mitochondrial function in cell death in pd. pathology loss of sn pigmented dopamine neurons lewy...

Mitochondrial function in Cell death in PD

Pathology

• Loss of SN pigmented dopamine neurons• Lewy bodies• Lewy neurites-multiple brain regions• Lewy bodies stain with antibodies to alpha synuclein,

ubiquitin, others• Also present in autonomic and submucosal ganglia• Clear that PD is more than just a disorder of dopamine

deficiency, but that SN cells for an unknown reason are even more sensitive to the stresses of the pathological abn than other parts of the brain

Environmental factors

• Post-encephalitic and post-traumatic PD

• MPTP (meperidine analog) 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, injected, metabolized to MPP+, taken up into dopaminergic neurons by transporter, concentrated as MPP+ in mitochondria

• Rotenone, paraquat

Pyruvate

Acetyl CoA

TCA cycle

NADH

H+

H+ leak controls basalmetabolic rate

ADP + PiATP

Respiratory enzyme complexes

NADH dehydrogenase

Succinate dehydrogenase

Cytochrome bCoQ

Cytochrome oxidase

ATP synthase

Lactate

AnaerobicGlycolysis

Glycolysis

FADH2

Oligomycin

X

Mitochondrial energy production

Inner mitochondrial membrane

H+

H+ H+

H+

H+ H+H+

H+

H+

Mito dysfunction

• In PD, SN neurons accumulate mito DNA deletions at an abn rate-suggests that oxidative stress is occurring.

• Impaired cell respiration results from mito DNA deficiency that causes respiratory chain deficiency

• A mutation in the gene for mito DNA polymerase assoc. with accumulation in deletions of mito DNA, SN loss, early PD

• Common feature of PD is evidence of Complex 1 deficiency

• Complex 1 also affected by rotenone and MPTP• When rotenone given chronically to rodents, it causes

complex 1 deficiency, dopaminergic cell loss in SN

Mito dysfunction

• 6-hydroxydopamine and paraquat cause oxidative stress, mimic mito toxicity seen with MPTP

• Findings led to trials of coenzyme Q, vit E, creatine, all anti-oxidant and pro-mitochondrial compounds

Mitochondria in PD

• Contributions to understanding the pathogenesis of PD by familial inherited forms of PD

Genetic mutations--synuclein

• First to be identified was -synuclein• Point mutations caused familial PD, rare AD form• Mice lacking gene for -synuclein show resistance to

MPTP-induced dopaminergic toxicity• In Lewy bodies it is present in aggregated form in

insoluble filaments that are hyperphosphorylated and ubiquitinated

• It is likely that misfolded synuclein is toxic to neurons• Factors that increase aggregation of synuclein are

genetic mutations, proteasome and mitochondrial dysfunction, oxidative stress, phosphorylation.

• Likely involved in synaptic vesicle function

Genetic mutations-Parkin

• Mutations in gene for Parkin cause aut. Recessive form of PD

• Most common genetic cause-50% with family history• Parkin is an E3 ligase-participates in addition of ubiquitin

molecules to target proteins, marking them for degradation by the proteasome

• Loss of parkin function therefore leads to an inability to break down toxic substances with subsequent neuronal dysfunction and cell death.

• Parkin substrates p38/JTV and FBP-1 accumulate in sporadic cases of PD and in Parkin K/O mice

• Role of ubiquitination in development of PD is a promising field of study

PINK-1• Mutations in this gene encoding PTEN (Phosphatase and tensin

homologue)-induced putative kinase 1(PINK-1) cause aut. recessive PD.

• Mitochondrial protein kinase, substrates unknown• Targets to mitochondria• K/O in Drosophila assoc. with mitochondrial dysfunction, reduced

respiratory chain activity, reduced mito DNA, reduced ATP content of tissues and increased propensity to apoptosis of affected cells such as muscle

• Parkin over-expression rescues the loss of function phenotype of PINK-1 K/O in Drosophila, Parkin downstream of PINK-1-links mitochondria to proteasome

• Patients with genetic mutations in Parkin or PINK-1 are clinically indistinguishable

Savitt et al., 2006

cytochrome c

VDAC

outer membrane

inner membrane

Intermembrane space

BCL-2 proteins induce apoptosis by releasing cytochrome cfrom mitochondria

caspase-9

caspase-3

Neuronal death

BAX

BAX

The mitochondrial permeability transition pore is a double membrane-spanning ion channelThe mitochondrial permeability transition pore is a double membrane-spanning ion channel

Outer mitochondrial membrane

Inner mitochondrial membrane

VDAC/BCL-xL

Ca2+ or Zn2+

mPTP

Cytochrome c

VDAC mPTP

CyD

Messenger

BAD

ANT

Inhibition of proteasome function may cause PD-like symptoms in

animal models

• We injected animals with a proteasome inhibitor (PSI)

• After 2 week of injections, animals had– Slowness of movement– Decreased dopamine metabolites

McNaught et al, Ann Neurol, 2004

1 5178241.600 5178241.600 9.252 .0160 9.252 .767

8 4477460.400 559682.550

DF Sum of Squares Mean Square F-Value P-Value Lambda Pow er

gruppi

Residual

ANOVA Table for DA (ng/g str)

5 6139.600 716.133 320.265

5 4700.400 778.793 348.287

Count Mean Std. Dev. Std. Err.

ctr

psi

Means Table for DA (ng/g str)Effect: gruppi

1439.200 1091.091 .0160 S

Mean Diff. Crit. Diff. P-Value

ctr, psi

Fisher's PLSD for DA (ng/g str)Effect: gruppiSignificance Level: 5 %

secondo esperimento: sono stati eliminati un controllo = 9262ed un PSI = 7121, discordanti con gli altri.

0

1000

2000

3000

4000

5000

6000

7000

ctr psi

DA

(n

g/g

str

iato

)

*

Assay of mitochondrial function

• Can protein aggregates produce or aggravate mitochondrial dysfunction?

• Can the mito dysfunction cause neuronal death of sensitive neurons?

• Organelle attached Patch Clamp Technique

• Mitochondria isolated from PSI treated rat basal ganglia, as early as one week after first PSI injection (i.e. before appearance of clinical phenotype)

rat brain

homogenize

low speed spin

high speed spin

digitonintreatment

Ficollgradient

hypo-osmotic treatment

Isolation of Mitochondria

Organelle attached Patch Clamp Technique

Measuring death channel activity with the mitochondrial recording technique

% activity

Closed Small Inter. Large0

20

40

60CTL Striatum

PSI Striatum

****

*

0

20

40

60CTL Cortex

PSI Cortex

Closed Small Inter. Large

Proteasome inhibitor injection into rats produces large conductance activityof mitochondrial membranes isolated from subcortex