Cyclin-dependent kinase 1 (CDK1) has been reported to phosphorylate Drp1 at the same site as the one reported here (Taguchiet al
Cyclin-dependent kinase 1 (CDK1) has been reported to phosphorylate Drp1 at the same site as the one reported here (Taguchiet al.,2007). function in cultured SH-SY5Y neuronal cells and in this rat model of hypertension-induced encephalopathy. Immunoprecipitation studies indicate that PKC binds Drp1, a major mitochondrial fission protein, and phosphorylates Drp1 at Ser 579, thus increasing mitochondrial fragmentation. Further, we found that Drp1 Ser 579 phosphorylation by PKC is associated with Drp1 translocation to the mitochondria under oxidative stress. Importantly, inhibition of PKC, using a selective PKC peptide inhibitor (V1-1), reduced mitochondrial fission and fragmentation and conferred neuronal protection in vivo and in culture. Our study suggests that PKC activation dysregulates the mitochondrial fission machinery and induces aberrant mitochondrial fission, thus contributing to neurological pathology. == INTRODUCTION == Mitochondria are critical for cell survival; mitochondrial dysfunction reduces ATP production, impairs calcium homeostasis, and enhances generation of reactive oxygen species (ROS), which, if left unchecked, can lead to cell death (DiMauro Pi-Methylimidazoleacetic acid and Schon,2008). Mitochondria are highly dynamic organelles that constantly change shape and number by fusion and fission in response to different stimuli and to changes in metabolic demands of the cell (Chen and Chan,2005; Chan,2006). These mitochondrial dynamic processes are required to preserve proper functioning of the cells; they enable mitochondrial recruitment to critical subcellular compartments, content exchange between mitochondria, control of mitochondrial shape and number, mitochondrial communication with the cytosol, and mitochondrial quality control (Chen and Chan,2009). Neurons are particularly sensitive to changes in mitochondrial dynamics due to their high energy demands (Chen and Chan,2009), and recent studies have highlighted a causal role of impaired mitochondrial dynamics (fusion and fission) in neuronal dysfunction and death (Franket al.,2001; Youle and Karbowski,2005; Barsoumet al.,2006; Cheunget al.,2007). Therefore drugs that correct aberration in mitochondrial dynamics may serve as new therapeutics for diverse neurological diseases. At least two proteinsdynamin-related protein 1 (Drp1) and the mitochondrial outer membrane protein Fis1are required for mitochondrial fission in mammalian cells (Labrousseet al.,1999; Smirnovaet al.,2001; Jameset al.,2003; Yoonet al.,2003). Similar to other membrane mechanoenzymes, Drp1 is a large GTPase located mostly in the cytosol. Upon activation, a pool of Drp1 translocates to the mitochondria, where it binds to Fis1, thus assembling future fission sites; Drp1 then enables severing of the mitochondrial membranes through a GTP hydrolysis-dependent mechanism (Smirnovaet al.,2001; Jameset al.,2003; Yoonet al.,2003). Cell culture studies demonstrated that excessive mitochondrial fission is associated with apoptosis, neuronal dysfunction, and cell death (Franket al.,2001; Youle and Karbowski,2005; Barsoumet al.,2006; Cheunget al.,2007). Inhibition of Drp1 by either expressing Ptgfrn a Drp1 dominant mutant or RNA interference leads to increased length and interconnectivity of mitochondrial tubules, thereby inhibiting the fission process and preventing cell death (Franket al.,2001; Jagasiaet al.,2005). Recently, an infant patient with a dominant negative Drp1 allele (Waterhamet al.,2007) and mice lacking Drp1 (Ishiharaet al.,2009; Wakabayashiet al.,2009) were found to have a wide range of brain developmental abnormalities. These findings collectively suggest a critical role of mitochondrial fission in the central nervous system (CNS). However, the signaling enzymes that regulate mitochondrial dynamics, the mechanisms by which excessive mitochondrial fragmentation and dysfunction are induced, and the roles of these processes in human diseases have not been identified. Hypertensive neuroencephalopathy (HTNE) is a neurological disease associated with cognitive and physical disabilities that can lead to death in patients with severe hypertension (Schwartz,2002). We recently reported that a 4-wk treatment of hypertensive rats with V1-1, a protein kinase C (PKC)selective peptide inhibitor (Chenet al.,2001a), reduced mortality from 50% in control-treated hypertensive rats to 8% (Qiet al.,2008). We showed that V1-1 treatment did not reduce blood pressure, but it improved bloodbrain barrier (BBB) function (Qiet al.,2008). Notably, we observed excessive mitochondrial fission and fragmentation, which was associated with ultrastructural damage of the organelle in brains of hypertensive rats with HTNE symptoms as well Pi-Methylimidazoleacetic acid as in the brains of humans who died of hypertension-induced neurological complications. In the animal model, these mitochondrial aberrations were prevented when the animals were treated with the PKC peptide inhibitor. Here we set out to identify the molecular basis of PKC-mediated regulation of mitochondrial fission and its role in neuropathogenesis. == RESULTS == == PKC inhibition reduces mitochondrial ultrastructural damage in Pi-Methylimidazoleacetic acid hypertensive rat brains == We induced pathological hypertension by keeping Dahl salt-sensitive (DS) rats on an 8% high-salt diet. These rats developed HTNE between the ages of 11 and 15 wk (Qiet al.,2008). Major neurological symptoms included seizures, head and forelimb repetitive twitching, forelimb and hind limb paralysis, and severe lethargy (Qiet al.,2008). To determine the extent of neuronal damage in these rat brains, we used transmission electron microscopy (TEM) and examined brain cortex sections of hypertensive rats treated with the PKC inhibitor V1-1 or with the control peptide carrier TAT4757, a component of V1-1 that is used for the delivery.