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Mic60/mitofilin overexpression alters mitochondrial dynamics and attenuates vulnerability of dopaminergic cells to dopamine and rotenone.

ABSTRACT: Mitochondrial dysfunction has been implicated in Parkinson's disease (PD) neuropathology. Mic60, also known as mitofilin, is a protein of the inner mitochondrial membrane and a key component of the mitochondrial contact site and cristae junction organizing system (MICOS). Mic60 is critical for maintaining mitochondrial membrane structure and function. We previously demonstrated that mitochondrial Mic60 protein is susceptible to both covalent modification and loss in abundance following exposure to dopamine quinone. In this study, we utilized neuronally-differentiated SH-SY5Y and PC12 dopaminergic cell lines to examine the effects of altered Mic60 levels on mitochondrial function and cellular vulnerability in response to PD-relevant stressors. Short hairpin RNA (shRNA)-mediated knockdown of endogenous Mic60 protein in neuronal SH-SY5Y cells significantly potentiated dopamine-induced cell death, which was rescued by co-expressing shRNA-insensitive Mic60. Conversely, in PC12 and SH-SY5Y cells, Mic60 overexpression significantly attenuated both dopamine- and rotenone-induced cell death as compared to controls. Mic60 overexpression in SH-SY5Y cells was also associated with increased mitochondrial respiration, and, following rotenone exposure, increased spare respiratory capacity. Mic60 knockdown cells exhibited suppressed respiration and, following rotenone treatment, decreased spare respiratory capacity. Mic60 overexpression also affected mitochondrial fission/fusion dynamics. PC12 cells overexpressing Mic60 exhibited increased mitochondrial interconnectivity. Further, both PC12 cells and primary rat cortical neurons overexpressing Mic60 displayed suppressed mitochondrial fission and increased mitochondrial length in neurites. These results suggest that altering levels of Mic60 in dopaminergic neuronal cells significantly affects both mitochondrial homeostasis and cellular vulnerability to the PD-relevant stressors dopamine and rotenone, carrying implications for PD pathogenesis.

SUBMITTER: Van Laar VS

PROVIDER: S-EPMC5298082 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Mic60/mitofilin overexpression alters mitochondrial dynamics and attenuates vulnerability of dopaminergic cells to dopamine and rotenone.

Van Laar Victor S VS Berman Sarah B SB Hastings Teresa G TG

Neurobiology of disease 20160319

Mitochondrial dysfunction has been implicated in Parkinson's disease (PD) neuropathology. Mic60, also known as mitofilin, is a protein of the inner mitochondrial membrane and a key component of the mitochondrial contact site and cristae junction organizing system (MICOS). Mic60 is critical for maintaining mitochondrial membrane structure and function. We previously demonstrated that mitochondrial Mic60 protein is susceptible to both covalent modification and loss in abundance following exposure ...[more]

PMID: 27001148

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Project description:BACKGROUND:Parkinson's disease (PD) is one of the neurodegeneration diseases characterized by the gradual loss of dopaminergic (DA) neurons in the substantia nigra region of the brain. Substantial evidence indicates that at the cellular level mitochondrial dysfunction is a key factor leading to pathological features such as neuronal death and accumulation of misfolded ?-synuclein aggregations. Autologous transplantation of healthy purified mitochondria has shown to attenuate phenotypes in vitro and in vivo models of PD. However, there are significant technical difficulties in obtaining large amounts of purified mitochondria with normal function. In addition, the half-life of mitochondria varies between days to a few weeks. Thus, identifying a continuous source of healthy mitochondria via intercellular mitochondrial transfer is an attractive option for therapeutic purposes. In this study, we asked whether iPSCs derived astrocytes can serve as a donor to provide functional mitochondria and rescue injured DA neurons after rotenone exposure in an in vitro model of PD. METHODS:We generated DA neurons and astrocytes from human iPSCs and hESCs. We established an astroglial-neuronal co-culture system to investigate the intercellular mitochondrial transfer, as well as the neuroprotective effect of mitochondrial transfer. We employed immunocytochemistry and FACS analysis to track mitochondria. RESULTS:We showed evidence that iPSCs-derived astrocytes or astrocytic conditioned media (ACM) can rescue DA neurons degeneration via intercellular mitochondrial transfer in a rotenone induced in vitro PD model. Specifically, we showed that iPSCs-derived astrocytes from health spontaneously release functional mitochondria into the media. Mito-Tracker Green tagged astrocytic mitochondria were detected in the ACM and were shown to be internalized by the injured neurons via a phospho-p38 depended pathway. Transferred mitochondria were able to significantly reverse DA neurodegeneration and axonal pruning following exposure to rotenone. When rotenone injured neurons were cultured in presence of ACM depleted of mitochondria (by ultrafiltration), the neuroprotective effects were abolished. CONCLUSIONS:Our studies provide the proof of principle that iPSCs-derived astrocytes can act as mitochondria donor to the injured DA neurons and attenuate pathology. Using iPSCs derived astrocytes as a donor can provide a novel strategy that can be further developed for cellular therapy for PD.

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Mic60/mitofilin overexpression alters mitochondrial dynamics and attenuates vulnerability of dopaminergic cells to dopamine and rotenone.

Publications

Mic60/mitofilin overexpression alters mitochondrial dynamics and attenuates vulnerability of dopaminergic cells to dopamine and rotenone.

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