Vellareddy Anantharam

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.

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.

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.

α-Synuclein Protects Against Manganese Neurotoxic Insult During the Early Stages of Exposure in a Dopaminergic Cell Model of Parkinson’s Disease

The pathological role of α-synuclein (α-Syn) aggregation in neurodegeneration is well recognized, but the physiological function of normal α-Syn remains unknown. As α-Syn protein contains multiple divalent metal binding sites, herein we conducted a comprehensive characterization of the role of α-Syn in manganese-induced dopaminergic neurotoxicity. We established transgenic N27 dopaminergic neuronal cells by stably expressing human wild-type α-Syn at normal physiological levels. α-Syn-expressing dopaminergic cells significantly attenuated Mn-induced neurotoxicity for 24-h exposures relative to vector control cells. To further explore cellular mechanisms, we studied the mitochondria-dependent apoptotic pathway. Analysis of a key mitochondrial apoptotic initiator, cytochrome c, revealed that α-Syn significantly reduces the Mn-induced cytochrome c release into cytosol. The downstream caspase cascade, involving caspase-9 and caspase-3 activation, during Mn exposure was also largely attenuated in Mn-treated α-Syn cells in a time-dependent manner. α-Syn cells also showed a dramatic reduction in the Mn-induced proteolytic activation of the pro-apoptotic kinase PKCδ. The generation of Mn-induced reactive oxygen species (ROS) did not differ between α-Syn and vector control cells, indicating that α-Syn exerts its protective effect independent of altering ROS generation. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed no significant differences in intracellular Mn levels between treated vector and α-Syn cells. Notably, the expression of wild-type α-Syn in primary mesencephalic cells also rescued cells from Mn-induced neurotoxicity. However, prolonged exposure to Mn promoted protein aggregation in α-Syn-expressing cells. Collectively, these results demonstrate that wild-type α-Syn exhibits neuroprotective effects against Mn-induced neurotoxicity during the early stages of exposure in a dopaminergic neuronal model of PD.

Fyn Kinase Regulates Microglial Neuroinflammatory Responses in Cell Culture and Animal Models of Parkinson's Disease.

Sustained neuroinflammation mediated by resident microglia is recognized as a key pathophysiological contributor to many neurodegenerative diseases, including Parkinsons disease (PD), but the key molecular signaling events regulating persistent microglial activation have yet to be clearly defined. In the present study, we examined the role of Fyn, a non-receptor tyrosine kinase, in microglial activation and neuroinflammatory mechanisms in cell culture and animal models of PD. The well-characterized inflammogens LPS and TNFα rapidly activated Fyn kinase in microglia. Immunocytochemical studies revealed that activated Fyn preferentially localized to the microglial plasma membrane periphery and the nucleus. Furthermore, activated Fyn phosphorylated PKCδ at tyrosine residue 311, contributing to an inflammogen-induced increase in its kinase activity. Notably, the Fyn-PKCδ signaling axis further activated the LPS- and TNFα-induced MAP kinase phosphorylation and activation of the NFκB pathway, implying that Fyn is a major upstream regulator of proinflammatory signaling. Functional studies in microglia isolated from wild-type (Fyn+/+) and Fyn knock-out (Fyn−/−) mice revealed that Fyn is required for proinflammatory responses, including cytokine release as well as iNOS activation. Interestingly, a prolonged inflammatory insult induced Fyn transcript and protein expression, indicating that Fyn is upregulated during chronic inflammatory conditions. Importantly, in vivo studies using MPTP, LPS, or 6-OHDA models revealed a greater attenuation of neuroinflammatory responses in Fyn−/− and PKCδ −/− mice compared with wild-type mice. Collectively, our data demonstrate that Fyn is a major upstream signaling mediator of microglial neuroinflammatory processes in PD. SIGNIFICANCE STATEMENT Parkinsons disease (PD) is a complex multifactorial disease characterized by the progressive loss of midbrain dopamine neurons. Sustained microglia-mediated neuroinflammation has been recognized as a major pathophysiological contributor to chronic degenerative processes in PD; however, the key molecular signaling mechanisms underlying microglial activation are not entirely clear. Herein, we identified a novel role for the non-receptor tyrosine kinase Fyn in regulating neuroinflammatory responses in microglia. Our data clearly suggest that the Fyn-PKCδ signaling axis acts as a major upstream signaling mediator of the sustained neuroinflammatory processes in cell culture and animal models of PD. Our finding has important clinical significance to PD because it identifies Fyn as a potential translational target for intervention of progressive neurodegenerative processes in PD.

Halothane acts on many potassium channels, including a minimal potassium channel

There has been considerable controversy over whether general anesthetics act directly on membrane proteins, and if so, whether there are uniquely sensitive protein targets upon which they act. Here, we examine the actions of halothane on a diverse collection of voltage-gated potassium channels expressed in Xenopus oocytes, and find that they are all sensitive at clinically relevant concentrations. To investigate further the molecular basis of this commonality, human and rat minimal potassium (minK) channels, which have exceedingly short amino acid sequences, were examined. Current through these channels is reversibly reduced to 68% of control values by 0.5% (0.34 mM) halothane. A double deletion mutant of the 130-amino acid minK protein, in which 30 amino acids of the N-terminus, thought to be extracellular, and 37 amino acids of the putative intracellular C-terminus are deleted (resulting in a protein in which more than half of both the extracellular and intracellular domains have been removed) responds to low halothane concentrations similarly to the parent channel. While alternative explanations are possible, this result is consistent with a model whereby halothane interacts with the channel protein from within the lipid bilayer.

Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling.

Although the prion protein is abundantly expressed in the CNS, its biological functions remain unclear. To determine the endogenous function of the cellular prion protein (PrP(c)), we compared the effects of oxidative stress and endoplasmic reticulum (ER) stress inducers on apoptotic signaling in PrP(c)-expressing and PrP(ko) (knockout) neural cells. H(2)O(2), brefeldin A (BFA), and tunicamycin (TUN) induced increases in caspase-9 and caspase-3, PKCdelta proteolytic activation, and DNA fragmentation in PrP(c) and PrP(ko) cells. Interestingly, ER stress-induced activation of caspases, PKCdelta, and apoptosis was significantly exacerbated in PrP(c) cells, whereas H(2)O(2)-induced proapoptotic changes were suppressed in PrP(c) compared to PrP(ko) cells. Additionally, caspase-12 and caspase-8 were activated only in the BFA and TUN treatments. Inhibitors of caspase-9, caspase-3, and PKCdelta significantly blocked H(2)O(2)-, BFA-, and TUN-induced apoptosis, whereas the caspase-8 inhibitor attenuated only BFA- and TUN-induced cell death, and the antioxidant MnTBAP blocked only H(2)O(2)-induced apoptosis. Overexpression of the kinase-inactive PKCdelta(K376R) or the cleavage site-resistant PKCdelta(D327A) mutant suppressed both ER and oxidative stress-induced apoptosis. Thus, PrP(c) plays a proapoptotic role during ER stress and an antiapoptotic role during oxidative stress-induced cell death. Together, these results suggest that cellular PrP enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases the vulnerability to oxidative insults, and that PKCdelta is a key downstream mediator of cellular stress-induced neuronal apoptosis.