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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 promitochondrial compounds
Mitochondria in PD
• Contributions to understanding the
pathogenesis of PD by familial inherited
forms of PD
Genetic mutations-a-synuclein
• First to be identified was a-synuclein
• Point mutations caused familial PD, rare AD form
• Mice lacking gene for a-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 a-synuclein is toxic to neurons
• Factors that increase aggregation of a-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
calcium
uniporter
inner membrane
Ca2+
outer membrane
sodium-calcium
exchanger
-160 mV
Ca2+
Na2+
permeability
transition
pore
Ca2+
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, DN-BCL-xL, and protease activity in post-ischemic
mitochondria, consistent with a role for cleaved BCL-xL in channel
formation.
PSI-treated mitochondria exhibit an opening of large/intermediate conductance channels.
1
2
3
4
200
150
100
IN #0
(pA)
50
0
-50
-100
-150
-200
0
10
20
Time (s)
1
30
3
4
2
100
80
60
40
IN #0
(pA)
20
0
-20
-40
-60
-80
-100
0
10
20
Time (s)
Recordings were made from organelle-attached patches on the membranes of isolated
PSI-treated and control diencephalic mitochondria. A. Sample recordings at -100 mV in
PSI-treated (left) and control (right) diencephalic mitochondria.
30
ANOVA Table for DA (ng/g str)
DF Sum of Squares
Mean Square
F-Value
P-Value
Lambda
Pow er
gruppi
1
5178241.600
5178241.600
9.252
.0160
9.252
.767
Residual
8
4477460.400
559682.550
Means Table for DA (ng/g str)
Effect: gruppi
Count
Mean Std. Dev.
Std. Err.
ctr
5 6139.600
716.133
320.265
psi
5 4700.400
778.793
348.287
7000
DA (ng/g striato)
6000
*
5000
4000
3000
2000
1000
0
ctr
psi
secondo esperimento: sono stati eliminati un controllo = 9262
ed un PSI = 7121, discordanti con gli altri.
Fisher's PLSD for DA (ng/g str)
Effect: gruppi
Significance Level: 5 %
Mean Diff. Crit. Diff.
ctr, psi
1439.200
1091.091
P-Value
.0160 S
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?
BCL-2 proteins induce apoptosis by releasing cytochrome c
from mitochondria
Intermembrane space
Neuronal death
inner membrane
outer membrane
VDAC
caspase-3
BAX
caspase-9
BAX
cytochrome c
The mitochondrial permeability transition pore
is a double membrane-spanning ion channel
BAD
VDAC/BCL-xL
VDAC
mPTP
mPTP
Outer
mitochondrial
membrane
Inner
mitochondrial
membrane
ANT
CyD
Cytochrome c
Ca2+ or Zn2+
Messenger
Measuring death channel activity with the mitochondrial recording technique
KNS-760704 [R(+)Pramipexole]
•
•
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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.
ABT-737 is a specific inhibitor of Bcl-xL,
Bcl-2, and Bcl-w
Oltersdorf et al., 2005
• 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 drug
ABT-737 inhibits cell death after ischemia in hippocampal CA1 neurons