Astrocyte-specific autophagy deficiency induces NADPH oxidase-mediated oxidative stress in motor neurons via mitophagy impairment

We hypothesize that autophagy dysfunction specifically in astrocytes induces motor neuron toxicity through impaired mitophagy, leading to accumulation of damaged mitochondria that release mitochondrial DAMPs and trigger NADPH oxidase (NOX2) activation in adjacent motor neurons. The resulting oxidative stress drives motor neuron death through protein oxidation, lipid peroxidation, and mitochondrial dysfunction. This non-cell autonomous mechanism can be tested by: (1) generating GFAP-Cre;ATG7flox/flox mice to model astrocyte-specific autophagy deficiency, (2) co-culturing mutant astrocytes with hiPSC-derived motor neurons to quantify oxidative stress markers (4-HNE, 8-OHdG) and cell death, and (3) blocking NOX2 activity with gp91dstat to determine if motor neuron toxicity is rescued. This pathway would explain how glial autophagy defects accelerate ALS progression independent of neuronal autophagy status.