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Neuronal Oxidative Injury in Parkinson's Disease: In vivo and in vitro studies J. Timothy Greenamyre Pittsburgh Institute for Neurodegenerative Diseases Pitt Collaborators: Outside Collaborators: Terri Hastings Chenjian Li - Weill Cornell

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Neuronal Oxidative Injury in Parkinson's Disease: In vivo and in vitro studies

  • J. Timothy Greenamyre

Pittsburgh Institute for Neurodegenerative Diseases

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Pitt Collaborators: Terri Hastings Ed Burton Sarah Berman David Hinkle Michael Palladino Ron Wetzel Charleen Chu Jun Chen Guodong Cao Valerian Kagan Current Funding: NINDS NIEHS Veterans Administration American Parkinson Disease Association Michael J. Fox Foundation Outside Collaborators: Chenjian Li - Weill Cornell Fabio Blandini - Mondino Institute Pier Mastroberardino - Erasmus MC Takao Yagi - Scripps

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Parkinson’s Disease

Prevalence: 1% of people over age 55 (1 million in North America) Inheritance: Sporadic and Familial Etiology: Environmental toxins Complex I defects? Single gene mutations α-synuclein dupli- & triplications Cardinal Signs: Tremor, rigidity, bradykinesia, postural instability Other Signs: Shuffling gait, masked facies, deceased blink rate

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  • Loss of dopamine neurons in the substantia nigra pars

compacta

  • Lewy bodies/neurites
  • Loss of neurons in locus ceruleus, dorsal vagal

nucleus, dorsal raphe and nucleus basalis of Meynert

  • Microglial activation

Classical Pathology:

Parkinson’s Disease

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Parkinson’s Disease

Nerve Terminals Cell Body Caudate & Putamen

Degeneration of nigrostriatal dopamine neurons

Substantia nigra

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Lewy Bodies

The pathological hallmark of Parkinson’s

  • disease. Among the proteins they contain:
  • Phosphorylated neurofilament proteins
  • Ubiquitin
  • α-Synuclein
  • Parkin
  • Proteasome subunits
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Parkinson’s Disease

  • Loss of reduced glutathione (GSH)
  • Increased levels of malondialdehyde & lipid

hydroperoxides

  • Oxidative DNA & protein damage
  • Oxidative (nitrative) modification of α-synuclein
  • Iron accumulation

Biochemical Pathology in Substantia Nigra:

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MPTP

Parkinson’s Disease

Etiology

Mendelian Genetics Toxic Exposure

α-synuclein parkin

Genetic Susceptibility

Environmental Exposure

+

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Parkinson’s Disease

Mutations and Mitochondria

PINK1 - a nuclear-encoded, mitochondrial protein kinase (Valente et al, 2004; Rohe et al, 2004) Parkin - mitochondrial quality control; knock-out results in disruption of mitochondrial function (Greene et al, 2003; Palacino et al, 2004) DJ-1 - under conditions of oxidative stress, DJ-1 translocates to mitochondria (Canet-Aviles et al, 2004) Omi - a mitochondrial protease (Strauss et al, 2005) POLG - mitochondrial DNA polymerase gamma

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Parkinson’s Disease

Etiology

Mendelian Genetics Toxic Exposure Genetic Susceptibility Environmental Exposure

+

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Parkinson’s Disease

Associated with pesticide exposure:

Butterfield et al., (1993) Neurology, 43, 1150-8. Fall et al., (1999) Mov Disord, 14, 28-37. Flemin et al., (1994) Ann Neurol, 36, 100-3. Hertzman et al., (1994) Mov Disord, 9, 69-75. Hubble et al., (1993) Neurology, 43, 1693-7. Liou et al., (1997) Neurology, 48, 1583-8. Menegon et al., (1998) Lancet, 352, 1344-6. Seidler et al., (1996) Neurology, 46, 1275-84. Fong et al., (2007) Clin Chim Acta 378, 136-41. Ascherio et al., (2006) Ann Neurol, 60, 197-203. Frigerio et al., (2006) Mov Disord, 21, 1688-1692. Tanner et al., Envir. Health Perspect, 2011

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MPTP Rotenone Pesticides

Parkinson’s Disease

Etiology

Mendelian Genetics Toxic Exposure

PINK1 DJ-1 Parkin

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MPTP

  • In 1982, IV drug users present with an acute

parkinsonian syndrome

  • Astute medical detective work identifies the toxin

as MPTP

  • MPTP is metabolized to MPP+, a substrate for

the dopamine uptake transporter (DAT)

  • Mechanism of action is inhibition of mitochondrial

respiration at complex I

  • Mitochondrial dysfunction can cause a

parkinsonian syndrome

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Parkinson’s Disease

A defect in mitochondrial complex I

After the discovery of MPTP and its mechanism:

  • 1989-92: A selective decrease in complex I

activity in PD brains (Mizuno et al, Schapira et al)

  • Complex I activity is reduced by 16 - 55% in

platelets of PD patients (Yoshino et al, Parker et al, Mann et al, Haas & Shults et al)

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Parkinson’s disease is associated with a systemic complex I defect, yet dopaminergic neurons of substantia nigra degenerate selectively. Is the complex I defect relevant? Hypothesis: An experimentally-induced, chronic, systemic inhibition of complex I can reproduce the behavioral, neurochemical and neuropathological features of PD in an animal model.

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Rotenone

  • Classical high-affinity inhibitor of complex I of the

mitochondrial electron transport chain

  • A natural product - from several plant species
  • Common pesticide; the “organic” (natural) alternative to

synthetic pesticides

  • Used to sample fish populations in reservoirs & kill

nuisance fish in lakes

  • Highly lipophilic; crosses biological membranes easily &

independent of transporters

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Substantia nigra (Ventral) Midbrain

Striatum

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Betarbet, Sherer et al, Nature Neuroscience

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Refinement of the rotenone model (3 mg/kg/d)

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TH α-Synuclein Poly-Ubiq Merge

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Proof of concept: Systemic mitochondrial impairment can cause alpha- synuclein accumulation & aggregation

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X

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Superoxide production in the mitochondria of rotenone- treated rats measured with electron spin resonance

100 200 300 400 500 600 100 200 300 400

Superoxide (pmole/mg)

RBM control RLM control RBM Rotenone RLM Rotenone

Time (seconds)

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Control Rotenone

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Why are dopamine neurons selectively vulnerable? Why does degeneration begin in nerve terminals? Is it dopamine itself?

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Ty

Terri Hastings

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* * *

Ty

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* * * * * *

Ty

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What is the effect of cytosolic DAQ on mitochondria?

DAQ DAQ DAQ DAQ DAQ DAQ DAQ

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DAQ penetrates intact mitochondria and binds covalently to complex I subunits

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DAQ penetrates intact mitochondria and inhibits complexes I & II

The effect of DAQ is blocked by glutathione

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Are these results relevant? Can DA inhibit mitochondrial function in vivo?

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Methamphetamine releases DA from vesicles

Relevance:

  • alpha-synuclein

increases cytosolic dopamine

  • Complex I dysfunction

increases cytosolic dopamine

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Cytosolic DA inhibits mitochondrial respiration in intact cells

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Cytosolic DA inhibits mitochondrial respiration in intact cells

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Parkinson’s Disease

The Rotenone Model

✔ Systemic mitochondrial impairment ✔ Pesticide exposure ✔ Selective nigrostriatal dopamine cell loss ✔ Lewy body formation (α-synuclein accumulation) ✔ Oxidative damage ✔ Microglial activation (inflammation) ✔ Proteasome dysfunction ✔ Cardiac sympathetic denervation ✔ GI pathology/constipation ✔ Iron accumulation

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