Transcriptomic Analysis of Drosophila Mushroom Body Neurons Lacking Amyloid-ß Precursor-Like Protein Activity.

The amyloid-ß protein precursor (AßPP) is subjected to sequential intramembrane proteolysis by α-, ß-, andγ-secretases, producing secreted amyloid-ß (Aß) peptides and a cytoplasmically released AßPP Intracellular Domain (AICD). AICD complexes with transcription factors in the nucleus, suggesting that this AßPP fragment serves as an active signaling effector that regulates downstream genes, although its nuclear targets are poorly defined. To further understand this potential signaling mechanism mediated by AßPP, we performed a transcriptomic identification of the Drosophila genome that is regulated by the fly AßPP orthologue in fly mushroom body neurons, which control learning- and memory-based behaviors. We find significant changes in expression of 245 genes, representing approximately 1.6% of the Drosophila genome, with the changes ranging from +6 fold to -40 fold. The largest class of responsive targets corresponds to non-protein coding genes and includes microRNAs that have been previously implicated in Alzheimer's disease pathophysiology. Several genes were identified in our Drosophila microarray analyses that have also emerged as putative AßPP targets in similar mammalian transcriptomic studies. Our results also indicate a role for AßPP in cellular pathways involving the regulation of Drosophila Casein Kinase II, mitochondrial oxidative phosphorylation, RNA processing, and innate immunity. Our findings provide insights into the intracellular events that are regulated by AßPP activity in healthy neurons and that might become dysregulated as a result of abnormal AßPP proteolysis in AD.

Disruption of Drosophila melanogaster lipid metabolism genes causes tissue overgrowth associated with altered developmental signaling.

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of many developmental pathways is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is critical for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila melanogaster, we recovered mutants that disrupt genes encoding serine palmitoyltransferase and acetyl-CoA carboxylase. Both mutants cause Notch, Wingless, the Epidermal Growth Factor Receptor (EFGR), and Patched to accumulate abnormally in endosomal compartments. In mosaic animals, mutant tissues exhibit an unusual non-cell-autonomous effect whereby mutant cells are functionally rescued by secreted activities emanating from adjacent wildtype tissue. Strikingly, both mutants display prominent tissue overgrowth phenotypes that are partially attributable to altered Notch and Wnt signaling. Our analysis of the mutants demonstrates genetic links between abnormal lipid metabolism, perturbations in developmental signaling, and aberrant cell proliferation.

Protein trafficking abnormalities in Drosophila tissues with impaired activity of the ZIP7 zinc transporter Catsup.

Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of the Notch signaling pathway is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is crucial for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila epithelial tissues, we recovered mutations that disrupt the Catsup gene, which encodes the Drosophila ortholog of the mammalian ZIP7 zinc transporter. Loss of Catsup function causes Notch to accumulate abnormally in the endoplasmic reticulum (ER) and Golgi compartments, resulting in impaired Notch signaling. In addition, Catsup mutant cells exhibit elevated ER stress, suggesting that impaired zinc homeostasis causes increased levels of misfolded proteins within the secretory compartment.

Dmon1 controls recruitment of Rab7 to maturing endosomes in Drosophila.

The small GTPases Rab5 and Rab7 are important organisers of endosome formation and maturation. In addition, they orchestrate the trafficking of cargo through the endosomal pathway. A crucial event during maturation of endosomes is the replacement of the early organiser Rab5 with the late organiser Rab7 in a process called Rab conversion. Rab conversion is a prerequisite for late events, chief among them the fusion of matured endosomes with the lysosome. Recent work identifies members of the Sand1/Mon1 protein family as crucial factors during this process. Here, we present an analysis of the function of the Drosophila ortholog of mon1/sand1, Dmon1. We found that loss of function of Dmon1 results in an enlargement of maturing endosomes and loss of their association with Rab7. The enlarged endosomes contain Notch and other trans-membrane proteins as cargo. We report the first electron microscopy analysis of Dmon1 cells in a metazoan and extend the analysis of the endosomes in mutant cells. Our results suggest that the phenotype can be explained by the loss of function of Rab7. Moreover, the endosomes of Dmon1 cells mature normally in many aspects, despite the loss of association with Rab7. Surprisingly, we did not observe overactive or ectopic signalling through receptors such as Notch and RTKs in Dmon1 mutant cells, as would have been expected because of the accumulation of receptors in the maturing endosomes of these cells. This was the case even when receptor uptake into intraluminal vesicles was suppressed.

Therapeutic approaches to modulating Notch signaling: current challenges and future prospects.

Dysregulated Notch signaling has been implicated in numerous human diseases, including a broad spectrum of cancers. Mutations in Notch1 are prevalent in T-cell acute lymphoblastic leukemia, and abnormal expression of different human Notch receptors contributes to B-cell tumors as well as cancers of the breast, lung, pancreas, skin, prostate, colon, brain and other tissues. Several γ-secretase inhibitors, small chemical compounds that were initially developed to inhibit the activity of the γ-secretase aspartyl protease in Alzheimer's disease, are now being explored for their potential chemotherapeutic applications in Notch-associated cancers. An alternative approach involves the development of antibodies to inhibit specific Notch receptors, their activating ligands, or other components of the Notch pathway in tumors. Here we review recent progress and current challenges in the use of these strategies to modulate Notch signaling for cancer therapy.

Deficient Notch signaling associated with neurogenic pecanex is compensated for by the unfolded protein response in Drosophila.

The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events, through local cell-cell communication. pecanex (pcx) encodes a multi-pass transmembrane protein of unknown function, widely found from Drosophila to humans. The zygotic and maternal loss of pcx in Drosophila causes a neurogenic phenotype (hyperplasia of the embryonic nervous system), suggesting that pcx might be involved in N signaling. Here, we established that Pcx is a component of the N-signaling pathway. Pcx was required upstream of the membrane-tethered and the nuclear forms of activated N, probably in N signal-receiving cells, suggesting that pcx is required prior to or during the activation of N. pcx overexpression revealed that Pcx resides in the endoplasmic reticulum (ER). Disruption of pcx function resulted in enlargement of the ER that was not attributable to the reduced N signaling activity. In addition, hyper-induction of the unfolded protein response (UPR) by the expression of activated Xbp1 or dominant-negative Heat shock protein cognate 3 suppressed the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes was induced by overexpression of O-fucosyltransferase 1, an N-specific chaperone. Taking these results together, we speculate that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER functions, which are compensated for by upregulation of the UPR and possibly by enhancement of N folding. Our results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity.

Pharmacological and genetic reversal of age-dependent cognitive deficits attributable to decreased presenilin function.

Alzheimer's disease (AD) is the leading cause of cognitive loss and neurodegeneration in the developed world. Although its genetic and environmental causes are not generally known, familial forms of the disease (FAD) are attributable to mutations in a single copy of the Presenilin (PS) and amyloid precursor protein genes. The dominant inheritance pattern of FAD indicates that it may be attributable to gain or change of function mutations. Studies of FAD-linked forms of presenilin (psn) in model organisms, however, indicate that they are loss of function, leading to the possibility that a reduction in PS activity might contribute to FAD and that proper psn levels are important for maintaining normal cognition throughout life. To explore this issue further, we have tested the effect of reducing psn activity during aging in Drosophila melanogaster males. We have found that flies in which the dosage of psn function is reduced by 50% display age-onset impairments in learning and memory. Treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium during the aging process prevented the onset of these deficits, and treatment of aged flies reversed the age-dependent deficits. Genetic reduction of Drosophila metabotropic glutamate receptor (DmGluRA), the inositol trisphosphate receptor (InsP(3)R), or inositol polyphosphate 1-phosphatase also prevented these age-onset cognitive deficits. These findings suggest that reduced psn activity may contribute to the age-onset cognitive loss observed with FAD. They also indicate that enhanced mGluR signaling and calcium release regulated by InsP(3)R as underlying causes of the age-dependent cognitive phenotypes observed when psn activity is reduced.

In vivo reconstitution of gamma-secretase in Drosophila results in substrate specificity.

The intramembrane aspartyl protease gamma-secretase plays a fundamental role in several signaling pathways involved in cellular differentiation and has been linked with a variety of human diseases, including Alzheimer's disease. Here, we describe a transgenic Drosophila model for in vivo-reconstituted gamma-secretase, based on expression of epitope-tagged versions of the four core gamma-secretase components, Presenilin, Nicastrin, Aph-1, and Pen-2. In agreement with previous cell culture and yeast studies, coexpression of these four components promotes the efficient assembly of mature, proteolytically active gamma-secretase. We demonstrate that in vivo-reconstituted gamma-secretase has biochemical properties and a subcellular distribution resembling those of endogenous gamma-secretase. However, analysis of the cleavage of alternative substrates in transgenic-fly assays revealed unexpected functional differences in the activity of reconstituted gamma-secretase toward different substrates, including markedly reduced cleavage of some APP family members compared to cleavage of the Notch receptor. These findings indicate that in vivo under physiological conditions, additional factors differentially modulate the activity of gamma-secretase toward its substrates. Thus, our approach for the first time demonstrates the overall functionality of reconstituted gamma-secretase in a multicellular organism and the requirement for substrate-specific factors for efficient in vivo cleavage of certain substrates.

Pharmacological analysis of Drosophila melanogaster gamma-secretase with respect to differential proteolysis of Notch and APP.

The gamma-secretase aspartyl protease is responsible for the cleavage of numerous type I integral membrane proteins, including amyloid precursor protein (APP) and Notch. APP cleavage contributes to the generation of toxic amyloid beta peptides in Alzheimer's disease, whereas cleavage of the Notch receptor is required for normal physiological signaling between differentiating cells. Mutagenesis studies as well as in vivo analyses of Notch and APP activity in the presence of pharmacological inhibitors indicate that these substrates can be differentially modulated by inhibition of mammalian gamma-secretase, although some biochemical studies instead show nearly identical dose-response inhibitor effects on Notch and APP cleavages. Here, we examine the dose-response effects of several inhibitors on Notch and APP in Drosophila melanogaster cells, which possess a homogeneous form of gamma-secretase. Four different inhibitors that target different domains of gamma-secretase exhibit similar dose-response effects for both substrates, including rank order of inhibitor potencies and effective concentration ranges. For two inhibitors, modest differences in inhibitor dose responses toward Notch and APP were detected, suggesting that inhibitors might be identified that possess some discrimination in their ability to target alternative gamma-secretase substrates. These findings also indicate that despite an overall conservation in inhibitor potencies toward different gamma-secretase substrates, quantitative differences might exist that could be relevant for the development of therapeutically valuable substrate-specific inhibitors.

Generation and characterization of monoclonal antibodies specific to Drosophila presenilin.

The development of a monoclonal antibody (MAb) specific to Drosophila presenilin (Psn) proteins in vivo was the major aim of this study, since the absence of specific antibodies recognizing Psn proteins hampered our progress in understanding Psn functions during development, differentiation, and pathogenesis. By dot blot and immunofluorescence screenings, we found that MAb Psn2G6 specifically recognized Psn proteins in wing imaginal discs and brains of wild-type control W1118 larvae. MAb Psn2G6 also transgenically expressed a long form of wild-type Psn (Psn + 14 WT) proteins in wing imaginal discs of two independent transgenic lines. Transgenic expression of Psn + 14 WT proteins in psn(B3) larvae completely rescued the expression patterns of Psn proteins and the development of wing imaginal discs. In addition, neural hyperplasia observed in wing imaginal discs of psn(B3) larvae was also suppressed.

Notch signaling: the core pathway and its posttranslational regulation.

Notch signaling controls numerous cell-fate specification events in multicellular organisms, and dysregulated Notch signaling causes several diseases with underlying developmental defects. A key step in Notch receptor activation is its intramembrane proteolysis, which releases an intracellular fragment that participates directly in transcriptional regulation of nuclear target genes. Despite the apparent simplicity of this mechanism, a host of posttranslational processes regulate Notch activity during its synthesis and secretion, ligand-dependent activation at the surface, endocytic trafficking, and degradation. This review describes the core developmental logic of Notch signaling and how regulatory mechanisms tailor Notch pathway outputs to specific developmental scenarios.

Endocytic regulation of Notch signaling.

Endocytosis and endosomal trafficking have emerged as important cell biological steps in the Notch developmental signaling pathway. Ligand endocytosis helps generate the physical forces needed to dissociate and activate the receptor, and activated receptors enter endosomes to signal productively. Endosomal trafficking is also responsible for downregulating Notch receptors that have not been activated by ligand. Recent studies have provided new insights into these Notch trafficking steps, and have uncovered additional endosomal mechanisms that contribute to asymmetric Notch activation and ligand-independent Notch signaling.

Inheritance of susceptibility to induction of nephroblastomas in the Noble rat.

Noble (Nb) strain rats are susceptible to nephroblastoma induction with transplacental exposure to direct-acting alkylating agent N-nitrosoethylurea (ENU), while F344 strain rats are highly resistant. To study the inheritance of susceptibility to induction of these embryonal renal tumors, fetal Nb and F344 rats and F1, F2 and reciprocal backcross hybrids were exposed transplacentally to ENU once on day 18 of gestation. Nephroblastomas developed in 53% of Nb offspring with no apparent gender difference, while no nephroblastomas developed in inbred F344 offspring. F1 and F2 hybrid offspring had intermediate responses, 28% and 30%, respectively. Nephroblastoma incidence in the offspring of F1 hybrids backcrossed to the susceptible strain Nb was 46%, while that in F1 hybrids backcrossed to resistant strain F344 was much lower (16%). Carcinogenic susceptibility is therefore consistent with the involvement of one major autosomal locus; the operation of a gene dosage effect; and a lack of simple Mendelian dominance for either susceptibility or resistance. Since established Wilms tumor-associated suppressor genes, Wt1 and Wtx, were not mutated in normal or neoplastic tissues, genomic profiling was performed on isolated Nb and F344 metanephric progenitors to identify possible predisposing factors to nephroblastoma induction. Genes preferentially elevated in expression in Nb rat progenitors included Wnt target genes Epidermal growth factor receptor, Inhibitor of DNA binding 2, and Jagged1, which were further increased in nephroblastomas. These studies demonstrate the value of this model for genetic analysis of nephroblastoma development and implicate both the Wnt and Notch pathways in its pathogenesis.

The big brain aquaporin is required for endosome maturation and notch receptor trafficking.

Activity of the big brain (bib) gene influences Notch signaling during Drosophila nervous system development. We demonstrate that Bib, which belongs to the aquaporin family of channel proteins, is required for endosome maturation in Drosophila epithelial cells. In the absence of Bib, early endosomes arrest and form abnormal clusters, and cells exhibit reduced acidification of endocytic trafficking organelles. Bib acts downstream of Hrs in early endosome morphogenesis and regulates biogenesis of endocytic compartments prior to the formation of Rab7-containing late endosomes. Abnormal endosome morphology caused by loss of Bib is accompanied by overaccumulation of Notch, Delta, and other signaling molecules as well as reduced intracellular trafficking of Notch to nuclei. Analysis of several endosomal trafficking mutants reveals a correlation between endosomal acidification and levels of Notch signaling. Our findings reveal an unprecedented role for an aquaporin in endosome maturation, trafficking, and acidification.

Endosomal entry regulates Notch receptor activation in Drosophila melanogaster.

Signaling through the transmembrane receptor Notch is widely used throughout animal development and is a major regulator of cell proliferation and differentiation. During canonical Notch signaling, internalization and recycling of Notch ligands controls signaling activity, but the involvement of endocytosis in activation of Notch itself is not well understood. To address this question, we systematically assessed Notch localization, processing, and signaling in a comprehensive set of Drosophila melanogaster mutants that block access of cargo to different endocytic compartments. We find that gamma-secretase cleavage and signaling of endogenous Notch is reduced in mutants that impair entry into the early endosome but is enhanced in mutants that increase endosomal retention. In mutants that block endosomal entry, we also uncover an alternative, low-efficiency Notch trafficking route that can contribute to signaling. Our data show that endosomal access of the Notch receptor is critical to achieve physiological levels of signaling and further suggest that altered residence in distinct endocytic compartments could underlie pathologies involving aberrant Notch pathway activation.

A role for presenilin in post-stress regulation: effects of presenilin mutations on Ca2+ currents in Drosophila.

It has been shown that presenilin is involved in maintaining Ca2+ homeostasis in neurons, including regulating endoplasmic reticulum (ER) Ca2+ storage. From studies of primary cultures and cell lines, however, its role in stress-induced responses is still controversial. In the present study we analyzed the effects of presenilin mutations on membrane currents and synaptic functions in response to stress using an in vivo preparation. We examined voltage-gated K+ and Ca2+ currents at the Drosophila larval neuromuscular junction (NMJ) with voltage-clamp recordings. Our data showed that both currents were generally unaffected by loss-of-function or Alzheimer's disease (AD) -associated presenilin mutations under normal or stress conditions induced by heat shock (HS) or ER stress. In larvae expressing the mutant presenilins, prolonged Ca2+ tail current, reflecting slower deactivation kinetics of Ca2+ channels, was observed 1 day after stress treatments were terminated. It was further demonstrated that the L-type Ca2+ channel was specifically affected under these conditions. Moreover, synaptic plasticity at the NMJ was reduced in larvae expressing the mutant presenilins. At the behavioral level, memory in adult flies was impaired in the presenilin mutants 1 day after HS. The results show that presenilin function is important during the poststress period and its impairment contributes to memory dysfunction observed during adaptation to normal conditions after stress. Our findings suggest a new stress-related mechanism by which presenilin may be implicated in the neuropathology of AD.