RESUMO
Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), and common genetic variation in LRRK2 modifies susceptibility to Crohn's disease and leprosy. High levels of LRRK2 expression in peripheral monocytes and macrophages suggest a role for LRRK2 in these cells, yet little is known about LRRK2 expression and function in immune cells of the brain. Here, we demonstrate a role for LRRK2 in mediating microglial proinflammatory responses and morphology. In a murine model of neuroinflammation, we observe robust induction of LRRK2 in microglia. Experiments with toll-like receptor 4 (TLR4)-stimulated rat primary microglia show that inflammation increases LRRK2 activity and expression, while inhibition of LRRK2 kinase activity or knockdown of protein attenuates TNFα secretion and nitric oxide synthase (iNOS) induction. LRRK2 inhibition blocks TLR4 stimulated microglial process outgrowth and impairs ADP stimulated microglial chemotaxis. However, actin inhibitors that phenocopy inhibition of process outgrowth and chemotaxis fail to modify TLR4 stimulation of TNFα secretion and inducible iNOS induction, suggesting that LRRK2 acts upstream of cytoskeleton control as a stress-responsive kinase. These data demonstrate LRRK2 in regulating responses in immune cells of the brain and further implicate microglial involvement in late-onset PD.
Assuntos
Microglia/enzimologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Células Cultivadas , Quimiotaxia/fisiologia , Feminino , Células HEK293 , Humanos , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Proteínas Serina-Treonina Quinases/genética , Distribuição Aleatória , RatosRESUMO
Proteolytic activity for [3H]elastin, pyro-Glu-Pro-Val-pNA(S-2484), and Suc-(Ala)3-pNA(AAApNA) was demonstrated in the bound fraction extracted with 2 M KSCN + 0.1% Triton X-100 from hypersensitivity-type murine lepromas in C57BL/6N mice, while elastase-inhibitor activity was separately observed in the soluble fraction extracted with a Tris-saline buffer. Sephacryl S-200 gel chromatography showed a peak of elastolytic activity with approximately 20,000 in molecular weight. The following DEAE-Sepharose chromatography demonstrated three fractions of elastolytic activity (E-I, II, III). The inhibitory profile showed that E-I is a thiol proteinase, while E-II and E-III belong to serine proteinase-type elastases. Both E-II and E-III showed different properties with neutrophil elastase or elastase secreted from cultured macrophages, but identical characteristics to membrane bound-type elastase of monocytes. A lower level of elastolytic activity was detected in the bound fraction of nonhypersensitivity-type murine lepromas in CBA/N mice, suggesting a more involvement of membrane bound-type elastase from monocytes/macrophages during the tissue remodelings of hypersensitivity-type granulomas.
Assuntos
Granuloma/enzimologia , Infecções por Mycobacterium/enzimologia , Elastase Pancreática/metabolismo , Inibidores de Proteases/análise , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Inflamação/enzimologia , Isoenzimas/análise , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Mycobacterium lepraemurium , Inibidores de Proteases/farmacologiaRESUMO
In order to investigate the behavior of hydrolytic enzymes in chronic infections, the activities of 17 hydrolytic enzymes were tested in limb muscles, heart muscle, spleen, liver, and kidney of lepromatous mice infected with Mycobacterium lepraemurium (M. lepraemurium) and their controls. Typical increases in those enzymatic activities were seen in spleen and liver, where pathological changes were the most pronounced, especially at the 11th week after the inoculation of the bacilli. At the 16th week, the enzymatic changes became less remarkable probably because of the decreased viability of tissues in these organs. The enzymatic changes observed could not be explained as due to bacterial enzymes. These findings are compatible with the notion that the increases in hydrolytic enzyme activities are related to tissue damage caused by murine leprosy.