Huntington's disease (HD) is a fetal inherited neurodegenerative disease and characterized by a selective loss of medium spiny neurons in the striatum. How to prevent the cell loss thus represents one of the most promising therapeutic strategies in treating HD. Our long-term goal is to characterize the molecular basis for mutant huntingtin (mhtt)-induced toxicity, which can lead us to identify potential targets for HD treatment. During the previous funding period, we clearly demonstrated in cell culture that mhtt up-regulates the expression of pro-apoptotic BH3-only protein BimEL, and BimEL is the critical molecule that functionally links mhtt aggregate formation to cell death. The current application is a logic extension of our research plan and aims to identify molecular causes for increased BimEL expression in two different HD models. Additionally, we will assess the physiological significance of BimEL knockdown in vivo using the R6/2 mouse model. Specifically, three aims are proposed in this application. First, we propose to test if reduced BDNF support observed in HD patients and animal models regulates BimEL expression to modulate striatal neuronal survival (Aim 1).
In Aim 1 a, we will reduce BDNF availability in cultured striatal cell lines that stably express full length murine htt with either 7 (STHdhQ7) and 111 (STHdhQ111) glutamines.
In Aim 1 b, we will test if increasing BDNF expression in vivo can reduce striatal BimEL levels in R6/2 mice. This will be achieved by crossing R6/2 mice with BDNF transgenic mice (BTg). In both experiments, changes in BimEL, phospho-BimEL and BDNF-mediated signaling will be analyzed by Western blot. Second, we propose to study the effect of BimEL phosphorylation on molecular interactions of BimEL with Bcl-2 family proteins (Aim 2). Using site-directed mutagenesis, we have created different BimEL phosphorylation mutants.
In Aim 2 a, these proteins will be used to test for altered interactions between mutant BimEL and Bcl-2 family proteins by co-immunoprecipitation from transfected Neuro-2a cells.
In Aim 2 b, we will test for protein-protein interactions between endogenous BimEL and pro-survival Bcl-2 family members using co-immunoprecipitation of striatal lysates prepared from WT and R6/2 mice. Last, we propose to test the hypothesis that BimEL gene knockout improves behavioral phenotypes in R6/2 mice (Aim 3).
In Aim 3 a, we will cross R6/2 HD mice with BimEL knockout mice to generate HD mice that lack BimEL expression.
In Aim 3 b, we will test if the crossed mice show improvements in behavioral symptoms compared to R6/2 mice using well-established behavioral measurements. At the conclusion of these studies, it will greatly improve our current understanding of the function of BimEL in HD pathogenesis and provide a valuable set of data to evaluate the promise of reducing BimEL expression as a potential novel therapeutic target.

Public Health Relevance

Huntington's disease (HD) is a devastating neurodegenerative disorder and HD patients usually die within 17 years of diagnosis. It has a prevalence of 3-10 affected subjects per 100,000 individuals in Western Europe and North America. Identifying the pathways that are responsible for mutant huntingtin-induced cell loss as proposed in the current application is crucial for understanding the cellular processes impacted by the disease and providing multiple points of therapeutic interventions in HD.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15NS066339-02A1
Application #
8366323
Study Section
Special Emphasis Panel (ZRG1-MDCN-E (96))
Program Officer
Sutherland, Margaret L
Project Start
2009-07-01
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2015-06-30
Support Year
2
Fiscal Year
2012
Total Cost
$428,694
Indirect Cost
$128,694
Name
Florida Atlantic University
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
004147534
City
Boca Raton
State
FL
Country
United States
Zip Code
33431
McCollum, Mark H; Leon, Rebecca T; Rush, Daniel B et al. (2013) Striatal oligodendrogliogenesis and neuroblast recruitment are increased in the R6/2 mouse model of Huntington's disease. Brain Res 1518:91-103
Rush, Daniel B; Leon, Rebecca T; McCollum, Mark H et al. (2012) Palmitoylation and trafficking of GAD65 are impaired in a cellular model of Huntington's disease. Biochem J 442:39-48
Leon, Rebecca; Bhagavatula, Nithya; Ulukpo, Onome et al. (2010) BimEL as a possible molecular link between proteasome dysfunction and cell death induced by mutant huntingtin. Eur J Neurosci 31:1915-25