Parkinson's disease (PD) is a major neurodegenerative disorder characterized by progressive and substantial loss of dopaminergic neurons in the substantia nigra compacta (SNc). Currently, no available drugs prevent the progressive loss of nigral dopaminergic neurons. The mechanisms underlying the dopaminergic degenerative process observed in PD are not well understood, which has hampered development of successful neuroprotective drugs. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine synthesis. Severely reduced TH positive neurons and TH fibers in dopaminergic terminal fields of PD patients and the successful application of L-DOPA therapy for Parkinson's disease suggest that this enzyme has a primary role in the progression of this disease. Previously, we found that the caspase-3 mediated proteolytic activation of Protein Kinase C δ (PKCδ), a member of the novel PKC isoform family, plays a critical role in oxidative stress-induced dopaminergic cell death in cell culture models of PD. In the present study, we report a novel interaction between PKCδ and TH, in which the kinase modulates dopamine synthesis by negatively regulating TH activity via protein phosphatase 2A (PP2A). We observed that PKCδ is highly expressed in nigral dopaminergic neurons and co-localizes with TH in the mouse brain. Interestingly, suppression of PKCδ activity with the kinase inhibitor rottlerin, PKCδ-siRNA, or with PKCδ dominant negative mutant effectively increased a number of key biochemical events in the dopamine pathway, including TH-ser40 phosphorylation, TH enzymatic activity, and dopamine synthesis in neuronal cell culture models. Additionally, we found that PKCδ not only physically associates with the PP2A catalytic subunit (PP2Ac) but also phosphorylates the phosphatase to increase its activity. Notably, inhibition of PKCδ reduced the dephosphorylation activity of PP2A and thereby increased TH-ser40 phosphorylation, TH activity, and dopamine synthesis. To further validate our findings, we used the PKCδ knockout (PKCδ -/-) mouse model. Consistent with other results, we found greater TH-ser40 phosphorylation and reduced PP2A activity in the substantia nigra of PKCδ -/- mice than wild-type mice. Importantly, this was accompanied by an increased dopamine level in the striatum of PKCδ -/- mice. Taken together, these results suggest that PKCδ phosphorylates PP2Ac to enhance its activity, and thereby reduces TH-ser40 phosphorylation and TH activity and ultimately dopamine synthesis.

Suppression of PKCδ activity with the kinase inhibitor rottlerin can increase dopamine synthesis in neuronal cell culture models. Thus, we treated C57 black mice with the PKCδ inhibitor rottlerin and found that rottlerin can effectively increase a number of key neurochemical events in the dopamine pathway, including TH phosphorylation, TH enzymatic activity, and striatal dopamine and DOPAC levels. Time course studies revealed that TH-ser31 and -ser40 phosphorylation and dopamine synthesis were increased within 1hr of rottlerin treatment. Increased dopamine synthesis was accompanied by stimulation of locomotor activity and stereotypic behavior. Consistent with the pharmacological effects of rottlerin, naïve PKCδ-knockout mice showed enhanced striatal dopamine levels and behavioral function as compared to wild-type mice. These results suggest that inhibition of PKCδ in the nigrostriatal dopaminergic system can enhance dopaminergic neurotransmission and neurobehavioral characteristics in animal models.

Proteolytic cleavage of PKCδ (74 kDa) by caspase-3 results in a 41-kDa catalytic subunit and a 38-kDa regulatory subunit, leading to a persistent activation of the kinase (Kaul et al., 2003; Yang et al., 2004). Blockade of proteolytic activation of PKCδ by overexpression of the kinase-dominant negative PKCδ mutant, cleavage-resistant PKCδ mutant, or siRNA directed against PKCδ almost completely prevented the dopaminergic cell death. Therefore, in the present study, we examined the neuroprotective efficacy of the PKCδ inhibitor rottlerin in both primary cell culture and animal models. Herein, we report that rottlerin not only protects against MPP+-induced degeneration of TH-positive neurons in primary mesencephalic culture models but, most importantly, is clearly neuroprotective in an MPTP (1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine) animal model of Parkinson's disease. Administration of rottlerin, either intraperitoneally or orally, to C57 black mice showed significant protection against MPTP-induced locomotor deficits and striatal depletion of dopamine and its metabolite DOPAC. Importantly, stereological analysis of nigral neurons revealed rottlerin treatment significantly protected against MPTP-induced TH-positive neuronal loss in the substantia nigra compacta.

Collectively, these results suggest that PKCδ phosphorylates PP2A to enhance its activity and thereby reduces TH-ser40 phosphorylation and TH activity and ultimately dopamine synthesis. Regulation of dopaminergic function through PKCδ and PP2A may be important for neurobehavioral function and metal-mediated neurological dopaminergic system disorders. Inhibition of PKCδ in the nigrostriatal dopaminergic system can offer dual benefits of neuroprotection and enhanced dopaminergic function in the development of therapeutic agents for treatment of Parkinson's disease. (Abstract shortened by UMI.)