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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-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