rapid-refined-cd11b-magnetic-isolation-primary-microglia-with

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. ...

Iowa State University

Sustained neuroinflammation mediated by resident microglia is recognized as a key pathophysiological contributor to many neurodegenerative diseases, including Parkinson's 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.

Fyn Kinase Regulates Microglial Neuroinflammatory Responses in Cell Culture and Animal 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.

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

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive motor deficits and degeneration of dopaminergic neurons. Caused by a number of genetic and environmental factors, mitochondrial dysfunction and oxidative stress play a role in neurodegeneration in PD. By selectively knocking out mitochondrial transcription factor A (TFAM) in dopaminergic neurons, the transgenic MitoPark mice recapitulate many signature features of the disease, including progressive motor deficits, neuronal loss, and protein inclusions. In the present study, we evaluated the neuroprotective efficacy of a novel mitochondrially targeted antioxidant, Mito-apocynin, in MitoPark mice and cell culture models of neuroinflammation and mitochondrial dysfunction.

Mito-Apocynin Prevents Mitochondrial Dysfunction, Microglial Activation, Oxidative Damage, and Progressive Neurodegeneration in MitoPark Transgenic Mice.

Souvarish Sarkar

Biomedical Sciences & Iowa Center for Advanced Neurotoxicology

Iowa State University

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

Souvarish Sarkar

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Iowa State University

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

Biomedical Sciences & Iowa Center for Advanced Neurotoxicology