Cyclin-dependent kinase 5 (Cdk5) and its regulatory subunit p35 are integral players in the proper development of the mammalian central nervous system. Proteolytic cleavage of p35 generates p25, leading to aberrant Cdk5 activation. The accumulation of p25 is implicated in several neurodegenerative diseases. In primary neurons, p25 causes apoptosis and tau hyperphosphorylation. Current mouse models expressing p25, however, fail to rigorously recapitulate these phenotypes in vivo. Here, we generated inducible transgenic mouse lines overexpressing p25 in the postnatal forebrain. Induction of p25 preferentially directed Cdk5 to pathological substrates. These animals exhibited neuronal loss in the cortex and hippocampus, accompanied by forebrain atrophy, astrogliosis, and caspase-3 activation. Endogenous tau was hyperphosphorylated at many epitopes, aggregated tau accumulated, and neurofibrillary pathology developed progressively in these animals. Our cumulative findings provide compelling evidence that in vivo deregulation of Cdk5 by p25 plays a causative role in neurodegeneration and the development of neurofibrillary pathology.

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Niemann-Pick type C (NPC) is a disease that affects intracellular cholesterol-trafficking pathways. By cloning the hamster ortholog of NPC1, we identified the molecular lesions in two independently isolated Chinese hamster ovary cell mutants, CT60 and CT43. Both mutants lead to premature translational terminations of the NPC1 protein. Transfecting hamster NPC1 cDNA complemented the defects of the mutants. Investigation of the CT mutants, their parental cells, and an NPC1-stable transfectant allow us to present evidence that NPC1 is involved in a post-plasma membrane cholesterol-trafficking pathway. We found that the initial movement of low density lipoprotein (LDL)-derived cholesterol to the plasma membrane (PM) did not require NPC1. After reaching the PM and subsequent internalization, however, cholesterol trafficking back to the PM did involve NPC1. Both LDL-derived cholesterol and cholesterol originating from the PM accumulated in a dense, intracellular compartment in the CT mutants. Cholesterol movement from this compartment to the PM or endoplasmic reticulum was defective in the CT mutants. Our results functionally distinguish the dense, intracellular compartment from the early endocytic hydrolytic organelle and imply that NPC1 is involved in sorting cholesterol from the intracellular compartment back to the PM or to the endoplasmic reticulum.

Aberrant processing of the amyloid precursor protein (APP) and the subsequent accumulation of amyloid ␤ (A␤) peptide has been widely established as a central event in Alzheimer's disease (AD) pathogenesis. The sequential cleavage steps required for the generation of A␤ are well outlined; however, there is a relative dearth of knowledge pertaining to signaling pathways and molecular mechanisms that can modulate this process. Here, we demonstrate a novel role for p25/cyclin-dependent kinase 5 (Cdk5) in regulating APP processing, A␤ peptide generation, and intraneuronal A␤ accumulation in inducible p25 transgenic and compound PD-APP transgenic mouse models that demonstrate deregulated Cdk5 activity and a neurodegenerative phenotype. Induction of p25 resulted in enhanced forebrain A␤ levels before any evidence of neuropathology in these mice. Intracellular A␤ accumulated in perinuclear regions and distended axons within the forebrains of these mice. Evidence for modulations in axonal transport or ␤-site APP cleaving enzyme 1 protein levels and activity are presented as mechanisms that may account for the A␤ accumulation caused by p25/Cdk5 deregulation. Collectively, these findings delineate a novel pathological mechanism involving aberrant APP processing by p25/Cdk5 and have important implications in AD pathogenesis.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT)is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl coenzyme A. The first gene encoding the enzyme, designated as ACAT-1, was identified in 1993 through an expression cloning approach. We isolated a Chinese hamster ovary cell line that stably expresses the recombinant human ACAT-1 protein bearing an N-terminal hexahistidine tag. We purified this enzyme approximately 7000-fold from crude cell extracts by first solubilizing the cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, then proceeding with an ACAT-1 monoclonal antibody affinity column and an immobilized metal affinity column. The final preparation is enzymologically active and migrates as a single band at 54 kDa on SDS-polyacrylamide gel electrophoresis. Pure ACAT-1 dispersed in mixed micelles containing sodium taurocholate, phosphatidylcholine, and cholesterol remains catalytically active. The cholesterol substrate saturation curves of the enzyme assayed either in mixed micelles or in reconstituted vesicles are both highly sigmoidal. The oleoyl-coenzyme A substrate saturation curves of the enzyme assayed under the same conditions are both hyperbolic. These results support the hypothesis that ACAT is an allosteric enzyme regulated by cholesterol. Specific polyclonal anti-ACAT-1 IgGs have been produced (19 -21). Immunoblot and immunodepletion analyses show that the ACAT-1 protein is present in homogenates from various human cells and tissues as a single 50-kDa protein band in SDS-PAGE (19) Furthermore, immunodepletion experiments using anti-ACAT-1 IgGs suggest that the ACAT-1 protein plays a major role in ACAT catalysis in human fibroblasts, HepG2 cells, human hepatocytes, macrophages, adrenal glands, and kidneys (22). In contrast, in human intestines, approximately 80% of total measurable ACAT activity is resistant to immunodepletion, suggesting that ACAT activity in this particular tissue may be largely due to the presence of a different ACAT protein (22). Judging from results currently available, it is possible that the physiological functions of ACAT-1 and ACAT-2 are different in different species. Whether ACAT-2 is responsible for most of the observed ACAT activity in human intestines is not clear at present. Acyl-coenzymeThe ACAT protein has never before been purified to homogeneity. The difficulty in doing so was largely due to its minute quantity and the lack of a suitable detergent for solubilizing the protein from the endoplasmic reticulum membrane with retention of enzyme activity (reviewed in Ref. 5). As described in this article, we have isolated a CHO cell line (HisACAT-1 cells) that stably expresses the human ACAT-1 (hACAT-1) protein bearing a hexahistidine tag at its N terminus. We report the use of this cell line as the starting material to develop a purification scheme that enables us to purify the enzyme to essential homogenei...

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