NEUROBIOLOGY OF DISEASE | 卷:159 |
Constitutive silencing of LRRK2 kinase activity leads to early glucocerebrosidase deregulation and late impairment of autophagy in vivo | |
Article | |
Albanese, Federica1  Mercatelli, Daniela1,2  Finetti, Luca3  Lamonaca, Giulia4  Pizzi, Sara4  Shimshek, Derya R.5  Bernacchia, Giovanni3  Morari, Michele1  | |
[1] Univ Ferrara, Dept Neurosci & Rehabil, Sect Pharmacol, Via Fossato di Mortara 17-19, I-44121 Ferrara, Italy | |
[2] LTTA Lab Adv Therapies, Technopole Ferrara, I-44121 Ferrara, Italy | |
[3] Univ Ferrara, Dept Life Sci & Biotechnol, I-44121 Ferrara, Italy | |
[4] Univ Lubeck, Inst Biomed, Eurac Res Affiliated Inst, I-39100 Bolzano, Italy | |
[5] Novartis Pharma AG, Dept Neurosci, Novartis Inst BioMed Res, CH-4002 Basel, Switzerland | |
关键词: Autophagy; Chaperone-mediated autophagy; Chloroquine; G20195 LRRK2; Glucocerebrosidase; LC3; MLi-2; pSer129 alpha-synudein; Parkinson's disease; TFEB; | |
DOI : 10.1016/j.nbd.2021.105487 | |
来源: Elsevier | |
【 摘 要 】
Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease. LRRK2 modulates the autophagy-lysosome pathway (ALP), a clearance process subserving the quality control of cellular proteins and organelles. Since dysfunctional ALP might lead to alpha-synuclein accumulation and, hence, Parkinson's disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 alpha-synuclein inclusions were investigated in vivo. Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G20195 knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D19945 knock-in (kinase-dead) mice and wild-type controls. The lysosomotmpic agent chlomquine was used to investigate the autophagic flux in vivo. Quantitative Real-time PCR was used to quantify the transcript levels of key ALP genes. The activity of the lysosomal enzyme glucocerebrosidase was measured using enzymatic assay. Immunohistochemistry was used to co-localize LC3B puncta with pSer129 alpha-synuclein inclusion in striatal and nigral neurons. No genotype differences in ALP markers were observed at 3 months. Conversely, increase of LC3-I, p62, LAMP2 and GAPDH levels, decrease of p-mTOR levels and downregulation of mTOR and TFEB expression was observed in 12-month-old kinase-dead mice. The LC3-II/I ratio was reduced following administration of chlomquine, suggesting a defective autophagic flux. G20195 knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAPILC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G20195 knock-in mice did not replicate the pattern of kinase-dead mice. Lysosomal glucocerebrosidase activity was increased in 3 and 12-month-old knock-out and kinase-dead mice. LC3B puncta accumulation and pSer129 alpha-synuclein inclusions were dissociated in striatal neurons of kinase-dead and G20195 knock-in mice. We conclude that constitutive LRRK2 kinase silencing results in early deregulation of GCase activity followed by late impairment of macroautophagy and chaperone-mediated autophagy.
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