SARS-CoV-2 Infection of Microglia Elicits Proinflammatory Activation and Apoptotic Cell Death.

Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes various neurological symptoms in patients with coronavirus disease 2019 (COVID-19). The most dominant immune cells in the brain are microglia. Yet, the relationship between neurological manifestations, neuroinflammation, and host immune response of microglia to SARS-CoV-2 has not been well characterized. Here, we reported that SARS-CoV-2 can directly infect human microglia, eliciting M1-like proinflammatory responses, followed by cytopathic effects. Specifically, SARS-CoV-2 infected human microglial clone 3 (HMC3), leading to inflammatory activation and cell death. RNA sequencing (RNA-seq) analysis also revealed that endoplasmic reticulum (ER) stress and immune responses were induced in the early, and apoptotic processes in the late phases of viral infection. SARS-CoV-2-infected HMC3 showed the M1 phenotype and produced proinflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor α (TNF-α), but not the anti-inflammatory cytokine IL-10. After this proinflammatory activation, SARS-CoV-2 infection promoted both intrinsic and extrinsic death receptor-mediated apoptosis in HMC3. Using K18-hACE2 transgenic mice, murine microglia were also infected by intranasal inoculation of SARS-CoV-2. This infection induced the acute production of proinflammatory microglial IL-6 and TNF-α and provoked a chronic loss of microglia. Our findings suggest that microglia are potential mediators of SARS-CoV-2-induced neurological problems and, consequently, can be targets of therapeutic strategies against neurological diseases in patients with COVID-19. IMPORTANCE Recent studies reported neurological and cognitive sequelae in patients with COVID-19 months after the viral infection with several symptoms, including ageusia, anosmia, asthenia, headache, and brain fog. Our conclusions raise awareness of COVID-19-related microglia-mediated neurological disorders to develop treatment strategies for the affected patients. We also indicated that HMC3 was a novel human cell line susceptible to SARS-CoV-2 infection that exhibited cytopathic effects, which could be further used to investigate cellular and molecular mechanisms of neurological manifestations of patients with COVID-19.

Interleukin 13 on Microglia is Neurotoxic in Lipopolysaccharide-injected Striatum in vivo.

To explore the potential function of interleukin-13 (IL-13), lipopolysaccharide (LPS) or PBS as a control was unilaterally microinjected into striatum of rat brain. Seven days after LPS injection, there was a significant loss of neurons and microglial activation in the striatum, visualized by immunohistochemical staining against neuronal nuclei (NeuN) and the OX-42 (complement receptor type 3, CR3), respectively. In parallel, IL-13 immunoreactivity was increased as early as 3 days and sustained up to 7 days post LPS injection, compared to PBS-injected control and detected exclusively within microglia. Moreover, GFAP immunostaining and blood brain barrier (BBB) permeability evaluation showed the loss of astrocytes and disruption of BBB, respectively. By contrast, treatment with IL-13 neutralizing antibody (IL-13NA) protects NeuN+ neurons against LPS-induced neurotoxicity in vivo . Accompanying neuroprotection, IL-13NA reduced loss of GFAP+ astrocytes and damage of BBB in LPS-injected striatum. Intriguingly, treatment with IL-13NA produced neurotrophic factors (NTFs) on survived astrocytes in LPS-injected rat striatum. Taken together, the present study suggests that LPS induces expression of IL-13 on microglia, which contributes to neurodegeneration via damage on astrocytes and BBB disruption in the striatum in vivo.

p70S6K on astrocytes protects dopamine neurons from 1-methyl-4-phenylpyridinium neurotoxicity.

Our recent finding has demonstrated that astrocytes confer neuroprotection by endogenously producing ciliary neurotrophic factor (CNTF) via transient receptor potential vanilloid 1 (TRPV1) in Parkinson's disease (PD). In this study, the possible molecular target for TRPV1-mediated CNTF production and its neuroprotective effects on dopamine neurons were further investigated. For comparison, glial cell-line derived neurotrophic factor (GDNF) was also examined. The results show that TRPV1-ribosomal protein 70 S6 kinase (p70S6K) signaling on astrocytes produces endogenous CNTF in the SN of MPP+ -lesioned rat. By marked contrast, the expression of GDNF on astrocytes is independent of TRPV1-p70S6K signaling. Administration of a TRPV1 agonist, capsaicin, increases levels of phosphorylated p70S6K (p-p70S6K; activation of p70S6K) on astrocytes, resulting in the survival of dopamine neurons and behavioral recovery through endogenous production of CNTF in the MPP+ -lesioned rat model of PD. Immunohistochemical analysis reveals expression of p-p70S6K on astrocytes in the SN of PD patients, indicating relevance to human PD. The present in vivo data is the first to demonstrate that astrocytic TRPV1-p70S6K signaling plays a pivotal role as endogenous neuroprotective, and it may constitute a novel therapeutic target for treating PD.

Interleukin-13 Propagates Prothrombin Kringle-2-Induced Neurotoxicity in Hippocampi In Vivo via Oxidative Stress.

The present study investigated expression of endogenous interleukin-13 (IL-13) and its possible function in the hippocampus of prothrombin kringle-2 (pKr-2)-lesioned rats. Here we report that intrahippocampal injection of pKr-2 revealed a significant loss of NeuN-immunopositive (NeuN+) and Nissl+ cells in the hippocampus at 7 days after pKr-2. In parallel, pKr-2 increased IL-13 levels, which reached a peak at 3 days post pKr-2 and sustained up to 7 days post pKr-2. IL-13 immunoreactivity was seen exclusively in activated microglia/macrophages and neutrophils, but not in neurons or astrocytes. In experiments designed to explore the involvement of IL-13 in neurodegeneration, IL-13 neutralizing antibody (IL-13Nab) significantly increased survival of NeuN+ and Nissl+ cells. Accompanying neuroprotection, immunohistochemical analysis indicated that IL-13Nab inhibited pKr-2-induced expression of inducible nitric oxide synthase and myeloperoxidase within activated microglia/macrophages and neutrophils, possibly resulting in attenuation of reactive oxygen species (ROS) generation and oxidative damage of DNA and protein. The current findings suggest that the endogenous IL-13 expressed in pKr-2 activated microglia/macrophages and neutrophils might be harmful to hippocampal neurons via oxidative stress.

Dynamics of T Lymphocyte between the Periphery and the Brain from the Acute to the Chronic Phase Following Ischemic Stroke in Mice.

Stroke causes systemic immunosuppression. T lymphocytes are involved in infarct size in the early stages of stroke. However, the phenotypes of T lymphocytes and their functions in peripheral immune organs and the brain have not been well analyzed in the acute and chronic phases of stroke. Here, we investigated pathological phenotypic alterations in the systemic immune response, especially changes in T lymphocytes, from one day to six months after ischemic stroke in mice. Impairment in thymocyte numbers, development, proliferation, and apoptosis were observed for up to two weeks. The number of mature T cells in the spleen and blood decreased and showed reduced interferon-γ production. Increased numbers of CD4-CD8-CD3+ double-negative T cells were observed in the mouse brain during the early stages of stroke, whereas interleukin (IL)-10+Foxp3+ regulatory T lymphocytes increased from two weeks during the chronic phase. These phenotypes correlated with body weight and neurological severity scores. The recovery of T lymphocyte numbers and increases in IL-10+Foxp3+ regulatory T lymphocytes may be important for long-term neurological outcomes. Dynamic changes in T lymphocytes between the acute and chronic phases may play different roles in pathogenesis and recovery. This study provides fundamental information regarding the T lymphocyte alterations from the brain to the peripheral immune organs following stroke.

Interleukin-4-Mediated Oxidative Stress Is Harmful to Hippocampal Neurons of Prothrombin Kringle-2-Lesioned Rat In Vivo.

The present study investigated the effects of reactive microglia/macrophages-derived interleukin-4 (IL-4) on hippocampal neurons in prothrombin kringle-2 (pKr-2)-lesioned rats. pKr-2 was unilaterally injected into hippocampus in the absence or presence of IL-4 neutralizing antibody (IL-4Nab). Immunohistochemical analysis showed a significant loss of Nissl+ and NeuN+ cells and activation of microglia/macrophages (increase in reactive OX-42+ and OX-6+ cells) in the hippocampus at 7 days after pKr-2 injection. The levels of IL-4 expression were upregulated in the reactive OX-42+ microglia/macrophages as early as 1 day, maximal at 3 days and maintained up to 7 days after pKr-2 injection. Treatment with IL-4Nab significantly increased neuronal survival in pKr-2-treated CA1 layer of hippocampus in vivo. Accompanying neuroprotection, IL-4 neutralization inhibited activation of microglia/macrophages, reactive oxygen species-derived oxidative damages, production of myeloperoxidase- and inducible nitric oxide synthase-derived reactive nitrogen species and nitrosative damages as analyzed by immunohistochemistry and hydroethidine histochemistry. These results suggest that endogenous IL-4 expressed on reactive microglia/macrophages mediates oxidative/nitrosative stress and play a critical role on neurodegeneration of hippocampal CA1 layer in vivo.

Functional Crosstalk between CB and TRPV1 Receptors Protects Nigrostriatal Dopaminergic Neurons in the MPTP Model of Parkinson's Disease.

The present study examined whether crosstalk between cannabinoid (CB) and transient potential receptor vanilloid type 1 (TRPV1) could contribute to the survival of nigrostriatal dopamine neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). MPTP induced a significant loss of nigrostriatal dopamine neurons and glial activation in the substantia nigra (SN) and striatum (STR) as visualized by tyrosine hydroxylase (TH) or macrophage antigen complex-1 (MAC-1) or glial fibrillary acidic protein (GFAP) immunocytochemistry, respectively. RT-PCR analysis shows the upregulation of inducible nitric oxide synthase, interleukin-1ß, and tumor necrosis factor-α in microglia in the SN in vivo, indicating the activation of the inflammatory system. By contrast, treatment with capsaicin (a specific TRPV1 agonist) increased the survival of dopamine neurons in the SN and their fibers and dopamine levels in the STR in MPTP mice. Capsaicin neuroprotection is accompanied by inhibiting MPTP-induced glial activation and production of inflammatory cytokines. Treatment with AM251 and AM630 (CB1/2 antagonists) abolished capsaicin-induced beneficial effects, indicating the existence of a functional crosstalk between CB and TRPV1. Moreover, treatment with anandamide (an endogenous agonist for both CB and TRVP1) rescued nigrostriatal dopamine neurons and reduced gliosis-derived neuroinflammatory responses in MPTP mice. These results suggest that the cannabinoid and vanilloid system may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with neuroinflammation.

Delayed Treatment of Capsaicin Produces Partial Motor Recovery by Enhancing Dopamine Function in MPP+-lesioned Rats via Ciliary Neurotrophic Factor.

Transient receptor potential vanilloid subtype 1 (TRPV1) on astrocytes prevents ongoing degeneration of nigrostriatal dopamine (DA) neurons in MPP+-lesioned rats via ciliary neurotrophic factor (CNTF). The present study determined whether such a beneficial effect of astrocytic TRPV1 could be achieved after completion of injury of DA neurons, rather than ongoing injury, which seems more relevant to therapeutics. To test this, the MPP+-lesioned rat model utilized here exhibited approximately 70~80% degeneration of nigrostriatal DA neurons that was completed at 2 weeks post medial forebrain bundle injection of MPP+. TRPV1 agonist, capsaicin (CAP), was intraperitoneally administered. CNTF receptor alpha neutralizing antibody (CNTFRαNAb) was nigral injected to evaluate the role of CNTF endogenously produced by astrocyte through TRPV1 activation on DA neurons. Delayed treatment of CAP produced a significant reduction in amphetamine-induced rotational asymmetry. Accompanying this behavioral recovery, CAP treatment increased CNTF levels and tyrosine hydroxylase (TH) activity in the substantia nigra pars compacta (SNpc), and levels of DA and its metabolites in the striatum compared to controls. Interestingly, behavioral recovery and increases in biochemical indices were not reflected in trophic changes of the DA system. Instead, behavioral recovery was temporal and dependent on the continuous presence of CAP treatment. The results suggest that delayed treatment of CAP increases nigral TH enzyme activity and striatal levels of DA and its metabolites by CNTF endogenously derived from CAP-activated astrocytes through TRPV1, leading to functional recovery. Consequently, these findings may be useful in the treatment of DA imbalances associated with Parkinson's disease.

Interleukin-4 and Interleukin-13 Exacerbate Neurotoxicity of Prothrombin Kringle-2 in Cortex In Vivo via Oxidative Stress.

The present study investigated the effects of activated microglia-derived interleukin-4 (IL-4) and IL-13 on neurodegeneration in prothrombin kringle-2 (pKr-2)-treated rat cortex. pKr-2 was unilaterally injected into the Sprague-Dawley rat cerebral cortex and IL-4 and IL-13 neutralizing antibody was used to block the function of IL-4 and IL-13. Immunohistochemical analysis showed a significant loss of NeuN+ and Nissl+ cells and an increase of OX-42+ cells in the cortex at seven days post pKr-2. The levels of IL-4 and IL-13 expression were upregulated in the activated microglia as early as 12 hours post pKr-2 and sustained up to seven days post pKr-2. Neutralization by IL-4 or IL-13 antibodies (NA) significantly increased neuronal survival in pKr-2-treated rat cortex in vivo by suppressing microglial activation and the production of reactive oxygen species, as analyzed by immunohisotochemistry and hydroethidine histochemistry. These results suggest that IL-4 and IL-13 that were endogenously expressed from reactive microglia may play a critical role on neuronal death by regulating oxidative stress during the neurodegenerative diseases, such as Alzheimer's disease and dementia.

Inhibition of Microglia-Derived Oxidative Stress by Ciliary Neurotrophic Factor Protects Dopamine Neurons In Vivo from MPP⁺ Neurotoxicity.

We demonstrated that capsaicin (CAP), an agonist of transient receptor potential vanilloid subtype 1 (TRPV1), inhibits microglia activation and microglia-derived oxidative stress in the substantia nigra (SN) of MPP⁺-lesioned rat. However, the detailed mechanisms how microglia-derived oxidative stress is regulated by CAP remain to be determined. Here we report that ciliary neurotrophic factor (CNTF) endogenously produced by CAP-activated astrocytes through TRPV1, but not microglia, inhibits microglial activation and microglia-derived oxidative stress, as assessed by OX-6 and OX-42 immunostaining and hydroethidine staining, respectively, resulting in neuroprotection. The significant increase in levels of CNTF receptor alpha (CNTFRα) expression was evident on microglia in the MPP⁺-lesioned rat SN and the observed beneficial effects of CNTF was abolished by treatment with CNTF receptor neutralizing antibody. It is therefore likely that CNTF can exert its effect via CNTFRα on microglia, which rescues dopamine neurons in the SN of MPP⁺-lesioned rats and ameliorates amphetamine-induced rotations. Immunohistochemical analysis revealed also a significantly increased expression of CNTFRα on microglia in the SN from human Parkinson's disease patients compared with age-matched controls, indicating that these findings may have relevance to the disease. These data suggest that CNTF originated from TRPV1 activated astrocytes may be beneficial to treat neurodegenerative disease associated with neuro-inflammation such as Parkinson's disease.

Modulation of M1/M2 polarization by capsaicin contributes to the survival of dopaminergic neurons in the lipopolysaccharide-lesioned substantia nigra in vivo.

The present study examined the neuroprotective effects of capsaicin (CAP) and explored their underlying mechanisms in a lipopolysaccharide (LPS)-lesioned inflammatory rat model of Parkinson's dieases (PD). LPS was unilaterally injected into the substantia nigra (SN) in the absence or presence of CAP or capsazepine (CZP, a TRPV1 antagonist). The SN tissues were prepared for immunohistochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, western blot analysis, blood-brain barrier (BBB) permeability evaluation, and reactive oxygen species (ROS) detection. We found that CAP prevented the degeneration of nigral dopamine neurons in a dose-dependent manner and inhibited the expression of proinflammatory mediators in the LPS-lesioned SN. CAP shifted the proinflammatory M1 microglia/macrophage population to an anti-inflammatory M2 state as demonstrated by decreased expression of M1 markers (i.e., inducible nitric oxide synthase; iNOS and interleukin-6) and elevated expression of M2 markers (i.e., arginase 1 and CD206) in the SN. RT-PCR, western blotting, and immunohistochemical analysis demonstrated decreased iNOS expression and increased arginase 1 expression in the CAP-treated LPS-lesioned SN. Peroxynitrate production, reactive oxygen species levels and oxidative damage were reduced in the CAP-treated LPS-lesioned SN. The beneficial effects of CAP were blocked by CZP, indicating TRPV1 involvement. The present data indicate that CAP regulated the M1 and M2 activation states of microglia/macrophage in the LPS-lesioned SN, which resulted in the survival of dopamine neurons. It is therefore likely that TRPV1 activation by CAP has therapeutic potential for treating neurodegenerative diseases, that are associated with neuroinflammation and oxidative stress, such as PD.

Norfluoxetine Prevents Degeneration of Dopamine Neurons by Inhibiting Microglia-Derived Oxidative Stress in an MPTP Mouse Model of Parkinson's Disease.

Neuroinflammation is the neuropathological feature of Parkinson's disease (PD) and causes microglial activation and activated microglia-derived oxidative stress in the PD patients and PD animal models, resulting in neurodegeneration. The present study examined whether norfluoxetine (a metabolite of fluoxetine) could regulate neuroinflammation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP) mouse model of PD and rescue dopamine neurons. Analysis by tyrosine hydroxylase (TH) immunohistochemistry demonstrated that norfluoxetine prevents degeneration of nigrostriatal dopamine neurons in vivo in MPTP-lesioned mice compared to vehicle-treated MPTP-lesioned control mice. MAC-1 immunostaining and hydroethidine histochemical staining showed that norfluoxetine neuroprotection is accompanied by inhibiting MPTP-induced microglial activation and activated microglia-derived reactive oxygen species production in vivo, respectively. In the separate experiments, treatment with norfluoxetine inhibited NADPH oxidase activation and nitrate production in LPS-treated cortical microglial cultures in vitro. Collectively, these in vivo and in vitro results suggest that norfluoxetine could be employed as a novel therapeutic agent for treating PD, which is associated with neuroinflammation and microglia-derived oxidative stress.

Injury-stimulated Sonic hedgehog expression in microglia contributes to neuroinflammatory response in the MPTP model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder in which dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) region are selectively destroyed. Sonic hedgehog (Shh) has been well known to play a key role in a variety of processes such as embryogenesis, cell proliferation and protection, and tissue repair during inflammation. However, the evidences for the innate role of Shh in adult brain injury are presently lacking and studies have been needed to unveil the importance of Shh in the process of neurodegeneration. Here, we investigated the role of Shh in the pathologic progress of Parkinson's disease in MPTP-induced animal model system. Interestingly, we observed that Shh expression was gradually increased in MPTP affected SNpc region. Activated microglia exclusively expressed SHH in vivo and we could recapitulate Shh induction in activated cultured primary microglia cells. Using the SHH responsive Cre-loxP binary genetic reporter transgenic mouse system, we also found that most of the cell types except for oligodendrocyte in the SNpc region reacted to the SHH by MPTP injection. Taken together, activated microglia induced Shh expression and most neural cells except oligodendrocyte responded to microglia-derived SHH in MPTP-treated SN. These results suggest that SHH in activated microglia by MPTP-injection might be involved in the innate processes of recovery from neurotoxin induced injury in the PD animal model system.

Epigenetic Modulators of Monocytic Function: Implication for Steady State and Disease in the CNS.

Epigenetic alterations are necessary for the establishment of functional and phenotypic diversity in the populations of immune cells of the monocytic lineage. The epigenetic status of individual genes at different time points defines their transcriptional responses throughout development and in response to environmental stimuli. Epigenetic states are defined at the level of DNA modifications, chromatin modifications, as well as at the level of RNA base changes through RNA editing. Drawing from lessons regarding the epigenome and epitranscriptome of cells of the monocytic lineage in the periphery, and from recently published RNAseq data deriving from brain-resident monocytes, we discuss the impact of modulation of these epigenetic states and how they affect processes important for the development of a healthy brain, as well as mechanisms of neurodegenerative disease and aging. An understanding of the varied brain responses and pathologies in light of these novel gene regulatory systems in monocytes will lead to important new insights in the understanding of the aging process and the treatment and diagnosis of neurodegenerative disease.

MMP-3 contributes to nigrostriatal dopaminergic neuronal loss, BBB damage, and neuroinflammation in an MPTP mouse model of Parkinson's disease.

The present study examined whether matrix metalloproteinase-3 (MMP-3) participates in the loss of dopaminergic (DA) neurons in the nigrostriatal pathway in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease with blood brain barrier (BBB) damage and infiltration of peripheral immune cells. Tyrosine hydroxylase (TH) immunostaining of brain sections from MPTP-treated mice showed that MPTP induced significant degeneration of nigrostriatal DA neurons. Moreover, FITC-labeled albumin detection and immunostaining revealed that MPTP caused damage to the BBB and increased the number of ED-1- and CD-3-immunopositive cells in the substantia nigra (SN). Genetic ablation of MMP-3 reduced the nigrostriatal DA neuron loss and improved motor function. This neuroprotective effect afforded by MMP-3 deletion was associated with the suppression of BBB disruption and a decrease in the number of ED-1- and CD-3-immunopositive cells in the SN. These data suggest that MMP-3 could play a crucial role in neurodegenerative diseases such as PD in which BBB damage and neuroinflammation are implicated.

Cannabinoids prevent lipopolysaccharide-induced neurodegeneration in the rat substantia nigra in vivo through inhibition of microglial activation and NADPH oxidase.

We investigated the effects of synthetic cannabinoids, WIN55,212-2 and HU210, on LPS-injected rat substantia nigra in vivo. Intranigral injection of LPS resulted in a significant loss of nigral dopaminergic (DA) neurons, as determined by Nissl staining and TH immunohistochemistry. LPS-induced neurotoxicity was accompanied by microglial activation, as demonstrated by OX-42 immunohistochemistry. In parallel, Western blot analysis, ELISA assay and hydroethidine histochemistry revealed activation of NADPH oxidase, as demonstrated by increased translocation of the cytosolic proteins p47(phox) and p67(phox), generation of reactive oxygen species (ROS) and increased level of proinflammatory cytokines (TNF-α and IL-1ß), where degeneration of nigral DA neurons was evident. Interestingly, WIN55,212-2 and HU210 increased the survival of nigral DA neurons at 7days post-LPS treatment. Consistent with these results, cannabinoids inhibited activation of NADPH oxidase, ROS production and production of proinflammatory cytokines in the rat SN. The present data suggest that cannabinoids may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with microglial activation.

The role of neuroinflammation on the pathogenesis of Parkinson's disease.

Parkinson's Disease (PD) is a common neurodegenerative disease characterized by the progressive degeneration of nigrostriatal dopaminergic (DA) neurons. Although the causative factors of PD remain elusive, many studies on PD animal models or humans suggest that glial activation along with neuroinflammatory processes contribute to the initiation or progression of PD. Additionally, several groups have proposed that dysfunction of the blood-brain barrier (BBB) combined with infiltration of peripheral immune cells play important roles in the degeneration of DA neurons. However, these neuroinflammatory events have only been investigated separately, and the issue of whether these phenomena are neuroprotective or neurotoxic remains controversial. We here review the current knowledge regarding the functions of these neuroinflammatory processes in the brain. Finally, we describe therapeutic strategies for the regulation of neuroinflammation with the goal of improving the symptoms of PD.

Prothrombin kringle-2 induces death of mesencephalic dopaminergic neurons in vivo and in vitro via microglial activation.

We have shown that prothrombin kringle-2 (pKr-2), a domain of human prothrombin distinct from thrombin could activate cultured rat brain microglia in vitro. However, little is known whether pKr-2-induced microglial activation could cause neurotoxicity on dopaminergic (DA) neurons in vivo. To address this question, pKr-2 was injected into the rat substantia nigra (SN). Tyrosine hydroxylase (TH) immunohistochemistry experiments demonstrate significant loss of DA neurons seven days after injection of pKr-2. In parallel, pKr-2-activated microglia were detected in the SN with OX-42 and OX-6 immunohistochemistry. Reverse transcription PCR and double-label immunohistochemistry revealed that activated microglia in vivo exhibit early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and several proinflammatory cytokines. The pKr-2-induced loss of SN DA neurons was partially inhibited by the NOS inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride, and the COX-2 inhibitor DuP-697. Extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were activated in the SN as early as 1 hr after pKr-2 injection, and localized within microglia. Inhibition of these kinases led to attenuation of mRNA expression of iNOS, COX-2 and several proinflammatory cytokines, and rescue of DA neurons in the SN. Intriguingly, following treatment with pKr-2 in vitro, neurotoxicity was detected exclusively in co-cultures of mesencephalic neurons and microglia, but not microglia-free neuron-enriched mesencephalic cultures, indicating that microglia are required for pKr-2 neurotoxicity. Our results strongly suggest that microglia activated by endogenous compound(s), such as pKr-2, are implicated in the DA neuronal cell death in the SN.