Inefficient autophagosome formation limits the temporal dynamics of OPTN-mediated mitophagy in neurons

Abstract

Mitophagy is an essential quality control mechanism that maintains neuronal health by selectively removing damaged mitochondria via autophagosomes. In neurons, mitophagy is mainly driven by Optineurin (OPTN), a selective autophagy receptor that is recruited to damaged mitochondria. Consistent with its role in maintaining mitochondrial integrity, OPTN-mediated mitophagy is upregulated in response to mild oxidative stress. However, many mechanistic studies of mitophagy have relied on non-neuronal systems and acute mitochondrial damage paradigms. Thus, it remains unclear how well these findings translate to physiological stress conditions in neurons. Here, we investigated the temporal dynamics of neuronal mitophagy under mild oxidative stress using live-cell imaging in primary rat hippocampal neurons. Surprisingly, we found that in neurons, autophagosomes failed to readily engulf OPTN-positive (OPTN+) mitochondria, revealing a novel rate-limiting step in neuronal mitophagy. Interestingly, this inefficient engulfment was specific to OPTN+ mitochondria at mitophagy events. Given the slow progression of mitophagy, we extended our time course to define the timescale of OPTN-regulated mitophagy. Using a pulse-chase assay to monitor long-term mitochondrial turnover, we found that OPTN+ mitochondria colocalized with acidified lysosomes over a timescale significantly longer than reported in non-neuronal cells and acute neuronal models. Since inefficient autophagosome engulfment appeared to limit mitophagy, we stimulated autophagosome formation via nutrient deprivation, which increased lysosomal colocalization with damaged mitochondria and enhanced mitophagy flux. Together, these findings indicate that mitophagy proceeds relatively slowly in neurons, a characteristic that may contribute to neuronal vulnerability in neurodegenerative disease by promoting the accumulation of dysfunctional mitochondria.