title: LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein.
abstract: BACKGROUND
  Human Immunodeficiency Virus-1 (HIV-1) associated neurocognitive disorders (HANDs) are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART). While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2) as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs.
  
  
  METHODS
  We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat) protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i). We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons.
  
  
  RESULTS
  We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence of Tat-activated microglia, as well as AnnexinV, a phosphatidylserine-binding protein. In addition, LRRK2i decreased brain-specific angiogenesis inhibitor 1 (BAI1) receptor expression on BV-2 cells after Tat-treatment, a key receptor in phosphatidylserine-mediated phagocytosis.
  
  
  CONCLUSIONS
  Taken together, these results implicate LRRK2 as a key player in microglial inflammation and, in particular, in the phagocytosis of neuronal elements. These studies show that LRRK2 kinase inhibition may prove an effective therapeutic strategy for HANDs, as well as other neuroinflammatory conditions.
authors.affiliation: Center for Neural Development and Disease, Department of Neurology, Child Neurology Division, University of Rochester, Rochester, NY, USA.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Marker
authors.forename: Jenna M
authors.initials: JM
authors.lastname: Puccini
authors.forename: Taryn E
authors.initials: TE
authors.lastname: Mockus
authors.forename: Justin
authors.initials: J
authors.lastname: Barbieri
authors.forename: Shao-Ming
authors.initials: SM
authors.lastname: Lu
authors.forename: Harris A
authors.initials: HA
authors.lastname: Gelbard
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D001369:Axons / Q000187:drug effects; D039481:CD11b Antigen / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D018920:Coculture Techniques; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D015497:HIV-1 / Q000187:drug effects / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D051379:Mice; D046210:Microfluidic Analytical Techniques; D017628:Microglia / Q000187:drug effects* / Q000378:metabolism*; D008863:Microspheres; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000494:pharmacology*
chemical_list: D017304:Annexin A5; D039481:CD11b Antigen; D016207:Cytokines; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D054322:tat Gene Products, Human Immunodeficiency Virus; D012694:Serine; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases