Functional characterization of novel presenilin-2 variants identified in human breast cancers.

We identified in breast cancer cases two germline alterations, R62H and R71W, in presenilin-2 (PS-2), a gene involved in familial Alzheimer's disease (FAD). The role of these alleles in FAD is unclear, but neither allele affected Abeta(42)/Abeta(40) ratio. However, both R62H and R71W alterations compromised PS-2 function in Notch signaling in Caenorhabditis elegans and cell growth inhibition in mouse embryonic fibroblasts, and these effects were dependent on gene dosage. We found that both alterations enhanced the degradation of the PS-2 full-length protein, indicating that they may have a loss-of function effect. The effect of the R71W alteration was noticeably stronger, and we observed an almost threefold higher frequency of this allele in breast cancer cases versus controls, but this difference did not reach statistical significance. Nonetheless, these results collectively suggest that the novel PS-2 alleles described here, especially R71W, affect PS-2 function and may potentially confer a moderate risk of susceptibility to breast cancer.

Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN.

A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.

Optical dysfunction of the crystalline lens in aquaporin-0-deficient mice.

Aquaporin-0 (AQP0), a water transport channel protein, is the major intrinsic protein (MIP) of lens fiber cell plasma membranes. Mice deficient in the gene for AQP0 (Aqp0, Mip) were generated from a library of gene trap embryo stem cells. Sequence analysis showed that the gene trap vector had inserted into the first exon of Aqp0, causing a null mutation as verified by RNA blotting and immunochemistry. At 3 wk of age (postnatal day 21), lenses from null mice (Aqp0(-/-)) contained polymorphic opacities, whereas lenses from heterozygous mice (Aqp0(+/-)) were transparent and did not develop frank opacities until approximately 24 wk of age. Osmotic water permeability values for Aqp0(+/-) and Aqp0(-/-) lenses were reduced to approximately 46% and approximately 20% of wild-type values, respectively, and the focusing power of Aqp0(+/-) lenses was significantly lower than that of wild type. These findings show that heterozygous loss of AQP0 is sufficient to trigger cataractogenesis in mice and suggest that this MIP is required for optimal focusing of the crystalline lens.

Upregulation of BiP and CHOP by the unfolded-protein response is independent of presenilin expression.

Presenilin 1 (PS1), a polytopic membrane protein, has a critical role in the trafficking and proteolysis of a selected set of transmembrane proteins. The vast majority of individuals affected with early onset familial Alzheimer's disease (FAD) carry missense mutations in PS1. Two studies have suggested that loss of PS1 function, or expression of FAD-linked PS1 variants, compromises the mammalian unfolded-protein response (UPR), and we sought to evaluate the potential role of PS1 in the mammalian UPR. Here we show that that neither the endoplasmic reticulum (ER) stress-induced accumulation of BiP and CHOP messenger RNA, nor the activation of ER stress kinases IRE1alpha and PERK, is compromised in cells lacking both PS1 and PS2 or in cells expressing FAD-linked PS1 variants. We also show that the levels of BiP are not significantly different in the brains of individuals with sporadic Alzheimer's disease or PS1-mediated FAD to levels in control brains. Our findings provide evidence that neither loss of PS1 and PS2 function, nor expression of PS1 variants, has a discernable impact on ER stress-mediated induction of the several established 'readouts' of the UPR pathway.

Mice lacking both presenilin genes exhibit early embryonic patterning defects.

Genetic studies in worms, flies, and humans have implicated the presenilins in the regulation of the Notch signaling pathway and in the pathogenesis of Alzheimer's Disease. There are two highly homologous presenilin genes in mammals, presenilin 1 (PS1) and presenilin 2 (PS2). In mice, inactivation of PS1 leads to developmental defects that culminate in a perinatal lethality. To test the possibility that the late lethality of PS1-null mice reflects genetic redundancy of the presenilins, we have generated PS2-null mice by gene targeting, and subsequently, PS1/PS2 double-null mice. Mice homozygous for a targeted null mutation in PS2 exhibit no obvious defects; however, loss of PS2 on a PS1-null background leads to embryonic lethality at embryonic day 9.5. Embryos lacking both presenilins, and surprisingly, those carrying only a single copy of PS2 on a PS1-null background, exhibit multiple early patterning defects, including lack of somite segmentation, disorganization of the trunk ventral neural tube, midbrain mesenchyme cell loss, anterior neuropore closure delays, and abnormal heart and second branchial arch development. In addition, Delta like-1 (Dll1) and Hes-5, two genes that lie downstream in the Notch pathway, were misexpressed in presenilin double-null embryos: Hes-5 expression was undetectable in these mice, whereas Dll1 was expressed ectopically in the neural tube and brain of double-null embryos. We conclude that the presenilins play a widespread role in embryogenesis, that there is a functional redundancy between PS1 and PS2, and that both vertebrate presenilins, like their invertebrate homologs, are essential for Notch signaling.

Cloning and characterization of Sel-1l, a murine homolog of the C. elegans sel-1 gene.

The Notch signaling pathway regulates specification and proliferation in a variety of cell lineages in invertebrates and vertebrates. We have cloned a murine homolog of SEL-1, a key negative regulator of the Notch pathway in Caenorhabditis elegans. Murine SEL-1L (mSEL-1L) protein exhibits a high degree of similarity to SEL-1, including a signal peptide and the C-terminal region required for SEL-1 function in C. elegans. This mammalian homolog of sel-1 is widely expressed in adult mouse and human tissues, with particularly high levels in the pancreas. RNA in situ analysis of developing mouse embryos indicates that mSEL-1L is moderately expressed throughout the neural tube and dorsal root ganglia, with particularly high levels in the floor plate of the neural tube beginning at E10.5 and increasing at E11.5. Expression is high at E14.5 and E17.5 in the acini of the pancreas, and moderate in the epithelial cells of the gut villi. We localized the SEL-1L protein to the cytosol, possibly in intracellular vesicles, in a beta-islet-derived tumor cell line (RinM).

Embryonic and adult expression patterns of the Tec tyrosine kinase gene suggest a role in megakaryocytopoiesis, blood vessel development, and melanogenesis.

The Tec cytoplasmic tyrosine kinase is a member of a family of src-like proteins that are thought to play important roles in hematopoiesis. Here we describe the temporal and spatial expression of the Tec gene during embryogenesis and in the adult. Our data demonstrate that embryonic Tec expression is restricted to distinct hematopoietic cells as well as structures and cell types that share a common feature of containing fluid in an enclosed cavity, e.g., endothelial cells. In addition, Tec is expressed in melanocytes late in gestation. The observed developmental expression pattern of Tec suggests a role for this gene in several aspects of hematopoiesis and/or blood vessel development as well as in late stages of melanogenesis.

Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.

Thrombospondin gene expression is associated with mitogenesis in 3T3 cells: induction by basic fibroblast growth factor.

Growth factor-depleted Swiss 3T3 cells responded to basic fibroblast growth factor (bFGF) with a burst of mitogenesis and with a rapid and marked increase in thrombospondin (TS) mRNA levels. mRNA levels for the alpha 1 chain of type I collagen and for fibronectin were unaffected. At early times following stimulation (0-2 h), "superinduction" of TS mRNA by inhibition of protein synthesis with cycloheximide was not observed, and the increase in TS mRNA could be attributed primarily to an increase in transcription rate of the TS gene. However, at later times (4-8 h) the combination of cycloheximide and bFGF superinduced TS mRNA levels, suggesting the existence of a labile inhibitor of transcription or a short-lived RNase that might be produced in response to prolonged treatment with bFGF. In contrast to its stimulatory effect on 3T3 cells, bFGF did not stimulate the proliferation of mouse muscle BC3H1 cells nor did it cause an increase in TS mRNA levels, but BC3H1 cells do respond to bFGF by inhibition of myogenic differentiation. We propose, on the basis of these and other findings, that TS facilitates the progression of some anchorage-dependent cells through the cell cycle.

Structural analysis and expression of the human thrombospondin gene promoter.

We have isolated a 34-kilobase pair genomic thrombospondin clone and determined the sequence of 2033 base pairs of the 5'-flanking region, the first entirely untranslated exon and the first intron. A number of interesting regulatory motifs were identified. The transcription start site, determined by primer extension and S1 nuclease analysis, was shown to be 20-30 bases 3' of a TATA-like sequence, TTTAAAA. We have also shown that a thrombospondin promoter-bovine growth hormone fusion gene is transcribed in transiently transfected cells. We conclude that the genomic clone contains the transcriptional signals of the thrombospondin gene and will therefore be useful in the analysis of cis-acting elements that regulate the expression of this gene.