Souvarish Sarkar

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.

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 Parkinsons 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.

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-1β 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-1β processing to IL-1β 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-1β 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.Microglia: Powerhouse breakdown aggravates neurodegenerationA team of American researchers demonstrate that disruption of mitochondria in microglia contributes to inflammation and neurodegeneration. Anumantha G. Kanthasamy at Iowa State University in Ames, IA and colleagues examined the effect of pesticides known to impair mitochondrial function on proinflammatory signaling pathways in microglia, the brain’s immune cells. They found that both rotenone and tebufenpyrad specifically stimulated the NLRP3 inflammasome, a multi-protein complex implicated in neuroinflammatory processes. The pesticide-treated microglia were able to cause more damage to neuronal cells than the untreated ones, indicating that mitochondrial dysfunction in microglia augments neurodegeneration. The authors also show that in rodents chronically exposed to rotenone, which causes many of the features of Parkinson’s disease (PD), the NLRP3 inflammasome is activated. These findings contribute to better understand the mechanisms driving chronic neuroinflammation in PD.

Involvement of c-Abl Kinase in Microglial Activation of NLRP3 Inflammasome and Impairment in Autolysosomal System

AbstractA growing body of evidence suggests that excessive microglial activation and pesticide exposure may be linked to the etiology of PD; however, the mechanisms involved remain elusive. Emerging evidence indicates that intracellular inflammasome complex namely NLRP3 complex is involved in the recognition and execution of host inflammatory response. Thus, in the present study, we investigated the hypothesis that NLRP3 inflammasome activation is linked to rotenone (ROT)-induced microglial activation which is dependent upon a priming stimulus by a pathogen-associated molecular pattern (PAMP) or damage associated molecular pattern (DAMP), respectively. Herein using both BV2 cells and primary microglial cells, we show that LPS priming and subsequent ROT stimulation enhanced NLRP3 inflammasome activation, c-Abl and PKCδ activation, mitochondrial dysfunction, NF-κB activation, and autophagic markers, while TFEB levels were decreased dramatically. Mechanistic studies revealed c-Abl acts as a proximal signal that exacerbated the activation of the afore mentioned markers. Intriguingly, siRNA-mediated depletion or pharmacological inhibition of c-Abl via dasatinib abrogated LPS and ROT-induced microglial activation response via attenuation of NLRP3 inflammasome activation, mitochondrial oxidative stress, and ALS dysfunction. Moreover, mitoTEMPO, a mitochondrial antioxidant, attenuated NLRP3 inflammasome activation effects via blockade of c-Abl and PKCδ activation. In LPS treated mice, dasatinib attenuated NLRP3 inflammasome activation, c-Abl and PKCδ activation; and sickness behavior. Together our findings identify an exaggerated ROS/c-Abl/NLRP3 signaling axis in the heightened microglial activation response evidenced in LPS-primed ROT-stimulated microglial cells and suggest that targeting c-Abl-regulated NLRP3 inflammasome signaling offers a novel therapeutic strategy for PD treatment. Graphical Abstractᅟ

Manganese exposure induces neuroinflammation by impairing mitochondrial dynamics in astrocytes

HighlightsMn induces mitochondrial dysfunction in astrocytes.Mn exposure induces inflammatory response in astrocytes and exacerbates &agr;Synagg‐induced inflammatory response in astrocytes.Mito‐apocynin attenuates Mn‐induced inflammatory response in astrocytes by reducing mitochondrial damage.Intranasal Mn exposure induces neuroinflammation and behavioral deficits in mouse model.Thus, Mn‐induced mitochondrial defects contribute to astroglial neuroinflammation. ABSTRACT 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 92.68 &mgr;M Mn at 24 h in primary mouse astrocytes (PMAs) and 50.46 &mgr;M 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 &agr;‐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.

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinsons, Alzheimers, and Huntingtons Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

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.

Cobinamide is effective for treatment of hydrogen sulfide–induced neurological sequelae in a mouse model

Hydrogen sulfide (H2S) is a highly neurotoxic gas. Acute exposure can lead to neurological sequelae among survivors. A drug for treating neurological sequelae in survivors of acute H2S intoxication is needed. Using a novel mouse model we evaluated the efficacy of cobinamide (Cob) for increasing survival of, and reducing neurological sequalae in, mice exposed to sublethal doses of H2S. There were two objectives: (1) to determine the dose–response efficacy of Cob and (2) to determine the effective therapeutic time window of Cob. To explore objective 1, mice were injected intramuscularly with Cob at 0, 50, or 100 mg/kg at 2 min after H2S exposure. For objective 2, mice were injected intramuscularly with 100 mg/kg Cob at 2, 15, and 30 min after H2S exposure. For both objectives, mice were exposed to 765 ppm of H2S gas. Cob significantly reduced H2S‐induced lethality in a dose‐dependent manner (P < 0.05). Cob‐treated mice exhibited significantly fewer seizures and knockdowns compared with the H2S‐exposed group. Cob also reversed H2S‐induced weight loss, behavioral deficits, neurochemical changes, cytochrome c oxidase enzyme inhibition, and neurodegeneration in a dose‐ and time‐dependent manner (P < 0.01). Overall, these findings show that Cob increases survival and is neuroprotective in a mouse model of H2S‐induced neurological sequelae.

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

HIGHLIGHTSBV2s have a high basal inflammatory state and are desensitized cells.MMCs are more suitable than BV2s for studying neuro‐inflammation and ‐toxicology.MMCs are more similar to neonatal primary mouse microglia than BV2s. ABSTRACT 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 &agr;‐synuclein) demonstrate that MMCs closely mimic neonatal PMGs, and are discernibly more suitable than BV2s for studying the neuroinflammatory mechanisms of neurotoxicants.

Role of the Fyn-PKCδ signaling in SE-induced neuroinflammation and epileptogenesis in experimental models of temporal lobe epilepsy

Status epilepticus (SE) induces neuroinflammation and epileptogenesis, but the mechanisms are not yet fully delineated. The Fyn, a non-receptor Src family tyrosine kinase (SFK), and its immediate downstream target, PKCδ are emerging as potential mediators of neuroinflammation. In order to first determine the role of Fyn kinase signaling in SE, we tested the efficacy of a SFK inhibitor, saracatinib (25mg/kg, oral) in C57BL/6J mouse kainate model of acute seizures. Saracatinib pretreatment dampened SE severity and completely prevented mortality. We further utilized fyn-/- and fyn+/+ mice (wildtype control for the fyn-/- mice on same genetic background), and the rat kainate model, treated with saracatinib post-SE, to validate the role of Fyn/SFK in SE and epileptogenesis. We observed significant reduction in SE severity, epileptiform spikes, and electrographic non-convulsive seizures in fyn-/- mice when compared to fyn+/+ mice. Interestingly, significant reductions in phosphorylated pSrc-416 and PKCδ (pPKCδ-507) and naive PKCδ were observed in fyn-/- mice as compared to fyn+/+ mice suggesting that PKCδ signaling is a downstream mediator of Fyn in SE and epileptogenesis. Notably, fyn-/- mice also showed a reduction in key proinflammatory mediators TNF-α, IL-1β, and iNOS mRNA expression; serum IL-6 and IL-12 levels; and nitro-oxidative stress markers such as 4-HNE, gp91phox, and 3-NT in the hippocampus. Immunohistochemistry revealed a significant increase in reactive microgliosis and neurodegeneration in the hippocampus and hilus of dentate gyrus in fyn+/+ mice in contrast to fyn-/- mice. Interestingly, we did not observe upregulation of Fyn in pyramidal neurons of the hippocampus during post-SE in fyn+/+ mice, but it was upregulated in hilar neurons of the dentate gyrus when compared to naïve control. In reactive microglia, both Fyn and PKCδ were persistently upregulated during post-SE suggesting that Fyn-PKCδ may drive neuroinflammation during epileptogenesis. Since disabling the Fyn kinase prior to SE, either by treating with saracatinib or fyn gene knockout, suppressed seizures and the subsequent epileptogenic events, we further tested whether Fyn/SFK inhibition during post-SE modifies epileptogenesis. Telemetry-implanted, SE-induced, rats were treated with saracatinib and continuously monitored for a month. At 2h post-diazepam, the saracatinib (25mg/kg) or the vehicle was administered orally and repeated twice daily for first three days followed by a single dose/day for the next four days. The saracatinib post-treatment prevented epileptogenesis in >50% of the rats and significantly reduced spontaneous seizures and epileptiform spikes in the rest (one animal did not respond) when compared to the vehicle treated group, which had >24 seizures in a month. Collectively, the findings suggest that Fyn/SFK is a potential mediator of epileptogenesis and a therapeutic target to prevent/treat seizures and epileptogenesis.