Xin Qi

Aberrant mitochondrial fission in neurons induced by protein kinase Cδ under oxidative stress conditions in vivo

Impaired mitochondrial fusion/fission plays a causal role in neuronal death. This study delineated a PKCδ-related signaling cascade in which excessive mitochondrial fission is induced during oxidative stress. Moreover, a selective peptide inhibitor of PKCδ inhibits impaired mitochondrial fission under these pathological conditions.

A novel Drp1 inhibitor diminishes aberrant mitochondrial fission and neurotoxicity

Summary Excessive 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 Parkinsons 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.

Inhibition of mitochondrial fragmentation diminishes Huntington's disease-associated neurodegeneration.

Huntingtons disease (HD) is the result of expression of a mutated Huntingtin protein (mtHtt), and is associated with a variety of cellular dysfunctions including excessive mitochondrial fission. Here, we tested whether inhibition of excessive mitochondrial fission prevents mtHtt-induced pathology. We developed a selective inhibitor (P110-TAT) of the mitochondrial fission protein dynamin-related protein 1 (DRP1). We found that P110-TAT inhibited mtHtt-induced excessive mitochondrial fragmentation, improved mitochondrial function, and increased cell viability in HD cell culture models. P110-TAT treatment of fibroblasts from patients with HD and patients with HD with iPS cell-derived neurons reduced mitochondrial fragmentation and corrected mitochondrial dysfunction. P110-TAT treatment also reduced the extent of neurite shortening and cell death in iPS cell-derived neurons in patients with HD. Moreover, treatment of HD transgenic mice with P110-TAT reduced mitochondrial dysfunction, motor deficits, neuropathology, and mortality. We found that p53, a stress gene involved in HD pathogenesis, binds to DRP1 and mediates DRP1-induced mitochondrial and neuronal damage. Furthermore, P110-TAT treatment suppressed mtHtt-induced association of p53 with mitochondria in multiple HD models. These data indicate that inhibition of DRP1-dependent excessive mitochondrial fission with a P110-TAT-like inhibitor may prevent or slow the progression of HD.

Acute Inhibition of Excessive Mitochondrial Fission After Myocardial Infarction Prevents Long-term Cardiac Dysfunction

Background Ischemia and reperfusion (IR) injury remains a major cause of morbidity and mortality and multiple molecular and cellular pathways have been implicated in this injury. We determined whether acute inhibition of excessive mitochondrial fission at the onset of reperfusion improves mitochondrial dysfunction and cardiac contractility postmyocardial infarction in rats. Methods and Results We used a selective inhibitor of the fission machinery, P110, which we have recently designed. P110 treatment inhibited the interaction of fission proteins Fis1/Drp1, decreased mitochondrial fission, and improved bioenergetics in three different rat models of IR, including primary cardiomyocytes, ex vivo heart model, and an in vivo myocardial infarction model. Drp1 transiently bound to the mitochondria following IR injury and P110 treatment blocked this Drp1 mitochondrial association. Compared with control treatment, P110 (1 μmol/L) decreased infarct size by 28±2% and increased adenosine triphosphate levels by 70+1% after IR relative to control IR in the ex vivo model. Intraperitoneal injection of P110 (0.5 mg/kg) at the onset of reperfusion in an in vivo model resulted in improved mitochondrial oxygen consumption by 68% when measured 3 weeks after ischemic injury, improved cardiac fractional shortening by 35%, reduced mitochondrial H2O2 uncoupling state by 70%, and improved overall mitochondrial functions. Conclusions Together, we show that excessive mitochondrial fission at reperfusion contributes to long‐term cardiac dysfunction in rats and that acute inhibition of excessive mitochondrial fission at the onset of reperfusion is sufficient to result in long‐term benefits as evidenced by inhibiting cardiac dysfunction 3 weeks after acute myocardial infarction.

Inhibition of excessive mitochondrial fission reduced aberrant autophagy and neuronal damage caused by LRRK2 G2019S mutation

LRRK2 G2019S mutation is the most common genetic cause of Parkinsons 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 PKCδ-Abl complex communicates ER stress to the mitochondria - an essential step in subsequent apoptosis

Conditions that compromise protein folding in the endoplasmic reticulum trigger the unfolded protein response (UPR), which either restores proper protein folding or results in cellular demise through apoptosis. In this study, we found that, in response to ER stress in vivo and in vitro, PKCδ translocates to the ER where it binds to the tyrosine kinase Abl. Tyrosine phosphorylation and kinase activity of PKCδ are required for PKCδ binding to Abl in the ER. Moreover, we found that inhibition of PKCδ by the PKCδ-specific peptide inhibitor δV1-1 or by silencing of PKCδ reduces ER-stress-induced JNK activation and inhibits ER-stress-mediated apoptosis. Furthermore, the inhibitor of PKCδ kinase activity rottlerin blocks the translocation of the PKCδ-Abl complex from the ER to the mitochondria and confers protection against apoptosis. Thus, PKCδ communicates ER stress to the mitochondria by binding to ER-localized Abl. The PKCδ-Abl complex then translocates to the mitochondria, communicating ER stress to this organelle, thereby, triggering apoptosis.

A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission

Drp1 catalyzes mitochondrial division, but the mechanisms remain elusive. The mitochondrial lipid cardiolipin stimulates Drp1 activity and supports membrane constriction. In addition, Drp1 populates two polymeric states that equilibrate via a dimeric intermediate. Dimers nucleate Drp1 reassembly on mitochondria for fission.

Sustained pharmacological inhibition of δPKC protects against hypertensive encephalopathy through prevention of blood-brain barrier breakdown in rats

Hypertensive encephalopathy is a potentially fatal condition associated with cerebral edema and the breakdown of the blood-brain barrier (BBB). The molecular pathways leading to this condition, however, are unknown. We determined the role of deltaPKC, which is thought to regulate microvascular permeability, in the development of hypertensive encephalopathy using deltaV1-1 - a selective peptide inhibitor of deltaPKC. As a model of hypertensive encephalopathy, Dahl salt-sensitive rats were fed an 8% high-salt diet from 6 weeks of age and then were infused s.c. with saline, control TAT peptide, or deltaV1-1 using osmotic minipumps. The mortality rate and the behavioral symptoms of hypertensive encephalopathy decreased significantly in the deltaV1-1-treated group relative to the control-treated group, and BBB permeability was reduced by more than 60%. Treatment with deltaV1-1 was also associated with decreased deltaPKC accumulation in capillary endothelial cells and in the endfeet of capillary astrocytes, which suggests decreased microvasculature disruption. Treatment with deltaV1-1 prevented hypertension-induced tight junction disruption associated with BBB breakdown, which suggests that deltaPKC may specifically act to dysregulate tight junction components. Together, these results suggest that deltaPKC plays a role in the development of hypertension-induced encephalopathy and may be a therapeutic target for the prevention of BBB disruption.

Cardiolipin's propensity for phase transition and its reorganization by dynamin-related protein 1 form a basis for mitochondrial membrane fission

Fluid cardiolipin (CL) promotes self-assembly of Drp1, a dynamin-family GTPase involved in mitochondrial fission. Drp1 sequesters CL into condensed membrane platforms and in a GTP-dependent manner increases the propensity of the lipid to undergo a nonbilayer phase transition. CL reorganization generates local membrane constriction for fission.

Drp1 stabilizes p53 on the mitochondria to trigger necrosis under oxidative stress conditions in vitro and in vivo

Oxidative-stress-induced necrosis is considered to be one of the main pathological mediators in various neurological disorders, such as brain ischaemia. However, little is known about the mechanism by which cells modulate necrosis in response to oxidative stress. In the present study, we showed that Drp1 (dynamin-related protein 1), a primary mitochondrial fission protein, stabilizes the well-known stress gene p53 and is required for p53 translocation to the mitochondria under conditions of oxidative stress. We found that Drp1 binding to p53 induced mitochondria-related necrosis. In contrast, inhibition of Drp1 hyperactivation by Drp1 siRNA reduced necrotic cell death in cell cultures exposed to oxidative stress. Most significantly, we demonstrated that inhibition of Drp1 by the Drp1 peptide inhibitor P110, which was developed recently by our group, abolished p53 association with the mitochondria and reduced brain infarction in rats subjected to brain ischaemia/reperfusion injury. Taken together, these findings reveal a novel mechanism of Drp1 hyperactivation in the induction of mitochondrial damage and subsequent cell death. We propose that a Drp1 inhibitor such as P110 is a possible therapeutic agent for diseases in which hyperactivated Drp1 contributes to the pathology.