Mitochondria likely play a role in Parkinson's disease (PD) neurodegeneration. We modelled PD by creating cytoplasmic hybrid (cybrid) cell lines in which endogenous mitochondrial DNA (mtDNA) from PD or control subject platelets was expressed within human teratocarcinoma (NT2) cells previously depleted of endogenous mtDNA. Complex I activity was reduced in both PD cybrid lines and in the platelet mitochondria used to generate them. Under basal conditions PD cybrids had less ATP, more LDH release, depolarized mitochondria, less mitochondrial cytochrome c, and higher caspase 3 activity. Equivalent MPP + exposures are more likely to trigger programmed cell death in PD cybrid cells than in control cybrid cells. Our data support a relatively upstream role for mitochondrial dysfunction in idiopathic PD.
Mitochondrial dysfunction, oxidative stress, and alpha-synuclein oligomerization occur in Parkinson disease (PD). We used an in vitro PD cybrid approach that models these three phenomena specifically to evaluate the impact of mitochondria-derived oxidative stress on alpha-synuclein oligomerization. Compared with control cybrid cell lines, reactive oxygen species (ROS) production and protein oxidative stress markers were elevated in PD cybrids. The antioxidants CoQ(10) and GSH attenuated changes in PD cybrid peroxide, protein carbonyl, and protein sulfhydryl levels. Elevated PD cybrid alpha-synuclein oligomer levels were also attenuated by CoQ(10) and GSH. In PD cybrids, alpha-synuclein oligomerization was activated via a complex I-mediated increase in the free tubulin/polymerized tubulin ratio. CoQ(10) but not GSH increased complex I activity, restored ATP to control levels, and normalized the PD cybrid free tubulin/polymerized tubulin ratio. Overall, we conclude that two different antioxidants can decrease alpha-synuclein oligomerization whether by improving mitochondrial function or by preventing protein carbonylation or both. We conclude that mitochondrial dysfunction can induce alpha-synuclein oligomerization via ATP depletion-driven microtubule depolymerization and via ROS increase-driven protein oxidation.
In Parkinson's disease mitochondrial dysfunction can lead to a deficient ATP supply to microtubule protein motors leading to mitochondrial axonal transport disruption. Compromised axonal transport will then lead to a disorganized distribution of mitochondria and other organelles in the cell, as well as, the accumulation of aggregated proteins like alpha-synuclein. Moreover, axonal transport disruption can trigger synaptic accumulation of autophagosomes packed with damaged mitochondria and protein aggregates promoting synaptic failure. We previously observed that neuronal-like cells with an inherent mitochondrial impairment derived from PD patients contain a disorganized microtubule network, as well as, alpha-synuclein oligomer accumulation. In this work we provide new evidence that an agent that promotes microtubule network assembly, NAP (davunetide), improves microtubule-dependent traffic, restores the autophagic flux and potentiates autophagosome-lysosome fusion leading to autophagic vacuole clearance in Parkinson's disease cells. Moreover, NAP is capable of efficiently reducing alpha-synuclein oligomer content and its sequestration by the mitochondria. Most interestingly, NAP decreases mitochondrial ubiquitination levels, as well as, increases mitochondrial membrane potential indicating a rescue in mitochondrial function. Overall, we demonstrate that by improving microtubule-mediated traffic, we can avoid mitochondrial-induced damage and thus recover cell homeostasis. These results prove that NAP may be a promising therapeutic lead candidate for neurodegenerative diseases that involve axonal transport failure and mitochondrial impairment as hallmarks, like Parkinson's disease and related disorders.
Multiple lines of evidence state a major role for mitochondrial dysfunction in sporadic Alzheimer's disease (AD) etiopathogenesis. However, the molecular mechanism(s) triggered by mitochondrial deficits that lead to neurodegeneration remain elusive. Herein, we propose a new mechanism by which mitochondrial loss of potential leads to a dysfunction in autophagy/mitophagy due to the overactivation of SIRT2, a tubulin deacetylase that regulates microtubule network acetylation, and provide insights into the association between metabolism, phosphorylation, and Aβ aggregation. We observed an increase in SIRT2 levels and a decrease in the acetylation of lys40 of tubulin in AD cells containing patient mtDNA as well as in AD brains. SIRT2 loss of function either with AK1 (a specific SIRT2 inhibitor) or by SIRT2 knockout recovers microtubule stabilization and improves autophagy, favoring cell survival through the elimination of toxic Aβ oligomers. Our data provide strong evidence for a functional role of tubulin acetylation on autophagic vesicle traffic and mitochondria degradation. We propose that SIRT2 inhibition may improve microtubule assembly thus representing a valid approach as disease-modifying therapy for AD.
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