Investigators/Authors: Yanping Li, PhD, PI; J. William Langston, MD, co-Investigator, Parkinson’s Institute and Clinical Center
Objective: The goal of this study is to generate a novel genetic model that has the exciting potential to replicate the wide spectrum of key PD clinical and neuropathological features. This model will provide a unique opportunity to explore the cause of nerve cell death in PD and lead to new strategies for therapeutic intervention.
Background: This grant will link two different mutations related to PD into a single mouse model to test the hypothesis that this will produce a more complete and more useful model of the disease than has been possible to date. The first of these is a mutation in the gene (GBA) encoding for an enzyme known as glucocerebrosidase (GCase).Typically these mutations cause lipid (fat) to accumulate in the brain and it is usually fatal at an early age (these are called “lipid storage diseases”. However, there is an adult form in which patients are relatively unaffected. While originally controversial, it is now clear that the adult form mutations in GBA not only increase the risk for PD, but they represent the most common genetic risk factor for PD. A recent report by Mazzulli and colleagues (2011) linking GCase to ?-synuclein provides a biologic connection to PD as ?-synuclein is a key player in PD neurodegeneration. Current theories suggest that toxic forms of a-synuclein are being transmitted from cell to cell causing spread of PD. The fatal even may be the inability to degrade this accumulating toxic synuclein by the part of the cell (known as the “lysosomal-autophagy degradation pathway”). Importantly, this same system is also affected by GBA mutations.
The other gene to be studied in combination with GBA mutations encodes a protein known as leucine-rich repeat kinase 2 (LRRK2). One mutation in this gene (known as G2019) is the most common known genetic cause for both familial and sporadic PD. In exploring the role of LRRK2 in PD, we have generated the first (and most widely used) LRRK2 mouse model to date; this model has expressed the abnormal LRRK2 protein associated with PD and it has shown some, but not the full spectrum of the symptoms of PD. Thus it seems likely that genetic interactions working in concert LRRK2 might be needed in order to obtain the complete replication of the key features of PD. Interestingly it has been reported that both LRRK2 and GBA share may share same degradation process. We suspect that the mutation of GBA might be “the straw” that breaks “the camel’s back” in LRRK2, resulting in the presence of the wide spectrum of key features of PD in the animal model containing both mutations of GBA and LRRK2.We therefore propose to explore whether there is mechanistic interplay between GBA and LRRK2 and how the interaction contributes to the full spectrum of key features of PD. This will be done generating a digenic mouse model (LRRK2-GBA) carrying human mutations of both GBA and LRRK2. To the best of our knowledge, this has never been done before. Importantly, now have a cohort of patients with both GBA and LRRK2 muations. Our new transgenic model will allow us to study the biologic effects in mice with these combined mutation in ways that would not be possible in humans.
Methods/Design: We will generate the above LRRK2-GBA digenic mouse model as following: First, we will create a transgenic mouse model carrying a mutant GBA associated with PD. Second, the GBA transgenic mice will cross breed with the aforementioned LRRK2 transgenic models to generate a digenic LRRK2-GBA transgenic mouse line. Full characterization of the new mouse model will be carried out.
Relevance to Diagnosis/Treatment of Parkinson’s disease: We anticipate that the digenic model may develop more severe and wider spectrum of key features of PD, some of which are absent in the current animal models. This digenic mouse model will provide a novel and valuable genetic tool for determining how the interaction of these two genes contributes to PD pathogenesis, and more importantly for drug screening. At the end of a year funding period, we expect to successfully establish the LRRK2_GBA digenic transgenic mouse lines, make them available to other investigators and for early stage characterization. The support from Unity Walk for this promising work is highly appreciated.
September 2013 Project Update:
With the generous support from the Unity Walk we are able to support our current program of creating a unique mouse model that carries a combination of mutant GBA and LRRK2, both of which are known to play a major role in sporadic and familial PD. We are using a transgenic strategy to create in a first step the GBA mouse model and then cross breed these mice with our previously generated LRRK2 mice to create a ‘digenic’ rodent model of PD.
The general workflow to generate the GBA mutant mice has involved first creating a vector or ‘DNA template’ containing the GBA mutation. We chose a mutation in the GBA gene which specifically encodes for a substitution at position Asn370 to Ser of the GBA gene, a change which is one of the common mutations in the GBA gene. We have isolated a clone encompassing the entire GBA gene and have inserted the GBA mutation into this clone. Our next step is to introduce this vector into fertilized eggs and then to identify animals that carry the mutation. The generation of these GBA mice is well under way and subsequent breeding with LRRK2 mutant mice is following. It is important to note that in our LRRK2 mutant mice, we can already observe accumulations of alpha-synuclein and we expect that LRRK2 mutants in combination with GBA mutants in the same mouse will
exacerbate this effect.
It is our hope that the utilization of transgenic technology, coupled with ‘digenic’ disease-causing mutations will drive the full spectrum of the PD phenotype in this model, something that has not been achieved to date. If we are successful, this model can be used for testing novel drugs and aid as a tool for elucidating mechanisms that underlie the process of neuronal degeneration in Parkinson’s disease.
September 2014 Project Update:
As noted above, virtually all mouse models of PD have limitations. The goal of this grant is to create a genetically engineered mouse model that carries not one but two (double hit) mutations that cause or predispose to typical Lewy body Parkinson’s disease in human. The first mutation is in the gene for LRRK2, which we already developed. The second is in a gene that causes Gaucher disease, in protein known as GBA. We first attempted to build a new GBA mutated mouse model from the ground up and then cross-bred this model with our already existent in-house LRRK2 mutant mouse. However, the presence of a so-called false gene (also known as a “pseudogene”) in the region of interest made this technical feat extremely costly and time consuming. We have now obtained a new GBA mutant mouse are now mating these GBA mutant mice with LRRK2 mutant transgenic mice to generate our “double hit mice”. If these mice exhibit a more faithful model of PD with Lewy bodies than has previously been possible, it could provide a transformative tool to investigate the mechanisms of Lewy body PD, and also provide an new exciting new model for drug discovery in PD.
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