title: Clozapine metabolites protect dopaminergic neurons through inhibition of microglial NADPH oxidase.
abstract: Clozapine, an atypical antipsychotic medication, has been effectively used to treat refractory schizophrenia. However, the clinical usage of clozapine is limited due to a high incidence of neutropenia or agranulocytosis. We previously reported that clozapine protected dopaminergic neurons through inhibition of microglial activation. The purpose of this study was to explore the neuroprotective effects of clozapine metabolites clozapine N-oxide (CNO) and N-desmethylclozapine (NDC), as well as their propensity to cause neutropenia.
  
  
  
  The primary midbrain neuron-glia culture was applied to detect the neuroprotective and anti-inflammatory effect of clozapine and its metabolites in lipopolysaccharide (LPS) and MPP(+)-induced toxicity. And the subsequent mechanism was demonstrated by gp91 (phox) mutant cell cultures as well as microgliosis cell lines. In vivo, to confirm the neuroprotective effect of clozapine and CNO, we measured the dopaminergic neuronal loss and rotarod motor deficits in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-generated mouse Parkinson's disease (PD) model. The neutropenia or agranulocytosis of clozapine and its metabolites was illustrated by white blood cell count of the treated mice.
  
  
  
  We found that, in midbrain neuron-glia cultures, CNO and NDC were more potent than clozapine in protecting dopaminergic neurons against LPS and MPP(+)-induced toxicity. CNO and NDC-afforded neuroprotection was linked to inhibition of microglia-mediated neuroinflammation, as demonstrated by abolished neuroprotection in microglia-depleted cultures and their capacity of inhibiting LPS-induced release of proinflammatory factors from activated microglia. NADPH oxidase (NOX2) was subsequently recognized as the main target of CNO and NDC since genetic ablation of gp91 (phox) , the catalytic subunit of NOX2, abolished their neuroprotective effects. CNO and NDC inhibited NOX2 activation through interfering with the membrane translocation of the NOX2 cytosolic subunit, p47 (phox) . The neuroprotective effects of CNO were further verified in vivo as shown by attenuation of dopaminergic neurodegeneration, motor deficits, and reactive microgliosis in MPTP-generated mouse PD model. More importantly, unlike clozapine, CNO did not lower the white blood cell count.
  
  
  
  Altogether, our results show that clozapine metabolites elicited neuroprotection through inactivation of microglia by inhibiting NOX2. The robust neuroprotective effects and lack of neutropenia suggest that clozapine metabolites may be promising candidates for potential therapy for neurodegenerative diseases.
authors.affiliation: Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA.|Institute of Toxicology, School of Public Health, Shandong University, Jinan, Shandong, 250012, China.
authors.forename: Lulu
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Department of Physiology, Dalian Medical University, Dalian, Liaoning, 116044, China.
authors.forename: Xuefei
authors.initials: X
authors.lastname: Wu
authors.affiliation: Institute of Toxicology, School of Public Health, Shandong University, Jinan, Shandong, 250012, China.
authors.forename: Shuo
authors.initials: S
authors.lastname: Wang
authors.affiliation: Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA.
authors.forename: Shih-Heng
authors.initials: SH
authors.lastname: Chen
authors.affiliation: Department of Occupational and Environmental Health, School of Public Health, Peking University, Beijing, 100191, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA.
authors.forename: Belinda
authors.initials: B
authors.lastname: Wilson
authors.affiliation: Center for Drug Discovery, Research Triangle Institute, Research Triangle Park, NC, 27709, USA.
authors.forename: Chun-Yang
authors.initials: CY
authors.lastname: Jin
authors.affiliation: Department of Psychiatry, Institute of Behavioral Medicine, Institute of Allied Health Sciences and Addiction Research Center, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, 70101, Taiwan.|Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, 35035, Taiwan.
authors.forename: Ru-Band
authors.initials: RB
authors.lastname: Lu
authors.affiliation: Institute of Toxicology, School of Public Health, Shandong University, Jinan, Shandong, 250012, China. keqinx@sdu.edu.cn.
authors.forename: Keqin
authors.initials: K
authors.lastname: Xie
authors.affiliation: Department of Occupational and Environmental Health, School of Public Health, Dalian Medical University, Dalian, Liaoning, 116044, China. wangq4@126.com.
authors.forename: Qingshan
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Neuropharmacology Section, Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA.
authors.forename: Jau-Shyong
authors.initials: JS
authors.lastname: Hong
mesh_terms: D000818:Animals; D014150:Antipsychotic Agents / Q000378:metabolism / Q000494:pharmacology; D003024:Clozapine / Q000378:metabolism* / Q000494:pharmacology; D018920:Coculture Techniques; D059290:Dopaminergic Neurons / Q000187:drug effects / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D008070:Lipopolysaccharides / Q000633:toxicity; D008297:Male; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017628:Microglia / Q000187:drug effects / Q000201:enzymology*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000378:metabolism* / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D011916:Rats, Inbred F344
chemical_list: D014150:Antipsychotic Agents; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D018696:Neuroprotective Agents; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D003024:Clozapine
keywords: Clozapine metabolites; NADPH oxidase; Neuroinflammation; Neuroprotection; Parkinson’s disease