Generation of Mitochondria-Targeted Lentivirus

Mitochondria targeted DsRed (Clontech, Mountain View, CA, USA) was inserted into lentivirus vector with human polyubiquitin promoter-C (Addgene, Cambridge, MA, USA). HEK293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were cultured in cell culture medium (DMEM + 10% FBS), one day before transfection, HEK293T cells were sub-cultured into 10 cm dish (Corning) at a density of 6 × 10⁶ cells, after 16 h, the cells were transiently co-transfected with packaging vector psPAX2 (Addgene) and envelope vector pMD2.G (Addgene) by using the standard calcium phosphate precipitation method^{14 (link)}. At 24 h post-transfection, the medium was replaced with fresh DMEM and lentivirus-containing medium was harvested 24 h later. The virus supernatant was then filtered using a 0.45 μm PVDF filter (Millipore) to remove cell debris, concentrated by ultracentrifugation (25,000 rpm at 4 °C for 2 h), resuspended in neuron culture medium and stored at − 80 °C until use.