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.

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.

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.