Figure 7.

Mitochondrial fission is required for neuronal cell death and mediated by Drp1 and Mfn1. Neurons were co-transfected with Mito-DsRed2 plus the plasmids encoding the indicated protein(s). (A) Representative fluorescence micrographs of mitochondrial morphology before and after SNOC (175 μM; 7 h) or rotenone (30 nM; 2 h) treatments and a comparison of the effects of Aβ_25–35 versus Aβ_35–25 (10 μM, 6 h), as indicated. Scale bar, 20 μm. (B) Percentage of mitochondrial fission and cell death at 18 h (^† and ^††† significance at P<0.05 and 0.001 as compared to control pcDNA3 transfection; ^*,** and ^*** significance at P<0.05 , 0.01 and 0.001, respectively, compared to SNOC treated, pcDNA3 transfected neurons; n=3 independent experiments). Dying neurons were recognized by their shrunken and condensed nuclei after Hoechst 33342 staining. (C) Enforced Drp1 or Fis1 expression (60 h) evokes fission and neuronal cell death (^*,** and ^*** significance at P<0.05, 0.01 and 0.001, respectively, compared to pcDNA3 transfected neurons; n=3 independent experiments). (D) Rotenone induces dose-dependent mitochondrial fission. The fraction of neurons displaying fissioned mitochondria is shown as the mean ±s.e.m. (^*significance at P<0.05, n=4). (E) Mfn1 or Drp1^K38A inhibits 100 nM rotenone-induced mitochondrial fission (4 h) and cell death (48 h). Data indicate means±s.e.m. of triplicate measurements (^†† and ^††† significance at P<0.01 and 0.001 as compared to untreated control; ^***significance at P<0.001 as compared to rotenone treatment with pcDNA3 transfection; n=3). (F) Mitochondrial fission by Aβ_35–25 or Aβ_25–35 exposure. The fraction of neurons exhibiting fragmented mitochondria is shown as the mean±s.e.m. (^††significance at P<0.01 compared to pcDNA3 transfected, Aβ₃₅₋₂₅ treated control; ^* and ^** significance at P<0.05 and 0.001, respectively, compared to pcDNA3 transfected, Aβ_25–35 treated neurons; n=3).