期刊论文详细信息
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
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【 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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