Epitranscriptomic Reader YTHDF2 Regulates SEK1( MAP2K4 )-JNK-cJUN Inflammatory Signaling in Astrocytes during Neurotoxic Stress.

As the most abundant glial cells in the CNS, astrocytes dynamically respond to neurotoxic stress, however, the key molecular regulators controlling the inflammatory status of these sentinels during neurotoxic stress have remained elusive. Herein, we demonstrate that the m6A epitranscriptomic mRNA modification tightly regulates the pro-inflammatory functions of astrocytes. Specifically, the astrocytic neurotoxic stresser, manganese (Mn), downregulated the m6A reader YTHDF2 in human and mouse astrocyte cultures and in the mouse brain. Functionally, YTHDF2 knockdown augmented, while its overexpression dampened, neurotoxic stress induced proinflammatory response, suggesting YTHDF2 serves as a key upstream regulator of inflammatory responses in astrocytes. Mechnistically, YTHDF2 RIP-sequencing identified MAP2K4 ( MKK4; SEK1) mRNA as a YTHDF2 target influencing inflammatory signaling. Our target validation revealed Mn-exposed astrocytes mediates proinflammatory response by activating the phosphorylation of SEK1, JNK, and cJUN signaling. Collectively, YTHDF2 serves a key upstream 'molecular switch' controlling SEK1( MAP2K4 )-JNK-cJUN proinflammatory signaling in astrocytes.

Kv1.3 modulates neuroinflammation and neurodegeneration in Parkinson's disease.

Characterization of the key cellular targets contributing to sustained microglial activation in neurodegenerative diseases, including Parkinson's disease (PD), and optimal modulation of these targets can provide potential treatments to halt disease progression. Here, we demonstrated that microglial Kv1.3, a voltage-gated potassium channel, was transcriptionally upregulated in response to aggregated α-synuclein (αSynAgg) stimulation in primary microglial cultures and animal models of PD, as well as in postmortem human PD brains. Patch-clamp electrophysiological studies confirmed that the observed Kv1.3 upregulation translated to increased Kv1.3 channel activity. The kinase Fyn, a risk factor for PD, modulated transcriptional upregulation and posttranslational modification of microglial Kv1.3. Multiple state-of-the-art analyses, including Duolink proximity ligation assay imaging, revealed that Fyn directly bound to Kv1.3 and posttranslationally modified its channel activity. Furthermore, we demonstrated the functional relevance of Kv1.3 in augmenting the neuroinflammatory response by using Kv1.3-KO primary microglia and the Kv1.3-specific small-molecule inhibitor PAP-1, thus highlighting the importance of Kv1.3 in neuroinflammation. Administration of PAP-1 significantly inhibited neurodegeneration and neuroinflammation in multiple animal models of PD. Collectively, our results imply that Fyn-dependent regulation of Kv1.3 channels plays an obligatory role in accentuating the neuroinflammatory response in PD and identify Kv1.3 as a potential therapeutic target for PD.

Enhanced differentiation of human dopaminergic neuronal cell model for preclinical translational research in Parkinson's disease.

Human-derived neuronal cell lines are progressively being utilized in understanding neurobiology and preclinical translational research as they are biologically more relevant than rodent-derived cells lines. The Lund human mesencephalic (LUHMES) cell line comprises human neuronal cells that can be differentiated to post-mitotic neurons and is increasingly being used as an in vitro model for various neurodegenerative diseases. A previously published 2-step differentiation procedure leads to the generation of post-mitotic neurons within 5-days, but only a small proportion (10%) of the total cell population tests positive for tyrosine hydroxylase (TH). Here we report on a novel differentiation protocol that we optimized by using a cocktail of neurotrophic factors, pleiotropic cytokines, and antioxidants to effectively generate proportionately more dopaminergic neurons within the same time period. Visualization and quantification of TH-positive cells revealed that under our new protocol, 25% of the total cell population expressed markers of dopaminergic neurons with the TH-positive neuron count peaking on day 5. These neurons showed spontaneous electrical activity and responded to known Parkinsonian toxins as expected by showing decreased cell viability and dopamine uptake and a concomitant increase in apoptotic cell death. Together, our results outline an improved method for generating a higher proportion of dopaminergic neurons, thus making these cells an ideal neuronal culture model of Parkinson's disease (PD) for translational research.

Exosomes as Mediators of Chemical-Induced Toxicity.

PURPOSE OF REVIEW: This review provides information regarding how exosomes may contribute to environmental chemical-induced pathogenesis of chronic diseases. In connecting exosome biology to environmental toxicology and disease pathogenesis, we address vital questions regarding what constitutes exosomal cargo, how toxicants influence exosomal cargo, and how environmental stimuli influence exosomal physiological and pathological functions. RECENT FINDINGS: Recent studies in the field demonstrate that exosomal cargo changes depending on external stimuli, which has consequences for the microenvironment of recipient cells. Based on recent findings, it is evident that exosomal cargo comprises various biological molecules including proteins, nucleic acids, and lipid molecules. Misfolded proteins and miRNA are examples of exosomal cargo molecules that can be altered by toxicants, ultimately changing the microenvironment of recipient cells in ways that are conducive to pathological processes. It will be crucial to map out the key signaling pathways that toxicants target to modulate exosomal cargo and their release.

Fyn kinase regulates misfolded α-synuclein uptake and NLRP3 inflammasome activation in microglia.

Persistent microglia-mediated neuroinflammation is a major pathophysiological contributor to the progression of Parkinson's disease (PD), but the cell-signaling mechanisms governing chronic neuroinflammation are not well understood. Here, we show that Fyn kinase, in conjunction with the class B scavenger receptor CD36, regulates the microglial uptake of aggregated human α-synuclein (αSyn), which is the major component of PD-associated Lewy bodies. αSyn can effectively mediate LPS-independent priming and activation of the microglial NLRP3 inflammasome. Fyn kinase regulates both of these processes; it mediates PKCδ-dependent NF-κB-p65 nuclear translocation, leading to inflammasome priming, and facilitates αSyn import into microglia, contributing to the generation of mitochondrial reactive oxygen species and consequently to inflammasome activation. In vivo experiments using A53T and viral-αSyn overexpression mouse models as well as human PD neuropathological results further confirm the role of Fyn in NLRP3 inflammasome activation. Collectively, our study identifies a novel Fyn-mediated signaling mechanism that amplifies neuroinflammation in PD.

Manganese promotes the aggregation and prion-like cell-to-cell exosomal transmission of α-synuclein.

The aggregation of α-synuclein (αSyn) is considered a key pathophysiological feature of certain neurodegenerative disorders, collectively termed synucleinopathies. Given that a prion-like, cell-to-cell transfer of misfolded αSyn has been recognized in the spreading of αSyn pathology in synucleinopathies, we investigated the biological mechanisms underlying the propagation of the disease with respect to environmental neurotoxic stress. Considering the potential role of the divalent metal manganese (Mn2+) in protein aggregation, we characterized its effect on αSyn misfolding and transmission in experimental models of Parkinson's disease. In cultured dopaminergic neuronal cells stably expressing wild-type human αSyn, misfolded αSyn was secreted through exosomes into the extracellular medium upon Mn2+ exposure. These exosomes were endocytosed through caveolae into primary microglial cells, thereby mounting neuroinflammatory responses. Furthermore, Mn2+-elicited exosomes exerted a neurotoxic effect in a human dopaminergic neuronal model (LUHMES cells). Moreover, bimolecular fluorescence complementation (BiFC) analysis revealed that Mn2+ accelerated the cell-to-cell transmission of αSyn, resulting in dopaminergic neurotoxicity in a mouse model of Mn2+ exposure. Welders exposed to Mn2+ had increased misfolded αSyn content in their serum exosomes. Stereotaxically delivering αSyn-containing exosomes, isolated from Mn2+-treated αSyn-expressing cells, into the striatum initiated Parkinsonian-like pathological features in mice. Together, these results indicate that Mn2+ exposure promotes αSyn secretion in exosomal vesicles, which subsequently evokes proinflammatory and neurodegenerative responses in both cell culture and animal models.

Manganese activates NLRP3 inflammasome signaling and propagates exosomal release of ASC in microglial cells.

Chronic, sustained inflammation underlies many pathological conditions, including neurodegenerative diseases. Divalent manganese (Mn2+) exposure can stimulate neurotoxicity by increasing inflammation. In this study, we examined whether Mn2+ activates the multiprotein NLRP3 inflammasome complex to promote neuroinflammation. Exposing activated mouse microglial cells to Mn2+ substantially augmented NLRP3 abundance, caspase-1 cleavage, and maturation of the inflammatory cytokine interleukin-1ß (IL-1ß). Exposure of mice to Mn2+ had similar effects in brain microglial cells. Furthermore, Mn2+ impaired mitochondrial ATP generation, basal respiratory rate, and spare capacity in microglial cells. These data suggest that Mn-induced mitochondrial defects drove the inflammasome signal amplification. We found that Mn induced cell-to-cell transfer of the inflammasome adaptor protein ASC in exosomes. Furthermore, primed microglial cells exposed to exosomes from Mn-treated mice released more IL-1ß than did cells exposed to exosomes from control-treated animals. We also observed that welders exposed to manganese-containing fumes had plasma exosomes that contained more ASC than did those from a matched control group. Together, these results suggest that the divalent metal manganese acts as a key amplifier of NLRP3 inflammasome signaling and exosomal ASC release.

Organophosphate pesticide chlorpyrifos impairs STAT1 signaling to induce dopaminergic neurotoxicity: Implications for mitochondria mediated oxidative stress signaling events.

The organophosphate (OP) pesticide chlorpyrifos (CPF), used in agricultural settings, induces developmental and neurological impairments. Recent studies using in vitro cell culture models have reported CPF exposure to have a positive association with mitochondria-mediated oxidative stress response and dopaminergic cell death; however, the mechanism by which mitochondrial reactive oxygen species (ROS) contribute to dopaminergic cell death remains unclear. Therefore, we hypothesized that STAT1, a transcription factor, causes apoptotic dopaminergic cell death via mitochondria-mediated oxidative stress mechanisms. Here we show that exposure of dopaminergic neuronal cells such as N27 cells (immortalized murine mesencephalic dopaminergic cells) to CPF resulted in a dose-dependent increase in apoptotic cell death as measured by MTS assay and DNA fragmentation. Similar effects were observed in CPF-treated human dopaminergic neuronal cells (LUHMES cells), with an associated increase in mitochondrial dysfunction. Moreover, CPF (10 µM) induced time-dependent increase in STAT1 activation coincided with the collapse of mitochondrial transmembrane potential, increase in ROS generation, proteolytic cleavage of protein kinase C delta (PKCδ), inhibition of the mitochondrial basal oxygen consumption rate (OCR), with a concomitant reduction in ATP-linked OCR and reserve capacity, increase in Bax/Bcl-2 ratio and enhancement of autophagy. Additionally, by chromatin immunoprecipitation (ChIP), we demonstrated that STAT1 bound to a putative regulatory sequence in the NOX1 and Bax promoter regions in response to CPF in N27 cells. Interestingly, overexpression of non-phosphorylatable STAT1 mutants (STAT1Y701F and STAT1S727A) but not STAT1 WT construct attenuated the cleavage of PKCδ and ultimately cell death in CPF-treated cells. Furthermore, small interfering RNA knockdown demonstrated STAT1 to be a critical regulator of autophagy and mitochondria-mediated proapoptotic cell signaling events after CPF treatment in N27 cells. Finally, oral administration of CPF (5 mg/kg) in postnatal rats (PNDs 27-61) induced motor deficits, and nigrostriatal dopaminergic neurodegeneration with a concomitant induction of STAT1-dependent proapoptotic cell signaling events. Conversely, co-treatment with mitoapocynin (a mitochondrially-targeted antioxidant) and CPF rescued motor deficits, and restored dopaminergic neuronal survival via abrogation of STAT1-dependent proapoptotic cell signaling events. Taken together, our study identifies a novel mechanism by which STAT1 regulates mitochondria-mediated oxidative stress response, PKCδ activation and autophagy. In this context, the phosphorylation of Tyrosine 701 and Serine 727 in STAT1 was found to be essential for PKCδ cleavage. By attenuating mitochondrial-derived ROS, mitoapocynin may have therapeutic applications for reversing CPF-induced dopaminergic neurotoxicity and associated neurobehavioral deficits as well as neurodegenerative diseases.

Characterization and comparative analysis of a new mouse microglial cell model for studying neuroinflammatory mechanisms during neurotoxic insults.

Microglia are the first responders of the central nervous system, acting as the key modulators of neuroinflammation observed during neurotoxic insults as well as in the pathophysiology of several neurodegenerative disorders including Alzheimer's (AD), Parkinson's (PD), and Huntington's diseases (HD). The number of publications on microglia has increased steadily throughout the past decade because of immense interests in the neuroinflammation that precedes the neurodegenerative process. To study microglial biology and its role in modulating neuroinflammation, immortalized microglial cell lines derived from mice, rats, and humans have been developed. Among these, the BV2 mouse microglial cell line is the most well characterized and widely used cell culture model. However, even unstimulated BV2 cells exhibit an amoeboid, hypertrophied morphology, indicating a highly activated and inflammatory state compared to primary microglia, thus making them less than ideal for studying the low-dose effects of toxicants on microglial activation. Therefore, we performed an in-depth characterization of a recently developed mouse microglial cell (MMC) line, which we compared with primary mouse microglia (PMG) and BV2s to identify which cell line was best suited for studying the microglial response to neurotoxicants. Comparative analyses reveal that MMCs are strikingly more similar to PMGs in basal activity, morphology, and sensitivity, than are BV2s. Furthermore, basal nitrite and inflammatory cytokine levels are significantly higher in BV2s compared to MMCs. BV2 cells are also less reactive to the inflammagen LPS compared to MMCs, due to the higher basal activation state of BV2s. Collectively, our in-depth analyses of morphology, basal activity, and responsivity to two different stimuli (LPS, aggregated α-synuclein) demonstrate that MMCs closely mimic neonatal PMGs, and are discernibly more suitable than BV2s for studying the neuroinflammatory mechanisms of neurotoxicants.

Environmental neurotoxicant manganese regulates exosome-mediated extracellular miRNAs in cell culture model of Parkinson's disease: Relevance to α-synuclein misfolding in metal neurotoxicity.

Many chronic neurodegenerative disorders share a common pathogenic mechanism involving the aggregation and deposition of misfolded proteins. Recently, it was shown that these aggregated proteins could be transferred from one cell to another via extracellular nanovesicles called exosomes. Initially thought to be a means of cellular waste removal, exosomes have since been discovered to actively participate in cell-to-cell communication. Importantly, various inflammatory and signaling molecules, as well as small RNAs are selectively packaged in these vesicles. Considering the important role of environmental manganese (Mn) in Parkinson's disease (PD)-like neurological disorders, we characterized the effect of Mn on exosome content and release using an MN9D dopaminergic cell model of PD, which was generated to stably express wild-type human α-synuclein (αSyn). Mn exposure (300µM MnCl2) for 24h induced the release of exosomes into the extracellular media prior to cytotoxicity, as determined by NanoSight particle analysis and electron microscopy. Strikingly, Western blot analysis revealed that Mn treatment in αSyn-expressing cells increases the protein Rab27a, which regulates the release of exosomes from cells. Moreover, next-generation sequencing showed more small RNAs in exosomes isolated from Mn-exposed cells than from control exosomes. Our miRNA profiling analysis led to the discovery of increased expression of certain miRNAs previously shown to regulate key biological pathways, including protein aggregation, autophagy, inflammation and hypoxia. Collectively, our results provide a glimpse of Mn's role in modulating extracellular miRNA content through exosomal release from dopaminergic neuronal cells and thus potentially contributing to progressive neurodegeneration. Further characterization of extracellular miRNAs and their targets will have major impacts on biomarker discovery and translational strategies for environmentally linked neurodegenerative diseases including PD.

Manganese exposure induces neuroinflammation by impairing mitochondrial dynamics in astrocytes.

Chronic manganese (Mn) exposure induces neurotoxicity, which is characterized by Parkinsonian symptoms resulting from impairment in the extrapyramidal motor system of the basal ganglia. Mitochondrial dysfunction and oxidative stress are considered key pathophysiological features of Mn neurotoxicity. Recent evidence suggests astrocytes as a major target of Mn neurotoxicity since Mn accumulates predominantly in astrocytes. However, the primary mechanisms underlying Mn-induced astroglial dysfunction and its role in metal neurotoxicity are not completely understood. In this study, we examined the interrelationship between mitochondrial dysfunction and astrocytic inflammation in Mn neurotoxicity. We first evaluated whether Mn exposure alters mitochondrial bioenergetics in cultured astrocytes. Metabolic activity assessed by MTS assay revealed an IC50 of 92.68µM Mn at 24h in primary mouse astrocytes (PMAs) and 50.46µM in the human astrocytic U373 cell line. Mn treatment reduced mitochondrial mass, indicative of impaired mitochondrial function and biogenesis, which was substantiated by the significant reduction in mRNA of mitofusin-2, a protein that serves as a ubiquitination target for mitophagy. Furthermore, Mn increased mitochondrial circularity indicating augmented mitochondrial fission. Seahorse analysis of bioenergetics status in Mn-treated astrocytes revealed that Mn significantly impaired the basal mitochondrial oxygen consumption rate as well as the ATP-linked respiration rate. The effect of Mn on mitochondrial energy deficits was further supported by a reduction in ATP production. Mn-exposed primary astrocytes also exhibited a severely quiescent energy phenotype, which was substantiated by the inability of oligomycin to increase the extracellular acidification rate. Since astrocytes regulate immune functions in the CNS, we also evaluated whether Mn modulates astrocytic inflammation. Mn exposure in astrocytes not only stimulated the release of proinflammatory cytokines, but also exacerbated the inflammatory response induced by aggregated α-synuclein. The novel mitochondria-targeted antioxidant, mito-apocynin, significantly attenuated Mn-induced inflammatory gene expression, further supporting the role of mitochondria dysfunction and oxidative stress in mediating astrogliosis. Lastly, intranasal delivery of Mn in vivo elevated GFAP and depressed TH levels in the olfactory bulbs, clearly supporting the involvement of astrocytes in Mn-induced dopaminergic neurotoxicity. Collectively, our study demonstrates that Mn drives proinflammatory events in astrocytes by impairing mitochondrial bioenergetics.

Mitochondrial impairment in microglia amplifies NLRP3 inflammasome proinflammatory signaling in cell culture and animal models of Parkinson's disease.

The NLRP3 inflammasome signaling pathway is a major contributor to the neuroinflammatory process in the central nervous system. Oxidative stress and mitochondrial dysfunction are key pathophysiological processes of many chronic neurodegenerative diseases, including Parkinson's disease (PD). However, the inter-relationship between mitochondrial defects and neuroinflammation is not well understood. In the present study, we show that impaired mitochondrial function can augment the NLRP3 inflammasome-driven proinflammatory cascade in microglia. Primary mouse microglia treated with the common inflammogen LPS increased NLRP3 and pro-IL-1ß expression. Interestingly, exposure of LPS-primed microglial cells to the mitochondrial complex-I inhibitory pesticides rotenone and tebufenpyrad specifically potentiated the NLRP3 induction, ASC speck formation and pro-IL-1ß processing to IL-1ß in a dose-dependent manner, indicating that mitochondrial impairment heightened the NLRP3 inflammasome-mediated proinflammatory response in microglia. The neurotoxic pesticide-induced NLRP3 inflammasome activation was accompanied by bioenergetic defects and lysosomal dysfunction in microglia. Furthermore, the pesticides enhanced mitochondrial ROS generation in primary microglia, while amelioration of mitochondria-derived ROS by the mitochondria-targeted antioxidant mito-apocynin completely abolished IL-1ß release, indicating mitochondrial ROS drives potentiation of the NLRP3 inflammasome in microglia. Exposure to conditioned media obtained from mitochondrial inhibitor-treated, LPS-primed microglial cells, but not unprimed cells, induced dopaminergic neurodegeneration in cultured primary mesencephalic and human dopaminergic neuronal cells (LUHMES). Notably, our in vivo results with chronic rotenone rodent models of PD further support the activation of proinflammatory NLRP3 inflammasome signaling due to mitochondrial dysfunction. Collectively, our results demonstrate that mitochondrial impairment in microglia can amplify NLRP3 inflammasome signaling, which augments the dopaminergic neurodegenerative process.

Exosomes in Toxicology: Relevance to Chemical Exposure and Pathogenesis of Environmentally Linked Diseases.

Chronic exposure to environmental toxins has been known to initiate or aggravate various neurological disorders, carcinomas and other adverse health effects. Uptake by naïve cells of pathogenic factors such as danger-associated molecules, mRNAs, miRNAs or aggregated proteins leads to disruption in cellular homeostasis further resulting in inflammation and disease propagation. Although early research tended to focus solely on exosomal removal of unwanted cellular contents, more recent reports indicate that these nano-vesicles play an active role in intercellular signaling. Not only is the exosomal cargo cell type-specific, but it also differs between healthy and dying cells. Moreover, following exosome uptake by naïve cells, the contents from these vesicles can alter the fate of recipient cells. Since exosomes can traverse long distances, they can influence distantly located cells and tissues. This review briefly explores the role played by environmental toxins in stimulating exosome release in the context of progressive neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's, as well as certain cancers such as lung, liver, ovarian, and tracheal carcinomas.

Role of neurotoxicants and traumatic brain injury in α-synuclein protein misfolding and aggregation.

Protein misfolding and aggregation are key pathological features of many neurodegenerative diseases including Parkinson's disease (PD) and other forms of human Parkinsonism. PD is a complex and multifaceted disorder whose etiology is not fully understood. However, several lines of evidence support the multiple hit hypothesis that genetic vulnerability and environmental toxicants converge to trigger PD pathology. Alpha-synuclein (α-Syn) aggregation in the brain is an important pathophysiological characteristic of synucleinopathies including PD. Epidemiological and experimental studies have shown that metals and pesticides play a crucial role in α-Syn aggregation leading to the onset of various neurodegenerative diseases including PD. In this review, we will emphasize key findings of several epidemiological as well as experimental studies of metal- and pesticide-induced α-Syn aggregation and neurodegeneration. We will also discuss other factors such as traumatic brain injury and oxidative insult in the context of α-Syn-related neurodegenerative processes.

Protein kinase Cδ upregulation in microglia drives neuroinflammatory responses and dopaminergic neurodegeneration in experimental models of Parkinson's disease.

Chronic microglial activation has been linked to the progressive degeneration of the nigrostriatal dopaminergic neurons evidenced in Parkinson's disease (PD) pathogenesis. The exact etiology of PD remains poorly understood. Although both oxidative stress and neuroinflammation are identified as co-contributors in PD pathogenesis, signaling mechanisms underlying neurodegenerative processes have yet to be defined. Indeed, we recently identified that protein kinase C delta (PKCδ) activation is critical for induction of dopaminergic neuronal loss in response to neurotoxic stressors. However, it remains to be defined whether PKCδ activation contributes to immune signaling events driving microglial neurotoxicity. In the present study, we systematically investigated whether PKCδ contributes to the heightened microglial activation response following exposure to major proinflammatory stressors, including α-synuclein, tumor necrosis factor α (TNFα), and lipopolysaccharide (LPS). We report that exposure to the aforementioned inflammatory stressors dramatically upregulated PKCδ with a concomitant increase in its kinase activity and nuclear translocation in both BV-2 microglial cells and primary microglia. Importantly, we also observed a marked upregulation of PKCδ in the microglia of the ventral midbrain region of PD patients when compared to age-matched controls, suggesting a role for microglial PKCδ in neurodegenerative processes. Further, shRNA-mediated knockdown and genetic ablation of PKCδ in primary microglia blunted the microglial proinflammatory response elicited by the inflammogens, including ROS generation, nitric oxide production, and proinflammatory cytokine and chemokine release. Importantly, we found that PKCδ activated NFκB, a key mediator of inflammatory signaling events, after challenge with inflammatory stressors, and that transactivation of NFκB led to translocation of the p65 subunit to the nucleus, IκBα degradation and phosphorylation of p65 at Ser536. Furthermore, both genetic ablation and siRNA-mediated knockdown of PKCδ attenuated NFκB activation, suggesting that PKCδ regulates NFκB activation subsequent to microglial exposure to inflammatory stimuli. To further investigate the pivotal role of PKCδ in microglial activation in vivo, we utilized pre-clinical models of PD. We found that PKCδ deficiency attenuated the proinflammatory response in the mouse substantia nigra, reduced locomotor deficits and recovered mice from sickness behavior in an LPS-induced neuroinflammation model of PD. Likewise, we found that PKCδ knockout mice treated with MPTP displayed a dampened microglial inflammatory response. Moreover, PKCδ knockout mice exhibited reduced susceptibility to the neurotoxin-induced dopaminergic neurodegeneration and associated motor impairments. Taken together, our studies propose a pivotal role for PKCδ in PD pathology, whereby sustained PKCδ activation drives sustained microglial inflammatory responses and concomitant dopaminergic neurotoxicity consequently leading to neurobehavioral deficits. We conclude that inhibiting PKCδ activation may represent a novel therapeutic strategy in PD treatment.