In this study, we demonstrated for the first time that PKCδ is a critical regulator of mitochondrial fission in a CNS disease. We show that PKCδ activation impairs neuronal mitochondrial morphology and increases neuronal cell death, at least in part, by inducing Drp1-dependent fission and fragmentation of the mitochondria under oxidative stress conditions ().
FIGURE 8: Summary scheme. Under control conditions, inactive PKCδ (green) is bound to Drp1, a component of the mitochondrial fission machinery in the cytosol (left). Hypertension (HT) and oxidative stress (OS) lead to PKCδ activation (brown) that (more ...)
Hypertension, which can cause vascular dementia in humans (Moretti et al.
), has been found to lead to oxidative stress and increased ROS production in neuronal cells (Iadecola and Davisson, 2008
), which trigger PKCδ activation (Cieslak and Lazou, 2007
; Qi et al.
). In a previous study, we showed that inhibition of PKCδ by sustained treatment with δV1-1 for 4 wk increases survival of hypertensive rats with HTNE symptoms partly by reversing BBB failure (Qi et al.
). Here we identified an additional PKCδ-mediated pathological mechanism, involving mitochondrial fission impairment in the neurons of hypertensive rat brains. Further, the selective mitochondrial damage in neurons (and lack of effect on glia, for example; Supplemental Figure 2) is consistent with the observation that neurons are particularly vulnerable to changes in mitochondrial dynamics because of their unique requirement for high levels of energy (Chen and Chan, 2005
; Cheung et al.
; Knott et al.
). Because prolonged treatment of normal rats with the PKCδ peptide inhibitor has no apparent adverse effects in any organ, including the CNS (Qi et al.
), and because treatment with δV1-1 in neurons under basal conditions has no effect on mitochondrial morphology ( and ), it is possible that PKCδ inhibition may provide a new means to reduce mitochondrial dysfunction and the resulting neuronal injury in hypertensive subjects.
In this study, we reported that PKCδ-mediated phosphorylation of Drp1 at Ser 579 promotes mitochondrial fragmentation under pathological conditions related to hypertensive brain injury. Phosphorylation of Drp1 at this site (Ser 579) by Cdk1 in the early mitotic phase of HeLa cells has a similar effect (Taguchi et al.
). Because neurons are postmitotic cells and the levels of Cdk1 in these cells are very low (Gompel et al.
), Cdk1 is unlikely to mediate mitochondrial fission under these pathological conditions. Indeed, we found that treatment with a CDK1 inhibitor has no effect on Drp1 phosphorylation under oxidative stress in cultured SH-SY5Y cells ( and Supplemental Figure 5), further supporting our hypothesis that PKCδ-induced Drp1 phosphorylation is likely to be a distinct pathway from that of CDK1. In addition, calcium/calmodulin-dependent kinase I (CaMKIα)–mediated Drp1 phosphorylation at another serine, Ser 600, was shown to induce mitochondrial fission in neurons in response to high potassium (Han et al.
). Phosphorylation of Drp1 by cAMP-dependent protein kinase A at the same residue (ser 600) as CaMKI appears to have opposing effects on mitochondrial morphology, as seen in PC12 cells (Cribbs and Strack, 2007
; Cereghetti et al.
). However, none of these studies determined the role of that protein kinase on mitochondrial function and neuronal integrity in animal models, as we report in the current study. If these kinases modify disease state in vivo, it should be determined whether they act synergistically to regulate mitochondrial dynamics under pathological conditions.
Because mitochondrial morphology is regulated by a balance between fission and fusion (Chan, 2006
), we cannot rule out the possibility that hypertension-induced mitochondrial fragmentation is associated with a disruption of mitochondrial fusion as well. However, using a variety of biochemical and molecular biological tools, we concluded that the fission process may be the main target of activated PKCδ in the mitochondria in response to oxidative stresses. We showed that activated PKCδ directly binds to and phosphorylates Drp1 in the mitochondria of neuronal cells, leading to mitochondrial fragmentation and subsequent neuronal cell death. Thus the mitochondrial fission impairment induced by HTNE in a hypertensive rat model is, at least in part, due to activation of PKCδ.
Because mitochondrial dynamics is necessary for neuronal functions such as synaptic maintenance (Li et al.
), neuronal energy generation (Barsoum et al.
), and brain development (Waterham et al.
; Ishihara et al.
), aberrant mitochondrial fission over time could lead to greater mitochondrial dysfunction and to energy deficits, which in turn impair neuronal dysfunction. Cell culture studies have recently suggested that impaired mitochondrial dynamics and excessive mitochondrial fission are connected to a number of neurological diseases, such as Parkinson’s diseases (Deng et al.
, 2008; Poole et al.
, 2008), Alzheimer’s diseases (Wang et al.
, 2008a, 2008b), and Huntington’s diseases (Liot et al.
; Reddy et al.
). Our findings of aberrant mitochondrial fission in neurons of hypertensive rats and its dependence on PKCδ activation suggest that PKCδ is a component of the mitochondrial fission machinery, at least under pathological conditions. It also provides the first evidence that the signal pathway could regulate mitochondrial dynamics in an animal model of neurological disorder. Thus PKCδ-induced aberrant mitochondrial fission may represent a common mechanism contributing to the pathology of these diseases. Moreover, analysis of human brains supports our findings that impaired mitochondrial dynamics is associated with brain disorders (Cho et al.
). Therefore a PKCδ-selective inhibitor, such as δV1-1, may be a useful treatment for the diseases in which impairment of mitochondrial dynamics occurs.