Protein kinase D1 (PKD1) activation mediates a compensatory protective response during early stages of oxidative stress-induced neuronal degeneration
© Asaithambi et al; licensee BioMed Central Ltd. 2011
Received: 28 January 2011
Accepted: 22 June 2011
Published: 22 June 2011
Oxidative stress is a key pathophysiological mechanism contributing to degenerative processes in many neurodegenerative diseases and therefore, unraveling molecular mechanisms underlying various stages of oxidative neuronal damage is critical to better understanding the diseases and developing new treatment modalities. We previously showed that protein kinase C delta (PKCδ) proteolytic activation during the late stages of oxidative stress is a key proapoptotic signaling mechanism that contributes to oxidative damage in Parkinson's disease (PD) models. The time course studies revealed that PKCδ activation precedes apoptotic cell death and that cells resisted early insults of oxidative damage, suggesting that some intrinsic compensatory response protects neurons from early oxidative insult. Therefore, the purpose of the present study was to characterize protective signaling pathways in dopaminergic neurons during early stages of oxidative stress.
Herein, we identify that protein kinase D1 (PKD1) functions as a key anti-apoptotic kinase to protect neuronal cells against early stages of oxidative stress. Exposure of dopaminergic neuronal cells to H2O2 or 6-OHDA induced PKD1 activation loop (PKD1S744/748) phosphorylation long before induction of neuronal cell death. Blockade of PKCδ cleavage, PKCδ knockdown or overexpression of a cleavage-resistant PKCδ mutant effectively attenuated PKD1 activation, indicating that PKCδ proteolytic activation regulates PKD1 phosphorylation. Furthermore, the PKCδ catalytic fragment, but not the regulatory fragment, increased PKD1 activation, confirming PKCδ activity modulates PKD1 activation. We also identified that phosphorylation of S916 at the C-terminal is a preceding event required for PKD1 activation loop phosphorylation. Importantly, negative modulation of PKD1 by the RNAi knockdown or overexpression of PKD1S916A phospho-defective mutants augmented oxidative stress-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 or constitutively active PKD1 plasmids attenuated oxidative stress-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury.
Collectively, our results demonstrate that PKCδ-dependent activation of PKD1 represents a novel intrinsic protective response in counteracting early stage oxidative damage in neuronal cells. Our results suggest that positive modulation of the PKD1-mediated compensatory protective mechanism against oxidative damage in dopaminergic neurons may provide novel neuroprotective strategies for treatment of PD.
Oxidative stress-induced neuronal damage has been implicated in many neurodegenerative disorders including Parkinson's disease (PD), Alzheimer's diseases, ALS, Huntington's diseases and stroke [1–7]. Neuronal cells maintain an oxidant/antioxidant homeostatic balance, and any breach in redox homeostasis causes excessive ROS production, resulting in oxidative damage [8–10]. Oxidative stress triggers apoptosis through activation of many signaling molecules including kinases and proteases [11–15], and the roles of these signaling molecules in dopaminergic cell death are being actively investigated. Recently, we demonstrated that the proteolytic activation of PKCδ, a novel PKC family member, mediates apoptosis in cell culture and animal models of PD [15–19].
PKCδ can be activated by membrane translocation, phosphorylation, or proteolytic cleavage by caspase-3, leading to persistently active catalytic fragments. We previously showed that various oxidative stressors like H2O2, MPP+ and 6-OHDA induce PKCδ cleavage to increase the kinase activity and apoptosis in dopaminergic cells [20, 15, 21, 16]. The time course studies revealed that the pro-apoptotic proteolytic activation of PKCδ occurs well before apoptotic cell death, and that cells resist early oxidative damage, suggesting that some key intrinsic compensatory responses protect neurons from the initial oxidative insult. Therefore, we speculated that the persistently active catalytic fragment of PKCδ might have other functions during the early stages of oxidative stress, and so we further explored downstream signaling mechanisms.
Protein kinase D1 (PKD1) is a calcium/calmodulin-dependent member of the CAMK kinase family and can be activated by dual phosphorylation of seine residues (Ser 744/748) in the catalytic domain by different PKCs, depending upon the cellular type and stimuli [22–24]. PKD1 is activated in response to multiple stimuli including growth factors, phorbol esters, G-protein coupled receptors, genotoxic stress and oxidative stress [25–28]. In non-neuronal cells, PKD1 activation has been shown to play a role in diverse cellular functions including proliferation, cytoskeletal reorganization, golgi function and immune response [27, 29–32]. PKD1 has been shown to regulate various cell signaling molecules and pathways including ERK1/2, JNK pathways [33–35], effector enzymes like MnSOD that scavenge ROS [31, 36], transcriptional regulators including NF-κB and MEF2 [37, 38], stress responsive chaperones like HSP27 , and some members of the HDAC family [26, 31, 40]. Recently, PKD1 was recognized as an important mitochondrial ROS sensor that translocates to the nucleus to switch on cell survival mechanisms . Also, PKD1 activation loop phosphorylation has been shown as an early marker of sympathetic neuronal DNA damage . In neuronal models, PKD1 regulates trafficking of dendritic membrane proteins to maintain neuronal polarity and synaptic plasticity .
While many biological functions of PKD1 are beginning to emerge, the role of PKD1 in the brain, specifically in the nigral dopaminergic system, remains unknown. The relationship between PKD1 signaling and neurodegeneration has not yet been examined in a single study. Herein, we demonstrate that PKD1 closely interacts with PKCδ and serves as a key compensatory protective mechanism in dopaminergic neuronal cells during the early stages of oxidative insult.
Role of PKCδ cleavage in the early stages of H2O2-induced oxidative stress in dopaminergic neuronal cells
Oxidative stress induces phosphorylation and activation of PKD1 in a time-dependent manner
To determine if oxidative stress can induce PKD1 Ser 744/Ser 748 phosphorylation in the activation loop, we examined the ability of H2O2 to induce time-dependent PKD1 activation loop phosphorylation in N27 dopaminergic cells. As shown in Figure 2C, 100 μM H2O2 induced transient PKD1 activation loop phosphorylation corresponding to the 120 kDa band starting at around 30 min, peaking at 60 min and returning to control levels after 90 min, with native protein levels remaining the same. We also observed a second band around 100 kDa, which might correspond to the other isoform, PKD2. According to the manufacturers (Cell Signaling Technology), the phospho-specific antibody can also detect PKD2 Ser 706/Ser 710 phosphorylation because of the conserved activation loop residues between PKD isoforms. However, the PKD2 activation loop phosphorylation does not follow the transient pattern of activation observed with PKD1 (Figure 2C). Furthermore, the activation loop phosphorylation of PKD1 increased the PKD1 kinase activity, as measured by a [32P] kinase assay using Syntide 2 substrate (Figure 2D). Collectively, these results demonstrate that oxidative stress activates PKD1 at early stages through phosphorylation of the dual phospho sites pS744/pS748.
Oxidative stress-induced PKD1 activation depends on PKCδ
The constitutively active catalytic fragment of PKCδ mediates PKD1 activation
PKD1 activation functions as an anti-apoptotic protective mechanism against oxidative stress
PKD1 C-terminal Ser 916 phosphorylation precedes PKD1 Ser 744/Ser 748 activation loop phosphorylation during oxidative stress
PKD1 activation acts as a protective compensatory mechanism during early stages of oxidative stress
Activated PKD1 translocates to nucleus during oxidative stress in cell culture models of neurodegeneration
Furthermore, we examined oxidative stress-induced PKD1 activation in primary mesencephalic dopaminergic neurons. Mouse primary mesencephalic neuronal cultures were treated with a low dose of 10 μM H2O2 to induce oxidative stress and then subcellular localization of PKD1 activation was monitored by TH/PKD1 double immunolabeling. Activated PKD1 (red) (PKD1 pS744/pS748) co-localized (pink) with the nuclear Hoechst stain (blue) following H2O2 treatment in primary mesencephalic neurons staining for TH (green), as visualized by fluorescence microscopy (Figure 8B). We also examined the activation profile of PKD1 pS916 C-terminal phosphorylation. PKD1pS916 (red) was localized (pink/yellow) in both cytosol and nucleus of TH +ve primary mesencephalic neurons staining for green during H2O2 -induced oxidative stress (Figure 8C).
Parkinsonian-specific toxicant causes PKD1 activation
The present study reveals a novel protective compensatory signaling mechanism via PKCδ-PKD1 molecular interaction in dopaminergic neuronal cells. Through our collective results, we report for the first time four key findings in a dopaminergic neuronal model pertinent to oxidative stress-mediated neurodegenerative processes: (i) A proteolytically activated catalytic PKCδ fragment (PKCδ-CF) phosphorylates and activates protein kinase D1 (PKD1); (ii) PKD1 activation counteracts early stage oxidative damage and protects dopaminergic neuronal cells from cytotoxicity; (iii) PKCδ-dependent phosphorylation of ser 916 residue precedes ser 744/ser 748; (iv) PKCδ - PKD1 crosstalk tightly regulates cell survival and cell death to maintain cellular homeostasis in response to oxidative damage. The elucidation of this compensatory signal transduction mechanism in neurodegenerative diseases may enhance understanding of degenerative processes and lead to development of novel treatment modalities.
H2O2-induced cytotoxicity causes apoptosis in neuronal and non-neuronal cells [15, 43, 44, 50]. Generally, oxidative stress-induced apoptosis can be classified into early and late stages. DNA fragmentation occurs in the late stage of apoptosis and is preceded by ROS generation, mitochondrial dysfunction and caspase-3 activation and membrane phosphatidyl exposure [10, 47]. In neurodegenerative disorders, especially PD, the signaling mechanisms that contribute to increased vulnerability of dopaminergic neurons to oxidative damage are still under investigation. Most current research focuses on cell death mechanisms in dopaminergic neurons. Some of the signaling kinases responsible for cell death mechanisms in PD include JNK, MLK, MAPK, LRRK2, etc. [51–54]. Earlier, the involvement of a novel biochemical mechanism for cell death in dopaminergic neurons through caspase-mediated proteolytic activation of PKCδ was demonstrated [15–19]. The high levels of persistently active PKCδ catalytic fragment mediate apoptosis during oxidative stress in both cell culture and animal models of PD [15–19]. We also have shown in our earlier study that a positive feedback loop exists during the late stages of oxidative stress, where the persistently active PKCδ catalytic fragment translocates to the mitochondria to promote cytochrome C release and apoptosis [16, 17, 55].
We previously demonstrated proteolytic activation of PKCδ occurs during the early stages of oxidative stress, even before cell death can occur, and coincides with the initiation of mitochondrial ROS generation/caspase-3 activation in dopaminergic neurons [15, 17]. Thus, we speculated that proteolytically activated PKCδ might play a regulatory role during the early stages of apoptosis. Previous research suggests the presence of a variety of protective compensatory mechanisms that counteract the early oxidative insult [8–10]. Since we observed in our present study a significant lag time before induction of cell death during the early stages of oxidative stress (Figure 1B), we hypothesize that proteolytically activated PKCδ might sense the extent of oxidative damage and act as a homeostatic regulator in response to oxidative stress, modulating cell survival and cell death mechanisms through interactions with protective signaling molecules.
Protein kinase D1 (PKD1) is emerging as an important signaling molecule associated with oxidative stress in non-neuronal cell lines [31, 35, 36]. Studies have shown that oxidative stress increases PKD1 activation loop phosphorylation (pS744/pS748) via full length PKCs, including PKCδ, in non-neuronal models [37, 56–59]. However, the functions of PKD1 during oxidative stress-induced neurodegeneration have not been studied previously. In the present study, we report that cleaved active PKCδ phosphorylates the activation loop of PKD1 and activates the kinase during the early stages of H2O2 -induced oxidative stress in dopaminergic neuronal cells. We also observed a similar activation pattern for PKD1 and PKCδ during oxidative stress caused by the parkinsonian-specific toxicant 6-OHDA (Figure 9). To our knowledge, this is the first report of a novel cell survival/cell death signal regulation by the cleaved catalytic fragment of PKCδ at two different stages of apoptosis based on the extent of oxidative damage.
PKD1 is mainly activated by a diacylglycerol-dependent PKCs mechanism [22, 60] or by PKD1 cleavage [61–63]. A recent study shows that PKD1 auto-inhibition is released through phosphorylation at the Y463 site in the regulatory domain, leading to the activation loop phosphorylation by PKCδ full length (PKCδ-FL) in Hela cells . PKD1 is in a closed conformation during the resting stage, with the regulatory fragment having an autoinhibitory effect on the catalytic fragment [46, 64]. Multiple phosphorylation sites on PKD1 seem to be important for its activation loop phosphorylation, depending on the cell types and stimuli. In human cancer cell lines, PKD1 can be phosphorylated at multiple sites including Y463, S910 (corresponding to murine Y469, S916) [24, 65]. Phosphorylation of Ser 916 (murine) autophosphorylation site correlated with PKD1 activation loop phosphorylation [58, 66]. During oxidative stress in non-neuronal models, Tyr 469 is phosphorylated by upstream kinases, which results in release of the Pleckstrin homology (PH) domain autoinhibition prior to activation loop phosphorylation; this mechanism does not involve C-terminus Ser 916 phosphorylation [24, 37]. In our dopaminergic neuronal models, oxidative stress failed to induce PKD1 Tyr 469 phosphorylation (Figure 6A), whereas PKD1 Tyr 469 phosphorylation was induced by the positive control desmopressin (Figure 6B). Our results demonstrate that the mechanism of PKD1 activation in dopaminergic neurons is distinct from the mechanisms in other non-neuronal models. We demonstrate that S916 phosphorylation, but not Tyr 469 phosphorylation, is a preceding event that occurs and is required for PKD1ser744/Ser748 activation loop phosphorylation (Figure 6). Our data suggest that Ser 916 phosphorylation on the C-terminal of PKD1 may open the conformation for full activation of the kinase through activation loop phosphorylation during oxidative stress in dopaminergic neurons. A detailed comparative analysis of PKCδ proteolytic activation, PKD1 activation loop phosphorylation and the extent of cell death during oxidative stress revealed an interesting functional relationship between activation of kinases and regulation of cell death. Comparison of PKD1 activation and cytotoxicity shows that PKD1 activation is maximal during the early oxidative stress stage when no measurable cytotoxicity is noted (Figure 7A). Interestingly, when PKD1 activation begins to decline at the end of the early stage, cell death begins to occur. Also, the level of PKCδ proteolytic activation directly correlates with the extent of cell death at the later stage of oxidative stress. When the constitutively active PKD1 mutant (PKD1S744E/S748E) is overexpressed, dopaminergic cells are resistant to H2O2 -induced neurotoxicity, even during the late stages of oxidative stress (Figure 7D), which is consistent with our hypothesis that PKD1 activation protects against oxidative damage. The downstream signaling mechanisms of PKD1 activation in dopaminergic neuronal cells are not known. PKD1 translocates to the nucleus and regulates phosphorylation of HDACs and various transcription factors in various non-neuronal cell lines including B cell, cardiomyocytes & oestoblasts [31, 23, 40, 67]. PKD1 translocation to the nucleus after activation in dopaminergic neurons is also noted in the present study (Figure 8), suggesting that nuclear translocation of PKD1 may activate key cell survival transcription factors and genes. Thus, we suggest that PKD1 functions as a cell survival switch and turns 'ON' a protective compensatory mechanism in dopaminergic neurons. Studies are underway to characterize the downstream protective response of PKD1 signaling in nigral dopaminergic neurons.
Materials and methods
The immortalized rat mesencephalic dopaminergic neuronal cell line (N27) was a kind gift from Dr. Kedar N. Prasad (University of Colorado Health Sciences Center, Denver, CO). N27 cells were grown in RPMI 1640 medium containing 10% fetal bovine serum, 2 mm l-glutamine, 50 units of penicillin, and 50 μg/ml streptomycin. Cells were maintained in a humidified atmosphere of 5% CO2 at 37°C, as described previously . N27 cells are used widely as a cell culture model for PD [20, 15, 21, 16, 68].
Primary mesencephalic neuronal culture
Primary mesencephalic neuronal cultures were prepared from the ventral mesencephalon of gestational 16- to 18-day-old mouse embryos, as described earlier . Tissues were dissected from E16 to E18 mouse embryos maintained in ice cold Ca2+-free Hanks' balanced salt solution and then dissociated in Hanks' balanced salt solution containing trypsin-0.25% EDTA for 30 min at 37°C. The dissociated cells were then plated at equal density (0.5 × 106 cells) on 12 mm coverslips precoated with 0.1 mg/ml poly-D-lysine. Cultures were maintained in neurobasal medium fortified with B-27 supplements, 500 μM l-glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The cells were maintained in a humidified CO2 incubator (5% CO2 and 37°C). Half of the culture medium was replaced every 2 days. Approximately 6- to 7-day-old cultures were used for experiments. Primary mesencephalic dopaminergic neuronal cells were exposed to 10 μM for 1 h.
N27 cells were exposed to H2O2 (100 μm) for 0-4 h at 37°C. Primary neurons were exposed to H2O2 (10 μm) for 1 h. Cell lysates were used for Western blotting and immunoprecipitation studies. Untreated cells were grown in the complete medium and used as the experimental control.
Cell death was determined using the Sytox green cytotoxicity assay, after exposing the N27 cells to H2O2 (100 μm), as described previously. This cytotoxicity assay was optimized for a multiwell format, which is more efficient and sensitive than other cytotoxicity measurements [70, 71]. Briefly, N27 cells were grown in 24-well cell culture plates at 100,000 cells per well and treated with H2O2 (100 μm) and 1 μm Sytox green fluorescent dye. The Sytox green assay allows dead cells to be viewed directly under a fluorescence microscope, as well as quantitatively measured with a fluorescence microplate reader (excitation 485 nm; emission 538 nm) (Biotek). Phase contrast and fluorescent images were taken after H2O2 exposure with a NIKON TE2000 microscope, and pictures were captured with a SPOT digital camera.
ROS Generation Assay
ROS generation was monitored by CM-DCFDA dye, as described previously [15, 72]. This is a non-fluorescent dye in its reduced form, but after oxidation in the cells, the acetate group is removed by cellular esterases, resulting in fluorescence. N27 cells were seeded in 48-well plates at a confluence of 40,000 cells/well for 24 h. First, cells were loaded with 10 μM CM-DCFDA dye (Invitrogen) at 37°C for 1 h in the dark. Cells were then treated with H2O2 in Hanks' balanced salt solution (HBSS) and the fluorescence of the cells was measured using the synergy 2 fluorescence plate reader (Biotek) at various time points (excitation 485 nm; emission 538 nm).
The primary mesencephalic neurons or N27 cells after H2O2 treatment were fixed with 4% paraformaldehyde and processed for immunocytochemical staining. First, nonspecific sites were blocked with 2% bovine serum albumin, 0.5% Triton and 0.05% Tween-20 in phosphate-buffered saline (PBS) for 20 min. The cells then were incubated with antibodies directed against TH, native PKD1 and PKD1-pS744/S748 in PBS containing 1% BSA at 4°C overnight, followed by incubation with Alexa 488 and Alexa 568 conjugated secondary antibodies in PBS containing 1% BSA. Secondary antibody treatments were followed by incubation with Hoechst 33342 dye for 5 min at room temperature to stain the nucleus. The coverslips containing stained cells were washed with PBS, mounted on slides, and viewed under a Nikon inverted fluorescence microscope (model TE-2000U; Nikon, Tokyo, Japan). Both fluorescence and confocal images were captured with a SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
Western Blot Analysis
Cells were lysed in either modified RIPA buffer or M-PER buffer (Thermo Scientific) for Western blot, immunoprecipitation and kinase assays. Lysates containing equal amounts of protein were loaded in each lane and separated on 10-12% SDS-PAGE, as described previously (Kaul et al., 2003). PKD1 polyclonal (1:1000), PKCδ polyclonal (1: 1000), PKD1-pS744/S748 (1:1000), PKD1-pS916 (1:1000), PKD1-pY469 (1:1000), - and β-actin (1:10000) antibodies were used to blot the membranes. IR dye-800 conjugated anti-rabbit (1:5000) and Alexa Fluor 680 conjugated anti-mouse (1:10000) were used for antibody detection with the Odyssey IR Imaging system (LICOR), as previously described.
PKCδ Kinase Assay
Immunoprecipitation and PKCδ kinase assay were performed as described earlier . After cell lysis, cell were immunoprecipitated using a polyclonal PKCδ rabbit antibody and protein A Sepharose, and washed three times with PKCδ kinase buffer (40 mM Tris (pH 7.4), 20 mM MgCl2, 20 μM ATP, 2.5 mM CaCl2). The reaction was started by adding 20 μl of buffer containing 0.4 mg histone and 5 μCi of [γ-32P]ATP (4,500 Ci/mM). After incubation for 10 min at 30°C, SDS loading buffer (2X) was added to the samples to terminate the reaction. The reaction products were separated on SDS-PAGE (12%), and the H1-phosphorylated bands were detected using a phosphoimager (Fujifilm FLA-5100) and quantified with MultiGauge V3.0 software.
Protein Kinase D1 Kinase Assay
The cells were exposed to H2O2 (100 μM) for 1 h and cell lysates were immunoprecipitated, as previously reported, with native PKD1 antibody (Santa Cruz). The kinase reaction was carried out at room temperature for 20 min after adding 10 μl of kinase substrate mix (0.1 mM ATP + 10 μci [γ-32P] ATP + 2 ug Syntide 2 peptide substrate in kinase buffer). Kinase buffer contains 20 mM Tris pH 7.5, 10 mM MgCl2, and 1 mM DTT. The samples were centrifuged to terminate the kinase reaction, and the supernatants containing the phosphorylated peptide were applied as spots to P81 phosphocellulose squares (Whatmann). The papers were washed four times with 0.75% phosphoric acid and once with acetone and dried, and activity was determined by liquid scintillation counting. The samples were also loaded on a SDS-PAGE and probed for native PKD1 to determine equal loading.
DNA Fragmentation Assay
DNA fragmentation was measured using the Cell Death Detection ELISA Plus assay kit (Roche), for the detection of early apoptotic death, as described previously [15, 72]. After 100 μm H2O2 treatment, the cells were spun down at 200 × g for 5 min and washed once with PBS. Then cells were lysed with lysis buffer provided with the kit. After lysis, the samples were spun down at 10,000 rpm for 10 min to collect the supernatant that was used to measure DNA fragmentation. The supernatants were further dispensed into the microtiter plates coated with streptavidin containing HRP-conjugated antibody cocktail that can detect the nucleosomes. After 2 h incubation, the HRP substrate provided in the kit was added. Measurements were taken in a Synergy 2 multiwell plate reader at 405 nm, with 490 nm as a reference reading.
Transient and Stable Transfections
cDNA encoding PKCδ catalytic fragment (PKCδ-CF), PKCδ regulatory fragment (PKCδ-RF) and PKCδ caspase-resistant mutant (PKCδ-CRM) (PKCδD327A) from the pEGFPN1 vector were subcloned into the lentiviral expression vector plenti6/V5-d-TOPO in our lab (herein referred to as V5-PKCδ-CF, V5-PKCδ-RF, V5-PKCδ-CRM) [15, 73]. ViraPower Lentiviral Expression System (Invitrogen) was used to establish stable transfections of a caspase-resistant mutant of PKCδD327A . Full-length human PKD1 plasmid (PKD1-FL), PKD1 activation loop, active PKD1S744E/S748E (PKD1-CA) and PKD1S916A mutants were obtained from Addgene, Inc. [74, 49, 37]. Electroporation was carried out with an Amaxa Nucleofector transfector instrument, as per the manufacturer's protocol. The transfected cells were then transferred to T-75 flasks or 6-well plates as desired and allowed to grow for a 24 h period before the treatment.
PKCδ-siRNA was prepared by an in vitro transcription method, as described previously . PKCδ-siRNA effectively suppressed > 80% of PKCδ protein expression levels within 24 h post-transfection. Predesigned PKD1-siRNA was purchased from IDT, Inc. PKD1-siRNA effectively suppressed > 80% of PKD1 protein expression levels after 36 h post-transfection. N27 cells (50-70% confluence) were transfected with siRNA duplexes using an Amaxa Nucleofector kit (Amaxa), as described in our previous study .
Data analysis was performed using Prism 3.0 software (GraphPad Software, San Diego, CA). Bonferroni's multiple comparison testing was used to find the significant differences between treatment and control groups. Differences with p < 0.05, p < 0.01, and p < 0.001 were considered significantly different from n ≥ 6 from two or more independent experiments, and are indicated in the figures.
Protein kinase D1
Mitogen-activated protein kinases
Protein kinase C delta
Ca 2+ /Calmodulin-Dependent Protein Kinase II
c-Jun N-terminal kinases
Leucine-rich repeat kinase 2 (LRRK2)
Reactive oxygen species
Manganese superoxide dismutase
Protein kinase C
Protein kinase C alpha.
The authors also acknowledge Ms. Mary Ann deVries for her assistance in the preparation of this manuscript. This work was supported by National Institutes of Health (NIH) [Grants NS 38644, ES10586, NS65167 and ES 19267]. The W. Eugene and Linda Lloyd Endowed Chair to A.G.K. is also acknowledged.
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