Study on the nature of the cerebral storage process. eventually, the cortex. Our data suggest that focal deregulation of cdk5/p25 in axons leads to cytoskeletal abnormalities and eventual neurodegeneration in NPC. The mouse is a valuablemodel for determining how and when GW-870086 cdk5 becomes deregulated and whether cdk5 inhibitors would be useful in blocking NPC neurodegeneration. (also referred to asgene encodes for a cholesterol transporter in late endosomes, and the gene encodes for a lysosomal cholesterol-binding protein. Neuropathologically, NPC is characterized by neurons distended with lipid storage material having a foamy appearance, dendritic and axonal abnormalities, demyelination, and widespread neuronal loss (Elleder et al., 1985; Love et al., 1995; Suzuki et al., 1995). In addition, neurofibrillary tangles (NFTs), a diagnostic lesion of Alzheimer’s disease (AD), are also a consistent finding, particularly in cases with a prolonged course of disease (Auer et al., 1995; Love et al., 1995;Suzuki et al., 1995) (H. H. Klnemann, B. Bu, J. Husseman, M. Elleder, K. Suzuki, S. Salamant, S. Love, H. Budka, C. Fligner, T. Bird, L.-W. Jin, D. Nochlin, and I. Vincent, unpublished observations). How these various neuropathologic features result from altered cholesterol metabolism in NPC is a mystery and a rather difficult one to resolve given the rarity of the disease. A tremendous asset for unraveling the neuropathologic effects ofmutations is the BALB/cNpc-1nih mouse, which harbors a spontaneous mutation in its gene (Loftus et al., 1997). Mice with homozygous mutations (mice) display extensive lipid storage accumulation, neuroaxonal dystrophy, and neuronal loss, similar to that of human NPC (Higashi et al., 1993; Suzuki et al., 1995; Sawamura et al., 2001). Cholesterol (Xie et al., 1999; Sawamura et al., 2001) and glycosphingolipids such as gangliosides GM2 and neutral glycolipids (Walkley, 1995; Zervas et al., 2001) are the predominant constituents of storage material GW-870086 in the mouse brain. Curiously, however, neither alleviation of cholesterol (Patterson et al., 1993; Erickson et al., 2000; Camargo et al., 2001) nor ganglioside storage (Liu et al., 2000) ameliorate the neurological phenotype or progressive neuronal loss in mice or feline NPC, although lipid storage was effectively reduced in neurons and other cells. Thus, it is yet unclear what mechanism underlies neuronal dysfunction and loss of neurons in NPC. A notable difference between the mouse and human NPC is the absence of NFTs in the mouse (German et al., 2001a; Sawamura et al., 2001). However, in light of the conspicuous axonal abnormalities in human, murine, and feline NPC (Elleder et al., 1985; Higashi et al., 1993; Ong et al., 2001), we wondered whether cytoskeletal abnormalities contribute to neuronal dysfunction and degeneration in NPC. Therefore, we have undertaken a detailed characterization of cytoskeletal protein phosphorylation in the brains of mice. MATERIALS AND METHODS All procedures in this study were approved by the Internal Review Board and Animal Use and Care Committee of the University of Washington. A breeding pair of heterozygousmice obtained from The Jackson Laboratory (Bar Harbor, ME) GW-870086 was bred to generate wild-type (+/+), heterozygous (+/?), and homozygous (?/?) mice, which were identified using an established PCR-based method (Loftus et GW-870086 al., 1997). Tail biopsies for genotyping were performed at the time of weaning (i.e., at 3 weeks). Only ?/? mice have been reported to display pathology (Tanaka et al., 1988). In initial studies, we screened +/? mice and confirmed the absence of cytoskeletal pathology in this genotype. Hence, all further study concentrated on comparisons of ?/? mice with +/+ siblings. Twenty-eight ?/? mice (4, 5, 7, and 9 weeks of age, = 3; 6 and 8 weeks of age, = 5; 10, 11, and 12 weeks of age,= 2) and a minimum of two age-matched (for each week), wild-type littermates were analyzed by immunohistochemistry and immunoblotting. Mice were killed by carbon dioxide exposure followed by decapitation. The brains were removed quickly and divided sagittally into halves. The right halves were immersion fixed with 4% SHCB paraformaldehyde/PBS for 1 week and then embedded in paraffin. Where indicated, some mice were transcardially perfused with 4% paraformaldehyde/PBS, and the brain was then processed for paraffin embedding. The paraffin-embedded blocks were sectioned at 6 m for histological analyses. The left halves were frozen at ?80C for biochemical study. In some cases, the forebrain, cerebellum, and brainstem were isolated and frozen separately GW-870086 for regional analysis. Frozen.