Proteomic analysis reveals co-ordinated alterations in protein synthesis and degradation pathways in LRRK2 knockout mice

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) segregate with familial Parkinson’s disease (PD) and genetic variation around LRRK2 contributes to risk of sporadic disease. Although knockout (KO) of Lrrk2 or knock-in of pathogenic mutations into the mouse germline does not result in a PD phenotype, several defects have been reported in the kidneys of Lrrk2 KO mice. To understand LRRK2 function in vivo, we used an unbiased approach to determine which protein pathways are affected in LRRK2 KO kidneys. We nominated changes in cytoskeletal-associated proteins, lysosomal proteases, proteins involved in vesicular trafficking and in control of protein translation. Changes were not seen in mice expressing the pathogenic G2019S LRRK2 mutation. Using cultured epithelial kidney cells, we replicated the accumulation of lysosomal proteases and demonstrated changes in subcellular distribution of the cation-independent mannose-6-phosphate receptor. These results show that loss of LRRK2 leads to co-ordinated responses in protein translation and trafficking and argue against a dominant negative role for the G2019S mutation.

Sequential screening nominates the Parkinson’s disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis

“…23,51 Recent proteomic analysis of aged LRRK2 KO kidneys characterised a number of changes to lysosomal, cytoskeletal and protein-translation related proteins. 33,52 Relative to their WT counterparts, aged KO animals also demonstrated a significant reduction in clathrin and AP2 heavy and medial subunits, α, β and μ respectively. These findings indicate AP2 is downregulated or degraded in the absence of LRRK2, although the precise mechanism by which this occurs is uncertain at this time.…”

“…47 LRRK2 has been previously suggested to regulate many facets of intracellular trafficking including, synaptic vesicle endocytosis and recycling, trafficking and degradation of EGFR, actin remodeling on endosomes, trafficking of mannose-6-phosphoate receptors from the TGN to lysosomes and the recycling of receptors from the membrane to the TGN through the retromer complex. 26,27,30,33,42,[48][49][50] However, the mechanistic details underpinning these observations remain uncertain with competing substrates and protein interactors nominated to mediate cellular effects.…”

“…For western blot analysis Kidney samples were prepared as described previously. 33 In brief, kidneys were dissected from 1-year-old mice and frozen prior to preparation and homogenized in HEPES lysis buffer as described above. Homogenates were centrifuged at 300g for 3 minutes, supernatant was collected and…”

“…investigate levels of multiple AP2 components in these tissues. 33 Kidney lysates from 1yr old. animals of WT and LRRK2 KO mice were processed as previously described in Pellegrini et al 33 Supernatant fractions were probed for all members of the AP2 complex as well as clathrin (figure 3b).…”

“…33 Kidney lysates from 1yr old. animals of WT and LRRK2 KO mice were processed as previously described in Pellegrini et al 33 Supernatant fractions were probed for all members of the AP2 complex as well as clathrin (figure 3b). Western blot analysis revealed a significant decrease in heavy and medium subunits of the AP2 complex (α, β & μ).…”