Kelly M. Hinkle

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

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 Parkinsons 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.

Adult neurogenesis and neurite outgrowth are impaired in LRRK2 G2019S mice

The generation and maturation of adult neural stem/progenitor cells are impaired in many neurodegenerative diseases, among them is Parkinsons disease (PD). In mammals, including humans, adult neurogenesis is a lifelong feature of cellular brain plasticity in the hippocampal dentate gyrus (DG) and in the subventricular zone (SVZ)/olfactory bulb system. Hyposmia, depression, and anxiety are early non-motor symptoms in PD. There are parallels between brain regions associated with non-motor symptoms in PD and neurogenic regions. In autosomal dominant PD, mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are frequent. LRRK2 homologs in non-vertebrate systems play an important role in chemotaxis, cell polarity, and neurite arborization. We investigated adult neurogenesis and the neurite development of new neurons in the DG and SVZ/olfactory bulb system in bacterial artificial chromosome (BAC) human Lrrk2 G2019S transgenic mice. We report that mutant human Lrrk2 is highly expressed in the hippocampus in the DG and the SVZ of adult Lrrk2 G2019S mice. Proliferation of newly generated cells is significantly decreased and survival of newly generated neurons in the DG and olfactory bulb is also severely impaired. In addition, after stereotactic injection of a GFP retrovirus, newly generated neurons in the DG of Lrrk2 G2019S mice exhibited reduced dendritic arborization and fewer spines. This loss in mature, developed spines might point towards a decrease in synaptic connectivity. Interestingly, physical activity partially reverses the decrease in neuroblasts observed in Lrrk2 G2010S mice. These data further support a role for Lrrk2 in neuronal morphogenesis and provide new insights into the role of Lrrk2 in adult neurogenesis.

LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors

Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation.We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis.Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.

In vivo silencing of alpha-synuclein using naked siRNA

BackgroundOverexpression of α-synuclein (SNCA) in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease post-mortem. Genetic variability in SNCA contributes to risk of idiopathic Parkinsons disease (PD), possibly as a result of overexpression. SNCA downregulation is therefore a valid therapeutic target for PD.ResultsWe have identified human and murine-specific siRNA molecules which reduce SNCA in vitro. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked), murine-specific siRNA into the hippocampus significantly reduces SNCA levels. Reduction of SNCA in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion.ConclusionWe have developed naked gene-specific siRNAs that silence expression of SNCA in vivo. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of SNCA, its role in disease, and eventually as a therapeutic strategy for α-synucleinopathies resulting from SNCA overexpression.

Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice

Mutations in the LRRK2 gene represent the most common genetic cause of late onset Parkinsons disease. The physiological and pathological roles of LRRK2 are yet to be fully determined but evidence points towards LRRK2 mutations causing a gain in kinase function, impacting on neuronal maintenance, vesicular dynamics and neurotransmitter release. To explore the role of physiological levels of mutant LRRK2, we created knock-in (KI) mice harboring the most common LRRK2 mutation G2019S in their own genome. We have performed comprehensive dopaminergic, behavioral and neuropathological analyses in this model up to 24months of age. We find elevated kinase activity in the brain of both heterozygous and homozygous mice. Although normal at 6months, by 12months of age, basal and pharmacologically induced extracellular release of dopamine is impaired in both heterozygous and homozygous mice, corroborating previous findings in transgenic models over-expressing mutant LRRK2. Via in vivo microdialysis measurement of basal and drug-evoked extracellular release of dopamine and its metabolites, our findings indicate that exocytotic release from the vesicular pool is impaired. Furthermore, profound mitochondrial abnormalities are evident in the striatum of older homozygous G2019S KI mice, which are consistent with mitochondrial fission arrest. We anticipate that this G2019S mouse line will be a useful pre-clinical model for further evaluation of early mechanistic events in LRRK2 pathogenesis and for second-hit approaches to model disease progression.

Digenic parkinsonism: investigation of the synergistic effects of PRKN and LRRK2.

The complex genetic etiology of Parkinsons disease (PD) is indicative of a multifactorial syndrome. A combination of gene-gene and gene-environment interactions may determine a variable phenotypic outcome. Recently a direct gene/protein interaction between two of the most common genetic causes of parkinsonism PRKN and LRRK2 has been postulated. We have identified three Spanish patients simultaneously harboring mutations in PRKN and LRRK2. In comparison to other Spanish patients with a single LRRK2 or PRKN mutation, the three double-mutation patients reported here do not present with an earlier age-at-onset or a faster progression of disease. Although the clinical findings do not support a synergistic effect of LRRK2 and PRKN, a potential genetic interplay might be concealed by the modulating effects of other genes. Nevertheless, this work demonstrates that the presence of mutations in one familial gene should not serve as exclusion criteria in a screen for further genetic variation. Direct interaction of Lrrk2 and parkin proteins was not observed in co-immunoprecipitation pull down experiments. However, in vivo studies are required to assess whether there is an indirect link between Lrrk2 and parkin in disease pathogenesis.

Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies

Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinsons disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinsons Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease.

Differential LRRK2 Expression in the Cortex, Striatum, and Substantia Nigra in Transgenic and Nontransgenic Rodents

Mutations in leucine‐rich repeat kinase 2 (LRRK2) are found in a significant proportion of late‐onset Parkinsons disease (PD) patients. Elucidating the neuroanatomical localization of LRRK2 will further define LRRK2 function and the molecular basis of PD. Here, we utilize recently characterized monoclonal antibodies to evaluate LRRK2 expression in rodent brain regions relevant to PD. In both mice and rats, LRRK2 is highly expressed in the cortex and striatum, particularly in pyramidal neurons of layer V and in medium spiny neurons within striosomes. Overall, rats have a more restricted distribution of LRRK2 compared with mice. Mice, but not rats, show high levels of LRRK2 expression in the substantia nigra pars compacta. Expression of the pathogenic LRRK2‐G2019S protein from mouse bacterial artificial chromosome (BAC) constructs closely mimics endogenous LRRK2 distribution in the mouse brain. However, LRRK2‐G2019S expression derived from human BAC constructs causes LRRK2 to be expressed in additional neuron subtypes in the rat such as striatal cholinergic interneurons and the substantia nigra pars compacta. The distribution of LRRK2 from human BAC constructs more closely resembles descriptions of LRRK2 in humans and nonhuman primates. Computational analyses of DNA regulatory elements in LRRK2 show a primate‐specific promoter sequence that does not exist in lower mammalian species. These noncoding regions may be involved in directing neuronal expression patterns. Together, these studies will aid in understanding the normal function of LRRK2 in the brain and will assist in model selection for future studies. J. Comp. Neurol. 522:2465–2480, 2014.

Leucine-rich repeat kinase 1: a paralog of LRRK2 and a candidate gene for Parkinson’s disease

Leucine-rich repeat kinase 1 gene (LRRK1) on chromosome 15q26.3 is a paralog of LRRK2 in which multiple substitutions were recently linked to Parkinson’s disease. We have examined the exon–intron structure of the gene and the expressed mRNA sequence in brain. LRRK1 sequencing analysis in 95 probands from families with autosomal dominant Parkinson’s disease identified 23 variants, 14 of which are novel, with four resulting in non-synonymous amino acid substitutions. These four substitutions are rare and do not clearly segregate with disease within our families or associate with sporadic Parkinson’s disease in a US case-control series. Subsequent sequencing of exon 26 encoding the kinase activation segment in an additional 360 probands identified one further synonymous variant, suggesting that LRRK1 variants are not a frequent cause of Parkinson’s disease. The relative absence of substitutions within LRRK1 highlights a greater conservation of sequence than observed for LRRK2. Comparison of evolutionary interspecies sequences of LRRK1 and LRRK2 suggests they diverged from a common founder gene.

Heterodimerization of Lrrk1-Lrrk2: Implications for LRRK2-associated Parkinson disease

LRRK2 mutations are recognized as the most frequent genetic cause of both familial and sporadic parkinsonism identified to date. A remarkable feature of this form of parkinsonism is the variable penetrance of symptom manifestation resulting in a wide range of age-at-onset in patients. Herein we use a functional approach to identify the Lrrk1 protein as a potential disease modifier demonstrating an interaction and heterodimer formation with Lrrk2. In addition, evaluation of LRRK1 variants in our large Lrrk2 p.G2019S-parkinsonism series from a Tunisian (n=145) identified a missense mutation (p.L416M) resulting in an average 6.2 years younger age at disease onset. In conclusion we show that the interaction of Lrrk1-Lrrk2 can form protein dimers and this interaction may influence the age of symptomatic manifestation in Lrrk2-parkinsonism patients.