Beclin 1-independent pathway of damage-induced mitophagy and autophagic stress: implications for neurodegeneration and cell death

Abstract

Growing evidence supports an active role for dysregulated macroautophagy (autophagic stress) in neuronal cell death and neurodegeneration. Alterations in mitochondrial function and dynamics are also strongly implicated in neurodegenerative diseases. Interestingly, whereas the core autophagy machinery is evolutionarily conserved and shared among constitutive and induced or selective autophagy, recent studies implicate distinct mechanisms regulating mitochondrial autophagy (mitophagy) in response to general autophagic stimuli. Little is known about pathways regulating selective, damage-induced mitophagy. We found that the parkinsonian neurotoxin MPP(+) induces autophagy and mitochondrial degradation that is inhibited by siRNA knockdown of autophagy proteins Atg5, Atg7 and Atg8, but occurs independently of Beclin 1, a component of the class III (PIK3C3/Vps34) phosphoinositide 3-kinase (PI3K) complex. Instead, MPP(+)-induced mitophagy is dependent upon MAPK signaling. Interestingly, all treatments that inhibited autophagy also conferred protection from MPP(+)-induced cell death. A prior human tissue study further supports a role for ERK/MAPK-regulated autophagy in Parkinson's and Lewy body diseases. As competition for limiting amounts of Beclin 1 may serve to prevent harmful overactivation of autophagy, understanding mechanisms that bypass or complement a requirement for PI3K-Beclin 1 activity could lead to strategies to modulate autophagic stress in injured or degenerating neurons.

Fig. 1. Schematic diagram of potential regulatory pathways for autophagy/mitophagy

The most studied pathways of autophagy regulation, including mTOR-mediated suppression of autophagy, which is reversed by amino acid deprivation, rapamycin, or AMP-kinase (1), and the Beclin 1-class III PI3K pathways (2) are shown. In addition, there is cell type-dependent stimulation of autophagy by ERK and JNK signaling (3), although kinase targets remain unidentified. Alternative mechanisms for generating PI(3)P exist in mammalian cells, including wortmannin-resistant class II PI3Ks and phosphoinositide phosphatases (4, see text). Recent studies also suggest a role for ROS acting downstream of Beclin 1 to reduce cleavage of LC3 from preautophagosomal membranes (5). The signals involved in generating mitochondrial ROS during starvation remain undefined, but ROS are involved in mitochondrial activation of ERK. It is unknown if ERK acts directly to phosphorylate mitochondrial targets or if its effects on autophagy reflect other cytoplasmic sites of action.

Fig. 2. Rapamycin co-treatment synergistically increases AV content and exacerbates MPP+-elicited TH neuron loss

Primary mouse embryonic midbrain cultures were treated with MPP+ (5 micromolar) in the presence or absence of rapamycin (50 nanomolar) and analyzed at 24 h for AVs by LC3 immunohistochemistry (A) and at 48 h for TH+ neuron number (B). * p < 0.05 vs. Ctrl; ** p < 0.05 vs. MPP+ alone; ANOVA/Fisher’s LSD.

Fig. 3. MPP+ elicits GFP-LC3 puncta that colocalize with mitochondria in neuronal processes

SH-SY5Y cells were co-transfected with mitochondrially-targeted dsRed and GFP-LC3 for two days followed by retinoic acid-induced differentiation for an additional three days prior to treatment. In control cultures, GFP-LC3 (green) is diffuse in neurites with no overlap with mitochondrial profiles (red), and only rare, small somatic puncta, as seen in an adjacent cell (left side of panel). MPP+-treated cells develop GFP-LC3 puncta adjacent to (arrowhead) or colocalized (yellow, arrows) with mitochondrial profiles along neurites.