A comparative study of Lrrk2 function in primary neuronal cultures☆
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.