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Role of Microglial Proton Channel Hv1 in Paraquat-Induced Neuroinflammation

Boyle, Alexa M
http://rave.ohiolink.edu/etdc/view?acc_num=kent1532970730744641

Abstract Details

2018, MS, Kent State University, College of Arts and Sciences / School of Biomedical Sciences.
Epidemiological studies have given rise to possible etiologies of Parkinsons disease (PD), especially farmers exposed to toxicants. The herbicide paraquat is similar in structure to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and redox cycles with microglial NADPH-oxidase to form reactive oxygen species (ROS). Excessive ROS and microglial activation can damage neurons and is thought to contribute to the initiation and progression of neurodegenerative diseases, including PD. Hv1 is a voltage-gated proton channel selectively found in microglia and other immune cells where it regulates NADPH-oxidase-dependent production of ROS. Deletion of Hv1 in mice has been found to protect neurons from oxidative damage during ischemic stroke, but there have been no studies of Hv1 in PD. The purpose of this study was to determine the role of Hv1 in paraquat-induced neuroinflammation. In vitro studies with the BV2 mouse microglial cell line and primary microglia demonstrated that while paraquat does not directly activate pro-inflammatory microglia, it induces Hv1 gene and protein expression by 2-fold and 60% respectively. Direct paraquat treatment also resulted in a 25% increase in ROS production both in the BV2 microglial cell line and C57 primary microglia. However, Hv1KO (Hv1 knockout) primary microglia show complete abolishment of paraquat-induced ROS production. Direct paraquat treatment was further shown to prime the microglial NLRP3 inflammasome via up-regulation of NLRP3 and IL-1beta gene expression allowing microglia to become sensitized to further insults. This paraquat-induced priming effect was shown to be abolished in Hv1KO microglia. To assess the effect of Hv1 in the microglial response to paraquat-induced neuronal damage, we exposed the BV2 mouse microglia cells and primary microglial cells from C57 and Hv1KO mice to conditioned media from N27 dopaminergic neuronal cells treated with paraquat. The paraquat-treated N27 conditioned media induced Hv1 gene expression by 1.5-fold, as well as, pro-inflammatory genes, iNOS, IL-1beta, and TNF-alpha by 1.7, 1.7, and 1.5-fold respectively in BV2 microglia. A more robust response was found in C57 primary microglia treated with N27 paraquat conditioned media exhibiting a 4-fold increase in Hv1 gene expression and 3, 2, and 2-fold induction of pro-inflammatory genes, iNOS, IL-1beta, and TNF-alpha respectively. However, this increase in pro-inflammatory cytokine gene expression was significantly attenuated or completely abolished in Hv1KO microglia treated with N27 paraquat-treated conditioned media. Furthermore, both the BV2 microglial cell line and C57 primary microglia exhibited increases in Hv1, TNF-alpha, and nitric oxide protein levels following treatment with N27 paraquat conditioned media, an effect attenuated in Hv1KO primary microglia. Similarly, Hv1KO primary microglia exhibited abolishment of ROS and H2O2 production, a result shown in C57 primary microglia treated with conditioned media from paraquat-treated N27 cells. This anti-inflammatory effect of Hv1KO microglia was also shown with an attenuation of DNA-bound pro-inflammatory nuclear transcription factor NF-kappaB p65 resulting from treatment of N27 paraquat conditioned media. A further role was found for Hv1 using in vivo studies with C57BL6/J and Hv1KO mice exposed to repeated injections of paraquat. Alongside in vitro studies, striatum from paraquat-treated C57 mice exhibit a 6-fold increase in Hv1 gene expression compared to saline controls. Alongside increases in Hv1 gene expression, C57 mice treated with paraquat show significantly increased pro-inflammatory cytokine gene expression, an effect completely abolished in Hv1KO mice. As the substantia nigra pars compacta (SNpc) neurons are shown to degenerate in paraquat-induced Parkinsons disease models, we further investigated the role of Hv1 in the SNpc. Hv1KO mice exhibit complete abolishment of paraquat-induced ROS production in the SNpc compared to C57 mice. These data demonstrate that paraquat regulates Hv1 expression and protein levels, Hv1 is involved in the paraquat-induced production of microglial ROS and nitric oxide, as well as, the paraquat-induced priming of the microglial NLRP3 inflammasome. Furthermore, genetic knockout of Hv1 reduces this microglial pro-inflammatory response and ROS production in vitro and in vivo. Thus, Hv1 appears to be a key regulator of microglial activation and dopamine neuron vulnerability to paraquat.
Jason Richardson (Advisor)
Samuel Crish (Committee Member)
Sheila Fleming (Committee Member)
153 p.
Biomedical Research; Neurosciences
Neuroinflammation; Neurodegeneration; Paraquat; Hv1; Parkinsons disease; Reactive oxygen species; NLRP3 inflammasome

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Citations

  • Boyle, A. M. (2018). Role of Microglial Proton Channel Hv1 in Paraquat-Induced Neuroinflammation [Master's thesis, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1532970730744641

    APA Style (7th edition)

  • Boyle, Alexa. Role of Microglial Proton Channel Hv1 in Paraquat-Induced Neuroinflammation. 2018. Kent State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1532970730744641.

    MLA Style (8th edition)

  • Boyle, Alexa. "Role of Microglial Proton Channel Hv1 in Paraquat-Induced Neuroinflammation." Master's thesis, Kent State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1532970730744641

    Chicago Manual of Style (17th edition)

kent1532970730744641
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This open access ETD is published by Kent State University and OhioLINK.