Elsevier

Neurobiology of Disease

Volume 40, Issue 3, December 2010, Pages 503-517
Neurobiology of Disease

Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice

https://doi.org/10.1016/j.nbd.2010.07.010Get rights and content

Abstract

Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene, first described in 2004 have now emerged as the most important genetic finding in both autosomal dominant and sporadic Parkinson's disease (PD). While a formidable research effort has ensued since the initial gene discovery, little is known of either the normal or the pathological role of LRRK2. We have created lines of mice that express human wild-type (hWT) or G2019S Lrrk2 via bacterial artificial chromosome (BAC) transgenesis. In vivo analysis of the dopaminergic system revealed abnormal dopamine neurotransmission in both hWT and G2019S transgenic mice evidenced by a decrease in extra-cellular dopamine levels, which was detected without pharmacological manipulation. Immunopathological analysis revealed changes in localization and increased phosphorylation of microtubule binding protein tau in G2019S mice. Quantitative biochemical analysis confirmed the presence of differential phospho-tau species in G2019S mice but surprisingly, upon dephosphorylation the tau isoform banding pattern in G2019S mice remained altered. This suggests that other post-translational modifications of tau occur in G2019S mice. We hypothesize that Lrrk2 may impact on tau processing which subsequently leads to increased phosphorylation. Our models will be useful for further understanding of the mechanistic actions of LRRK2 and future therapeutic screening.

Research highlights

► LRRK2 transgenic mice have reduced extracellular dopamine levels in the striatum. ► LRRK2 G2019S transgenic mice show alterations in tau processing. ► LRRK2 G2019S mice display anxiety like behavior.

Introduction

LRRK2 mutations represent a unique opportunity for the development of genetic model systems for Parkinson's disease (PD). LRRK2 mutations are the most common form of familial parkinsonism and account for up to 40% of sporadic parkinsonism in certain populations (reviewed by Farrer, 2006). Clinically and pathologically, the features of LRRK2-associated parkinsonism are often indistinguishable from idiopathic PD; although pathologic variability exists even within PARK8-linked kindreds, ranging from nigral neuronal loss only, to neuronal loss with α-synuclein, ubiquitin or tau inclusions (reviewed by Whaley et al., 2006). To date, four different domains (ROC, C-terminal of ROC, kinase and WD40) of Lrrk2 are known to be affected by mutation/risk factors. While the ultimate downstream consequence of LRRK2 mutation in humans is parkinsonism, it is still unclear if the variants share common pathogenic mechanisms or if individual variants exert specific effects.

Until recently, hypotheses about Lrrk2 dysfunction were based on data from lower model organisms and cellular systems. Several lines of evidence now point towards an important role of Lrrk2 in neuronal outgrowth and guidance (MacLeod et al., 2006, Plowey et al., 2008, Sakaguchi-Nakashima et al., 2007, van Egmond et al., 2008). Mechanistic studies have repeatedly observed increased kinase activity for the most common G2019S mutation, however the mode of action for the other mutations has remained conflicting (Greggio and Cookson, 2009). With the emergence of several rodent mutant LRRK2 models, in vivo insight into the mechanistic actions is now forthcoming. ROC domain mutant human R1441G bacterial artificial chromosome (BAC) and R1441C knock-in mice both exhibit impaired dopamine release (Li et al., 2009, Tong et al., 2009), with additional behavioral and pathological abnormalities seen in the R1441G BAC mice. Mice over-expressing a murine LRRK2 BAC containing a G2019S mutation have also been shown to have decreased dopamine release whereas mice over-expressing a murine wild-type LRRK2 BAC have increased dopamine release and are hyperactive (Li et al., 2010). Inducible human G2019S and wild-type over-expressing mice, while absent of phenotype alone, both show synergistic effects when crossed with mutant A53T alpha-synuclein mice (Lin et al., 2009). Curiously, A53T mice on a murine LRRK2 knockout background have an ameliorated phenotype (Lin et al., 2009) whereas the kidneys of LRRK2 knockout mice accumulate alpha-synuclein (Tong et al., 2010). Interestingly, although extracellular dopamine release was not measured in LRRK2 knockout mice, in two independent lines levels of striatal dopamine were normal and the knockout mice are without overt brain phenotype (Andres-Mateos et al., 2009, Tong et al., 2010).

In this study we report human wild type (hWT) and mutant G2019S mice that were generated via BAC transgenesis. We have performed a comprehensive analysis of the dopaminergic system in these mice as well as behavioral and pathological analysis. We show that human kinase domain G2019S over-expressing mutants, like ROC mutants and murine G2019S BAC mutants, have reduced extracellular dopamine levels, which can be detected without pharmacological intervention. Importantly, we demonstrate that BAC mice over-expressing human wild type Lrrk2 also exhibit decreased extracellular dopamine levels, lending support to gain of function mechanism. Furthermore, we show that G2019S mice exhibit a number of age related changes in tau protein, including mislocalization and increased tau phosphorylation. We present novel biochemical data that suggests that the tau banding pattern is altered in G2019S mice compared to age matched controls. Finally, we show that G2019S mice, but not hWT mice, display anxiety like behaviors. Our results both support and extend data recently obtained in other LRRK2 models. The human G2019S and wild-type BAC mice will provide further insight into understanding basic mechanisms of LRRK2 biology and aid future therapeutic design.

Section snippets

Animals

All animal procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee and were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996.

Generation of BAC transgenic mice

A BAC (RP-11 568G5) was identified in silico to contain the entire human LRRK2 gene (NM_198578.3) and regulatory sequences, and was purchased from the Children's Hospital Oakland Research Institute (Oakland, CA). Library RP-11 genomic DNA

Generation of Human LRRK2 wild type and G2019S BAC mice

The F1 offspring from transgenic hWT and G2019S founders were characterized for LRRK2 expression and full length human LRRK2 cDNA transcript was found in one hWT and in four G2019S BAC lines. The data for G2019S mice presented in this manuscript are from the highest expressing G2019S line. The presence of the G  A base change was confirmed in human LRRK2 cDNA isolated from G2019S mice (Fig. 1A). Regional localization of transgenic mRNA with a Taqman human specific probe (Fig. 1B) and finer

Discussion

Our data indicate that over-expression of human wild type LRRK2 significantly reduces extracellular dopamine levels in the striatum. Likewise, the reduction in extracellular dopamine levels (~ 33%) in our G2019S mice complements findings in mutant R1441G BAC transgenic mice (Li et al., 2009) which exhibit a 35% decrease in extracellular dopamine compared to NT following administration of the DAT blocker nomifensine. This is additionally supported by a 50% deficit in dopamine release in

Conclusions

In summary we describe two new LRRK2 models, created using BAC transgenesis to over-express human WT or G2019S Lrrk2 in mice. As for previous LRRK2 models, we confirm a deficit in extracellular dopamine levels in our mutant G2019S model, but importantly we demonstrate this dopamine impairment is also present in mice expressing high levels of human wild-type Lrrk2. Furthermore, we show that while tau alterations occur in both models, mutant G2019S leads to a much more pronounced effect. Finally

Acknowledgments

We would like to thank Linda Rousseau and Virginia Philips for technical assistance and Naru Sahara and Michael DeTure for helpful discussions. Funding support was provided by the Mayo Clinic, NIH Grants NIA AG17216, NINDS NS40256, NIA AG11762 and AG17586, Lundbeck A/S, The Pacific Alzheimer's Research Foundation, Michael J Fox Foundation and the Robert H and Clarice Smith/ML Simpson Foundation Trust Fellowship.

References (63)

  • H. Melrose

    Anatomical localization of leucine-rich repeat kinase 2 in mouse brain

    Neuroscience

    (2006)
  • H.L. Melrose

    A comparative analysis of leucine-rich repeat kinase 2 (Lrrk2) expression in mouse brain and Lewy body disease

    Neuroscience

    (2007)
  • G.E. Meredith

    Modeling PD pathogenesis in mice: advantages of a chronic MPTP protocol

    Parkinsonism Relat. Disord.

    (2008)
  • M. Nakano et al.

    Age-related changes in the metabolism of neurotransmitters in rat striatum: a microdialysis study

    Mech. Ageing Dev.

    (1996)
  • M. Palkovits

    Isolated removal of hypothalamic or other brain nuclei of the rat

    Brain Res.

    (1973)
  • W. Pope

    Phosphorylated tau epitope of Alzheimer's disease is coupled to axon development in the avian central nervous system

    Exp. Neurol.

    (1993)
  • S. Puglisi-Allegra et al.

    Psychopharmacology of dopamine: the contribution of comparative studies in inbred strains of mice

    Prog. Neurobiol.

    (1997)
  • H. Rosenmann

    A novel transgenic mouse expressing double mutant tau driven by its natural promoter exhibits tauopathy characteristics

    Exp. Neurol.

    (2008)
  • A. Sakaguchi-Nakashima

    LRK-1, a C. elegans PARK8-related kinase, regulates axonal-dendritic polarity of SV proteins

    Curr. Biol.

    (2007)
  • K. Schindowski

    Alzheimer's disease-like tau neuropathology leads to memory deficits and loss of functional synapses in a novel mutated tau transgenic mouse without any motor deficits

    Am. J. Pathol.

    (2006)
  • N. Shin

    LRRK2 regulates synaptic vesicle endocytosis

    Exp. Cell Res.

    (2008)
  • P. Simon

    Thigmotaxis as an index of anxiety in mice. Influence of dopaminergic transmissions

    Behav. Brain Res.

    (1994)
  • T.L. Spires-Jones

    Tau pathophysiology in neurodegeneration: a tangled issue

    Trends Neurosci.

    (2009)
  • W.N. van Egmond

    Intramolecular activation mechanism of the Dictyostelium LRRK2 homolog Roco protein GbpC

    J. Biol. Chem.

    (2008)
  • J.P. Allen

    Somatostatin receptor 2 knockout/lacZ knockin mice show impaired motor coordination and reveal sites of somatostatin action within the striatum

    Eur. J. Neurosci.

    (2003)
  • E. Andres-Mateos

    Unexpected lack of hypersensitivity in LRRK2 knock-out mice to MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine)

    J. Neurosci.

    (2009)
  • J.C. Augustinack

    Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer's disease

    Acta Neuropathol.

    (2002)
  • S. Biskup

    Localization of LRRK2 to membranous and vesicular structures in mammalian brain

    Ann. Neurol.

    (2006)
  • C.D. Clelland

    A functional role for adult hippocampal neurogenesis in spatial pattern separation

    Science

    (2009)
  • G. Di Chiara

    Estimation of in-vivo neurotransmitter release by brain microdialysis: the issue of validity

    Behav. Pharmacol.

    (1996)
  • G. Drewes

    Microtubule-associated protein/microtubule affinity-regulating kinase (p110mark). A novel protein kinase that regulates tau-microtubule interactions and dynamic instability by phosphorylation at the Alzheimer-specific site serine 262

    J. Biol. Chem.

    (1995)
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