Transcript Cell death in PD-the case for mitochondria

Mitochondrial function in Cell death
in PD
Pathology
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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-4phenyl-1,2,3,6-tetrahydropyridine, injected,
metabolized to MPP+, taken up into
dopaminergic neurons by transporter,
concentrated as MPP+ in mitochondria
• Rotenone, paraquat
Glycolysis
Mitochondrial energy production
Pyruvate
Inner mitochondrial membrane
Acetyl CoA
Lactate
Anaerobic
Glycolysis
TCA cycle
NADH
H+
H+ leak controls basal
metabolic rate
Oligomycin
FADH2
H+
H+
H+
X
Respiratory enzyme complexes
ATP synthase
Succinate
dehydrogenase
NADH
dehydrogenase
H+
ADP + Pi
ATP
Cytochrome oxidase
Cytochrome b
CoQ
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 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
Savitt et al., 2006
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
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
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
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)
Isolation of Mitochondria
homogenize
rat brain
low speed
spin
high speed
spin
digitonin
treatment
Ficoll
gradient
hypo-osmotic
treatment
Organelle attached Patch Clamp
Technique
Measuring death channel activity with the mitochondrial recording technique
Proteasome inhibitor injection into rats produces large conductance activity
of mitochondrial membranes isolated from subcortex
CTL Striatum
60
CTL Cortex
60
PSI Cortex
PSI Striatum
%
activity
40
40
***
*
*
20
0
20
Closed
Small
Inter.
Large
0
Closed
Small
Inter.
Large