5 research outputs found
Molecular and cellular insights into mitochondrial contributions to neuronal autophagy: links to energetics and mitophagy
© 2016 Dr. Yea Seul ShinNeurones are essential for brain homeostasis and as highly metabolic cells rely on mitochondrial oxidative phosphorylation (OXPHOS) for energy. The integrity and functionality of mitochondria are critical for neuronal survival, and the involvement of dysfunctional mitochondria is recognized as a common theme amongst various neuropathologies. New evidence has suggested that the inappropriate clearance of dysfunctional mitochondria via autophagy (termed mitophagy) determines the pathogenesis of neurodegenerative diseases such as Parkinson’s disease. Mechanistic studies of mitophagy have been undertaken using mammalian cell lines, but this research lacks relevance to neuropathologies. This thesis investigates triggers of autophagy/mitophagy in primary neurones, and specifically if disruption of mitochondrial bioenergetics triggers neuronal autophagy, and mitophagy in particular.
Cultures of primary cerebellar granule cells (CGCs) were utilized and inhibitors of the OXPHOS complexes (rotenone, 3-Nitropropionic acid, antimycin A, potassium cyanide and oligomycin targeting complex I-V, respectively), were employed to induce bioenergetic dysfunction of mitochondria. Initial investigations using MTT cell viability assay, phase contrast microscopy and cellular membrane permeabilization detected by propidium iodide staining, determined appropriate concentrations of OXPHOS inhibitors which induced effective mitochondrial damage producing slow neuronal degeneration. From this baseline adverse effects of OXPHOS inhibitors on mitochondrial bioenergetics were documented by monitoring reductions in cellular ATP level, mitochondrial membrane potential (ΔΨm) and oxygen consumption rate (OCR) of CGCs. ΔΨm was rapidly dissipated in CGCs exposed to the inhibitors of complexes I, III and IV (rotenone, antimycin A and potassium cyanide, respectively), whilst the inhibitor of complex II, 3- Nitropropionic acid, produced a much slower reduction of ΔΨm. Employing Seahorse XF24 technology allowed an incisive readout of mitochondrial functional changes where significant bioenergetic impairment was observed subsequent to inhibition of complexes I and II, which are core components of energy metabolism regulating the redox balance (NAD+/NADH levels) and TCA cycle.
Existent evidence indicates depolarization of ΔΨm triggers mitophagy in mammalian cell lines, however CGCs display contrasting results where ΔΨm depolarization via complex III and IV inhibition was insufficient to elicit mitophagy despite the inducer of mitophagy, PINK1, being mobilized to mitochondria. In contrast, inhibition of complexes I and II induced mitophagy, as indicated by PINK1 mobilization and disappearance of the pH-sensitive fluorescence mitophagy reporter, mt-Rosella. Western immunoblotting of the general autophagy marker, LC3, and monitoring of acidic vesicles with monodansylcadaverine revealed activation of autophagic flux in CGCs exposed to inhibitors of complexes I-IV, indicating general autophagy in response to bioenergetic impairment irrespective of mitophagy induction.
Results presented herein reveal the complexity of neuronal mitophagy and that ΔΨm may not be a necessary trigger of neuronal mitophagy. Thus inhibition of individual respiratory complexes, and notably complexes I and II, may underlie the triggering of mitophagy in primary neurones, where different mechanisms induce mitophagy in neurones compared to immortalized cell lines. This difference may be due to the unique bioenergetic dependence of neurones. Understanding the mechanisms of mitophagy and autophagy in primary neurones provides valuable insights into therapeutic approaches for neurodegenerative diseases
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LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson’s disease
International audienceMutations of LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore carbonylcyanide m-chlorophenylhydrazone. Förster resonance energy transfer imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a 'kinase-dead' LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only. Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy