Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice
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)
Genotypic differences in central neurotransmitter responses to aging in mice
Neurobiol. Aging
(1987)Altered depression-related behavior and neurochemical changes in serotonergic neurons in mutant R406W human tau transgenic mice
Brain Res.
(2005)Lrrk2-associated parkinsonism is a major cause of disease in Northern Spain
Parkinsonism Relat. Disord.
(2007)Staging of neurofibrillary degeneration caused by human tau overexpression in a unique cellular model of human tauopathy
Am. J. Pathol.
(2001)GSK-3 dependent phosphoepitopes recognized by PHF-1 and AT-8 antibodies are present in different tau isoforms
Neurobiol. Aging
(2003)LRRK2 Gly2019Ser penetrance in Arab-Berber patients from Tunisia: a case-control genetic study
Lancet Neurol.
(2008)Constitutive Alzheimer's-type tau epitopes in a neuritogenic rat CNS cell line
Neurobiol. Aging
(1995)Leucine-rich repeat kinase 2 regulates the progression of neuropathology induced by Parkinson's-disease-related mutant alpha-synuclein
Neuron
(2009)3, 4-Dihydroxyphenylacetic acid concentrations in the intermediate lobe and neural lobe of the posterior pituitary gland as an index of tuberohypophysial dopaminergic neuronal activity
Brain Res.
(1990)The familial Parkinsonism gene LRRK2 regulates neurite process morphology
Neuron
(2006)