Complement receptor 3-mediated neurotoxic glial activation contributes to rotenone-induced cognitive decline in mice.

Microglia-mediated chronic neuroinflammation has been associated with cognitive decline induced by rotenone, a well-known neurotoxic pesticide used in agriculture. However, the mechanisms remain unclear. This work aimed to elucidate the role of complement receptor 3 (CR3), a highly expressed receptor in microglia, in cognitive deficits induced by rotenone. Rotenone up-regulated the expression of CR3 in the hippocampus and cortex area of mice. CR3 deficiency markedly ameliorated rotenone-induced cognitive impairments, neurodegeneration and phosphorylation (Ser129) of α-synuclein in mice. CR3 deficiency also attenuated rotenone-stimulated microglial M1 activation. In microglial cells, siRNA-mediated knockdown of CR3 impeded, while CR3 activation induced by LL-37 exacerbated, rotenone-induced microglial M1 activation. Mechanistically, CR3 deficiency blocked rotenone-induced activation of nuclear factor κB (NF-κB), signal transducer and activator of transcription 1 (STAT1) and STAT3 signaling pathways. Pharmacological inhibition of NF-κB or STAT3 but not STAT1 was confirmed to suppress microglial M1 activation elicited by rotenone. Further study revealed that CR3 deficiency or knockdown also reduced rotenone-induced expression of C3, an A1 astrocyte marker, and production of microglial C1q, TNFα and IL-1α, a cocktail for activated microglia to induce neurotoxic A1 astrocytes, via NF-κB and STAT3 pathways. Finally, a small molecule modulator of CR3 efficiently mitigated rotenone-elicited cognitive deficits in mice even administered after the establishment of cognitive dysfunction. Taken together, our findings demonstrated that CR3 is a key factor in mediating neurotoxic glial activation and subsequent cognitive impairments in rotenone-treated mice, giving novel insights into the immunopathogenesis of cognitive impairments in pesticide-related Parkinsonism.

Integrin Mac1 mediates paraquat and maneb-induced learning and memory impairments in mice through NADPH oxidase-NLRP3 inflammasome axis-dependent microglial activation.

INTRODUCTION: The mechanisms of cognitive impairments in Parkinson's disease (PD) remain unknown. Accumulating evidence revealed that brain neuroinflammatory response mediated by microglial cells contributes to cognitive deficits in neuropathological conditions and macrophage antigen complex-1 (Mac1) is a key factor in controlling microglial activation. OBJECTIVES: To explore whether Mac1-mediated microglial activation participates in cognitive dysfunction in PD using paraquat and maneb-generated mouse PD model. METHODS: Cognitive performance was measured in wild type and Mac1-/- mice using Morris water maze test. The role and mechanisms of NADPH oxidase (NOX)-NLRP3 inflammasome axis in Mac1-mediated microglial dysfunction, neuronal damage, synaptic degeneration and phosphorylation (Ser129) of α-synuclein were explored by immunohistochemistry, Western blot and RT-PCR. RESULTS: Genetic deletion of Mac1 significantly ameliorated learning and memory impairments, neuronal damage, synaptic loss and α-synuclein phosphorylation (Ser129) caused by paraquat and maneb in mice. Subsequently, blocking Mac1 activation was found to mitigate paraquat and maneb-elicited microglial NLRP3 inflammasome activation in both in vivo and in vitro. Interestingly, stimulating activation of NOX by phorbol myristate acetate abolished the inhibitory effects of Mac1 blocking peptide RGD on paraquat and maneb-provoked NLRP3 inflammasome activation, indicating a key role of NOX in Mac1-mediated NLRP3 inflammasome activation. Furthermore, NOX1 and NOX2, two members of NOX family, and downstream PAK1 and MAPK pathways were recognized to be essential for NOX to regulate NLRP3 inflammasome activation. Finally, a NLRP3 inflammasome inhibitor glybenclamide abrogated microglial M1 activation, neurodegeneration and phosphorylation (Ser129) of α-synuclein elicited by paraquat and maneb, which were accompanied by improved cognitive capacity in mice. CONCLUSIONS: Mac1 was involved in cognitive dysfunction in a mouse PD model through NOX-NLRP3 inflammasome axis-dependent microglial activation, providing a novel mechanistic basis of cognitive decline in PD.

Microglial Activation Damages Dopaminergic Neurons through MMP-2/-9-Mediated Increase of Blood-Brain Barrier Permeability in a Parkinson's Disease Mouse Model.

Chronic neuroinflammation has been considered to be involved in the progressive dopaminergic neurodegeneration in Parkinson's disease (PD). However, the mechanisms remain unknown. Accumulating evidence indicated a key role of the blood-brain barrier (BBB) dysfunction in neurological disorders. This study is designed to elucidate whether chronic neuroinflammation damages dopaminergic neurons through BBB dysfunction by using a rotenone-induced mouse PD model. Results showed that rotenone dose-dependently induced nigral dopaminergic neurodegeneration, which was associated with increased Evans blue content and fibrinogen accumulation as well as reduced expressions of zonula occludens-1 (ZO-1), claudin-5 and occludin, three tight junction proteins for maintaining BBB permeability, in mice, indicating BBB disruption. Rotenone also induced nigral microglial activation. Depletion of microglia or inhibition of microglial activation by PLX3397 or minocycline, respectively, greatly attenuated BBB dysfunction in rotenone-lesioned mice. Mechanistic inquiry revealed that microglia-mediated activation of matrix metalloproteinases-2 and 9 (MMP-2/-9) contributed to rotenone-induced BBB disruption and dopaminergic neurodegeneration. Rotenone-induced activation of MMP-2/-9 was significantly attenuated by microglial depletion and inactivation. Furthermore, inhibition of MMP-2/-9 by a wide-range inhibitor, SB-3CT, abrogated elevation of BBB permeability and simultaneously increased tight junctions expression. Finally, we found that microglial depletion and inactivation as well as inhibition of MMP-2/-9 significantly ameliorated rotenone-elicited nigrostriatal dopaminergic neurodegeneration and motor dysfunction in mice. Altogether, our findings suggested that microglial MMP-2/-9 activation-mediated BBB dysfunction contributed to dopaminergic neurodegeneration in rotenone-induced mouse PD model, providing a novel view for the mechanisms of Parkinsonism.

Rotenone impairs learning and memory in mice through microglia-mediated blood brain barrier disruption and neuronal apoptosis.

Rotenone is a neurotoxic pesticide widely used in agriculture. Dopaminergic neuron has long been considered as the target of rotenone. We recently reported that rotenone exposure also resulted in hippocampal and cortical neurodegeneration and cognitive dysfunction in mice. However, the mechanisms remain unknown. Here, we elucidated whether blood brain barrier (BBB) disruption and subsequent neuronal apoptosis mediated by microglial activation were involved in rotenone-elicited cognitive impairments. Results showed that rotenone dose-dependently elevated evens blue extravasation, fibrinogen accumulation and reduced expressions of tight junction proteins in the hippocampus and cortex of mice. Interestingly, microglial depletion and inactivation by PLX3397 and minocycline, respectively, markedly attenuated rotenone-elicited increase of BBB permeability, indicating a critical role of microglia. Furthermore, microglial depletion and inactivation were shown to abrogate rotenone-induced activation of matrix metalloproteinases 2 and 9 (MMP-2/-9), two important factors to regulate tight junction degradation and BBB permeability, in mice. Moreover, SB-3CT, a widely used MMP-2/-9 inhibitor, increased BBB integrity and simultaneously elevated expressions of tight junction proteins in rotenone-intoxicated mice. Finally, we found that SB-3CT significantly mitigated rotenone-induced neuronal apoptosis and synaptic loss as well as learning and memory impairments in mice. Altogether, this study revealed that rotenone elicited cognitive impairments in mice through microglia-mediated BBB disruption and neuronal apoptosis via MMP-2/-9, providing a novel aspect for the pathogenesis of pesticide-induced neurotoxicity and Parkinson's disease (PD)-related dementia.

Microglial Activation Mediates Noradrenergic Locus Coeruleus Neurodegeneration via Complement Receptor 3 in a Rotenone-Induced Parkinson's Disease Mouse Model.

BACKGROUND: Chronic exposure to the insecticide rotenone can damage dopaminergic neurons and lead to an increased risk of Parkinson's disease (PD). Whereas it is not clear whether rotenone induces neurodegeneration of noradrenergic locus coeruleus (LC/NE) neurons. Chronic neuroinflammation mediated by microglia has been involved in the pathogenesis of PD. Evidence shows that complement receptor 3 (CR3) is a crucial regulator of microglial activation and related neurodegeneration. However, it is not clear whether CR3 mediates rotenone-elicited degeneration of LC/NE neurons through microglia-mediated neuroinflammation. MATERIALS AND METHODS: Wild type (WT) and CR3 knockout (KO) mice were treated with rotenone. PLX3397 and minocycline were used to deplete or inactivate the microglia. Leukadherin-1 (LA-1) was used to modulate CR3. LC/NE neurodegeneration, microglial phenotype, and expression of CR3 were determined by using immunohistochemistry, Western blot and real-time polymerase chain reaction (PCR) techniques. The glutathione (GSH) and malondialdehyde (MDA) contents were measured by using commercial kits. RESULTS: Rotenone exposure led to dose- and time-dependent LC/NE neuronal loss and microglial activation in mice. Depletion of microglia by PLX3397 or inhibition of microglial activation by minocycline significantly reduced rotenone-induced LC/NE neurodegeneration. Mechanistic studies revealed that CR3 played an essential role in the rotenone-induced activation of microglia and neurodegeneration of LC/NE neurons. Rotenone elevated the expression of CR3, and genetic ablation of CR3 markedly reduced rotenone-induced microglial activation and M1 polarization. LA-1 also suppressed rotenone-induced toxic microglial M1 activation. Furthermore, lack of CR3 or treatment with LA-1 reduced oxidative stress in the brainstem of rotenone-intoxicated mice. Finally, we found that mice deficient in CR3 or treated with LA-1 were more resistant to rotenone-induced LC/NE neurodegeneration than WT or vehicle-treated mice, respectively. CONCLUSION: Our results indicate that CR3-mediated microglial activation participates in rotenone-induced LC/NE neurodegeneration, providing novel insight into environmental toxin-induced neurotoxicity and related Parkinsonism.

Microglial activation contributes to cognitive impairments in rotenone-induced mouse Parkinson's disease model.

BACKGROUND: Cognitive decline occurs frequently in Parkinson's disease (PD), which greatly decreases the quality of life of patients. However, the mechanisms remain to be investigated. Neuroinflammation mediated by overactivated microglia is a common pathological feature in multiple neurological disorders, including PD. This study is designed to explore the role of microglia in cognitive deficits by using a rotenone-induced mouse PD model. METHODS: To evaluate the role of microglia in rotenone-induced cognitive deficits, PLX3397, an inhibitor of colony-stimulating factor 1 receptor, and minocycline, a widely used antibiotic, were used to deplete or inactivate microglia, respectively. Cognitive performance of mice among groups was detected by Morris water maze, objective recognition, and passive avoidance tests. Neurodegeneration, synaptic loss, α-synuclein phosphorylation, glial activation, and apoptosis were determined by immunohistochemistry and Western blot or immunofluorescence staining. The gene expression of inflammatory factors and lipid peroxidation were further explored by using RT-PCR and ELISA kits, respectively. RESULTS: Rotenone dose-dependently induced cognitive deficits in mice by showing decreased performance of rotenone-treated mice in the novel objective recognition, passive avoidance, and Morris water maze compared with that of vehicle controls. Rotenone-induced cognitive decline was associated with neurodegeneration, synaptic loss, and Ser129-phosphorylation of α-synuclein and microglial activation in the hippocampal and cortical regions of mice. A time course experiment revealed that rotenone-induced microglial activation preceded neurodegeneration. Interestingly, microglial depletion by PLX3397 or inactivation by minocycline significantly reduced neuronal damage and α-synuclein pathology as well as improved cognitive performance in rotenone-injected mice. Mechanistically, PLX3397 and minocycline attenuated rotenone-induced astroglial activation and production of cytotoxic factors in mice. Reduced lipid peroxidation was also observed in mice treated with combined PLX3397 or minocycline and rotenonee compared with rotenone alone group. Finally, microglial depletion or inactivation was found to mitigate rotenone-induced neuronal apoptosis. CONCLUSIONS: Taken together, our findings suggested that microglial activation contributes to cognitive impairments in a rotenone-induced mouse PD model via neuroinflammation, oxidative stress, and apoptosis, providing novel insight into the immunopathogensis of cognitive deficits in PD.

2,5-hexanedione induces NLRP3 inflammasome activation and neurotoxicity through NADPH oxidase-dependent pathway.

Chronic exposure to n-hexane causes sensorimotor neuropathy, which is mediated by 2,5-hexanedione (HD), a toxic metabolite of n-hexane. Activation of the nucleotide-binding and oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome is involved in multiple neurodegenerative diseases. However, whether the NLRP3 inflammasome contributes to HD-induced neurotoxicity remains unclear. In this study, the effects of HD on NLRP3 inflammasome activation and the underlying mechanisms were determined by using HD-treated rat and cell culture models. Increased NLRP3 expression, caspase-1 activation and interleukin-1ß production were observed in both the brain and spinal cord of HD-treated rats. Double-immunofluorescence staining showed that ASC speck formation and caspase-1 expression were mainly localized in microglia. HD-induced activation of the NLRP3 inflammasome was further mirrored in BV2 microglial cells and was associated with NADPH oxidase activation. Interestingly, inhibition of NADPH oxidase by apocynin or specific siRNAs significantly mitigated HD-induced NLRP3 inflammasome activation. Furthermore, apocynin suppressed activation of the MAPK and NF-κB signaling pathways. Blocking activation of p38-MAPK and NF-κB significantly reduced HD-induced capase-1 activation and interleukin-1ß maturation, indicating a critical role of NADPH oxidase and downstream MAPK and NF-κB pathways in regulating activation of NLRP3 inflammasome, in HD-treated microglia. Finally, we found that inhibition of microglial NLRP3 inflammasome and NADPH oxidase activation abrogated HD-induced microglial activation and neurodegeneration in both SHSY5Y neuronal cells and primary cortical neuron-glia cultures. Altogether, our findings suggest that NADPH oxidase-dependent activation of microglial NLRP3 inflammasome contributes to HD-induced neurotoxicity, providing novel insight into the mechanisms of this solvent-induced neuropathy.

NLRP3 inflammasome mediates 2,5-hexanedione-induced neurotoxicity through regulation of macrophage infiltration in rats.

Currently, whether nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation contributes to neuropathy induced by 2,5-Hexanedione (HD), the toxic metabolite of n-hexane, remains unknown. In this study, we found that HD intoxication elevated NLRP3 expression, caspase-1 activation and interleukin-1ß production in sciatic nerve of rats, indicating activation of NLRP3 inflammasome. The increased cleavage of gasdermin D (GSDMD) protein, an important mediator of pyroptosis, and axon degeneration were also observed in sciatic nerves of HD-intoxicated rats. Interestingly, glybenclamide, a widely used inhibitor of NLRP3 inflammasome, significantly reduced NLRP3 inflammasome activation, which was associated with decreased GSDMD cleavage and axon degeneration as well as improved motor performance of HD-intoxicated rats. Subsequently, we found that inhibition of NLRP3 inflammasome by glybenclamide attenuated macrophage infiltration, activation and M1 polarization in sciatic nerves of HD-intoxicated rats. Furthermore, decreased malondialdehyde (MDA) contents and increased glutathione (GSH) level and total anti-oxidative capacity were also observed in sciatic nerves of rats treated with combined glybenclamide and HD compared with HD alone group. Altogether, our findings suggest that NLRP3 inflammasome activation contributes to HD-induced neurotoxicity by enhancing macrophage infiltration and activation as well as oxidative stress, providing a novel mechanism of neuropathy induced by this neurotoxicant.