Constitutive silencing of LRRK2 kinase activity leads to early glucocerebrosidase deregulation and late impairment of autophagy in vivo

Neurobiology of Disease

Luca Finetti¹  Michele Morari²  Giulia Lamonaca²  Daniela Mercatelli³  Federica Albanese³  Giovanni Bernacchia⁴  Sara Pizzi⁵  Derya R. Shimshek⁵ 

[1] Technopole of Ferrara, LTTA Laboratory for Advanced Therapies, 44121 Ferrara, Italy;Department of Life Sciences and Biotechnology, University of Ferrara, 44121 Ferrara, Italy;Department of Neuroscience and Rehabilitation, Section of Pharmacology, University of Ferrara, 44121 Ferrara, Italy;Department of Neuroscience, Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4002 Basel, Switzerland;Institute for Biomedicine, Eurac Research-Affiliated Institute of the University of Lübeck, 39100 Bolzano, Italy;

关键词: Autophagy;    Chaperone-mediated autophagy;    Chloroquine;    G2019S LRRK2;    Glucocerebrosidase;    LC3;   

DOI  :  

来源: DOAJ

【 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 α-synuclein accumulation and, hence, Parkinson's disease, LRRK2 kinase modulation of ALP, its age-dependence and relation with pSer129 α-synuclein inclusions were investigated in vivo. Striatal ALP markers were analyzed by Western blotting in 3, 12 and 20-month-old LRRK2 G2019S knock-in mice (bearing enhanced kinase activity), LRRK2 knock-out mice, LRRK2 D1994S knock-in (kinase-dead) mice and wild-type controls. The lysosomotropic agent chloroquine 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 α-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 chloroquine, suggesting a defective autophagic flux. G2019S knock-in mice showed LAMP2 accumulation and downregulation of ALP key genes MAP1LC3B, LAMP2, mTOR, TFEB and GBA1. Subacute administration of the LRRK2 kinase inhibitor MLi-2 in wild-type and G2019S 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 α-synuclein inclusions were dissociated in striatal neurons of kinase-dead and G2019S 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.

【 授权许可】

Unknown