Despite recent identification of a gene (npc1) that is mutated in Niemann- Pick Type C (NPC) disease, little is known about the mechanism(s) by which this error leads to the devastating, neurodegenerative changes and childhood neuropathology characteristic of this disorder. We recently provided new insight into the functional implications of this mutation by showing that neurons from the brain of embryonic mice, with NPC disease (npc/nih mice), exhibit not only abnormal cholesterol metabolism, but also deficient morphological and biochemical responses to the neurotrophin, BDNF. In additional experiments, we found that the insensitivity to BDNF stems from a lack of TrkB activation, despite expression of these receptors on the cell surface. Guided by these results, we propose to extend and expand our analysis of this disorder. First, we will define further the nature and extent of the defects in neuronal membrane signaling in NPC disease. We will evaluate the morphological responses to other growth factors, and use cell fractionation and gradient centrifugation to determine if the localization of TrkB and other growth factor receptors to low density, cholesterol-enriched microdomains (""""""""lipid rafts"""""""") in the plasma membrane is disrupted in neurons from npc/nih mice. Western blot analysis will also be used to assay the distribution of signaling intermediates often found in lipid rafts, such as Ras and Src, as well as the distribution of the GM1, GM2, and GM3 gangliosides which, in some cases, exhibit altered expression in NPC disease, and in other studies have been shown to be localized to lipid rafts and influence growth factor receptor function. The influence of NPC disease on membrane signaling, through ion channels and synaptic connections, will also be assessed using patch clamp measures of spontaneous activity, evoked activity, and synaptic currents in Purkinje cells in slices of the cerebellum, a region our in situ hybridization studies indicate express high-levels of the npc1 gene. Second, we will test the potential of adenoviral-mediated approaches to retard, halt, or even reverse the neurological deficits observed in NPC disease. To begin, we will determine if viral-mediated expression of an Npc1/GFP fusion protein can restore cholesterol metabolism and BDNF responsiveness to cultured striatal neurons from npc/nih mice. Furthermore, using different modes of infection, including intravenous administration and direct injection into the brain of npc/nih mice, we will assess the extent and duration of Npc1/GFP expression in vivo using confocal microscopic analysis of brain sections, and determine if viral-mediated expression influences aspects of physiology and behavior compromised in NPC disease, including weight, life-span, reproductive capacity, and motor performance. The proposed studies represent novel ideas and approaches with regard to NPC disease, and will both increase our understanding of the neurological deficits that occur and explore a potential therapeutic strategy for what is, at present, an incurable disorder.
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