Cell death in PD:the case for mitochondria
Background
• defects in the capacity of the ubiquitin-proteasome system (UPS) to degrade unwanted proteins is considered to play a role in the pathogenesis of PD
• Synthetic PSI (Z-lle-Glu(OtBu)-Ala-Leu-al), an inhibitor of the proteasome, selectively impairs the enzyme in a reversible manner
• A rat model of PD based on inhibition of proteasomal function and UPS-mediated protein degradation has been recently proposed (McNaught KSP, et al. Ann Neurol 2004;56:149–162).
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
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
• 18 rats were injected with 6.0mg/kg (SC) PSI (Peptides International) dissolved in 10% dimethyl sulfoxide (DMSO) and 14 rats with 10% DMSO (SC) as a control. In each case, six injections were made over the course of 2 weeks (Mon, Wed, Fri, Mon, Wed, Fri).
Inner membrane channels
Ca2+
Ca2+
Ca2+
permeabilitytransition pore
calcium uniporter
sodium-calciumexchanger
inner membrane
outer membrane
Na2+
-160 mV
In a previous study [Bonanni L, et al. J Neurosci. 2006 Jun 21;26(25):6851-62] we found a large mitochondrial channel activity to be present in a rat model of global ischemia and it was associated with appearance of the pro-apoptotic N-terminal proteolytic cleavage fragment, N-BCL-xL, and protease activity in post-ischemic mitochondria, consistent with a role for cleaved BCL-xL in channel formation.
3020100Time (s )
IN #
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A)
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-80
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-40
-20
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1001 243
3020100Time (s )
IN #
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2002 3 41
Recordings were made from organelle-attached patches on the membranes of isolatedPSI-treated and control diencephalic mitochondria. A. Sample recordings at -100 mV inPSI-treated (left) and control (right) diencephalic mitochondria.
PSI-treated mitochondria exhibit an opening of large/intermediate conductance channels.
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
)
*
Savitt et al., 2006
Assay of mitochondrial function
• Can protein aggregates produce or aggravate mitochondrial dysfunction?
• Can the mito dysfunction cause neuronal death of sensitive neurons?
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
Measuring death channel activity with the mitochondrial recording technique
KNS-760704 [R(+)Pramipexole]
• Optical enantiomer of Mirapex without the dopaminergic component
• Mirapex has utility for neuroprotection in PD (and other diseases) that is limited by its dopaminergic side-effects.
• Both compounds are neuroprotective by an unknown mechanism-likely mitochondrial, independent of dopamine receptor affinity.
• Rationally designed BAD mimetic binds to hydrophobic cleft of BCL-xL
• Displaces pro-apoptotic BH3-domain proteins to initiate cell death pathway in non-neuronal cells
• Stage II clinical trials as anti-cancer drugOltersdorf et al., 2005
ABT-737 is a specific inhibitor of Bcl-xL, Bcl-2, and Bcl-w
ABT-737 inhibits cell death after ischemia in hippocampal CA1 neurons