Isoflurane facilitates synaptic NMDA receptor endocytosis in mice primary neurons.

Inhalation anesthetic isoflurane has been reported to induce caspase activation and accumulation of ß-amyloid (Aß), however, the down-stream consequences of these effects are largely unknown. Isoflurane has also been shown to impair learning and memory, however, the up-stream mechanisms of these effects remain largely to be determined. Facilitation of synaptic NMDA receptor endocytosis can reduce synaptic function, leading to learning and memory impairment. We therefore set out to determine the effects of isoflurane on synaptic NMDA receptor endocytosis. Primary neurons from wild-type and Alzheimer's disease transgenic mice were treated with 2% isoflurane for six hours. Synaptic surface levels of NMDA receptor 2B (NR2B) and NR2B internalization were determined by surface and cleavable biotinylation assay, western blot analysis and immunofluorescence. Here we show that isoflurane can induce caspase-3 activation, increase levels of ß-site amyloid precursor protein-cleaving enzyme and cause accumulation of Aß in the primary neurons. Isoflurane facilitates synaptic NR2B endocytosis as evidenced by reducing surface NR2B levels, increasing NR2B internalization, and decreasing the ratio of synaptic surface NR2B to synapsin in mice primary neurons. Moreover, caspase activation inhibitor Z-VAD and γ-secretase inhibitor L-685,458 attenuated the isoflurane-facilitated NR2B endocytosis. These results suggest that isoflurane induces caspase activation and Aß accumulation, leading to facilitation of synaptic NMDA receptor endocytosis, which potentially serve as the upstream mechanism of the isoflurane-induced impairment of learning and memory. These findings will encourage further studies to determine the underlying mechanism by which isoflurane and other anesthetics promote Alzheimer's disease neuropathogenesis and induce cognitive dysfunction.

Acyl-coenzyme A: cholesterol acyltransferase modulates the generation of the amyloid beta-peptide.

The pathogenic event common to all forms of Alzheimer's disease is the abnormal accumulation of the amyloid beta-peptide (Abeta). Here we provide strong evidence that intracellular cholesterol compartmentation modulates the generation of Abeta. Using genetic, biochemical and metabolic approaches, we found that cholesteryl-ester levels are directly correlated with Abeta production. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyses the formation of cholesteryl esters, modulates the generation of Abeta through the tight control of the equilibrium between free cholesterol and cholesteryl esters. We also show that pharmacological inhibitors of ACAT, developed for the treatment of atherosclerosis, are potent modulators of Abeta generation, indicating their potential for use in the treatment of Alzheimer's disease.

Alzheimer disease: 100 years later.

Almost 100 years since the first clinical report of a case of Alzheimer disease (AD), three early-onset and two late-onset AD genes have been identified. While rare mutations in the early-onset genes (amyloid precursor protein, and presenilins 1 and 2) lead to increased generation of specific forms of the amyloid beta protein (A,beta), common polymorphisms in the late-onset genes (apolipoprotein E and alpha 2-macroglobulin) are thought to alter the clearance and degradation of A,beta in brain. Although definite proof for a direct link between altered A beta generation/clearance and neurodegeneration has not yet been attained, mechanism-based approaches for the therapeutic treatment of AD based on lowering levels of the potentially pathogenic A beta are currently underway. The recent discovery of the enzymes (secretases) responsible for generating A beta have paved the way for the development of such drugs and increase the prospects for successful therapeutic intervention to arrest AD neuropathogenesis.

alpha2-macroglobulin in late-onset Alzheimer's disease.

alpha2-macroglobulin (alpha(2)M) is an abundant plasma protein similar in structure and function to a group of proteins called alpha-macroglobulins. alpha(2)M is also produced in the brain where it binds multiple extracellular ligands and is internalized by neurons and astrocytes. In the brain of Alzheimer's disease (AD) patients, alpha(2)M has been localized to diffuse amyloid plaques. alpha(2)M also binds soluble beta-amyloid, of which it mediates degradation. However, an excess of alpha(2)M can also have neurotoxic effects. Based on genetic evidence, is now recognized as one of the two confirmed late onset AD genes. As for the three early onset genes (the amyloid beta-protein precursor and the two presenilins) and for the other late onset gene (ApoE), DNA polymorphisms in the A2M gene associated with AD result in significantly increased accumulation of amyloid plaques in AD brains. These data support an important role for A2M in AD etiopathology.

Inhibitors of beta-amyloid formation based on the beta-secretase cleavage site.

A series of inhibitors of beta-amyloid formation have been developed based on the beta-secretase cleavage site (VNL-DA) of the Swedish mutant Amyloid Precursor Protein. A simple tripeptide aldehyde was found to be the most potent (IC(50) = 700 nM) in the series displaying an inhibitory profile which is different from reported inhibitors of beta-amyloid formation.

Staurosporine-induced activation of caspase-3 is potentiated by presenilin 1 familial Alzheimer's disease mutations in human neuroglioma cells.

Familial Alzheimer's disease (FAD) mutant forms of presenilin 1 (PS1) and 2 have been shown to sensitize cells to apoptotic cell death. Here we explore the effects of FAD mutant forms of PS1 on caspase activation during apoptosis. We show that caspase activation leads to increased generation of alternative C-terminal fragments (CTFs) from mutant as compared to wild-type (wt) PS1. For this purpose, very low expression levels of wt, A246E, L286V, and deltaE10 FAD mutant PS1 proteins in stably transfected human H4 neuroglioma cells were used to avoid artifactual induction of spontaneous apoptosis due to overexpression of PS1. Staurosporine treatment of these cells resulted in increased cell death and up to a 10-fold increase in caspase-3 activation in mutant versus wt PS1-expressing cell lines. Correspondingly, relative levels of caspase-cleaved PS1 CTFs were increased by five- to sixfold in the FAD mutant versus wt PS1 cells. Elevated caspase activation and caspase cleavage of FAD mutant PS1 suggest the possibility of either a direct proapoptotic effect of mutant PS1 or interference of mutant PS1 with antiapoptotic effects of wt PS1.

Monogenic determinants of familial Alzheimer's disease: presenilin-1 mutations.

Presenilin-1 (PS1) mutations account for the greatest portion of early onset familial Alzheimer's disease (FAD) cases. The exact cellular function of PS1 is not known. To date, PS1 mutations have been shown to alter two potential biological roles of the protein, either of which could make neurons more susceptible to neurodegeneration. First, PS1 mutations result in elevated A beta 42/A beta 40 ratios in plasma of FAD patients, in transgenic mice and in transfected cell lines. A beta 42 is the more hydrophobic and most neurotoxic form of the peptide. A common molecular event that has been associated with all of the known early onset FAD genes is the excessive production or accumulation of the A beta peptide in the brain. PS1 mutations have also been found to alter the Notch signalling pathway, but the mechanism by which this may affect neurodegeneration remains to be determined. Future studies will be needed to elucidate whether PS1 mutations lead directly to neuronal dysfunction and degeneration or cause cell death by increasing A beta 42 generation and deposition.

Alternative cleavage of Alzheimer-associated presenilins during apoptosis by a caspase-3 family protease.

Most cases of early-onset familial Alzheimer's disease (FAD) are caused by mutations in the genes encoding the presenilin 1 (PS1) and PS2 proteins, both of which undergo regulated endoproteolytic processing. During apoptosis, PS1 and PS2 were shown to be cleaved at sites distal to their normal cleavage sites by a caspase-3 family protease. In cells expressing PS2 containing the asparagine-141 FAD mutant, the ratio of alternative to normal PS2 cleavage fragments was increased relative to wild-type PS2-expressing cells, suggesting a potential role for apoptosis-associated cleavage of presenilins in the pathogenesis of Alzheimer's disease.

Evidence for phosphorylation and oligomeric assembly of presenilin 1.

Pathogenic mutations in presenilin 1 (PS1) are associated with approximately 50% of early-onset familial Alzheimer disease. PS1 is endoproteolytically cleaved to yield a 30-kDa N-terminal fragment (NTF) and an 18-kDa C-terminal fragment (CTF). Using COS7 cells transfected with human PS1, we have found that phorbol 12, 13-dibutyrate and forskolin increase the state of phosphorylation of serine residues of the human CTF. Phosphorylation of the human CTF resulted in a shift in electrophoretic mobility from a single major species of 18 kDa to a doublet of 20-23 kDa. This mobility shift was also observed with human PS1 that had been transfected into mouse neuroblastoma (N2a) cells. Treatment of the phosphorylated CTF doublet with phage lambda protein phosphatase eliminated the 20- to 23-kDa doublet while enhancing the 18-kDa species, consistent with the interpretation that the electrophoretic mobility shift was due to the addition of phosphate to the 18-kDa species. The NTF and CTF eluted from a gel filtration column at an estimated mass of over 100 kDa, suggesting that these fragments exist as an oligomerized species. Upon phosphorylation of the PS1 CTF, the apparent mass of the NTF- or CTF-containing oligomers was unchanged. Thus, the association of PS1 fragments may be maintained during cycles of phosphorylation/dephosphorylation of the PS1 CTF.

Behavioral, affective, and social correlates of involvement in cross-sex friendship in elementary school.

The purpose of this study was to compare children with and without cross-sex friends on measures of social and cognitive competence, endorsement of sex-role stereotypes, and family composition. Subjects were 723 third and fourth graders (377 girls, 346 boys) from diverse socioeconomic backgrounds; 35% were African American. Measures included sociometric assessments of peer acceptance, friendship, and behavioral reputation, as well as self-reports of perceived self-competence and endorsement of sex-role stereotypes. In addition, teachers completed ratings of children's social and cognitive competence. In all, 92 children, about 14% of the sample, had one or more reciprocal opposite-sex friends; for 21 of these children, their cross-sex friendships were their primary or only friendships. African American children were more likely than European American children to have opposite-sex friends. Involvement in cross-sex friendships was unrelated to the gender make-up of the classroom but was related to family structure. Comparisons of the children who had primarily or only cross-sex friends to matched groups of children who had only same-sex friends and to children who had cross-sex friends secondarily to same-sex ones revealed a number of differences between the groups in social competence and relationships with peers. Overall, children with primarily opposite-sex friends had poorer social skills than other children with friends, although they were less stereotyped about sex roles than other children, and were better adjusted than children with no friends on most measures. In contrast, children involved in opposite-sex friendship secondarily to same-sex friendship were as well adjusted socially as children with only same-sex friendships. These results suggest that children with cross-sex friends differ among themselves, depending on the primacy of the cross-sex relationship.

The gene defects responsible for familial Alzheimer's disease.

Four different genes have now been found to contain AD-associated mutations or polymorphisms. While the pathogenic mutations in the early-onset FAD genes, APP, PS1, and PS2 directly cause AD with nearly 100% penetrance, in a larger subset of AD cases with onset over 60 years (maximally for onset at 61-65 years), inheritance of the APOE4 allele confers increased risk for AD but is not sufficient to cause the disease. Together, these four genes appear to account for approximately 50% of FAD cases. We are actively screening the genome for additional FAD loci by genotyping markers in over 400 FAD nuclear pedigrees and affected sib-pairs (83% late-onset and 17% early-onset). We have recently discovered genetic linkage to a novel FAD locus on chromosome 12 as well as another putative locus on chromosome 3 (unpublished findings). Positional cloning strategies are currently under way to identify these potentially novel FAD genes. A common event which is associated with all of the known FAD genes is the excessive accumulation of the A beta peptide and deposition of beta-amyloid in the brain. Thus, a common pathogenic pathway for AD neuropathogenesis appears to center around the cellular trafficking, maturation, and processing of APP, and the subsequent generation, aggregation, and deposition of A beta (or more specifically, A beta 1-42). APP and presenilin gene mutations most likely act as either gain-of-function or dominant negative gene defects which may ultimately lead to the transport of APP into intracellular compartments that promote the enhanced production of A beta or A beta 1-42. AD patients who carry an APOE4 allele experience increased amyloid burden in their brains compared to APOE4-negative AD cases. Thus, the presence of APOE4 would also appear to lead to abnormal generation, aggregation, or clearance of A beta in the brain A beta, perhaps by working in concert with its neuronal receptor, LRP. While the exact mechanisms by which the known FAD gene changes lead to the onset of AD remain unclear, the available data indicate that novel therapies aimed at curbing the generation, aggregation, and deposition of A beta would appear to carry the greatest potential for the effective treatment of this formidable disease.

Alzheimer-associated presenilins 1 and 2: neuronal expression in brain and localization to intracellular membranes in mammalian cells.

Mutations in two recently identified genes appear to cause the majority of early-onset familial Alzheimer's disease (FAD). These two novel genes, presenilin 1 (PS1) and presenilin 2 (PS2) are members of an evolutionarily conserved gene family. The normal biological role(s) of the presenilins and the mechanism(s) by which the FAD-associated mutations exert their effect remain unknown. Employing in situ hybridization, we demonstrate that the expression patterns of PS1 and PS2 in the brain are extremely similar to each other and that messages for both are primarily detectable in neuronal populations. Immunochemical analyses indicate that PS1 and PS2 are similar in size and localized to similar intracellular compartments (endoplasmic reticulum and Golgi complex). FAD-associated mutations in PS1 and PS2 do not significantly modify either their migration patterns on SDS-polyacrylamide gel electrophoresis or their overall subcellular localization, although subtle differences in perinuclear staining were noted for mutant PS1.

The upstream stimulatory factor functionally interacts with the Alzheimer amyloid beta-protein precursor gene.

The amyloid beta-protein precursor (APP) gives rise to the A beta peptide, which is deposited in the brains of patients with Alzheimer's disease and Down's syndrome. Overexpression of APP due to a third copy of the gene appears to correlate with very early onset of Alzheimer's disease neuropathology in the brains of Down's syndrome patients. Thus, the identification of the factors involved with transcriptional regulation of the APP gene could provide critical clues regarding the events leading to the formation of amyloid deposits. An overlapping AP-1/AP-4 site in the proximal promoter region (-39 to -49) of the human APP gene has previously been shown to increase transcription 4-fold. Here we identify the factor binding specifically to this element as the upstream stimulatory factor USF, unrelated to the c-fos/c-jun complex or the AP-4 factor. In vitro transcription and co-transfection studies show that USF activates transcription from the APP promoter and that the AP-1/AP-4 element participates in this activation. Modulation of APP expression via regulation of USF could potentially ameliorate the production of Alzheimer-augmented beta-amyloid.

Discordant estimates of heterologous promoter activity as determined by reporter gene mRNA levels and enzyme activity.

In this study, the human cytomegalovirus (CMV) promoter fused to the lacZ (beta-gal) reporter gene was transfected into neuroblastoma SK-N-BE(2)-C cells, and phorbol ester-stimulated promoter activity assessed by both PCR quantitation of reporter gene mRNA levels and enzyme activity. Surprisingly, significant differences were observed in the induction profile of CMV promoter activity as judged by these two independent methods of analysis. For example, at 24 hrs post-transfection beta-gal activity was elevated 7.3-fold in phorbol ester-treated cells, whereas 2.4-fold increases were observed in the cognate mRNA levels. These findings demonstrate the efficacy of quantitative PCR methodology to evaluate promoter activity in DNA-mediated cell transfection analyses, and raise a cautionary note on the reliance of reporter gene enzyme activity to estimate the transcriptional activity of heterologous promoters.

Cold-induced alterations in the binding of adrenomedullary nuclear proteins to the promoter region of the tyrosine hydroxylase gene.

It is well documented that cold stress induces a rapid trans-synaptically mediated increase in the relative abundance of rat adrenomedullary tyrosine hydroxylase (TH) mRNA. To investigate the transcriptional mechanisms regulating the cold stress response, we have employed a gel mobility shift assay, using DNA fragments prepared from the proximal 5' flanking region of the bovine TH gene as a heterologous molecular probe. In pilot studies, this region of the bovine TH promoter (nucleotides -246 to +21) was fused to the bacterial reporter gene, chloramphenicol acetyltransferase, and the chimeric construct transfected into human neuroblastoma SK-N-BE(2)-C, hepatoma HepG2, and rat pheochromocytoma PC-12 cells. Results of this analysis indicate that the proximal 5' flanking region of the bovine TH gene contains sufficient information to drive transient reporter gene expression in both human and rat catecholaminergic clonal cell lines. The findings derived from the gel mobility shift studies demonstrate that cold exposure causes rapid and selective alterations in the binding of adrenomedullary nuclear proteins to the proximal 5' flanking region of the TH gene. The most striking cold stress-induced alteration in DNA/nucleoprotein binding occurs in a region of the TH promoter (nucleotides -246 to -189) which contains an element bearing marked sequence similarity to an AP1 binding site and is highly conserved among animal species. This alteration occurs within 1 hr of cold exposure and persists for up to 48 hr after the onset of stress. The results of adrenal denervation experiments indicate that the cold-induced change in DNA/nucleoprotein binding is neurally mediated, requiring intact sympathetic innervation of the gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of amyloid precursor protein (APP): study with antisense transfection of human neuroblastoma cells.

The function of amyloid precursor protein (APP) was investigated in human neuroblastoma La-N-1 cells by stable transfection with a DNA construct encoding antisense APP mRNA. Levels of APP mRNA, as well as proteins, were reduced by 80-90% in antisense APP transfected (ASAT) cells. ASAT cells exhibited three main features as a result of APP gene expression deprivation: (1) a 30% reduction in cell proliferation, (2) reduced cell adhesion that could be reversed by the addition of La-N-1 conditioned media as a source of secreted APP, and (3) a two- and four-fold increase in neurite-bearing cells suggesting that cellular APP may be involved in neurite extension. The first two features confirm previously reported functions for APP in proliferation and adhesion of non-neuronal cell types but the use of neuroblastoma cells in this study disclose a novel role for cellular APP in neurite extension.