Beta-site APP-cleaving enzyme (BACE) is required for production of the Alzheimer's disease (AD)-associated Abeta protein. BACE levels are elevated in AD brain, and increasing evidence reveals BACE as a stress-related protease that is upregulated following cerebral ischemia. However, the molecular mechanism responsible is unknown. We show that increases in BACE and beta-secretase activity are due to posttranslational stabilization following caspase activation. We also found that during cerebral ischemia, levels of GGA3, an adaptor protein involved in BACE trafficking, are reduced, while BACE levels are increased. RNAi silencing of GGA3 also elevated levels of BACE and Abeta. Finally, in AD brain samples, GGA3 protein levels were significantly decreased and inversely correlated with increased levels of BACE. In summary, we have elucidated a GGA3-dependent mechanism regulating BACE levels and beta-secretase activity. This mechanism may explain increased cerebral levels of BACE and Abeta following cerebral ischemia and existing in AD.

Converging lines of evidence implicate the beta-amyloid peptide (Ab) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce Ab production by functionally inhibiting g-secretase, the activity responsible for the carboxy-terminal cleavage required for Ab production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon Ab production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-di¯uoro-phenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP V717F reduces brain levels of Ab in a dose-dependent manner within 3 h. These studies represent the ®rst demonstration of a reduction of brain Ab in vivo. Development of such novel functional g-secretase inhibitors will enable a clinical examination of the Ab hypothesis that Ab peptide drives the neuropathology observed in Alzheimer's disease.

Estrogen receptors (ERs) are believed to be ligand-activated transcription factors belonging to the nuclear receptor superfamily, which on ligand binding translocate into the nucleus and activate gene transcription. To date, two ERs have been identified: ER␣ and ER␤. ER␣ plays major role in the estrogen-mediated genomic actions in both reproductive and nonreproductive tissue, whereas the function of ER␤ is still unclear. In this study, we used immunocytochemistry, immunoblotting, and proteomics to demonstrate that ER␤ localizes to the mitochondria. In immunocytochemistry studies, ER␤ was detected with two ER␤ antibodies and found to colocalize almost exclusively with a mitochondrial marker in rat primary neuron, primary cardiomyocyte, and a murine hippocampal cell line. The colocalization of ER␤ and mitochondrial markers was identified by both fluorescence and confocal microscopy. No translocation of ER␤ into the nucleus on 17␤-estradiol treatment was seen by using immunocytochemistry. Immunoblotting of purified human heart mitochondria showed an intense signal of ER␤, whereas no signals for nuclear and other organelle markers were found. Finally, purified human heart mitochondrial proteins were separated by SDS͞PAGE. The 50,000 -65,000 Mr band was digested with trypsin and subjected to matrix-assisted laser desorption͞ionization mass spectrometric analysis, which revealed seven tryptic fragments that matched with those of ER␤. In summary, this study demonstrated that ER␤ is localized to mitochondria, suggesting a role for mitochondrial ER␤ in estrogen effects on this important organelle.nuclear receptor ͉ mitochondria E strogens play an important role in development, growth, and differentiation of both female and male secondary sex characteristics. Estrogen receptors (ERs) were the first identified nuclear receptor family member (1). The first ER, now called ER␣, was cloned in 1986 (2, 3). A second ER␤, was identified and cloned a decade later (4, 5). Like other members of the nuclear receptor superfamily, both ERs have a modular structure consisting of distinct functional domains (1). The DNA-binding domain (DBD) enables the receptor to bind its cognate target site consisting of an inverted repeat of two half-sites with the consensus motif AG-GTCA spaced by 3 bp, referred to as an estrogen response element (ERE). The ligand-binding domain enables estrogen binding to the receptors. ERs are highly conserved between ER␣ and ER␤, with Ͼ95% homology for the DBD and Ϸ50% homology for the ligand-binding domain. Less homology is observed for the transactivational domain between ER␣ and ER␤ (5, 6).Genomic actions of ER␣ are well described (7). On binding to ER␣, estrogens induce a conformational change in the ER␣ proteins, which is accompanied by the dissociation of the accessory protein, heat shock protein 90, thereby exposing the DBD. In the nucleus, the receptor-ligand complex binds to DNA and modulates gene transcription. This transcriptional͞translational activation is comparatively slow and sensitive to cyclohexi...

Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a ''CAAX protein'' that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by 2 statistic). These studies suggest a possible treatment strategy for HGPS.aging ͉ lamin A͞C ͉ laminopathy H utchinson-Gilford progeria syndrome (HGPS) is a progeroid syndrome characterized by a host of aging-like phenotypes, including a wizened appearance of the skin, osteoporosis, alopecia, and premature atherosclerosis (1). Children with HGPS die at the mean age of 13, generally from myocardial infarctions or strokes (1). This disease is caused by the accumulation of a mutant form of prelamin A that cannot be processed to mature lamin A (1). In normal cells, wild-type prelamin A is virtually undetectable because it is fully converted to mature lamin A, a structural protein of the nuclear lamina (2, 3). The nuclear lamina is an intermediate filament meshwork adjacent to the inner nuclear membrane that provides structural support for the nucleus (2, 3).Prelamin A contains a nuclear localization signal and terminates with a CAAX motif (2), in which C is a cysteine, A residues are usually aliphatic amino acids, and X can be one of many different residues. CAAX motifs are also found on lamin B1, lamin B2, the Ras family of proteins, and many other cellular proteins. The CAAX motif triggers three sequential enzymatic posttranslational modifications, beginning with protein prenylation. In the case of prelamin A, the first processing step is carried out by protein farnesyltransferase (FTase) and involves the addition of a 15-carbon farnesyl lipid to the thiol group of the cysteine within the CAAX motif. Second, the last 3 aa of the protein (i.e., ϪAAX) are removed by a prenylprotein-specific endoprotease. For p...