Isolation of Novel Transcripts & Proteins in Hypocretin-Containing Cells

Stanford University, Stanford CA

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Abstract

Project E: Isolation of Novel Transcripts &Proteins in Hypocretin-Containing Cells The etiology of narcolepsy-cataplexy is now known to involve a degeneration of hypocretin-containing neurons in the hypothalamus. The cause of the hypocretin cell loss is unknown but, considering the well known HLA-narcolepsy association, likely to be autoimmune. Preliminary studies have not identified any specific autoantibodies against hypocretin peptides themselves, suggesting the existence of other factors Simple western blots and immunochemical staining experiments have also failed to reveal evidence fo hypothalamic-directed autoimmunity. Novel experimental approaches are needed to identify additiona factors. The goal of our revised proposal is to systematically isolate and study genes and gene products preferentially expressed in hypocretin-producing neurons. We believe that the identification of these factors will further our understanding of the physiology of these cells and provide further candidate proteins for the presumed autoimmune origin of narcolepsy. To isolate and study these factors, we will (1) Use gene expression array studies in hypocretin neuron deficient versus wild-type littermate mouse brains to establish a collection of transcripts potentially expressed specifically in hypocretin neurons. Hypocretin knockout mice will be used as an additional control. (2) Use subtractive hybridization to generate a collection of low abundant transcripts enriched in mouse hypocretin neurons. (3) Use gene expression arrays and quantitative mmunohistochemistry with brain tissue obtained from narcoleptic patients with and without cataplexy and com control individuals to establish key differences among these groups. (4) Validate all possible ranscriptional candidates by quantitative real time (RT)-PCR and in situ hybridization and explore their nvolvement in the pathophysiology of narcolepsy. This will involve verification of primary expression in hypocretin cells and basic characterization of diurnal variation. Depending on the candidate genes, other ypes of functional validation such as immunological studies, generation of genetically modified animals or candidate gene analysis in human narcolepsy DMA samples may be carried out. Whether or not narcolepsy s an autoimmune disease, the identification of products specific for hypocretin-containing cells is likely to shed new light into the pathophysiology of narcolepsy. These studies are the logical next step toward understanding why hypocretin cell loss occurs in human narcolepsy.