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 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 impact 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 LRRK2 (leucine-rich repeat kinase 2) 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

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 patient

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

Modeling Parkinson's Disease in Adult Rats Using Viral Vectors as Gene Delivery Tools

Animal models of human pathologies remain invaluable tools for unraveling disease mechanisms and evaluating potential therapeutic strategies. For a number of diseases, the lack of a reliable animal model represents an important limiting step towards the development of efficient treatments. This holds particularly true for Parkinson's disease (PD), a major neurodegenerative disorder for which only symptomatic treatments currently exist. The difficulties encountered by researchers to reproduce PD pathology in animals stem primarily from an incomplete understanding of the disease. Indeed, the cause of the disease remains unknown in 90% of cases, referred to as sporadic or idiopathic. The discovery of familial forms of the disease, however, has led to the development of a large number of transgenic mice models based on genetic modifications that play a direct causative role in a significant proportion of human PD cases. Unfortunately, these transgenic mice fail to recapitulate the robust neurodegeneration of dopaminergic (DAergic) neurons of the substantia nigra pars compacta (SNpc) and concomitant loss of DAergic projections to the striatum, the neuropathological hallmark of the human condition. The lack of nigral pathology severely limits the usefulness of such models for pre-clinical evaluation of potential therapeutics. Viral vector gene delivery tools represent an interesting alternative to classical transgenesis as they allow for targeted and high-level transgene expression in the nigrostriatal system of adult animals. During the course of this thesis we have developed two new viral vector-based rodent models of PD. In our first model, we have used a recombinant adeno-associated virus (rAAV) vector, with a high tropism towards nigral DAergic neurons, to drive overexpression of the parkin-associated endothelin receptor-like receptor (Pael-R) in the SNpc of adult rats. Indeed, accumulation of Pael-R is implicated in the pathogenesis of autosomal-recessive juvenile parkinsonism (AR-JP), a young-onset familial form of PD. We show that insoluble accumulation of Pael-R in rats induces a rapidly progressing degeneration of nigral DAergic neurons and a loss of DAergic fibers and terminals in the striatum. Lesioned animals also displayed spontaneous behavioral abnormalities linked to depletion of striatal dopamine (DA) and persisting up to 6 months post-injection. Chronic accumulation of Pael-R in the nigrostriatal system of adult rats therefore represents a robust and highly reproducible model of PD, recapitulating key pathological and phenotypical features of the human condition. The second model developed was based on nigral delivery of the PD-associated mutant G2019S leucine-rich repeat kinase 2 (LRRK2) protein. Indeed, the G2019S mutation in the LRRK2 gene is the most important genetic determinant of PD, accounting for a significant proportion of both familial and sporadic PD cases. Due to the large size of the LRRK2 coding sequence, an adenoviral system with a high packaging capacity was used to drive expression of the protein. Recombinant adenoviral (rAd) vectors are potentially pro-inflammatory and less efficient tools as rAAV vectors for long-term gene delivery to the SNpc. Nevertheless, through retrograde axonal delivery of rAd-LRRK2 particles, we achieved robust and neuron-specific expression of full-length wild-type or mutant G2019S human LRRK2 in nigral DAergic neurons of adult rats. Expression persisted up to 6 weeks post-injection, with no visible signs of inflammation in the SNpc. We demonstrate that the wild-type form of LRRK2 does not induce neuronal loss when expressed in the SNpc. In contrast, under the same conditions and levels of expression as the wild-type form, the PD-associated G2019S mutation in LRRK2 is sufficient to cause a progressive loss of nigral dopaminergic neurons. This is the first demonstration of frank dopaminergic neuronal degeneration in rodents induced by the expression of G2019S mutant LRRK2. Our data also provide a new rodent model of LRRK2-linked PD which recapitulates one of the cardinal pathological features of the disease. In the absence of a clear understanding of human PD pathogenesis, the development of multiple transgenic models may help to identify common disease mechanisms and drug targets. A potential treatment identified in one model may be effective only in the corresponding subset of PD patients, carrying this particular genetic modification. On the other hand, the pathogenic pathway targeted may also be activated in sporadic PD. Ultimately, whether or not these models stand the test of time will depend on how effective newly-identified drugs will be, not only on AR-JP or LRRK2-linked PD patients, but above all on sporadic PD, which represents the vast majority of PD patients