SummaryExcessive mitochondrial fission is associated with the pathology of a number of neurodegenerative diseases. Therefore, inhibitors of aberrant mitochondrial fission could provide important research tools in addition to potential leads for drug development. Using a rational approach, we designed a novel and selective peptide inhibitor, P110, of excessive mitochondrial fission. P110 inhibits Drp1 enzyme activity and blocks Drp1/Fis1 interaction in vitro and in cultured neurons, whereas it has no effect on the interaction between Drp1 and other mitochondrial adaptors, as demonstrated by co-immunoprecipitation. Furthermore, using a model of Parkinson's disease (PD) in culture, we demonstrated that P110 is neuroprotective by inhibiting mitochondrial fragmentation and reactive oxygen species (ROS) production and subsequently improving mitochondrial membrane potential and mitochondrial integrity. P110 increased neuronal cell viability by reducing apoptosis and autophagic cell death, and reduced neurite loss of primary dopaminergic neurons in this PD cell culture model. We also found that P110 treatment appears to have minimal effects on mitochondrial fission and cell viability under basal conditions. Finally, P110 required the presence of Drp1 to inhibit mitochondrial fission under oxidative stress conditions. Taken together, our findings suggest that P110, as a selective peptide inhibitor of Drp1, might be useful for the treatment of diseases in which excessive mitochondrial fission and mitochondrial dysfunction occur.

LRRK2 G2019S mutation is the most common genetic cause of Parkinson's disease (PD). Cellular pathology caused by this mutant is associated with mitochondrial dysfunction and augmented autophagy. However, the underlying mechanism is not known. In this study, we determined whether blocking excessive mitochondrial fission could reduce cellular damage and neurodegeneration induced by the G2019S mutation. In both LRRK2 G2019S-expressing cells and PD patient fibroblasts carrying this specific mutant, treatment with P110, a selective peptide inhibitor of fission dynamin-related protein 1 (Drp1) recently developed in our lab, reduced mitochondrial fragmentation and damage, and corrected excessive autophagy. LRRK2 G2019S directly bound to and phosphorylated Drp1 at Threonine595, whereas P110 treatment abolished this phosphorylation. A site-directed mutant, Drp1(T595A), corrected mitochondrial fragmentation, improved mitochondrial mass and suppressed excessive autophagy in both cells expressing LRRK2 G2019S and PD patient fibroblasts carrying the mutant. Further, in dopaminergic neurons derived from LRRK2 G2019S PD patient-induced pluripotent stem cells, we demonstrated that either P110 treatment or expression of Drp1(T595A) reduced mitochondrial impairment, lysosomal hyperactivity and neurite shortening. Together, we propose that inhibition of Drp1-mediated excessive mitochondrial fission might be a strategy for treatment of PD relevant to LRRK2 G2019S mutation.

The functions of the Betanodavirus non-structural protein B1 is still unknown. We examined B1 expression patterns and investigated novel cell death regulatory functions for this viral protein following RGNNV infection in fish cells. The B1 gene (336 nt) was cloned from the redspotted grouper nervous necrosis virus (RGNNV) genome. B1 mRNA was rapidly expressed in the fish cells from viral RNA3 at 12 h post-infection (p.i.). At the protein level, expression was low at 12 h p.i., and then increased rapidly between 24 h and 72 h p.i. In RGNNV-infected, B1-containing fish cells, over expression of RGNNV B1 reduced Annexin-V positive cells by 50% and 65% at 48 h and 72 h p.i., respectively, and decreased loss of mitochondrial membrane potential (MMP) by 20% and 70% at 48 h and 72 h p.i., respectively. Finally, B1 knockdown during RGNNV infection using anti-sense RNA increased necrotic cell death and reduced cell viability during the early replication cycle (24 h p.i.). Our results suggest that B1 is an early expression protein that has an anti-necrotic cell death function which reduces the MMP loss and enhances viral host cell viability. This finding provides new insights into RNA viral pathogenesis and disease control.

The Betanodavirus non-structural protein B2 plays a role in silencing RNA interference (RNAi), which mediated regulation of animal and plant innate immune responses, but little is known regarding the role of B2 in cell death. The present study examined the effects of B2 on mitochondria-mediated necrotic cell death in grouper liver (GL-av) cells. B2 was expressed at 12 h post-infection (pi), with increased expression between 24 and 72 h pi by Western blot. B2 was transiently expressed to investigate possible novel protein functions. Transient expression of B2 in GL-av cells resulted in apoptotic cell features and positive TUNEL assays (28%) at 24 h post-transfection (pt). During mechanistic studies of cell death, B2 upregulated expression of the proapoptotic gene Bax (2.8 fold at 48 h pt) and induced loss of mitochondria membrane potential (MMP) but not mitochondrial cytochrome c release. Furthermore, over expression of Bcl-2 family member zfBcl-xL effectively prevented B2-induced, mitochondria-mediated necrotic cell death. Finally, using RNA interference to reduce B2 expression, both B2 and Bax expression were downregulated and RGNNV-infected cells were rescued from secondary necrosis. Taken together, our results suggest that B2 upregulates Bax and triggers mitochondria-mediated necrotic cell death independent of cytochrome c release.