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
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