A comparative study of Lrrk2 function in primary neuronal cultures

https://doi.org/10.1016/j.parkreldis.2010.08.018Get rights and content

Abstract

Objective

To assess the contribution of wild-type, mutant and loss of leucine-rich repeat kinase-2 (LRRK2; Lrrk2) on dendritic neuronal arborization.

Background

LRRK2 mutations are recognized as the major genetic determinant of susceptibility to Parkinson’s disease for which a cellular assay of Lrrk2 mutant function would facilitate the development of targeted molecular therapeutics.

Methods

Dendritic neuronal arborization (neurite length, branching and the number of processes per cell) was quantified in primary hippocampal and midbrain cultures derived from five lines of recombinant LRRK2 mice, including human BAC wild-type and mutant overexpressors (Y1699C and G2019S), murine knock-out and G2019S knock-in animals.

Results

Neuronal arborization in cultures from BAC Lrrk2 wild-type animals is comparable to non-transgenic littermate controls, despite high levels of human transgene expression. In contrast, primary neurons from both BAC mutant overexpressors presented with significantly reduced neuritic outgrowth and branching, although the total number of processes per cell remained comparable. The mutant-specific toxic gain-of-function observed in cultures from BAC mutant mice may be partially rescued by staurosporine treatment, a non-specific kinase inhibitor. In contrast, neuronal arborization is far more extensive in neuronal cultures derived from murine knock-out mice that lack endogenous Lrrk2 expression. In Lrrk2 G2019S knock-in mice, arguably the most physiologically relevant system, neuritic arborization is not impaired.

Conclusions

Impairment of neuritic arborization is an exaggerated, albeit mutant specific, consequence of Lrrk2 over-expression in primary cultures. The phenotype and assay described provides a means to develop therapeutic agents that modulate the toxic gain-of-function conferred by mutant Lrrk2.

Introduction

Autosomal-dominant mutations in leucine-rich repeat kinase-2 (LRRK2) have been identified as an important cause for familial and sporadic Parkinson’s disease (PD) [1]. LRRK2 encodes a 286KDa protein, previously classified as a member of the ROCO superfamily [2], [3], characterized by the presence of tandem Ras-related GTPase (Roc), C-terminal of Roc (COR) and kinase domains. Additional domains include armadillo and ankyrin repeats at the N-terminus, a central leucine-rich repeat (LRR), and a WD40 domain towards the C-terminus.

Given the epidemiological importance of LRRK2 mutations [4], [5], [6] intensive efforts have been made to elucidate Lrrk2-specific signaling pathways and the mechanisms underlying PD. Rare pathogenic mutations focused initial studies on the enzymatic activity and regulation of Lrrk2 kinase and GTPase domains [7], [8]. Several studies now report Lrrk2 is involved in the regulation of neurite maintenance and survival [9], [10], [11]. Cellular over-expression of the LRRK2 transgene (wild-type and mutant) has been achieved using viral transduction, transfection of cDNA constructs, and the ex-vivo culture of neurons derived from mice expressing LRRK2 cDNA with heterologous promoters. Each of the published models represents a valuable tool to investigate Lrrk2 biology but nevertheless none recapitulate endogenous wild-type or mutant Lrrk2 expression in brain [12]. The changes in neuritic arborization observed in ex-vivo transfected cultures are non-physiologic, thus their relevance to age-associated human disease is not intuitive.

We have used a comparative approach to assess the role of Lrrk2 in bacterial artificial chromosome (BAC) wild-type (hWT) and mutant over-expressing [G2019S (GS) and Y1699C (YC)] mice as well as in murine LRRK2 knock-out (KO) and more physiologic G2019S knock-in animals (KI). Using primary neuronal cultures from our BAC models, we have assessed whether deficits in neuritic outgrowth can be recapitulated, and whether they are a consequence of high levels of transgene expression, are mutant specific or a combination. Subsequently, in primary cultures from KO mice we have examined whether regulation of neuritic outgrowth, morphology and cellular homeostasis is an inherent function of Lrrk2. Finally, using KI animals, we have assessed whether mutant Lrrk2 expressed at endogenous levels is sufficient to induce similar morphological changes.

Section snippets

hWT, GS and YC LRRK2 mice

A BAC (RP-11 568G5) containing the entire human wild-type LRRK2 gene and regulatory sequences was identified and recombination-based BAC mutagenesis performed to generate mutant G2019S BAC and Y1699C clones. Following confirmation of integrity, purified BAC DNA was injected into FVB/N (Taconic) fertilized oocytes and transplanted into pseudo-pregnant ICR (Harlan) female mice. Subsequent offspring were genotyped to identify founders. Transgenic founders were bred to FVB mice and transgenic F1

Results

To explore whether we can recapitulate impaired neuritic outgrowth and branching in a LRRK2 over-expression model we prepared primary cultures from BAC LRRK2 transgenic animals and non-transgenic littermate controls. Human BAC LRRK2 transgenic animals express the transgene under the endogenous promoter, to mimic the temporal as well as regional expression patterns of the endogenous gene. Based on the expression pattern of full length hWT, GS and YC Lrrk2 proteins in various brain regions (Fig. 1

Discussion

Ex-vivo experiments in human BAC models revealed that over-expression of mutant but not wild-type Lrrk2 protein leads to pronounced deficits in mean process length and branching. These results compliment recent in-vivo studies where over-expression of human LRRK2 R1441G in mice lead to a marked diminution in the number of tyrosine hydroxylase positive dendrites in the pars reticulata of the substantia nigra [17]. In both cases, toxic gain-of-function appears to be a mutant-specific phenotype.

Acknowledgement

We would like to thank Gunnar P. Dietz and Karina Fog for their valuable advice and helpful discussions. Funding support was provided by the Mayo Clinic, NIH Grants NIA AG17216, NINDS NS40256, the Michael J Fox Foundation and H. Lundbeck A/S. Matt Farrer is a Canada Excellence Research Chairholder.

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The review of this paper was entirely handled by an Associate Editor, Robert Rodnitzky.

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