Arthi Kanthasamy

Caspase-3 dependent proteolytic activation of protein kinase Cδ mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration

1‐Methyl‐4‐phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase‐3 mediated proteolytic activation of protein kinase Cδ (PKCδ) is critical in MPP+‐induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time‐dependent increases in reactive oxygen species generation, cytochrome c release, and caspase‐9 and caspase‐3 activation. Interestingly, MPP+ induced proteolytic cleavage of PKCδ (72–74 kDa) into a 41‐kDa catalytic and a 38‐kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase‐3 inhibitor Z‐DEVD‐fmk effectively blocked MPP+‐induced PKCδ cleavage and kinase activity, suggesting that the proteolytic activation is caspase‐3 mediated. Similar results were seen in MPP+‐treated rat midbrain slices. Z‐DEVD‐fmk and the PKCδ specific inhibitor rottlerin almost completely blocked MPP+‐induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+‐induced caspase‐3 activation, PKCδ cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+‐induced caspase‐3 activity without affecting basal activity, suggesting positive feedback activation of caspase‐3 by PKCδ. Intracellular delivery of catalytically active recombinant PKCδ significantly increased caspase‐3 activity, further indicating that PKCδ regulates caspase‐3 activity. Finally, over‐expression of a kinase inactive PKCδK376R mutant prevented MPP+‐induced caspase activation and DNA fragmentation, confirming the pro‐apoptotic function of PKCδ in dopaminergic cell death. Together, we demonstrate for the first time that MPP+‐induced oxidative stress proteolytically activates PKCδ in a caspase‐3‐dependent manner to induce apoptosis and up‐regulate the caspase cascade in dopaminergic neuronal cells.

Mitochondria-targeted antioxidants for treatment of Parkinson's disease: preclinical and clinical outcomes.

Parkinsons disease is a progressive neurodegenerative disease in the elderly, and no cure or disease-modifying therapies exist. Several lines of evidence suggest that mitochondrial dysfunction and oxidative stress have a central role in the dopaminergic neurodegeneration of Parkinsons disease. In this context, mitochondria-targeted therapies that improve mitochondrial function may have great promise in the prevention and treatment of Parkinsons disease. In this review, we discuss the recent developments in mitochondria-targeted antioxidants and their potential beneficial effects as a therapy for ameliorating mitochondrial dysfunction in Parkinsons disease.

Role of Proteolytic Activation of Protein Kinase Cδ in Oxidative Stress-Induced Apoptosis

Protein kinase Cdelta (PKCdelta), a member of the novel PKC family, is emerging as a redox-sensitive kinase in various cell types. Oxidative stress activates the PKCdelta kinase by translocation, tyrosine phosphorylation, or proteolysis. During proteolysis, caspase-3 cleaves the native PKCdelta (72-74 kDa) into 41-kDa catalytically active and 38-kDa regulatory fragments to persistently activate the kinase. The proteolytic activation of PKCdelta plays a key role in promoting apoptotic cell death in various cell types, including neuronal cells. Attenuation of PKCdelta proteolytic activation by antioxidants suggests that the cellular redox status can influence activation of the proapoptotic kinase. PKCdelta may also amplify apoptotic signaling via positive feedback activation of the caspase cascade. Thus, the dual role of PKCdelta as a mediator and amplifier of apoptosis may be important in the pathogenesis of major neurodegenerative disorders, such as Parkinsons disease, Alzheimers disease, and Huntington disease.

Neuroprotective Effect of Protein Kinase Cδ Inhibitor Rottlerin in Cell Culture and Animal Models of Parkinson's Disease

Recent studies from our laboratory demonstrated that the protein kinase C (PKC) δ isoform is an oxidative stress-sensitive kinase and a key mediator of apoptotic cell death in Parkinsons Disease (PD) models (Eur J Neurosci 18:1387–1401, 2003; Mol Cell Neurosci 25:406–421, 2004). We showed that native PKCδ is proteolytically activated by caspase-3 and that suppression of PKCδ 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 PKCδ, we systematically characterized the neuroprotective effect of a PKCδ inhibitor, rottlerin, in 1-methyl-4-phenylpyridinium (MPP+)-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+-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 PKCδ kinase activity. Importantly, stereological analysis of nigral neurons revealed rottlerin treatment significantly protected against MPTP-induced 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 PKCδ may offer a novel therapeutic strategy for treatment of PD.

Environmental neurotoxic pesticide increases histone acetylation to promote apoptosis in dopaminergic neuronal cells: relevance to epigenetic mechanisms of neurodegeneration.

Pesticide exposure has been implicated in the etiopathogenesis of Parkinsons disease (PD); in particular, the organochlorine insecticide dieldrin is believed to be associated with PD. Emerging evidence indicates that histone modifications play a critical role in cell death. In this study, we examined the effects of dieldrin treatment on histone acetylation and its role in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells. In mesencephalic dopaminergic neuronal cells, dieldrin induced a time-dependent increase in the acetylation of core histones H3 and H4. Histone acetylation occurred within 10 min of dieldrin exposure indicating that acetylation is an early event in dieldrin neurotoxicity. The hyperacetylation was attributed to dieldrin-induced proteasomal dysfunction, resulting in accumulation of a key histone acetyltransferase (HAT), cAMP response element-binding protein. The novel HAT inhibitor anacardic acid significantly attenuated dieldrin-induced histone acetylation, Protein kinase C δ proteolytic activation and DNA fragmentation in dopaminergic cells protected against dopaminergic neuronal degeneration in primary mesencephalic neuronal cultures. Furthermore, 30-day exposure of dieldrin in mouse models induced histone hyperacetylation in the striatum and substantia nigra. For the first time, our results collectively demonstrate that exposure to the neurotoxic pesticide dieldrin induces acetylation of core histones because of proteasomal dysfunction and that hyperacetylation plays a key role in dopaminergic neuronal degeneration after exposure of dieldrin.

α-Synuclein negatively regulates protein kinase Cδ expression to suppress apoptosis in dopaminergic neurons by reducing p300 histone acetyltransferase activity.

We recently demonstrated that protein kinase Cδ (PKCδ), an important member of the novel PKC family, is a key oxidative stress-sensitive kinase that can be activated by caspase-3-dependent proteolytic cleavage to induce dopaminergic neuronal cell death. We now report a novel association between α-synuclein (αsyn), a protein associated with the pathogenesis of Parkinsons disease, and PKCδ, in which αsyn negatively modulates the p300- and nuclear factor-κB (NFκB)-dependent transactivation to downregulate proapoptotic kinase PKCδ expression and thereby protects against apoptosis in dopaminergic neuronal cells. Stable expression of human wild-type αsyn at physiological levels in dopaminergic neuronal cells resulted in an isoform-dependent transcriptional suppression of PKCδ expression without changes in the stability of mRNA and protein or DNA methylation. The reduction in PKCδ transcription was mediated, in part, through the suppression of constitutive NFκB activity targeted at two proximal PKCδ promoter κB sites. This occurred independently of NFκB/IκBα (inhibitor of κBα) nuclear translocation but was associated with decreased NFκB-p65 acetylation. Also, αsyn reduced p300 levels and its HAT (histone acetyltransferase) activity, thereby contributing to diminished PKCδ transactivation. Importantly, reduced PKCδ and p300 expression also were observed within nigral dopaminergic neurons in αsyn-transgenic mice. These findings expand the role of αsyn in neuroprotection by modulating the expression of the key proapoptotic kinase PKCδ in dopaminergic neurons.

Protein Kinase Cδ Negatively Regulates Tyrosine Hydroxylase Activity and Dopamine Synthesis by Enhancing Protein Phosphatase-2A Activity in Dopaminergic Neurons

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 Cδ (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 colocalizes with TH. Interestingly, suppression of PKCδ activity with the kinase inhibitor rottlerin, PKCδ-small interfering RNA, 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 knock-out (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 in wild-type mice. Importantly, this was accompanied by an increased dopamine level in the striatum of PKCδ−/− mice. Collectively, these results suggest that PKCδ phosphorylates PP2Ac to enhance its activity and thereby reduces TH-ser40 phosphorylation and TH activity and ultimately dopamine synthesis.

Manganese nanoparticle activates mitochondrial dependent apoptotic signaling and autophagy in dopaminergic neuronal cells

The production of man-made nanoparticles for various modern applications has increased exponentially in recent years, but the potential health effects of most nanoparticles are not well characterized. Unfortunately, in vitro nanoparticle toxicity studies are extremely limited by yet unresolved problems relating to dosimetry. In the present study, we systematically characterized manganese (Mn) nanoparticle sizes and examined the nanoparticle-induced oxidative signaling in dopaminergic neuronal cells. Differential interference contrast (DIC) microscopy and transmission electron microscopy (TEM) studies revealed that Mn nanoparticles range in size from single nanoparticles (~25 nM) to larger agglomerates when in treatment media. Manganese nanoparticles were effectively internalized in N27 dopaminergic neuronal cells, and they induced a time-dependent upregulation of the transporter protein transferrin. Exposure to 25-400 μg/mL Mn nanoparticles induced cell death in a time- and dose-dependent manner. Mn nanoparticles also significantly increased ROS, accompanied by a caspase-mediated proteolytic cleavage of proapoptotic protein kinase Cδ (PKCδ), as well as activation loop phosphorylation. Blocking Mn nanoparticle-induced ROS failed to protect against the neurotoxic effects, suggesting the involvement of other pathways. Further mechanistic studies revealed changes in Beclin 1 and LC3, indicating that Mn nanoparticles induce autophagy. Primary mesencephalic neuron exposure to Mn nanoparticles induced loss of TH positive dopaminergic neurons and neuronal processes. Collectively, our results suggest that Mn nanoparticles effectively enter dopaminergic neuronal cells and exert neurotoxic effects by activating an apoptotic signaling pathway and autophagy, emphasizing the need for assessing possible health risks associated with an increased use of Mn nanoparticles in modern applications.

Vanadium induces dopaminergic neurotoxicity via protein kinase Cdelta dependent oxidative signaling mechanisms: Relevance to etiopathogenesis of Parkinson's disease

Environmental exposure to neurotoxic metals through various sources including exposure to welding fumes has been linked to an increased incidence of Parkinsons disease (PD). Welding fumes contain many different metals including vanadium typically present as particulates containing vanadium pentoxide (V2O5). However, possible neurotoxic effects of this metal oxide on dopaminergic neuronal cells are not well studied. In the present study, we characterized vanadium-induced oxidative stress-dependent cellular events in cell culture models of PD. V2O5 was neurotoxic to dopaminergic neuronal cells including primary nigral dopaminergic neurons and the EC50 was determined to be 37 microM in N27 dopaminergic neuronal cell model. The neurotoxic effect was accompanied by a time-dependent uptake of vanadium and upregulation of metal transporter proteins Tf and DMT1 in N27 cells. Additionally, vanadium resulted in a threefold increase in reactive oxygen species generation, followed by release of mitochondrial cytochrome c into cytoplasm and subsequent activation of caspase-9 (>fourfold) and caspase-3 (>ninefold). Interestingly, vanadium exposure induced proteolytic cleavage of native protein kinase Cdelta (PKCdelta, 72-74 kDa) to yield a 41 kDa catalytically active fragment resulting in a persistent increase in PKCdelta kinase activity. Co-treatment with pan-caspase inhibitor Z-VAD-FMK significantly blocked vanadium-induced PKCdelta proteolytic activation, indicating that caspases mediate PKCdelta cleavage. Also, co-treatment with Z-VAD-FMK almost completely inhibited V2O5-induced DNA fragmentation. Furthermore, PKCdelta knockdown using siRNA protected N27 cells from V2O5-induced apoptotic cell death. Collectively, these results demonstrate that vanadium can exert neurotoxic effects in dopaminergic neuronal cells via caspase-3-dependent PKCdelta cleavage, suggesting that metal exposure may promote nigral dopaminergic degeneration.

Microarray Analysis of Oxidative Stress Regulated Genes in Mesencephalic Dopaminergic Neuronal Cells: Relevance to Oxidative Damage in Parkinson’s Disease

Oxidative stress and apoptotic cell death have been implicated in the dopaminergic cell loss that characterizes Parkinsons disease. While factors contributing to apoptotic cell death are not well characterized, oxidative stress is known to activate an array of cell signaling molecules that participate in apoptotic cell death mechanisms. We investigated oxidative stress-induced cytotoxicity of hydrogen peroxide (H2O2) in three cell lines, the dopaminergic mesencephalon-derived N27 cell line, the GABAergic striatum-derived M213-20 cell line, and the hippocampal HN2-5 cell line. N27 cells were more sensitive to H2O2-induced cell death than M213-20 and HN2-5 cells. H2O2 induced significantly greater increases in caspase-3 activity in N27 cells than in M213-20 cells. H2O2-induced apoptotic cell death in N27 cells was mediated by caspase-3-dependent proteolytic activation of PKCdelta. Gene expression microarrays were employed to examine the specific transcriptional changes in N27 cells exposed to 100 microM H2O2 for 4 h. Changes in genes encoding pro- or anti-apoptotic proteins included up-regulation of BIK, PAWR, STAT5B, NPAS2, Jun B, MEK4, CCT7, PPP3CC, and PSDM3, while key down-regulated genes included BNIP3, NPTXR, RAGA, STK6, YWHAH, and MAP2K1. Overall, the changes indicate a modulation of transcriptional activity, chaperone activity, kinase activity, and apoptotic activity that appears highly specific, coordinated and relevant to cell survival. Utilizing this in vitro model to identify novel oxidative stress-regulated genes may be useful in unraveling the molecular mechanisms underlying dopaminergic degeneration in Parkinsons disease.