parkinson‘s disease treatment and models. definition parkinson‘s disease (pd) is the second most...

PARKINSON‘S DISEASE

Treatment and models

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N• Parkinson‘s disease (PD) is the second

most common neurodegenerative disorder after Alzheimer‘s disease.

• It is characterized by the loss of substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons.

• Parkinson is affecting 1% of the population older than 50 years.

DEFIN

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N• Characteristic are also the presence

of intraneuronal cytoplasmic inclusions, called Lewy Bodies (LBs).

• PD is also called Paralysis agitans and Shaking palsy because it causes resting tremor, muscular rigidity and the loss of postural reflexes.

• The sense and intellect remains quite normal.

DOPAMIN

EDOPAMIN

E• Is a neurotransmitter in the

substantia nigra, caudate nucleus and putamen (basalganglia)

• It is essential for a normal movement and transmit the signal between cortex and thalamus.

Neocortex

Basalganglia

Thalamus

Development:

Dopa Dopamine + CO2

• MAO and COMT destroy Dopamine.

• Dopamine cannot cross blood-brain-barrier.

DOPAMIN

EDOPAMIN

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Bra

in-b

loo

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2 forms:αα

-SY

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EIN

-SY

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EIN α-syn:

• Is a 140 amino acid protein, very abundant in the brain.

• its exact function is not known yet.

• It has a natural tendency to clump together to form insoluble aggregates (LBs, LNs) and this tendency is likely increased in the mutated form.

αα-S

YN

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IN-S

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IN• Detected immunohistochemically in

LBs, LNs. (LB Dementia, Parkinson‘s, Alzheimer‘s, NBIA1).

• In Drosophila: similar perinuclear and neuritic filamentous inclusions like LBs & LNs.

• Is the precursor protein of a nonamyloid-β-protein component (NAC) isolated from Alzheimer‘s plaques.

• Induces selective and progressive loss of DA neurons.

ββ-S

YN

UC

LE

IN-S

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INβ-syn: homologue to α-syn

• Is not able to form insoluble LBs or LNs.

• Aminoacids 73-83 in the NAC region are absent

= nonamyloidogenic

• This region (aa 73-83) is essential for This region (aa 73-83) is essential for the typical aggregations in the brain.the typical aggregations in the brain.

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Selective Insolubility of Selective Insolubility of αα-syn in -syn in human Lewy Body diseaseshuman Lewy Body diseases

Lewy Body diseases:

- Parkinson‘s Disease

- Alzheimer‘s Disease

- LB Dementias

- Hallervorden – Spatz Disease (NBIA type1)

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Transgenic mice expressing human Transgenic mice expressing human

αα-syn-syn

Wild type: [wt]

Point mutation: [A30P]- α-syn

Point mutation: [A53T]- α-syn

Promotor: THY1 (brain neuron-specific)

Investigation of solubility of „syns“ in human LB Diseases

transgenic animals expressing human [wt] and mutant [A30P]- α-syn,

[A53T]- α-syn

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ION • [wt] and mutant α-syn assembled into LB-like

fibrils in transgenic Drosophila:

locomotor deficit became apparent with increasing age.

2 missense mutations in the α-syn gene (rare) dominantly inherited PD.

• In transgenic mice:

somal and neuritic accumulations of [wt] and mutant α-syn were observed in transgenice mouse brain.

modest reduction of locomotor performance, due to age-dependent degeneration of neuromuscular junctions.

IMP

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ION

IMP

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ION α-syn and β-syn: both in synaptosomal

fractions of rodent + human brain.

• α-syn: highly soluble in aqueous buffer

In case of LB diseases:

detergent- insoluble α-syn + aggregates

were found in urea extracts.

Human α-syn was detergent-insoluble in

transgenic mouse brains, but not

endogenous mouse α-syn and β-syn!

IMP

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IMP

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ION • β-syn: no aggregates in vitro (no 73-83)

Conclusion:Conclusion: specific accumulation of

detergent-insoluble α-syn in transgenic

mice recapitulates a pivotal feature of

human LB diseases.

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• Antibodies (rat, mouse) against LB recognizing epitopes

• Brain fractionating and Western Bloting• Generation and Characterization of

transgenic mice• In vitro aggregation

of recombinant syns

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• Drosophila:Drosophila:– short generation time– fully sequenced genome– vast array of genetic information– availability of huge number of stocks

containing mutations in almost every genes

Complete control about the experiment

– short life-span: investigations of age-dependent disorders (neurodegeneration)

– expression of human α-syn-gene causes several characteristics of human PD:

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Late-onset progressive degeneration of subsets of DA neurons.

Develop filamentous protein aggregates rich in α-synuclein within cell bodies and neurites of DA neurons.

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• Heat shock proteins • Up-regulated during stress response• Help to refold misfolded proteins

Auluck et al. – aggregation = disorder involving

protein misfolding.

Boost the amount of chaperones in neurons

• Expression of gene encoding human Hsp70 completely prevents late-onset loss of DA neurons (induced by human α-syn).

• Decreasing endogenous chaperone activity accelerates the neuronal loss in PD flies.

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ES Human disease

Linkage to specific genes

Fly modelof disease

Fly genetics to develop new treatments

α-synuclein

Fly Prakinson‘s disease

Prakinson‘s disease

Treatment of Treatment of Prakinson‘s Prakinson‘s diseasedisease

Treatment Treatment of human of human diseasedisease

Chaperones

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• Polyglutamine disease: same neuronal

degeneration (expanded polyglutamine stretches)• Abnormal polyglutamine proteins suppressed by

Hsp70

Coexpression of Hsp70 with α-syn • Normal numbers of DA neurons in aged flies

control: β-galactosidase coexpressed with α-syn • 50% loss of DA neurons in aged flies

(α-syn – concentration did not alter [immunoblot] )

Interaction between endogenous chaperone Interaction between endogenous chaperone activity and activity and αα-synuclein.-synuclein.

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Influencing of α-syn confirmation? α-syn interferes with chaperone‘s activity by

sequestration?

• Chaperones can „detoxify“ the protein aggregation in a more subtle way.

• Risk factors may hamper normal activity of chaperones (smoking)

• Inducing general stress response: (for treatment)Increases production of chaperonesAdvertantly protect neurons?

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Main goal:Main goal: increase dopamine level

• Dopamine precursors: can cross blood-brain-barrier, changed to dopamine– i.e. Levodopa, Sinemet, Madopar

• Dopamine agonists: act directly on dopamine receptors (D1, D2), replace dopamine– i.e. Apomorphine, Mirapex

• Inhibitors of dopamine metabolism:– i.e. Selegiline, Eldepryl

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• Blocking the reuptake of dopamine/ increasing the release of dopamine: – i.e. Symmetrel

• COMT inhibitors (Catechol-O-methyltransferase): are taken with L-Dopa, slow the break down of it. – i.e. tolcapone, entacapone

• Anticholinergics: act not directly on dopamine, help to decrease the activity of Ach, which is balancing neurotransmitter– i.e. trihexyphenidyl

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• Pallidotomy: tiny hole drilled in the skull, electric probe is used to destroy a small part of the global pallidus to reduce dyskinesia

• Deep brain stimulation (DBS): very thin electrode is implanted into the brain, small electric pulses are used to stimulate the brain , blocks signals that cause PD symptoms.

• Stem cell therapy: stem cells are undifferentiated cells, can be manipulated to differentiate into dopamine producing neurons (still experimental stage)

Neuron developed from stem cell

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• Science #295: Auluck et al.

„Suppression of α.synuclein toxicity in a Drosophila model for Parkinson‘s disease“

• Science #295: Helfand„Chaperones take flight“

• Am J Pathol #159: Kahle et al.„Selective insolubility of alpha-synuclein in human Lewy body diseases is recapitulated in a transgenic mouse model“

• www.allsciencestuff.com/mbiology/research/parkinsons

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