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Recent studies from our laboratory demonstrated that the protein kinase C (PKC) ␦ isoform is an oxidative stress-sensitive kinase and a key mediator of apoptotic cell death in Parkinson's Disease (PD) models (Eur J Neurosci 18:1387-1401, 2003; Mol Cell Neurosci 25: 406 -421, 2004). We showed that native PKC␦ is proteolytically activated by caspase-3 and that suppression of PKC␦ by dominant-negative mutant or small interfering RNA against the kinase can effectively block apoptotic cell death in cellular models of PD. In an attempt to translate the mechanistic studies to a neuroprotective strategy targeting PKC␦, we systematically characterized the neuroprotective effect of a PKC␦ inhibitor, rottlerin, in 1-methyl-4-phenylpyridinium (MPP ϩ )-treated primary mesencephalic neuronal cultures as well as in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. Rottlerin treatment in primary mesencephalic cultures significantly attenuated MPP ϩ -induced tyrosine hydroxylase (TH)-positive neuronal cell and neurite loss. 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 3,4-dihydroxyphenylacetic acid. Notably, rottlerin post-treatment was effective even when MPTP-induced depletion of dopamine and its metabolites was greater than 60%, demonstrating its neurorescue potential. Furthermore, the dose of rottlerin used in neuroprotective studies effectively attenuated the MPTP-induced PKC␦ kinase activity. Importantly, stereological analysis of nigral neurons revealed rottlerin treatment significantly protected against MPTPinduced TH-positive neuronal loss in the substantia nigra compacta. Collectively, our findings demonstrate the neuroprotective effect of rottlerin in both cell culture and preclinical animal models of PD, and they suggest that pharmacological modulation of PKC␦ may offer a novel therapeutic strategy for treatment of PD.Parkinson's disease (PD) is a major neurodegenerative disorder characterized by progressive and substantial loss of dopaminergic neurons in the substantia nigra compacta (SNc), resulting in debilitating motor signs including tremors, bradykinesia, and rigidity. PD affects more than 1% of the population over the age of 60 in the United States (West et al., 2005), ranking it as the second most common neurodegenerative disorder. Although the existing approaches to PD treatment alleviate the signs, they fail to prevent the progression of the neurodegenerative process. 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. Several clinical studies in post-mortem PD human brain tissues and experimental studies in animal PD models indicate that oxidative stress, mitochondrial and ubiquitin proteasomal dysfunction, ...

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Tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, can be regulated by phosphorylation at multiple serine residues, including serine-40. In the present study, we report a novel interaction between a key member of the novel PKC family, protein kinase C␦ (PKC␦), and TH, in which the kinase modulates dopamine synthesis by negatively regulating TH activity via protein phosphatase 2A (PP2A). We observed that PKC␦ is highly expressed in nigral dopaminergic neurons and colocalizes with TH. Interestingly, suppression of PKC␦ activity with the kinase inhibitor rottlerin, PKC␦-small interfering RNA, or with PKC␦ 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 PKC␦ not only physically associates with the PP2A catalytic subunit (PP2Ac) but also phosphorylates the phosphatase to increase its activity. Notably, inhibition of PKC␦ 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 PKC␦ knock-out (PKC␦ Ϫ/Ϫ) mouse model. Consistent with other results, we found greater TH-ser40 phosphorylation and reduced PP2A activity in the substantia nigra of PKC␦ Ϫ/Ϫ mice than in wild-type mice. Importantly, this was accompanied by an increased dopamine level in the striatum of PKC␦Ϫ/Ϫ mice. Collectively, these results suggest that PKC␦ phosphorylates PP2Ac to enhance its activity and thereby reduces TH-ser40 phosphorylation and TH activity and ultimately dopamine synthesis.

L-dopa-induced dyskinesias (LIDs) are a serious complication of L-dopa therapy for Parkinson's disease. Emerging evidence indicates that the nicotinic cholinergic system plays a role in LIDs, although the pathways and mechanisms are poorly understood. Here we used optogenetics to investigate the role of striatal cholinergic interneurons in LIDs. Mice expressing cre-recombinase under the control of the choline acetyltransferase promoter (ChAT-Cre) were lesioned by unilateral injection of 6-hydroxydopamine. AAV5-ChR2-eYFP or AAV5-control-eYFP was injected into the dorsolateral striatum, and optical fibers implanted. After stable virus expression, mice were treated with L-dopa. They were then subjected to various stimulation protocols for 2 h and LIDs rated. Continuous stimulation with a short duration optical pulse (1-5 ms) enhanced LIDs. This effect was blocked by the general muscarinic acetylcholine receptor (mAChR) antagonist atropine indicating it was mAChR-mediated. By contrast, continuous stimulation with a longer duration optical pulse (20 ms to 1 s) reduced LIDs to a similar extent as nicotine treatment (~50%). The general nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine blocked the decline in LIDs with longer optical pulses showing it was nAChR-mediated. None of the stimulation regimens altered LIDs in control-eYFP mice. Lesion-induced motor impairment was not affected by optical stimulation indicating that cholinergic transmission selectively regulates LIDs. Longer pulse stimulation increased the number of c-Fos expressing ChAT neurons, suggesting that changes in this immediate early gene may be involved. These results demonstrate that striatal cholinergic interneurons play a critical role in LIDs and support the idea that nicotine treatment reduces LIDs via nAChR desensitization.

Background Dyskinesias are a disabling motor complication that arises with prolonged L-dopa treatment. Studies using D1 receptor drugs and genetically modified mice suggest that medium spiny neurons expressing D1 receptors play a primary role in L-dopa-induced dyskinesias. However, the specific role of these neurons in dyskinesias is not fully understood. Methods We used optogenetics, which allows for precise modulation of select neurons in vivo, to investigate whether striatal D1-expressing medium spiny neuron activity regulates abnormal involuntary movements or dyskinesia in parkinsonian mice. D1-cre mice unilaterally lesioned with 6-hydroxydopamine received striatal injections of cre-dependent channelrhodopsin2-virus or control-virus. After stable virus expression, the effect of optical stimulation on dyskinesia was tested in L-dopa-naïve and L-dopa-primed mice. Results Single pulse and burst optical stimulation of D1-expressing medium spiny neurons induced dyskinesias in L-dopa-naïve channelrhodopsin2 mice. In stably dyskinetic mice, L-dopa injection induced dyskinesia to a similar or somewhat greater extent than optical stimulation. Combined L-dopa administration and stimulation resulted in an additive increase in dyskinesias, indicating that other mechanisms also contribute. Molecular studies indicate that changes in extracellular signal-regulated kinase phosphorylation in D1-expressing medium spiny neurons are involved. Optical stimulation did not ameliorate parkinsonism in L-dopa-naïve mice. However, it improved parkinsonism in L-dopa-primed mice to a similar extent as L-dopa administration. None of the stimulation paradigms enhanced dyskinesia or modified parkinsonism in L-dopa-naïve or L-dopa-primed control-virus mice. Conclusion The data provide direct evidence that striatal D1-expressing medium spiny neuron stimulation is sufficient to induce dyskinesias and contributes to the regulation of motor control.

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Accumulating evidence suggests that CNS α7 nicotinic acetylcholine receptors (nAChRs) are important targets for the development of therapeutic approaches for Parkinson’s disease. This progressive neurodegenerative disorder is characterized by debilitating motor deficits, as well as autonomic problems, cognitive declines, changes in affect and sleep disturbances. Currently L-dopa is the gold standard treatment for Parkinson’s disease motor problems, particularly in the early disease stages. However, it does not improve the other symptoms, nor does it reduce the inevitable disease progression. Novel therapeutic strategies for Parkinson’s disease are therefore critical. Extensive pre-clinical work using a wide variety of experimental models shows that nicotine and nAChR agonists protect against damage to nigrostriatal and other neuronal cells. This observation suggests that nicotine and/or nAChR agonists may be useful as disease modifying agents. Additionally, studies in several parkinsonian animal models including nonhuman primates show that nicotine reduces L-dopa-induced dyskinesias, a side effect of L-dopa therapy that may be as incapacitating as Parkinson’s disease itself. Work with subtype selective nAChR agonists indicate that α7 nAChRs are involved in mediating both the neuroprotective and antidyskinetic effects, thus offering a targeted strategy with optimal beneficial effects and minimal adverse responses. Here, we review studies demonstrating a role for α7 nAChRs in protection against neurodegenerative effects and for the reduction of L-dopa-induced dyskinesias. Altogether, this work suggests that α7 nAChRs may be useful targets for reducing Parkinson’s disease progression and for the management of the dyskinesias that arise with L-dopa therapy.