Gunter Merdes

Homo‐ and heterodimerization of APP family members promotes intercellular adhesion

The amyloid precursor protein (APP) plays a central role in Alzheimers disease, but its physiological function and that of its mammalian paralogs, the amyloid precursor‐like proteins 1 and 2 (APLPs), is still poorly understood. APP has been proposed to form dimers, a process that could promote cell adhesion via trans‐dimerization. We investigated the dimerization and cell adhesion properties of APP/APLPs and provide evidence that all three paralogs are capable of forming homo‐ and heterocomplexes. Moreover, we show that trans‐interaction of APP family proteins promotes cell–cell adhesion in a homo‐ and heterotypic fashion and that endogenous APLP2 is required for cell–cell adhesion in mouse embryonic fibroblasts. We further demonstrate interaction of all the three APP family members in mouse brain, genetic interdependence, and molecular interaction of APP and APLPs in synaptically enriched membrane compartments. Together, our results provide evidence that homo‐ and heterocomplexes of APP/APLPs promote trans‐cellular adhesion in vivo.

A combined ex vivo and in vivo RNAi screen for notch regulators in Drosophila reveals an extensive notch interaction network.

Notch signaling plays a fundamental role in cellular differentiation and has been linked to human diseases, including cancer. We report the use of comprehensive RNAi analyses to dissect Notch regulation and its connections to cellular pathways. A cell-based RNAi screen identified 900 candidate Notch regulators on a genome-wide scale. The subsequent use of a library of transgenic Drosophila expressing RNAi constructs enabled large-scale in vivo validation and confirmed 333 of 501 tested genes as Notch regulators. Mapping the phenotypic attributes of our data on an interaction network identified another 68 relevant genes and revealed several modules of unexpected Notch regulatory activity. In particular, we note an intriguing relationship to pyruvate metabolism, which may be relevant to cancer. Our study reveals a hitherto unappreciated diversity of tissue-specific modulators impinging on Notch and opens new avenues for studying Notch regulation and function in development and disease.

Interference of human and Drosophila APP and APP-like proteins with PNS development in Drosophila

The view that only the production and deposition of Aβ plays a decisive role in Alzheimers disease has been challenged by recent evidence from different model systems, which attribute numerous functions to the amyloid precursor protein (APP). To investigate the potential cellular functions of APP and its paralogs, we use transgenic Drosophila as a model. Upon overexpression of the APP‐family members, transformations of cell fates during the development of the peripheral nervous system were observed. Genetic analysis showed that APP, APLP1 and APLP2 induce Notch gain‐of‐function phenotypes, identified Numb as a potential target and provided evidence for a direct involvement of Disabled and Neurotactin in the induction of the phenotypes. The severity of the induced phenotypes not only depended on the dosage and the particular APP‐family member but also on particular domains of the molecules. Studies with Drosophila APPL confirmed the results obtained with human proteins and the analysis of flies mutant for the appl gene further supports an involvement of APP‐family members in neuronal development and a crosstalk between the APP family and Notch.

Rhomboid cleaves Star to regulate the levels of secreted Spitz.

Intracellular trafficking of the precursor of Spitz (Spi), the major Drosophila EGF receptor (EGFR) ligand, is facilitated by the chaperone Star, a type II transmembrane protein. This study identifies a novel mechanism for modulating the activity of Star, thereby influencing the levels of active Spi ligand produced. We demonstrate that Star can efficiently traffic Spi even when present at sub‐stoichiometric levels, and that in Drosophila S2R+ cells, Spi is trafficked from the endoplasmic reticulum to the late endosome compartment, also enriched for Rhomboid, an intramembrane protease. Rhomboid, which cleaves the Spi precursor, is now shown to also cleave Star within its transmembrane domain both in cell culture and in flies, expanding the repertoire of known Rhomboid substrates to include both type I and type II transmembrane proteins. Cleavage of Star restricts the amount of Spi that is trafficked, and may explain the exceptional dosage sensitivity of the Star locus in flies.

Axonal accumulation of synaptic markers in APP transgenic Drosophila depends on the NPTY motif and is paralleled by defects in synaptic plasticity.

Alzheimers disease (AD) is characterized by neurofibrillary tangles and extracellular plaques, which consist mainly of β‐amyloid derived from the β‐amyloid precursor protein (APP). An additional feature of AD is axonopathy, which might contribute to impairment of cognitive functions. Specifically, axonal transport defects have been reported in AD animal models, including mice and flies that overexpress APP and tau. Here we demonstrate that the APP‐induced traffic jam of vesicles in peripheral nerves of Drosophila melanogaster larvae depends on the four residues NPTY motif in the APP intracellular domain. Furthermore, heterologous expression of Fe65 and JIP1b, scaffolding proteins interacting with the NPTY motif, also perturb axonal transport. Together, these data indicate that JIP1b or Fe65 may be involved in the APP‐induced axonal transport defect. Moreover, we have characterized neurotransmission at the neuromuscular junction in transgenic larvae that express human APP. Consistent with the observation that these larvae do not show any obvious movement deficits, we found no changes in basal synaptic transmission. However, short‐term synaptic plasticity was affected by overexpression of APP. Together, our results show that overexpression of APP induces partial stalling of axonal transport vesicles, paralleled by abnormalities in synaptic plasticity, which may provide a functional link to the deterioration of cognitive functions observed in AD.

Cell‐type‐specific processing of the amyloid precursor protein by Presenilin during Drosophila development

The cleavage of proteins within their transmembrane domain by Presenilin (PS) has an important role in different signalling pathways and in Alzheimers disease. Nevertheless, not much is known about the regulation of PS activity. It has been suggested that substrate recognition by the PS complex depends only on the size of the extracellular domain independent of the amino‐acid sequence and that PS activity is constitutive in all cells that express the minimal components of the complex. We report here the development of an in vivo reporter system that allowed us to analyse the processing of human amyloid precursor protein (APP) and the Notch receptor tissue specifically during Drosophila development in the living organism. Using this system, we demonstrate differences between APP and Notch processing and show that PS‐mediated cleavage of APP can be regulated in different cell types independent of the size of the extracellular domain.

In Vivo Reconstitution of γ-Secretase in Drosophila Results in Substrate Specificity

ABSTRACT The intramembrane aspartyl protease γ-secretase plays a fundamental role in several signaling pathways involved in cellular differentiation and has been linked with a variety of human diseases, including Alzheimers disease. Here, we describe a transgenic Drosophila model for in vivo-reconstituted γ-secretase, based on expression of epitope-tagged versions of the four core γ-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 γ-secretase. We demonstrate that in vivo-reconstituted γ-secretase has biochemical properties and a subcellular distribution resembling those of endogenous γ-secretase. However, analysis of the cleavage of alternative substrates in transgenic-fly assays revealed unexpected functional differences in the activity of reconstituted γ-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 γ-secretase toward its substrates. Thus, our approach for the first time demonstrates the overall functionality of reconstituted γ-secretase in a multicellular organism and the requirement for substrate-specific factors for efficient in vivo cleavage of certain substrates.

Shedding of APP limits its synaptogenic activity and cell adhesion properties

The amyloid precursor protein (APP) plays a central role in Alzheimer’s disease (AD) and has essential synapse promoting functions. Synaptogenic activity as well as cell adhesion properties of APP presumably depend on trans-cellular dimerization via its extracellular domain. Since neuronal APP is extensively processed by secretases, it raises the question if APP shedding affects its cell adhesion and synaptogenic properties. We show that inhibition of APP shedding using cleavage deficient forms of APP or a dominant negative α-secretase strongly enhanced its cell adhesion and synaptogenic activity suggesting that synapse promoting function of APP is tightly regulated by α-secretase mediated processing, similar to other trans-cellular synaptic adhesion molecules.

A Deterministic Analysis of Genome Integrity during Neoplastic Growth in Drosophila

The development of cancer has been associated with the gradual acquisition of genetic alterations leading to a progressive increase in malignancy. In various cancer types this process is enabled and accelerated by genome instability. While genome sequencing-based analysis of tumor genomes becomes increasingly a standard procedure in human cancer research, the potential necessity of genome instability for tumorigenesis in Drosophila melanogaster has, to our knowledge, never been determined at DNA sequence level. Therefore, we induced formation of tumors by depletion of the Drosophila tumor suppressor Polyhomeotic and subjected them to genome sequencing. To achieve a highly resolved delineation of the genome structure we developed the Deterministic Structural Variation Detection (DSVD) algorithm, which identifies structural variations (SVs) with high accuracy and at single base resolution. The employment of long overlapping paired-end reads enables DSVD to perform a deterministic, i.e. fragment size distribution independent, identification of a large size spectrum of SVs. Application of DSVD and other algorithms to our sequencing data reveals substantial genetic variation with respect to the reference genome reflecting temporal separation of the reference and laboratory strains. The majority of SVs, constituted by small insertions/deletions, is potentially caused by erroneous replication or transposition of mobile elements. Nevertheless, the tumor did not depict a loss of genome integrity compared to the control. Altogether, our results demonstrate that genome stability is not affected inevitably during sustained tumor growth in Drosophila implying that tumorigenesis, in this model organism, can occur irrespective of genome instability and the accumulation of specific genetic alterations.

About Combs, Notches, and Tumors: Epigenetics Meets Signaling

The identities of cells, determined by differential gene expression, are heritably maintained by the antagonistic functions of Polycomb group (PcG) and Trithorax group proteins. Two recent papers shed new light on tumor suppressive functions of PcG by reporting direct silencing of the Notch and JAK/STAT signaling pathways in Drosophila melanogaster.