For the ATP synthesis experiments of Fig. 6, forebrains from P0 pups were dissected as described above. Cells (2 × 107) were harvested for high-purity mitochondrial isolation using the Qiagen Qproteome kit. The mitochondrial pellet was resuspended in 500 μl of mitochondria storage buffer (provided by kit) and kept on ice until further use. Mitochondrial protein content was determined to be within 0.3 to 0.4 μg/μl.

Whole brains were harvested from two mice per group of 9-month-old C57BL/6J mice receiving dyclonine (25 mg/kg)–supplemented or standard water for 7 months. Mitochondria were isolated as described in (38), method “A” with modifications. Briefly, fresh brain tissue was minced and homogenized in a 40-ml Dounce homogenizer with cold isolation buffer [10 mM tris, 1 mM EDTA, and sucrose (110 g/liter) (pH 7.4)], performing all subsequent steps on ice. The supernatant from two consecutive 5-min centrifugations at 1300g were combined and centrifuged for 10 min at 21,000g. The harvested pellet was resuspended in 15% Percoll and layered above a 23% over 40% Percoll gradient. The gradient was centrifuged at 30,700g for 15 min in an SW41Ti rotor, and the mitochondrial fraction (a band at the 23%/40% Percoll interface) was aspirated. Mitochondria were washed and pelleted at 16,900g for 10 min, and the pellet was precipitated by adding bovine serum albumin (BSA) and centrifuging at 6700g for 10 min. The final mitochondrial pellet was resuspended in cold mitochondrial assay solution (MAS) without BSA [70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM Hepes, and 1 mM EGTA (pH 7.2 adjusted using KOH)]. The yield of mitochondrial protein was determined by bicinchoninic acid (BCA) assay.

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