Isolation of Neuronal Mitochondria and Cytosol

First, the cell suspension was prepared according to a previously described method^{97 (link)}. Briefly, brain tissues were dissociated with 300 μL papain solution (28 units/mL, Worthington) containing 1% DNase and 12 mg/mL L-cysteine in DMEM/F12. The mixture was incubated at 37 °C for 15 min with gentle agitation. Dissociated cells were then washed twice with washing buffer (100 mL: 650 μL 45% glucose, 500 μL 1 M HEPES and 5 mL FBS into 93.85 mL 1×DPBS). Subsequently, a mitochondria isolation kit for tissue (Abcam, #ab110168) was used to obtain the cytoplasmic and mitochondrial fractions according to the manufacturer’s instructions with minor changes. Neuronal cells were homogenized in ice-cold mitochondria isolation buffer containing protease inhibitor cocktail after resuspension and then centrifuged at 1000 × g for 10 min twice at 4 °C to remove debris. The supernatant was centrifuged at 12,000 × g for 15 min twice to isolate the mitochondrial fraction. Finally, the resulting pellet containing mitochondria was washed twice with isolation buffer, and the remaining supernatant served as the cytosolic fraction.