Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice
Introduction
Mutations and genetic variability in the LRRK2 gene represent the most common genetic cause of Parkinson's disease (PD). The frequency of the pathogenic mutations is rare at around 2% overall (Di Fonzo et al., 2006, Farrer et al., 2007), however the most common mutation G2019S is found in up to 40% of patients in certain ethnic populations (Kachergus et al., 2005, Ozelius et al., 2006, Ishihara et al., 2007). In addition to pathogenic mutations, common genetic variability in LRRK2 is a risk factor for sporadic PD (Tan, 2006, Ross et al., 2008, Ross et al., 2011).
LRRK2 Parkinsonism has some unique features, including an age-dependent penetrance (Healy et al., 2008, Hulihan et al., 2008), with some aged carriers escaping disease (Kay et al., 2005) suggesting that disease manifestation is subject to other genetic or environmental modifiers, and potentially that the course of the disease may be altered by therapy. At the neuropathological level, LRRK2 Parkinsonism typically resembles idiopathic PD, exhibiting dopamine neuronal loss with synucleinopathy. Exceptions do exist in some kindreds, with patients that carry the same mutations having differential pathologies, including neuronal loss only and filamentous tau inclusions (Zimprich et al., 2004). The presence of pathologies that overlap with other neurodegenerative diseases such as Alzheimer's disease and Progressive Supranuclear Palsy has led to speculation that LRRK2 dysfunction may be upstream of several important neuronal signaling cascades relevant to other neurodegenerative diseases, and as such, a LRRK2 based therapeutic may have wider applications than just LRRK2 PD.
The physiological and pathological roles of LRRK2 protein are not yet fully understood but it is generally accepted that it functions as a kinase, with an important role in neuronal maintenance, vesicular trafficking and neurotransmitter release in the brain. The overwhelming data from rodent models with near-physiologic levels of transgenic expression suggest that mutant LRRK2 impairs dopamine neurotransmission, in the absence of neuronal loss (Li et al., 2009, Li et al., 2010, Melrose et al., 2010, Zhou et al., 2011, Beccano-Kelly et al., 2014b, Liu et al., 2014, Tsika et al., 2014, Walker et al., 2014, Lee et al., 2015) whereas higher levels of expression of LRRK2, via heterologous promoters or viral delivery, lead to dopamine neuronal death in mice and rats (Lee et al., 2010, Dusonchet et al., 2011, Ramonet et al., 2011). Nigro-striatal dopamine alterations were not found in two previously reported gene-targeted LRRK2 mutant models (Tong et al., 2009, Herzig et al., 2011). However, stimulated catecholamine release from adrenal chromaffin cells was reduced in the R1441C knock-in mice, and mutant mice displayed differential responses to pharmacologically induced behaviors (Tong et al., 2009). G2019S knock-in mice did not display altered dopamine drug-induced locomotor behaviors, but peripheral phenotypes were evident, including a moderate decrease in diastolic blood pressure and changes in mTOR signaling in the kidney (Herzig et al., 2011).
We have created a G2019S knock-in (KI) mouse model and performed an extensive dopaminergic and behavioral evaluation in heterozygous (HET) and homozygous (HOMO) animals. We show that both HET and HOMO G2019S mice have elevated kinase activity in the brain from a young age. Similar to the two previously described LRRK2 KI models, we do not observe loss of dopamine neurons. However, by utilizing microdialysis to measure extracellular release of monoamines in freely moving mice, we are able to demonstrate a progressive dopaminergic phenotype in HET and HOMO G2019S KI mice, which is characterized by a decrease in basal and evoked dopamine release in the striatum. Additionally, by measuring extracellular dopamine metabolism, we also provide evidence that suggests that the amount of packaged dopamine available release by exocytosis is less. Finally, we reveal that HOMO KI mice display progressive changes in mitochondria which are consistent with arrested fission. We anticipate that this G2019S KI model will be useful for further studies of pre-clinical mechanism of Parkinson's disease and assessment of neuroprotective therapies.
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 G2019S KI mice
G2019S KI mice were generated at Ozgene PLC (Australia) utilizing a construct designed to replace two bases in LRRK2 exon 41. The base ‘G’ at LRRK2 cDNA position 6055 is conserved in mouse and human (as is the Gly amino acid codon). The ‘G’
Generation of G2019S knock-in mice
The targeting strategy for generation of LRRK2 G2019S KI mice is shown in Fig. 1a. Homozygous mice received from Ozgene PLC were bred to Jackson C57BL 6/J mice and subsequent heterozygous offspring were bred together to obtain WT, HET and HOMO KI animals. Sequencing of cDNA was performed to confirm the presence of the G2019S mutation at the RNA level in the mouse genome (Fig. 1b). To determine if LRRK2 mRNA levels were equivalent across genotypes we used quantitative Taqman assays, using cDNA
Discussion
Herein, we demonstrate that mice expressing just one or two copies of mutant G2019S LRRK2 in their own genome have significantly elevated kinase activity and progressive dopaminergic alterations. To our knowledge, this is the first report showing that kinase activity is increased in a gene-targeted LRRK2 mouse model and the first comprehensive in vivo investigation of extracellular striatal dopamine release of dopamine and its metabolites in a LRRK2 knock-in model.
Using brain microdialysis to
Acknowledgments
We would like to thank Monica Castanedes-Casey, Linda Rousseau and Virginia Philips for technical assistance. Funding support was provided by the Mayo Clinic, NIH Grants NINDS NS065860 (HLM), NINDS NS40256, NS072187 (DWD, MJF), NIEHS ES020715 (ET), NINDS NS085070 (WS) and NINDS NS073740 (PJM), the Alzheimer's Association (JDF), and the Michael J. Fox Foundation (HLM, WS). WS is partially supported by the GHR Foundation, the Marriot Family Foundation and the Gerstner Family Career Development
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