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PubMed2003

Sequential activation of Notch family receptors during mouse spermatogenesis.

The expression pattern of Notch family receptors during mouse spermatogenesis was examined by immunohistochemistry. The entire cytoplasm of spermatogonia, spermatocytes and spermatids showed staining with antibodies against extracellular domains of Notch1, 2 and 4. In contrast, the nuclei of spermatogonia showed staining with an antibody against the intracellular domain of Notch3, and the nuclei of spermatocytes and spermatids showed staining with antibodies against the intracellular domains of Notch1 and 4. During regeneration of spermatogonia in busulfan-treated mice, the nuclei of all proliferating cells showed staining for the intracellular domain of Notch3. Western blot analysis showed that the molecular weights of the intracellular domains of Notch1 and 3 localizing in the nuclear fraction were smaller than those in the cytoplasmic fraction. This was consistent with the theory that the intracellular domain of Notch was cleaved in the cytoplasm and translocated to the nucleus. These results suggest that different Notch signals are sequentially activated during mouse spermatogenesis and control the proliferation and differentiation of spermatogenic stem cells.

Alkylating Agents↗
PubMed2002

Conservation of Pax 6 function and upstream activation by Notch signaling in eye development of frogs and flies.

Loss of Pax 6 function leads to an eyeless phenotype in both mammals and insects, and ectopic expression of both the Drosophila and the mouse gene leads to the induction of ectopic eyes in Drosophila, which suggested to us that Pax 6 might be a universal master control gene for eye morphogenesis. Here, we report the reciprocal experiment in which the RNAs of the Drosophila Pax 6 homologs, eyeless and twin of eyeless, are transferred into a vertebrate embryo; i.e., early Xenopus embryos at the 2- and 16-cell stages. In both cases, ectopic eye structures are formed. To understand the genetic program specifying eye morphogenesis, we have analyzed the regulatory mechanisms of Pax 6 expression that initiates eye development. Previously, we have demonstrated that Notch signaling regulates the expression of eyeless and twin of eyeless in Drosophila. Here, we show that in Xenopus, activation of Notch signaling also induces eye-related gene expression, including Pax 6, in isolated animal caps. In Xenopus embryos, the activation of Notch signaling causes eye duplications and proximal eye defects, which are also induced by overexpression of eyeless and twin of eyeless. These findings indicate that the gene regulatory cascade is similar in vertebrates and invertebrates.

Animals↗
PubMed2000

Notch signaling and the determination of appendage identity.

The Notch signaling pathway defines an evolutionarily conserved cell-cell interaction mechanism that throughout development controls the ability of precursor cells to respond to developmental signals. Here we show that Notch signaling regulates the expression of the master control genes eyeless, vestigial, and Distal-less, which in combination with homeotic genes induce the formation of eyes, wings, antennae, and legs. Therefore, Notch is involved in a common regulatory pathway for the determination of the various Drosophila appendages.

Animals↗
PubMed2002

Presenilin-1 mutations of leucine 166 equally affect the generation of the Notch and APP intracellular domains independent of their effect on Abeta 42 production.

The Alzheimer's disease (AD)-associated presenilin (PS) proteins are required for the gamma-secretase cleavages of the beta-amyloid precursor protein and the site 3 (S3) protease cleavage of Notch. These intramembrane cleavages release amyloid-beta peptide (Abeta), including the pathogenic 42-aa variant (Abeta(42)), as well as the beta-amyloid precursor protein and the Notch intracellular domains (AICD, NICD). Whereas Abeta is generated by endoproteolysis in the middle of the transmembrane domain, AICD and NICD are generated by cleavages at analogous positions close to the cytoplasmic border of the transmembrane domain. Numerous mutations causing familial AD (FAD) that all cause increased production of Abeta(42) have been found in the PS1 gene. Here we have investigated the previously uncharacterized, very aggressive FAD mutation L166P that causes onset of AD in adolescence. Strikingly, the PS1 L166P mutation not only induces an exceptionally high increase of Abeta(42) production but also impairs NICD production and Notch signaling, as well as AICD generation. Thus, FAD-associated PS mutants cannot only affect the generation of NICD, but also that of AICD. Moreover, further analysis with artificial L166 mutants revealed that the gamma-secretase cleavage at position 40/42 and the S3-like gamma-secretase cleavage at position 49 of the Abeta domain are both differentially affected by PS1 L166 mutants. Finally, we show that PS1 L166 mutants affect the generation of NICD and AICD in a similar manner, supporting the concept that S3 protease and S3-like gamma-secretase cleavages are mediated by identical proteolytic activities.

Alzheimer Disease↗
PubMed1993

Further evidence for function of the Drosophila Notch protein as a transmembrane receptor.

N locus mutations associated with unusual mutant phenotypes were found to alter the structure of the encoded protein. Two mutations, NCo and N60g11, eliminate much of the cytoplasmic domain. NCo can act as a null allele or as a competitive inhibitor of N+ function, whereas N60g11 produces dominant gain of function in some cell types. This difference in function can be attributed to retention of cdc10/SWI6 repeats in the Notch60g11 protein. The results suggest a role for these repeats in intracellular signaling and are consistent with action of Notch as a receptor. nd3 and l(1)NB alter extracellular epidermal growth factor-like and lin-12/Notch elements, respectively. nd3 eliminates a conserved cysteine residue, so the mutation may result in complete loss of function for a single Notch epidermal growth factor element. N60g11 and l(1)NB produce related gain-of-function phenotypes. It is proposed that l(1)NB produces an extracellular modification of the protein that stimulates aberrant intracellular signaling by the Notch cytoplasmic domain.

Amino Acid Sequence↗
PubMed1999

A loss of function mutation of presenilin-2 interferes with amyloid beta-peptide production and notch signaling.

Presenilin-1 (PS1) facilitates gamma-secretase cleavage of the beta-amyloid precursor protein and the intramembraneous cleavage of Notch1. Although Alzheimer's disease-associated mutations in the homologous presenilin (PS2) gene elevate amyloid beta-peptide (Abeta42) production like PS1 mutations, here we demonstrate that a gene ablation of PS2 (unlike that of PS1) in mice does not result in a severe phenotype resembling that of Notch-ablated animals. To investigate the amyloidogenic function of PS2 more directly, we mutagenized a conserved aspartate at position 366 to alanine, because the corresponding residue of PS1 is known to be required for its amyloidogenic function. Cells expressing the PS2 D366A mutation exhibit significant deficits in proteolytic processing of beta-amyloid precursor protein indicating a defect in gamma-secretase activity. The reduced gamma-secretase activity results in the almost complete inhibition of Abeta and p3 production in cells stably expressing PS2 D366A, whereas cells overexpressing the wild-type PS2 cDNA produce robust levels of Abeta and p3. Using highly sensitive in vivo assays, we demonstrate that the PS2 D366A mutation not only blocks gamma-secretase activity but also inactivates PS2 activity in Notch signaling by inhibiting the proteolytic release of the cytoplasmic Notch1 domain. These data suggest that PS2 is functionally involved in Abeta production and Notch signaling by facilitating similar proteolytic cleavages.

Amyloid beta-Peptides↗
PubMed1999

Mouse jagged1 physically interacts with notch2 and other notch receptors. Assessment by quantitative methods.

The Delta/Serrate/LAG-2 (DSL) domain containing proteins are considered to be ligands for Notch receptors. However, the physical interaction between DSL proteins and Notch receptors is poorly understood. In this study, we cloned a cDNA for mouse Jagged1 (mJagged1). To identify the receptor interacting with mJagged1 and to gain insight into its binding characteristics, we established two experimental systems using fusion proteins comprising various extracellular parts of mJagged1, a "cell" binding assay and a "solid-phase" binding assay. mJagged1 physically bound to mouse Notch2 (mNotch2) on the cell surface and to a purified extracellular portion of mNotch2, respectively, in a Ca(2+)-dependent manner. Scatchard analysis of mJagged1 binding to BaF3 cells and to the soluble Notch2 protein demonstrated dissociation constants of 0.4 and 0.7 nM, respectively, and that the number of mJagged1-binding sites on BaF3 is 5,548 per cell. Furthermore, deletion mutant analyses showed that the DSL domain of mJagged1 is a minimal binding unit and is indispensable for binding to mNotch2. The epidermal growth factor-like repeats of mJagged1 modulate the affinity of the interaction, with the first and second repeats playing a major role. Finally, solid-phase binding assay showed that Jagged1 binds to Notch1 and Notch3 in addition to Notch2, suggesting that mJagged1 is a ligand for multiple Notch receptors.

Amino Acid Sequence↗
PubMed2002

Presenilin 1 mutations activate gamma 42-secretase but reciprocally inhibit epsilon-secretase cleavage of amyloid precursor protein (APP) and S3-cleavage of notch.

The presenilin 1 (PS1) and presenilin 2 (PS2) proteins are necessary for proteolytic cleavage of the amyloid precursor protein (APP) within its transmembrane domain. One of these cleavage events (termed gamma-secretase) generates the C-terminal end of the Abeta-peptide by proteolysis near residue 710 or 712 of APP(770). Another event (termed gamma-like or epsilon-secretase cleavage) cleaves near residue 721 at approximately 2-5 residues inside the cytoplasmic membrane boundary to generate a series of stable, C-terminal APP fragments. This latter cleavage is analogous to S3-cleavage of Notch. We report here that specific mutations in the N terminus, loop, or C terminus of PS1 all increase the production of Abeta(42) but cause inhibition of both epsilon-secretase cleavage of APP and S3-cleavage of Notch. These data support the hypothesis that epsilon-cleavage of APP and S3-cleavage of Notch are similar events. They also argue that, although both the gamma-site and the epsilon-site cleavage of APP are presenilin-dependent, they are likely to be independent catalytic events.

Amino Acid Sequence↗
PubMed2002

Mammalian APH-1 interacts with presenilin and nicastrin and is required for intramembrane proteolysis of amyloid-beta precursor protein and Notch.

Presenilin and nicastrin are essential components of the gamma-secretase complex that is required for the intramembrane proteolysis of an increasing number of membrane proteins including the amyloid-beta precursor protein (APP) and Notch. By using co-immunoprecipitation and nickel affinity pull-down approaches, we now show that mammalian APH-1 (mAPH-1), a conserved multipass membrane protein, physically associates with nicastrin and the heterodimers of the presenilin amino- and carboxyl-terminal fragments in human cell lines and in rat brain. Similar to the loss of presenilin or nicastrin, the inactivation of endogenous mAPH-1 using small interfering RNAs results in the decrease of presenilin levels, accumulation of gamma-secretase substrates (APP carboxyl-terminal fragments), and reduction of gamma-secretase products (amyloid-beta peptides and the intracellular domains of APP and Notch). These data indicate that mAPH-1 is probably a functional component of the gamma-secretase complex required for the intramembrane proteolysis of APP and Notch.

Amino Acid Sequence↗
PubMed2005

Generation and characterization of mutant cell lines defective in gamma-secretase processing of Notch and amyloid precursor protein.

Several type I integral membrane proteins, such as the Notch receptor or the amyloid precursor protein, are cleaved in their intramembrane domain by a gamma-secretase enzyme, which is carried within a multiprotein complex. These cleavages generate molecules that are involved in intracellular or extracellular signaling. At least four transmembrane proteins belong to the gamma-secretase complex: presenilin, nicastrin, Aph-1, and Pen-2. It is still unclear whether these proteins are the only components of the complex and whether a unique complex is involved in the different gamma-secretase cleavage events. We have set up a genetic screen based on the permanent acquisition or loss of an antibiotic resistance depending on the presence of an active gamma-secretase able to cleave a Notch-derived substrate. We selected clones deficient in gamma-secretase activity using this screen on mammalian cells after random mutagenesis. We further analyzed two of these clones and identified previously undescribed mutations in the nicastrin gene. The first mutation abolishes nicastrin production, and the second mutation, a point mutation in the ectodomain, abolishes nicastrin maturation. In both cases, gamma-secretase activity on Notch and APP is impaired.

Amino Acid Sequence↗
PubMed2006

Neuralized-2 regulates a Notch ligand in cooperation with Mind bomb-1.

Mutations in Drosophila neuralized (Dneur) result in a variety of developmental defects that closely resemble those of Notch mutants and other Notch pathway mutants. However, mice with disrupted neur1 do not show any aberrant cell fate specifications in neurogenesis and somitogenesis. Thus, we speculated that other vertebrate neur homolog(s) might compensate for loss of the neur gene. Here, we report the paralog of mouse Neur1, named Neuralized-2 (Neur2), which is a ubiquitin-protein isopeptide ligase (E3) that interacts with and ubiquitinates Delta. Both murine Neur1 and Neur2 have similar degrees of homology to DNeur, and neur2 is expressed in patterns similar to those of neur1 in embryos, suggesting potential functional redundancy. Interestingly, two distinct classes of E3 ligases, Mind bomb-1 (Mib1) and Neur2, have cooperative but distinct roles in Delta endocytosis to Hrs-positive vesicles, i.e. Mib1 functions in the initial step of Delta endocytosis, and Neur2 is required for targeting endocytosed Delta to Hrs-positive vesicles. Thus, our study provides a new insight into how distinct E3 ligases work together in the endocytic pathways for Notch signaling.

Amino Acid Sequence↗
PubMedDate not supplied

Signal integration during development: mechanisms of EGFR and Notch pathway function and cross-talk.

Metazoan development relies on a highly regulated network of interactions between conserved signal transduction pathways to coordinate all aspects of cell fate specification, differentiation, and growth. In this review, we discuss the intricate interplay between the epidermal growth factor receptor (EGFR; Drosophila EGFR/DER) and the Notch signaling pathways as a paradigm for signal integration during development. First, we describe the current state of understanding of the molecular architecture of the EGFR and Notch signaling pathways that has resulted from synergistic studies in vertebrate, invertebrate, and cultured cell model systems. Then, focusing specifically on the Drosophila eye, we discuss how cooperative, sequential, and antagonistic relationships between these pathways mediate the spatially and temporally regulated processes that generate this sensory organ. The common themes underlying the coordination of the EGFR and Notch pathways appear to be broadly conserved and should, therefore, be directly applicable to elucidating mechanisms of information integration and signaling specificity in vertebrate systems.

Amyloid Precursor Protein Secretases↗
PubMed2000

The notch ligand jagged-1 represents a novel growth factor of human hematopoietic stem cells.

The Notch ligand, Jagged-1, plays an essential role in tissue formation during embryonic development of primitive organisms. However, little is known regarding the role of Jagged-1 in the regulation of tissue-specific stem cells or its function in humans. Here, we show that uncommitted human hematopoietic cells and cells that comprise the putative blood stem cell microenvironment express Jagged-1 and the Notch receptors. Addition of a soluble form of human Jagged-1 to cultures of purified primitive human blood cells had modest effects in augmenting cytokine-induced proliferation of progenitors. However, intravenous transplantation of cultured cells into immunodeficient mice revealed that human (h)Jagged-1 induces the survival and expansion of human stem cells capable of pluripotent repopulating capacity. Our findings demonstrate that hJagged-1 represents a novel growth factor of human stem cells, thereby providing an opportunity for the clinical utility of Notch ligands in the expansion of primitive cells capable of hematopoietic reconstitution.

Animals↗
PubMed2001

Presenilin-dependent gamma-secretase processing of beta-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch.

The presenilin (PS)-dependent site 3 (S3) cleavage of Notch liberates its intracellular domain (NICD), which is required for Notch signaling. The similar gamma-secretase cleavage of the beta-amyloid precursor protein (betaAPP) results in the secretion of amyloid beta-peptide (Abeta). However, little is known about the corresponding C-terminal cleavage product (CTFgamma). We have now identified CTFgamma in brain tissue, in living cells, as well as in an in vitro system. Generation of CTFgamma is facilitated by PSs, since a dominant-negative mutation of PS as well as a PS gene knock out prevents its production. Moreover, gamma-secretase inhibitors, including one that is known to bind to PS, also block CTFgamma generation. Sequence analysis revealed that CTFgamma is produced by a novel gamma-secretase cut, which occurs at a site corresponding to the S3 cleavage of Notch.

Amino Acid Sequence↗
PubMed2002

Keeping a good pathway down: transcriptional repression of Notch pathway target genes by CSL proteins.

CSL [CBF-1, Su(H), Lag-1]-type transcription factors are the primary effectors of the Notch pathway, a signal transduction cascade that is essential for the development of all metazoan organisms. Interestingly, CSL proteins were originally classified as transcriptional repressors in vertebrates, but as transcriptional activators in model invertebrate organisms. Resolution of this paradox came with the realization that repression and activation by CSL proteins occurs in both systems and that the switch involves recruitment of distinct co-repressor and co-activator complexes. Although CSL proteins appear to utilize a common co-activator complex of largely similar constitution, recent studies have demonstrated that vertebrate and Drosophila CSL interact with a variety of distinct co-repressor complexes. This review highlights differences in composition and similarities in function of different CSL co-repressor complexes, which actively repress Notch pathway target genes in the absence of Notch pathway activity.

Animals↗
PubMed1996

Scalloped wings is the Lucilia cuprina Notch homologue and a candidate for the modifier of fitness and asymmetry of diazinon resistance.

The Scalloped wings (Scl) gene of the Australian sheep blowfly, Lucilia cuprina, is shown to be the homologue of the Drosophila melanogaster Notch gene by comparison at the DNA sequence and genetic levels. A L. cuprina genomic fragment, which shows strong identity with the Notch (N) gene at the molecular level, hybridizes to the location of the Scl gene on polytene chromosomes. The two genes are functionally homologous; the dominant and recessive Notch-like phenotypes produced by mutations in the Scl gene allow these alleles to be classed as N-like or Abruptex-like. The Scl gene is under investigation as a candidate for the fitness and asymmetry Modifier (M) of diazinon resistance. We show that M affects the penetrance of wing and bristle phenotypes associated with two Scl alleles in a manner consistent with the M being an allele of Scl. In addition, we report a phenotypic interaction between the diazinon-resistance mutation, Rop-1, and the same alleles of Scl. We propose that the product of Rop-1, an esterase, may be involved in cell adhesion in developmental processes involving the Scl gene product.

Alleles↗
PubMed2006

Transcriptional regulation of APH-1A and increased gamma-secretase cleavage of APP and Notch by HIF-1 and hypoxia.

The proteolytic cleavage of Alzheimer beta-amyloid precursor protein (APP) and signaling receptor Notch is mediated by the PS/gamma-secretase complex, which consists of presenilins, nicastrin, APH-1, and PEN-2. Although the four components are known to coordinately regulate each other at the protein level, information regarding their transcription regulation is scarce. Here we characterized the 5'-flanking region of the human APH-1A gene and identified a 271-bp fragment containing the transcription initiation site for the promoter activity. Sequence analysis, mutagenesis, and gel shift studies revealed a binding of AP4 and HIF-1 to the promoter, which affects the promoter activity. Activation of HIF-1 by short-term NiCl2 treatments (a condition of chemical hypoxia) dramatically increased APH-1A mRNA and protein expression, accompanied by increased secretion of Abeta and decreased APP CTFs formation, indicative of an increase in gamma-secretase activity. NiCl2 treatments had little effect on APP and the other three components of the gamma-secretase complex. The cellular concentration of Notch intracellular domain (NICD) was also increased by the hypoxic treatment. Our results demonstrate that APH-1A expression and the gamma-secretase mediated Abeta and Notch NICD generation are regulated by HIF-1, and the specific control of APH-1A expression may imply physiological functions uniquely assigned to APH-1A.

Amyloid Precursor Protein Secretases↗
PubMed2004

Notch expression in developing olfactory neuroepithelium.

Notch genes encode receptors for signaling pathways that regulate neurogenesis in various tissues. To better understand the roles of Notch genes in olfactory neurogenesis, we studied the expression of Notch family in developing mouse olfactory epithelium. In the earliest stage of olfactory development, Notch1 was observed in the mesenchyme lateral to the olfactory placode. During the developmental stage, Notch1 was mainly observed around the basal membrane, while Notch3 was observed in the lower compartment of the olfactory neuroepithelium. Notch2 was not detected during the entire observation period. As the olfactory neuroepithelium grew mature, both Notch1 and Notch3 gradually disappeared. These results suggest distinct roles of Notch1 and Notch3 in the neurogenesis of the peripheral olfactory system.

Animals↗