Objective/Rationale:
LRRK2 is a human gene encoding a protein kinase whose normal function in healthy subjects is still unknown. However, mutations in the LRRK2 gene have recently been recognized as the cause of a familial form of PD. Our goal is to express PD-causing mutant forms of the LRRK2 protein in the substantia nigra neurons of rats to elucidate the link between the mutated protein and the development of PD.
Project Description:
To express the LRRK2 protein we will take advantage of unique properties offered by adenoviral vectors. The choice of adenoviral vectors is dictated by the size of the LRRK2 gene which largely exceeds what can be accommodated in adeno-associated viral vectors or lentiviral vectors. To avoid inflammation and to achieve long term expression the helper dependent version of the adenoviral system will be used. Since the gene delivery system derived from the canine adenovirus 2 has a strong preference for neurons and is retrogradely transported from the striatum to the substantia nigra much more efficiently than human adenovirus, we will use the canine system. Beside the normal LRRK2 protein, a mutated variant with an increased kinase activity and a variant with no kinase activity will be tested.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
By providing an animal model of the LRRK2-associated PD, our project will contribute a key biological system to discover and test novel therapies.
Anticipated Outcome:
The expression of the mutated LRRK2 protein should induce in the rat dopaminergic neurons a disease process closely resembling the one affecting human patients.
Final Outcome
We have generated second-generation human adenoviral vectors (Ad5) coding for LRRK2. Following injection in the brains of pre-clinical models, these vectors drive robust expression of human LRRK2 in more than 30% of substantia nigra dopamine neurons. Overexpressed LRRK2 can be detected for at least 42 days following vector injection, in spite of a progressive decline in expression from two weeks onward. This model is currently being explored to directly compare the pathogenic effect of G2019S mutant LRRK2 vs. wild-type LRRK2. In order to generate an alternative vector allowing for stable long-term expression, we have produced canine adenoviral particles coding for LRRK2 and its mutant form. The functionality of this vector system, which has been reported to induce expression in neurons that can last a year, will be tested in an experimental paradigm similar to the one established for Ad5-LRRK2. These vector systems will complement transgenic pre-clinical models to investigate in vivo the role of LRRK2 in the demise of adult substantia nigra dopamine neurons.
The investigators published the following paper based on this work
Dusonchet J, Kochubey O, Stafa K, Young SM Jr, Zufferey R, Moore DJ, Schneider BL, Aebischer P. A pre-clinical model of progressive nigral neurodegeneration induced by the Parkinson's disease-associated G2019S mutation in LRRK2. Journal of Neuroscience. 31(3):907-12, 2011.
\Note: This project is associated with a follow-up grant from Dr. Darren Moore who is characterizing pre-clinical models developed using this viral vector delivery of LRRK2.