LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

Journal of Neuroinflammation

Harris A Gelbard¹  Shao-Ming Lu¹  Justin Barbieri¹  Taryn E Mockus¹  Jenna M Puccini¹  Daniel F Marker¹ 

[1] Center for Neural Development and Disease, Department of Neurology, Child Neurology Division, University of Rochester, Rochester, NY, USA

关键词: Brain-specific angiogenesis inhibitor 1 (BAI1);    Parkinson’s disease;    AnnexinV;    Leucine-rich repeat kinase 2 (LRRK2);    Tat;    HIV-1;    Microglia;    Phagocytosis;   

Others  :  1160134
DOI  :  10.1186/1742-2094-9-261

 received in 2012-09-07, accepted in 2012-11-09,  发布年份 2012

【 摘 要 】

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.

Conclusion

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.

【 授权许可】

   
2012 Marker et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Heaton RK, Clifford DB, Franklin DR Jr, Woods SP, Ake C, Vaida F, Ellis RJ, Letendre SL, Marcotte TD, Atkinson JH, Rivera-Mindt M, Vigil OR, Taylor MJ, Collier AC, Marra CM, Gelman BB, McArthur JC, Morgello S, Simpson DM, McCutchan JA, Abramson I, Gamst A, Fennema-Notestine C, Jernigan TL, Wong J, Grant I, CHARTER Group: HIV-associated neurocognitive disorders persist in the era of potent antiretroviral therapy: CHARTER Study. Neurology 2010, 75:2087-2096.
• [2]Anderson E, Zink W, Xiong H, Gendelman HE: HIV-1-associated dementia: a metabolic encephalopathy perpetrated by virus-infected and immune-competent mononuclear phagocytes. J Acquir Immune Defic Syndr 2002, 31(Suppl 2):S43-S54.
• [3]Kure K, Lyman WD, Weidenheim KM, Dickson DW: Cellular localization of an HIV-1 antigen in subacute AIDS encephalitis using an improved double-labeling immunohistochemical method. Am J Pathol 1990, 136:1085-1092.
• [4]Mothobi NZ, Brew BJ: Neurocognitive dysfunction in the highly active antiretroviral therapy era. Curr Opin Infect Dis 2012, 25:4-9.
• [5]Cowley D, Gray LR, Wesselingh SL, Gorry PR, Churchill MJ: Genetic and functional heterogeneity of CNS-derived tat alleles from patients with HIV-associated dementia. J Neurovirol 2011, 17:70-81.
• [6]Li W, Li G, Steiner J, Nath A: Role of Tat protein in HIV neuropathogenesis. Neurotox Res 2009, 16:205-220.
• [7]Sui Z, Fan S, Sniderhan L, Reisinger E, Litzburg A, Schifitto G, Gelbard HA, Dewhurst S, Maggirwar SB: Inhibition of mixed lineage kinase 3 prevents HIV-1 Tat-mediated neurotoxicity and monocyte activation. J Immunol 2006, 177:702-711.
• [8]Lu SM, Tremblay ME, King IL, Qi J, Reynolds HM, Marker DF, Varrone JJ, Majewska AK, Dewhurst S, Gelbard HA: HIV-1 Tat-induced microgliosis and synaptic damage via interactions between peripheral and central myeloid cells. PLoS One 2011, 6:e23915.
• [9]Seol W: Biochemical and molecular features of LRRK2 and its pathophysiological roles in Parkinson’s disease. BMB Rep 2010, 43:233-244.
• [10]Gloeckner CJ, Boldt K, von Zweydorf F, Helm S, Wiesent L, Sarioglu H, Ueffing M: Phosphopeptide analysis reveals two discrete clusters of phosphorylation in the N-terminus and the Roc domain of the Parkinson-disease associated protein kinase LRRK2. J Proteome Res 2010, 9:1738-1745.
• [11]Dzamko N, Deak M, Hentati F, Reith AD, Prescott AR, Alessi DR, Nichols RJ: Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J 2010, 430:405-413.
• [12]Deng X, Dzamko N, Prescott A, Davies P, Liu Q, Yang Q, Lee JD, Patricelli MP, Nomanbhoy TK, Alessi DR, Gray NS: Characterization of a selective inhibitor of the Parkinson’s disease kinase LRRK2. Nat Chem Biol 2011, 7:203-205.
• [13]Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, Rioux JD, Brant SR, Silverberg MS, Taylor KD, Barmada MM, Bitton A, Dassopoulos T, Datta LW, Green T, Griffiths AM, Kistner EO, Murtha MT, Regueiro MD, Rotter JI, Schumm LP, Steinhart AH, Targan SR, Xavier RJ, Libioulle C, Sandor C, Lathrop M, Belaiche J, Dewit O, Gut I, Heath S, et al.: Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease. Nat Genet 2008, 40:955-962.
• [14]Zhang FR, Huang W, Chen SM, Sun LD, Liu H, Li Y, Cui Y, Yan XX, Yang HT, Yang RD, Chu TS, Zhang C, Zhang L, Han JW, Yu GQ, Quan C, Yu YX, Zhang Z, Shi BQ, Zhang LH, Cheng H, Wang CY, Lin Y, Zheng HF, Fu XA, Zuo XB, Wang Q, Long H, Sun YP, Cheng YL, et al.: Genomewide association study of leprosy. N Eng J Med 2009, 361:2609-2618.
• [15]Zimprich A, Biskup S, Leitner P, Lichtner P, Farrer M, Lincoln S, Kachergus J, Hulihan M, Uitti RJ, Calne DB, Stoessl AJ, Pfeiffer RF, Patenge N, Carbajal IC, Vieregge P, Asmus F, Müller-Myhsok B, Dickson DW, Meitinger T, Strom TM, Wszolek ZK, Gasser T: Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 2004, 44:601-607.
• [16]Hakimi M, Selvanantham T, Swinton E, Padmore RF, Tong Y, Kabbach G, Venderova K, Girardin SE, Bulman DE, Scherzer CR, LaVoie MJ, Gris D, Park DS, Angel JB, Shen J, Philpott DJ, Schlossmacher MG: Parkinson’s disease-linked LRRK2 is expressed in circulating and tissue immune cells and upregulated following recognition of microbial structures. J Neural Transm 2011, 118:795-808.
• [17]Liu Z, Lee J, Krummey S, Lu W, Cai H, Lenardo MJ: The kinase LRRK2 is a regulator of the transcription factor NFAT that modulates the severity of inflammatory bowel disease. Nat Immunol 2011, 12:1063-1070.
• [18]Moehle MS, Webber PJ, Tse T, Sukar N, Standaert DG, DeSilva TM, Cowell RM, West AB: LRRK2 inhibition attenuates microglial inflammatory responses. J Neurosci 2012, 32:1602-1611.
• [19]Tufekci KU, Meuwissen R, Genc S, Genc K: Inflammation in Parkinson’s disease. Adv Protein Chem Struct Biol 2012, 88:69-132.
• [20]Gardet A, Benita Y, Li C, Sands BE, Ballester I, Stevens C, Korzenik JR, Rioux JD, Daly MJ, Xavier RJ, Podolsky DK: LRRK2 is involved in the IFN-gamma response and host response to pathogens. J Immunol 2010, 185:5577-5585.
• [21]Kim B, Yang MS, Choi D, Kim JH, Kim HS, Seol W, Choi S, Jou I, Kim EY, Joe EH: Impaired inflammatory responses in murine lrrk2-knockdown brain microglia. PLoS One 2012, 7:e34693.
• [22]Greggio E, Civiero L, Bisaglia M, Bubacco L: Parkinson’s disease and immune system: is the culprit LRRKing in the periphery? J Neuroinflammation 2012, 9:94. BioMed Central Full Text
• [23]Gandhi PN, Chen SG, Wilson-Delfosse AL: Leucine-rich repeat kinase 2 (LRRK2): a key player in the pathogenesis of Parkinson’s disease. J Neurosci Res 2009, 87:1283-1295.
• [24]Rudenko IN, Chia R, Cookson MR: Is inhibition of kinase activity the only therapeutic strategy for LRRK2-associated Parkinson’s disease? BMC Med 2012, 10:20. BioMed Central Full Text
• [25]Chen CY, Weng YH, Chien KY, Lin KJ, Yeh TH, Cheng YP, Lu CS, Wang HL: (G2019S) LRRK2 activates MKK4-JNK pathway and causes degeneration of SN dopaminergic neurons in a transgenic mouse model of PD. Cell Death Differ 2012, 19:1623-1633.
• [26]Winner B, Melrose HL, Zhao C, Hinkle KM, Yue M, Kent C, Braithwaite AT, Ogholikhan S, Aigner R, Winkler J, Farrer MJ, Gage FH: Adult neurogenesis and neurite outgrowth are impaired in LRRK2 G2019S mice. Neurobiol Dis 2011, 41:706-716.
• [27]Melrose H, Lincoln S, Tyndall G, Dickson D, Farrer M: Anatomical localization of leucine-rich repeat kinase 2 in mouse brain. Neuroscience 2006, 139:791-794.
• [28]Tong Y, Yamaguchi H, Giaime E, Boyle S, Kopan R, Kelleher RJ 3rd, Shen J: Loss of leucine-rich repeat kinase 2 causes impairment of protein degradation pathways, accumulation of alpha-synuclein, and apoptotic cell death in aged mice. Proc Natl Acad Sci U S A 2010, 107:9879-9884.
• [29]Kim WG, Mohney RP, Wilson B, Jeohn GH, Liu B, Hong JS: Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J Neurosci 2000, 20:6309-6316.
• [30]Gillardon F, Schmid R, Draheim H: Parkinson’s disease-linked leucine-rich repeat kinase 2(R1441G) mutation increases proinflammatory cytokine release from activated primary microglial cells and resultant neurotoxicity. Neuroscience 2012, 208:41-48.
• [31]Chan D, Citro A, Cordy JM, Shen GC, Wolozin B: Rac1 protein rescues neurite retraction caused by G2019S leucine-rich repeat kinase 2 (LRRK2). J Biol Chem 2011, 286:16140-16149.
• [32]Kim HJ, Martemyanov KA, Thayer SA: Human immunodeficiency virus protein Tat induces synapse loss via a reversible process that is distinct from cell death. J Neurosci 2008, 28:12604-12613.
• [33]Glynn MW, McAllister AK: Immunocytochemistry and quantification of protein colocalization in cultured neurons. Nat Protoc 2006, 1:1287-1296.
• [34]Park JW, Vahidi B, Taylor AM, Rhee SW, Jeon NL: Microfluidic culture platform for neuroscience research. Nat Protoc 2006, 1:2128-2136.
• [35]Edelstein A, Amodaj N, Hoover K, Vale R, Stuurman N, et al.: Computer control of microscopes using microManager. In Current Protocols in Molecular Biology, Chapter 14:Unit14 20. Edited by Ausubel FM. Hoboken, NJ: Wiley-Blackwell; 2010.
• [36]Schneider CA, Rasband WS, Eliceiri KW: NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012, 9:671-675.
• [37]Meijering E, Jacob M, Sarria JC, Steiner P, Hirling H, Unser M: Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry A 2004, 58:167-176.
• [38]Bocchini V, Mazzolla R, Barluzzi R, Blasi E, Sick P, Kettenmann H: An immortalized cell line expresses properties of activated microglial cells. J Neurosci Res 1992, 31:616-621.
• [39]Tyor WR, Glass JD, Griffin JW, Becker PS, McArthur JC, Bezman L, Griffin DE: Cytokine expression in the brain during the acquired immunodeficiency syndrome. Ann Neurol 1992, 31:349-360.
• [40]Xing HQ, Hayakawa H, Izumo K, Kubota R, Gelpi E, Budka H, Izumo S: In vivo expression of proinflammatory cytokines in HIV encephalitis: an analysis of 11 autopsy cases. Neuropathology 2009, 29:433-442.
• [41]El-Hage N, Wu G, Wang J, Ambati J, Knapp PE, Reed JL, Bruce-Keller AJ, Hauser KF: HIV-1 Tat and opiate-induced changes in astrocytes promote chemotaxis of microglia through the expression of MCP-1 and alternative chemokines. Glia 2006, 53:132-146.
• [42]Eugenin EA, Osiecki K, Lopez L, Goldstein H, Calderon TM, Berman JW: CCL2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (HIV)-infected leukocytes across the blood–brain barrier: a potential mechanism of HIV-CNS invasion and NeuroAIDS. J Neurosci 2006, 26:1098-1106.
• [43]Wu Y, Tibrewal N, Birge RB: Phosphatidylserine recognition by phagocytes: a view to a kill. Trends Cell Biol 2006, 16:189-197.
• [44]Kiss RS, Elliott MR, Ma Z, Marcel YL, Ravichandran KS: Apoptotic cells induce a phosphatidylserine-dependent homeostatic response from phagocytes. Curr Biol 2006, 16:2252-2258.
• [45]Park D, Tosello-Trampont AC, Elliott MR, Lu M, Haney LB, Ma Z, Klibanov AL, Mandell JW, Ravichandran KS: BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature 2007, 450:430-434.
• [46]Masliah E, Heaton RK, Marcotte TD, Ellis RJ, Wiley CA, Mallory M, Achim CL, McCutchan JA, Nelson JA, Atkinson JH, Grant I: Dendritic injury is a pathological substrate for human immunodeficiency virus-related cognitive disorders. HNRC Group. The HIV Neurobehavioral Research Center. Ann Neurol 1997, 42:963-972.
• [47]Buscemi L, Ramonet D, Geiger JD: Human immunodeficiency virus type-1 protein Tat induces tumor necrosis factor-alpha-mediated neurotoxicity. Neurobiol Dis 2007, 26:661-670.
• [48]Brown GC, Neher JJ: Eaten alive! Cell death by primary phagocytosis: ‘phagoptosis’. Trends Biochem Sci 2012, 37:325-332.
• [49]Neher JJ, Neniskyte U, Brown GC: Primary phagocytosis of neurons by inflamed microglia: potential roles in neurodegeneration. Front Pharmacol 2012, 3:27.
• [50]Neher JJ, Neniskyte U, Zhao JW, Bal-Price A, Tolkovsky AM, Brown GC: Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death. J Immunol 2011, 186:4973-4983.
• [51]Neniskyte U, Neher JJ, Brown GC: Neuronal death induced by nanomolar amyloid beta is mediated by primary phagocytosis of neurons by microglia. J Biol Chem 2011, 286:39904-39913.
• [52]Giunta B, Zhou Y, Hou H, Rrapo E, Fernandez F, Tan J: HIV-1 TAT inhibits microglial phagocytosis of Abeta peptide. Int J Clin Exp Pathol 2008, 1:260-275.
• [53]Dzamko N, Inesta-Vaquera F, Zhang J, Xie C, Cai H, Arthur S, Tan L, Choi H, Gray N, Cohen P, Pedrioli P, Clark K, Alessi DR: The IkappaB Kinase family phosphorylates the Parkinson’s disease Kinase LRRK2 at Ser935 and Ser910 during toll-like receptor signaling. PLoS One 2012, 7:e39132.
• [54]Biskup S, Moore DJ, Celsi F, Higashi S, West AB, Andrabi SA, Kurkinen K, Yu SW, Savitt JM, Waldvogel HJ, Faull RL, Emson PC, Torp R, Ottersen OP, Dawson TM, Dawson VL: Localization of LRRK2 to membranous and vesicular structures in mammalian brain. Ann Neurol 2006, 60:557-569.
• [55]Friedman LG, Lachenmayer ML, Wang J, He L, Poulose SM, Komatsu M, Holstein GR, Yue Z: Disrupted autophagy leads to dopaminergic axon and dendrite degeneration and promotes presynaptic accumulation of alpha-synuclein and LRRK2 in the Brain. J Neurosci 2012, 32:7585-7593.
• [56]Plowey ED, Cherra SJ 3rd, Liu YJ, Chu CT: Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells. J Neurochem 2008, 105:1048-1056.
• [57]Ramonet D, Daher JP, Lin BM, Stafa K, Kim J, Banerjee R, Westerlund M, Pletnikova O, Glauser L, Yang L, Liu Y, Swing DA, Beal MF, Troncoso JC, McCaffery JM, Jenkins NA, Copeland NG, Galter D, Thomas B, Lee MK, Dawson TM, Dawson VL, Moore DJ: Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2. PLoS One 2011, 6:e18568.
• [58]Kawakami F, Yabata T, Ohta E, Maekawa T, Shimada N, Suzuki M, Maruyama H, Ichikawa T, Obata F: LRRK2 phosphorylates tubulin-associated tau but not the free molecule: LRRK2-mediated regulation of the tau-tubulin association and neurite outgrowth. PLoS One 2012, 7:e30834.
• [59]MacLeod D, Dowman J, Hammond R, Leete T, Inoue K, Abeliovich A: The familial Parkinsonism gene LRRK2 regulates neurite process morphology. Neuron 2006, 52:587-593.
• [60]Meixner A, Boldt K, Van Troys M, Askenazi M, Gloeckner CJ, Bauer M, Marto JA, Ampe C, Kinkl N, Ueffing M: A QUICK screen for Lrrk2 interaction partners--leucine-rich repeat kinase 2 is involved in actin cytoskeleton dynamics. Mol Cell Proteomics 2011, 10:M110 001172.
• [61]Abd-el-Basset EM, Fedoroff S: Dynamics of actin filaments in microglia during Fc receptor-mediated phagocytosis. Acta Neuropathol 1994, 88:527-537.