Mitochondrial function in Cell death in PD. Pathology Loss of SN pigmented dopamine neurons Lewy...
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Mitochondrial function in Cell death in PD
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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
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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
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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+
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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
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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
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Mitochondria in PD
• Contributions to understanding the pathogenesis of PD by familial inherited forms of PD
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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
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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
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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
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Savitt et al., 2006
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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
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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
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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
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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
)
*
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Assay of mitochondrial function
• Can protein aggregates produce or aggravate mitochondrial dysfunction?
• Can the mito dysfunction cause neuronal death of sensitive neurons?
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• 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)
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rat brain
homogenize
low speed spin
high speed spin
digitonintreatment
Ficollgradient
hypo-osmotic treatment
Isolation of Mitochondria
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Organelle attached Patch Clamp Technique
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Measuring death channel activity with the mitochondrial recording technique
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% 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