LRRK2 BAC transgenic rats develop progressive, L-DOPA-responsive motor impairment, and deficits in dopamine circuit function

Human Molecular Genetics, 2016, Vol. 25, No. 5 951–963 doi: 10.1093/hmg/ddv628 Advance Access Publication Date: 6 January 2016 Original Article ORIGINAL ARTICLE LRRK2 BAC transgenic rats develop progressive, L-DOPA-responsive motor impairment, and deficits in dopamine circuit function 1 Oxford Parkinson’s Disease Centre, 2Department of Physiology, Anatomy and Genetics, 3Medical Research Council Brain Network Dynamics Unit, Department of Pharmacology and 4Department of Experimental Psychology, University of Oxford, Oxford, UK *To whom correspondence should be addressed at: Oxford Parkinson’s Disease Centre, Department of Physiology, Anatomy and Genetics, Le Gros Clark Building, South Parks Road, Oxford OX1 3QX, UK; Tel: +44 1865 282837; Fax: +44 1865 272420; Email: (S.J.C); (P.D.D); Email: (R.W.-M.) Abstract Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson’s disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson’s disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson’s disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18–21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson’s disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson’s therapeutics. † These authors contributed equally to this work. Received: November 24, 2015. Revised: December 21, 2015. Accepted: December 29, 2015 © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 951 Max Sloan1,2,†, Javier Alegre-Abarrategui1,2,†, Dawid Potgieter1,2,†, Anna-Kristin Kaufmann1,3,†, Richard Exley1,2, Thierry Deltheil1,2, Sarah Threlfell1,2, Natalie Connor-Robson1,2, Katherine Brimblecombe1,2, Rebecca Wallings1,2, Milena Cioroch1,2, David M. Bannerman4, J. Paul Bolam1,3, Peter J. Magill1,3, Stephanie J. Cragg1,2, *, Paul D. Dodson1,3,* and Richard Wade-Martins1,2, * 952 | Human Molecular Genetics, 2016, Vol. 25, No. 5 Introduction Results Molecular characterization of LRRK2 BAC transgenic rats LRRK2 transgenic rats were generated using previously developed BAC constructs (25) consisting of the entire 144 kb human LRRK2 locus, fused to a YPet reporter tag (Supplementary Material, Fig. S1A). Three LRRK2 BAC transgenic lines were generated on a Sprague–Dawley background: one expressing human wild-type (hWT) LRRK2 and two mutant lines, expressing either the G2019S or R1441C mutant forms of the human gene. Anatomical transgene protein expression patterns were similar between lines throughout the brain (Fig. 1A). In agreement with previous Mutant LRRK2 rats display progressive motor and cognitive deficits Cohorts of G2019S, R1441C and non-transgenic (nTG) littermates, and of hWT and nTG littermates, were generated and underwent in-depth phenotyping. To determine whether LRRK2 rats develop motor impairment, we analysed performance on the accelerating rotarod (Supplementary Material, Fig. S3). Compared with nTG controls at a young age (3–6 months), all transgenic lines showed no impairment on the rotarod, whereas G2019S showed an enhancement, as previously reported (23,28). At an old age (18–21 months) G2019S and R1441C mutant, but not hWT, transgenic animals showed a significant impairment in performance (Supplementary Material, Fig. S3). The non-transgenic controls in each experimental cohort showed equivalent performance allowing us to integrate data from the two cohorts to perform additional analysis directly comparing all lines (G2019S v R1441C v hWT versus nTG). Importantly, we show that aged (18–21 months) G2019S and R1441C rats exhibited a significant age-dependent impaired performance compared with both nTG and hWT controls (Fig. 2A). Gait disturbances play a major role in the motor manifestation of Parkinson’s disease patients (29). We therefore examined the gait of mutant LRRK2 transgenic rats; aged R1441C, but not G2019S rats displayed abnormal gait compared with nTG controls (Supplementary Material, Fig. S4G–I). Motor dysfunction in Parkinson’s disease is clinically responsive to -DOPA treatment owing to the loss of nigrostriatal dopamine function. To investigate whether motor impairment in the rats was -DOPA-responsive, we administered either -DOPA or saline to aged G2019S, R1441C and nTG rats. -DOPA reversed the rotarod performance deficits seen in both G2019S and R1441C rats (Fig. 2B). LRRK2 transgenic animals used for rotarod tests were not different in weight when compared with nTG controls (Supplementary Material, Fig. S4A–D). Transgenic rats did not display impaired grip strength (Supplementary Material, Fig. S4E–F), suggesting that motor deficits were likely not a consequence of muscle weakness, motor neuron alterations or motor plate neurotransmission that Mutations in the leucine-rich repeat kinase 2 (LRRK2/PARK8) gene lead to the development of autosomal dominant Parkinson’s disease with pathology and characteristic motor and non-motor features highly similar to sporadic forms of the disease (1,2). The LRRK2 gene encodes a large (286 kDa), multidomain protein consisting of several repeat-containing regions, followed by ROCCOR GTPase, kinase and WD40 domains (3). Parkinson’s disease-causing mutations lie in either the GTPase (R1441C/G/H), COR (Y1699C) or kinase (G2019S and I2020T) domains of the LRRK2 protein (4, (...truncated)