Mitochondrial division inhibitor 1 reduces dynamin-related protein 1 and mitochondrial fission activity.

Abstract

The purpose of our study was to better understand the effects of mitochondrial-division inhibitor 1 (Mdivi-1) on mitochondrial fission, mitochondrial biogenesis, electron transport activities and cellular protection. In recent years, researchers have found excessive mitochondrial fragmentation and reduced fusion in a large number of diseases with mitochondrial dysfunction. Therefore, several groups have developed mitochondrial division inhibitors. Among these, Mdivi-1 was extensively studied and was found to reduce dynamin-related protein 1 (Drp1) levels and excessive mitochondrial fission, enhance mitochondrial fusion activity and protect cells. However, a recent study by Bordt et al. (1) questioned earlier findings of the beneficial, inhibiting effects of Mdivi-1. In the current study, we studied the protective effects of Mdivi-1 by studying the following: mRNA and protein levels of electron transport chain (ETC) genes; mitochondrial dynamics and biogenesis genes; enzymatic activities of ETC complexes I, II, III and IV; the mitochondrial network; mitochondrial size & number; Drp1 GTPase enzymatic activity and mitochondrial respiration (1) in N2a cells treated with Mdivi-1, (2) overexpressed with full-length Drp1 + Mdivi-1-treated N2a cells and (3) Drp1 RNA silenced+Mdivi-1-treated N2a cells. We found reduced levels of the fission genes Drp1 and Fis1 levels; increased levels of the fusion genes Mfn1, Mfn2 and Opa1; and the biogenesis genes PGC1α, nuclear respiration factor 1, nuclear respiratory factor 2 and transcription factor A, mitochondrial. Increased levels mRNA and protein levels were found in ETC genes of complexes I, II and IV genes. Immunoblotting data agreed with mRNA changes. Transmission electron microscopy analysis revealed reduced numbers of mitochondria and increased length of mitochondria (1) in N2a cells treated with Mdivi-1, (2) cells overexpressed with full-length Drp1 + Mdivi-1-treated N2a cells and (3) Drp1 RNA silenced+Mdivi-1-treated N2a cells. Immunofluorescence analysis revealed that mitochondrial network was increased. Increased levels of complex I, II and IV enzymatic activities were found in all three groups of cells treated with low concentration of Mdivi-1. Mitochondrial function was increased and GTPase Drp1 activity was decreased in all three groups of N2a cells. These observations strongly suggest that Mdivi-1 is a Drp1 inhibitor and directly reduces mitochondrial fragmentation and further, Mdivi-1 is a promising molecule to treat human diseases with ETC complexes, I, II and IV.