO-B inhibitors
- Prolong effects of L-DOPA
- Can be used alone in early stages of disease (increase DA enough)
- It has not been found to reduce progression of the disease or be neuroprotective (block MO,
reduce amount of free radicals production..)

COMT inhibitors
Entacapone
- reduce breakdown of DA
- may increase peripheral side effects of L-DOPA.

Dopamine release enhancers

Amantadine (initially antiviral)
- useful in early stages of disease
- fast acting but effects wears off after 6-8 weeks (unknown why)
- amantadine can be used in combination with submaximal L-DOPA, useful in patients for side
effects
- Budipine, appears to have multiple mechanisms of action, novel
Antimuscarinic
Not used very much.

Benzhexol, Benztropine and Orphenadrine are most used; the latter two also have histamine H1
blocking effects.
- Centrally acting muscarinic receptor blocking drugs have a modest therapeutic effect
- Reduction in tremor and rigidity.
- They can be added to L-DOPA therapy.
- Predictable peripheral side-effects occur.
- Mental confusion and hallucinations may require cessation of anticholinergics.

Surgical treatments for PD

- In the past, lesioning of GP and SthN but lots of side effects and irreversible
- Now, Deep brain stimulation on the same areas.
- Position and stimulation parameters may be fine-tuned, reversible.
- Effective against all 3 cardinal symptoms.

Foetal tissue transplantation: a disappointment

- grafted tissue was shown to develop Lewy bodies
- Possible misfolded synuclein is acting like a transmissible prion to infect neighbouring neurons.
- Sustained improvement only in some patient.
 No real controlled trials, never allowed in the UK
 -synuclein working like a prion

New treatment avenues for PD

 Genetically engineered cells expressing TH
 Viral vector to introduce DNA for TH into brain cells
 Stem-cells could replace foetal cells for transplantation
 Agents that reduce protein aggregation
 Agents that reduce inflammation - tumour necrosis factor antagonists

Use of Ca2+ channel blockers in PD

- DHPs done in animal models and in retrospective study in patients that took DHP in hypertension.
- Seems that fewer people on DHPs had less susceptibility
- Clinical trial just started.