Protein synthesis is suppressed in sporadic and familial Parkinson’s Disease by LRRK2

Abstract

Gain of function LRRK2-G2019S is the most common mutation associated with both familial and sporadic Parkinson's disease. It is expected therefore that understanding the cellular function of LRRK2 will provide much needed insight on the pathological mechanism of sporadic Parkinson's, which is the most common form. Here we show that constitutive LRRK2 activity represses nascent protein synthesis in rodent neurons. Specifically, enzymatic inhibition of LRRK2, gene silencing or gene knockout of Lrrk2 increase translation. In the rotenone model of Parkinson's, LRRK2 activity increases, leading to repression of translation and dopaminergic neuron atrophy both of which are prevented by LRRK2 inhibition. This is accompanied by altered phosphorylation of eIF2a-S52(↑), eIF2s2-S2(↓) and eEF2-T57(↑) in striatum/substantia nigra in a direction that signifies inhibition of protein synthesis. Significantly, LRRK2 is activated and translation is 40% reduced in Parkinson's patient fibroblasts (G2019S and sporadic) and LRRK2 inhibition restores normal translation. In contrast, translation is unchanged in cells from multiple system atrophy patients, implying disease specificity. These findings suggest that LRRK2-dependent repression of translation may be a proximal function of LRRK2 in Parkinson's pathology. Words: 178Table 1. Demographic and clinical characteristics of patients and healthy controls -The source of material either Turku University Hospital (TUH), Telethon network of genetic biobanks (TNGB) or NINDS Coriell repository (NINDS) is indicated. *Unified Parkinson's Disease rating scale (UPDRS). Gender male (m)and female (f), and age in years when the biopsy was taken, are indicated. Estimated age of onset refers to the age when motor symptoms were first reported. For patients where the relevant information was not available, a hyphen is shown.

Regulators of proteostasis are translationally repressed in fibroblasts from patients with sporadic and LRRK2-G2019S Parkinson’s disease

“…are described in Table 1. To label nascent proteins, cells were metabolically labeled according to the FUNCAT method as previously described 12,19 . In this procedure, endogenous methionine is replaced with the L-Azidohomoalanine (AHA) analog of methionine.…”

“…Isolation and LC-MS/MS identification of newly synthesized proteins from LRRK2-G2019S carriers and sporadic patients We set out to identify which mRNA transcripts were differentially regulated at the level of translation in PD patient cells, investigating both sporadic and LRRK2-G2019S cases. Skin punches were isolated from 10 sporadic PD cases and 6 healthy donors from patients attending TUH and from an accompanying family member (usually a spouse or sibling), as previously described 12 . In addition, we analyzed five fibroblast samples from LRRK-G2019S patients and six from healthy controls using material from the NINDS and TNGB repositories.…”

“…Emerging evidence from cellular and rodent models as well as patient-induced pluripotent cells suggests that protein synthesis is deregulated in PD [7][8][9][10][11] . We have found that de novo protein synthesis is reduced in fibroblasts from PD patients with a receiver operating characteristic area under the curve of 0.925, suggesting good predictive power of this function as a biomarker readout 12 . Moreover, fibroblasts derived from patient skin biopsies have been shown to harbor disease-associated features of PD.…”

A Robust Bayesian Approach to Bulk Gene Expression Deconvolution with Noisy Reference Signatures

“…This pseudo-compartmentalized translation within neural cells has been shown to create spatial variability in protein expression that is critical for neural development, function, and plasticity (34). Disruptions to localized translation have been linked to various neurodevelopmental and neurodegenerative disorders (35,36). Combined with the results linking ER/translation GO terms in these same cells, these findings indicate that dysregulation of translation and protein folding, whether at the ER and/or at distal sites, might contribute to the clinical presentation of pediatric DPD deficiency.…”

Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity

“…Prior studies have suggested that LRRK2 kinase activity, even when overexpressed in cells, may be very low. Exposure of cells to oxidative stress has been reported to enhance LRRK2 activation [22,[42][43][44][45]. Since endogenous pT73-RAB10 signal in transfected HEK293T cells overexpressing LRRK2 was low (Supplementary Figure 1), we co-transfected cells with flag-tagged WT LRRK2 and GFP-tagged RAB10 (Fig.…”