Exogenous LRRK2G2019S induces parkinsonian-like pathology in a nonhuman primate.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease among the elderly. To understand its pathogenesis and to test therapies, animal models that faithfully reproduce key pathological PD hallmarks are needed. As a prelude to developing a model of PD, we tested the tropism, efficacy, biodistribution, and transcriptional effect of canine adenovirus type 2 (CAV-2) vectors in the brain of Microcebus murinus, a nonhuman primate that naturally develops neurodegenerative lesions. We show that introducing helper-dependent (HD) CAV-2 vectors results in long-term, neuron-specific expression at the injection site and in afferent nuclei. Although HD CAV-2 vector injection induced a modest transcriptional response, no significant adaptive immune response was generated. We then generated and tested HD CAV-2 vectors expressing leucine-rich repeat kinase 2 (LRRK2) and LRRK2 carrying a G2019S mutation (LRRK2G2019S), which is linked to sporadic and familial autosomal dominant forms of PD. We show that HD-LRRK2G2019S expression induced parkinsonian-like motor symptoms and histological features in less than 4 months.

Adenoviral-mediated expression of G2019S LRRK2 induces striatal pathology in a kinase-dependent manner in a rat model of Parkinson's disease.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). LRRK2 contains functional GTPase and kinase domains. The most common G2019S mutation enhances the kinase activity of LRRK2 in vitro whereas G2019S LRRK2 expression in cultured neurons induces toxicity in a kinase-dependent manner. These observations suggest a potential role for kinase activity in LRRK2-associated PD. We have recently developed a novel rodent model of PD with progressive neurodegeneration induced by the adenoviral-mediated expression of G2019S LRRK2. In the present study, we further characterize this LRRK2 model and determine the contribution of kinase activity to LRRK2-mediated neurodegeneration. Recombinant human adenoviral vectors were employed to deliver human wild-type, G2019S or kinase-inactive G2019S/D1994N LRRK2 to the rat striatum. LRRK2-dependent pathology was assessed in the striatum, a region where LRRK2 protein is normally enriched in the mammalian brain. Human LRRK2 variants are robustly expressed throughout the rat striatum. Expression of G2019S LRRK2 selectively induces the accumulation of neuronal ubiquitin-positive inclusions accompanied by neurite degeneration and the altered distribution of axonal phosphorylated neurofilaments. Importantly, the introduction of a kinase-inactive mutation (G2019S/D1994N) completely ameliorates the pathological effects of G2019S LRRK2 in the striatum supporting a kinase activity-dependent mechanism for this PD-associated mutation. Collectively, our study further elucidates the pathological effects of the G2019S mutation in the mammalian brain and supports the development of kinase inhibitors as a potential therapeutic approach for treating LRRK2-associated PD. This adenoviral rodent model provides an important tool for elucidating the molecular basis of LRRK2-mediated neurodegeneration.

Interaction of LRRK2 with kinase and GTPase signaling cascades.

LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson's disease (PD). Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however, studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in Caenorhabditis elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the mitogen activated protein kinase (MAPK) pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GTPase-activating proteins and GTPase exchange factors are another strong theme in LRRK2 biology, with LRRK2 binding to rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1, and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy. These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term PD.

A Parkinson's disease gene regulatory network identifies the signaling protein RGS2 as a modulator of LRRK2 activity and neuronal toxicity.

Mutations in LRRK2 are one of the primary genetic causes of Parkinson's disease (PD). LRRK2 contains a kinase and a GTPase domain, and familial PD mutations affect both enzymatic activities. However, the signaling mechanisms regulating LRRK2 and the pathogenic effects of familial mutations remain unknown. Identifying the signaling proteins that regulate LRRK2 function and toxicity remains a critical goal for the development of effective therapeutic strategies. In this study, we apply systems biology tools to human PD brain and blood transcriptomes to reverse-engineer a LRRK2-centered gene regulatory network. This network identifies several putative master regulators of LRRK2 function. In particular, the signaling gene RGS2, which encodes for a GTPase-activating protein (GAP), is a key regulatory hub connecting the familial PD-associated genes DJ-1 and PINK1 with LRRK2 in the network. RGS2 expression levels are reduced in the striata of LRRK2 and sporadic PD patients. We identify RGS2 as a novel interacting partner of LRRK2 in vivo. RGS2 regulates both the GTPase and kinase activities of LRRK2. We show in mammalian neurons that RGS2 regulates LRRK2 function in the control of neuronal process length. RGS2 is also protective against neuronal toxicity of the most prevalent mutation in LRRK2, G2019S. We find that RGS2 regulates LRRK2 function and neuronal toxicity through its effects on kinase activity and independently of GTPase activity, which reveals a novel mode of action for GAP proteins. This work identifies RGS2 as a promising target for interfering with neurodegeneration due to LRRK2 mutations in PD patients.

A rat model of progressive nigral neurodegeneration induced by the Parkinson's disease-associated G2019S mutation in LRRK2.

The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is the most common genetic cause of Parkinson's disease (PD), accounting for a significant proportion of both autosomal dominant familial and sporadic PD cases. Our aim in the present study is to generate a mammalian model of mutant G2019S LRRK2 pathogenesis, which reproduces the robust nigral neurodegeneration characteristic of PD. We developed adenoviral vectors to drive neuron-specific expression of full-length wild-type or mutant G2019S human LRRK2 in the nigrostriatal system of adult rats. Wild-type LRRK2 did not induce any significant neuronal loss. In contrast, under the same conditions and levels of expression, G2019S mutant LRRK2 causes a progressive degeneration of nigral dopaminergic neurons. Our data provide a novel rat model of PD, based on a prevalent genetic cause, that reproduces a cardinal feature of the disease within a rapid time frame suitable for testing of neuroprotective strategies.

Targeted overexpression of the parkin substrate Pael-R in the nigrostriatal system of adult rats to model Parkinson's disease.

Loss of function of parkin, an ubiquitin ligase, is responsible for autosomal recessive juvenile parkinsonism (AR-JP). Parkin-associated endothelin receptor-like receptor (Pael-R) was identified as an authentic substrate of parkin and is thought to accumulate abnormally following loss of parkin activity, causing neurodegeneration of nigral dopaminergic neurons in AR-JP patients. Our aim is therefore to generate a model of AR-JP through overexpression of Pael-R in the nigrostriatal system of adult rats. Using recombinant adeno-associated virus pseudotyped with the serotype 6 capsid (rAAV2/6) as a gene delivery tool, we achieved targeted and robust overexpression of rat Pael-R in nigral neurons and their striatal terminals. Overexpressed Pael-R was shown to accumulate very rapidly in an insoluble form. Accumulation of the receptor triggered a rapid and severe loss of nigral neurons and nigrostriatal fibers and terminals. No cell recovery was observed for up to 6 months post-injection. GABAergic neurons of the globus pallidus were unaffected by Pael-R overexpression, highlighting the selective vulnerability of nigral dopaminergic neurons to abnormal levels of Pael-R. Pael-R-induced degeneration also led to a depletion of striatal dopamine resulting in spontaneous motor impairments, as measured in the cylinder and stepping tests for forelimb akinesia. Interestingly, behavioral deficits of individual animals were correlated with the extent of the nigrostriatal lesion. Insoluble accumulation of Pael-R in the nigrostriatal system of adult rats represents, therefore, a chronic, rapidly progressing and specific model of AR-JP, which recapitulates major pathological hallmarks of the disease.

Metabolic regulation of the pancreatic beta-cell ATP-sensitive K+ channel: a pas de deux.

Closure of ATP-sensitive K+ channels (KATP channels) is a key step in glucose-stimulated insulin secretion. The precise mechanism(s) by which glucose metabolism regulates KATP channel activity, however, remains controversial. It is widely believed that the principal determinants are the intracellular concentrations of the metabolic ligands, ATP and ADP, which have opposing actions on KATP channels, with ATP closing and MgADP opening the channel. However, the sensitivity of the channel to these nucleotides in the intact cell, and their relative contribution to the regulation of channel activity, remains unclear. The precise role of phosphoinositides and long-chain acyl-CoA esters, which are capable of modulating the channel ATP sensitivity, is also uncertain. Furthermore, it is still a matter of debate whether it is changes in the concentration of ATP, of MgADP, or of other agents, which couples glucose metabolism to KATP channel activity. In this article, we review current knowledge of the metabolic regulation of the KATP channel and provide evidence that MgADP (or MgATP hydrolysis), acting at the regulatory subunit of the channel, shifts the ATP concentration-response curve into a range in which the channel pore can respond to dynamic changes in cytosolic ATP. This metabolic pas de deux orchestrates the pivotal role of ATP in metabolic regulation of the KATP channel.