Jang-Ho J. Cha - US grants

Glutamate receptors (GluR's) mediate the majority of excitatory signal transduction within the mammalian central nervous system. Excitotoxicity, a process in which abnormal activation of GluR's leads to neuronal death, is thought to play a role in numerous neurological conditions. The role of GluR's linked to ion channels ("ionotropic" glutamate receptors, iGluR's) in excitotoxicity has been well documented. Activation of GluR's linked to second messenger systems ("metabotropic" glutamate receptors, mGluR's) can also influence excitotoxicity. mGluR's which stimulate phosphoinositide metabolism may exacerbate excitotoxicity, whereas those mGluR's which inhibit cyclic AMP production may attenuate excitotoxicity. While the mechanisms by which mGluR's influence excitotoxicity remain unclear, one possibility is that mGluR's influence excitotoxic processes by altering the function of iGluR's, for example, through phosphorylation of iGluR's. mGluR's thus offer an attractive potential target for the development of therapeutic neuroprotective agents. The objective of this proposal will be to determine how mGluR's affect iGluR-mediated striatal excitotoxicity. Three main areas will be investigated: 1) the ability of mGluR agonists and antagonists as well as of antisense oligonucleotides directed against specific mGluR's to influence in vivo excitotoxicity, 2) the ability of antagonists, activators, and antisense oligonucleotides targeted against mGluR-linked second messenger systems to influence in vivo excitotoxicity, and 3) the effect of mGluR receptor stimulation on iGluR phosphorylation. The determination of in vivo excitotoxicity will involve lesion size analysis in a well characterized model: stereotaxic intrastriatal injection in the rat. In addition, reverse transcription-PCR, in situ hybridization, receptor binding, Western blotting, striatal cell culture, and functional biochemical assays will be employed to assess both the efficacy of anti sense oligonucleotide treatment, as well as regulation of receptor function. Taken together, these studies are designed to shed light on the important interactions between different types of GluR's, especially as they pertain to excitotoxicity. Once the relationships have been elucidated, mGluR's themselves may become logical targets for the development of novel neuroprotective agents.

DESCRIPTION: (Verbatim from the Applicant's Abstract) Recently Huntington's disease (HD) and other triplet repeat disorders were found to be caused by expansions in the length of CAG codon repeat stretches in the mutated genes and mice transgenic for these mutations were created. The deleterious, biochemical consequences of this type of mutation remain unknown. Any clues to the biochemical effects of the mutation may, therefore, be vital clues to the toxic consequences of the mutation. We have found striking changes in dopamine D1 and D2, adenosine A2a and metabotropic glutamate type 2 receptor mRNA expression in three strains of mice transgenic for exon 1 of the HD gene with increased polyglutamines. The observed changes in receptor mRNA suggest that the truncated HD protein alters gene transcription. Few other biochemical changes have been found in HD transgenic mice. As such, it is one of the very few clues we can use to determine the pathologic mechanism of the disease. While receptor changes may themselves not cause disease, our data strongly suggest that receptor changes reflect a crucial, deleterious process which may include dysregulation of gene expression. Further, the magnitude of the change we have found (up to 90% decrease in presymptomatic mice) provides a strong readout that can be used in mechanistic and therapeutic studies. We will determine the generalizability and validity of these changes by measuring the levels of mRNA and protein expression for these receptors and control receptors in identified neuronal types of cortex, striatum, hippocampus and cerebellum in HD transgenic and littermate control mice as well as post-mortem presymptomatic HD and control human brains using techniques we have developed over the last 15 years; double label isotopic and nonisotopic in situ hybridization, immunoblotting, ligand binding assays and immuno-cytochemistry. We also will determine the relationship of the changes to the development of the neuronal intranuclear inclusions. (NII) that have been seen in both these mice and in human HD, whether they occur in cultured cells transfected with the mutant HD gene and the degree to which receptor changes correlate with CAG repeat number in human, mouse and cultured cells.

DESCRIPTION (provided by applicant): Huntington's disease is a progressive neurodegenerative disease for which no effective treatment exists. Transcriptional dysregulation is now considered to be an important mechanism in the pathogenesis of Huntington's disease (HD) and other polyglutamine diseases. Alteration of mRNA populations, including those encoding for neurotransmitter receptors, is a hallmark of murine and cellular models of HD as well as human HD. Although numerous studies have confirmed that mRNA populations are altered in HD models, the mechanism underlying such changes remains unknown. Transcription factors, including specificity protein 1 (Sp1), have been implicated in HD pathogenesis, but the role of altered Sp1 function in producing mRNA alterations has not been answered. In this application, we take advantage of a well-described set of gene alterations--those occurring in neurotransmitter receptors--as a starting point for determining the molecular mechanisms of transcriptional dysregulation. We propose a series of hypotheses that will yield critical mechanistic insight into the processes that alter mRNA populations and cause disease pathogenesis. Specific Aim 1 will test the hypothesis that mutant huntingtin selectively alters the association of Sp1 with the promoters of genes that are downregulated in HD. Chromatin Immunoprecipitation (CHIP) assays will assess the degree of Sp1-gene binding in cellular, murine and human HD tissues. Specific Aim 2 will test the hypothesis that decreased Sp1 binding to selected gene promoters causes HD phenotypes. Sp1 levels will be manipulated through RNA interference, dominant negative constructs and overexpression, and the effects of these manipulations on mRNA and cellular toxicity will be assessed. Specific Aim 3 tests the hypothesis that interference in Sp 1 function by huntingtin causes abnormal histone modification. Histone modifications will be examined in mouse and cell models of HD. Taken together, these proposed experiments will systematically address several key molecular loci of potential damage by mutant huntingtin. Such fundamental mechanistic information is critical to the eventual development of effective therapy for HD.

DESCRIPTION (provided by applicant): This application requests funding for the 2009 Gordon Research Conference on CAG Triplet Repeat Disorders to be held at Waterville Valley Resort, New Hampshire from May 31 to June 5, 2009 as well as the associated Graduate Research Seminar to be held May 30-31, 2009. This will be the fifth Gordon Research Conference on CAG Triplet Repeat Disorders, with the previous four conferences having alternated between American (Mount Holyoke College, 2001 and 2005) and European (Il Ciocco, Italy 2003 and Aussois, France 2007) sites. This is the first year that there will be an associated Graduate Research Seminar. The CAG Triplet Repeat Disorders are a group of largely untreatable inherited neurological disorders which result from an expansion in a CAG trinucleotide repeat in the mutant genes. This group of diseases includes Huntington's disease (HD), spinal and bulbar muscular atrophy (SBMA, Kennedy's disease), spinocerebellar ataxias types 1, 2, 3, 6, 7, and 17, and dentatorubropallidoluysian atrophy (DRPLA). In each case, the CAG repeat lies within the coding region of a gene and results in an abnormally long polyglutamine tract within the mutant protein. Marked similarities in the underlying genetics and neuropathology suggest common pathologic mechanisms among these disorders. Differences in the anatomical distribution of selective neuronal degeneration also make it imperative to unravel the distinguishing factors. Since the identification of the genetic defects, significant insights have been gained into the pathogenesis of these diseases such that the development of therapeutic interventions is now a reality. In order not only to increase the pace of basic research discovery but also to move the basic science into the clinic, a multidisciplinary research effort is required. It is essential that collaborative projects between scientists from diverse disciplines ranging from organic chemistry and fruit fly genetics to neurology and human clinical trials be established. The conference on CAG Triplet Repeat Disorders will gather together young investigators and established senior scientist to deliver provoking lectures on the cutting-edge of science. In keeping with the Gordon Research Conference format, there will be generous time allocated for structured discussions led by peers and for informal discussion and social interactions to facilitate collaboration. Strong emphasis is placed on mentoring of young scientists, and time will be devoted to career issues. All participants will be required to present posters. Priority will be given to women, minorities and persons with disabilities when selecting participants. PUBLIC HEALTH RELEVANCE: The 2009 Gordon Research Conference on CAG Triplet Repeat Disorders and its associated Graduate Research Seminar will bring together researchers and clinicians to discuss cutting edge information on disease mechanisms and therapeutic interventions for these devastating neurological diseases. In addition, the format of the Gordon Research Conference and the funding sought herein will promote and ensure the attendance and enhanced education of junior scientists, including graduate students, postdoctoral fellows, and junior faculty.