Indirect evidence for Delta-dependent intracellular processing of Notch in Drosophila embryos

AbstractCell–cell signalling mediated by the receptor Notch regulates the differentiation of a wide variety of cell types in invertebrate and vertebrate species [1], but the mechanism of signal transduction following receptor activation is unknown. A recent model proposes that ligand binding induces intracellular processing of Notch [2–4]; the processed intracellular form of Notch then translocates to the nucleus and interacts with DNA-bound Suppressor of Hairless (Su(H)), a transcription factor required for target gene expression [5–8]. As intracellular processing of endogenous Notch has so far escaped immunodetection [1], we devised a sensitive nuclear-activity assay to monitor indirectly the processing of an engineered Notch in vivo. First, we show that the intracellular domain of Notch, fused to the DNA-binding domain of Gal4, regulated transcription, in a Delta-independent manner. Second, we show that full-length Notch, containing the Gal4 DNA-binding domain inserted 27 amino acids carboxy terminal to the transmembrane domain, activated transcription in a Delta-dependent manner. These results provide indirect evidence for a ligand-dependent intracellular processing event in vivo, supporting the view that Su(H)-dependent Notch signalling involves intracellular cleavage, and transcriptional regulation by processed Notch

Trisomy for Synaptojanin1 in Down syndrome is functionally linked to the enlargement of early endosomes

Enlarged early endosomes have been observed in neurons and fibroblasts in Down syndrome (DS). These endosome abnormalities have been implicated in the early development of Alzheimer's disease (AD) pathology in these subjects. Here, we show the presence of enlarged endosomes in blood mononuclear cells and lymphoblastoid cell lines (LCLs) from individuals with DS using immunofluorescence and confocal microscopy. Genotype-phenotype correlations in LCLs carrying partial trisomies 21 revealed that triplication of a 2.56 Mb locus in 21q22.11 is associated with the endosomal abnormalities. This locus contains the gene encoding the phosphoinositide phosphatase synaptojanin 1 (SYNJ1), a key regulator of the signalling phospholipid phosphatidylinositol-4,5-biphosphate that has been shown to regulate clathrin-mediated endocytosis. We found that SYNJ1 transcripts are increased in LCLs from individuals with DS and that overexpression of SYNJ1 in a neuroblastoma cell line as well as in transgenic mice leads to enlarged endosomes. Moreover, the proportion of enlarged endosomes in fibroblasts from an individual with DS was reduced after silencing SYNJ1 expression with RNA interference. In LCLs carrying amyloid precursor protein (APP) microduplications causing autosomal dominant early-onset AD, enlarged endosomes were absent, suggesting that APP overexpression alone is not involved in the modification of early endosomes in this cell type. These findings provide new insights into the contribution of SYNJ1 overexpression to the endosomal changes observed in DS and suggest an attractive new target for rescuing endocytic dysfunction and lipid metabolism in DS and in A

Inhibition of proteasome and Shaggy/Glycogen synthase kinase-3β kinase prevents clearance of phosphorylated tau inDrosophila

International audienceTauopathies, including Alzheimer's disease (AD), are a group of neurodegenerative disorders characterized by the presence of intraneuronal filamentous inclusions of abnormally phosphorylated tau protein. In AD brains, it has been shown that the level of abnormally phosphorylated tau is higher than in age-matched control brains, suggesting that abnormally phosphorylated tau is resistant to degradation. By using a Drosophila model of tauopathy, we studied the relationship between tau phosphorylation and degradation. We showed that in vivo reduction of proteasome activity results in an accumulation of high-molecular-weight forms of hyperphosphorylated tau. We also found that glycogen synthase kinase (GSK)-3beta-mediated hyperphosphorylated forms of tau are degradable by the proteasomal machinery. Unexpectedly, GSK-3beta inactivation resulted in a very large accumulation of high-molecular-weight species consisting of hyperphosphorylated tau, suggesting that, depending on the kinase(s) involved, tau phosphorylation state affects its degradation differently. We thus propose a model for tauopathies in which, depending on toxic challenges (e.g., oxidative stress, exposure to amyloid peptide, etc.), abnormal phosphorylation of tau by kinases distinct from GSK-3beta leads to progressive accumulation of hyperphosphorylated tau oligomers that are resistant to degradation

Moderate Overexpression of Tau in Drosophila Exacerbates Amyloid-β-Induced Neuronal Phenotypes and Correlates with Tau Oligomerization

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SorLA Protective Function Is Restored by Improving SorLA Protein Maturation in a Subset of Alzheimer’s Disease-Associated SORL1 Missense Variants

International audienceSORL1 loss of function is associated with Alzheimer’s disease (AD) risk through increased Aβ peptide secretion. We expressed 10 maturation-defective rare missense SORL1 variants in HEK cells and showed that decreasing growing temperature led to a significant increase in the maturation of the encoded protein SorLA for 6/10. In edited hiPSC carrying two of these variants, maturation of the protein was restored partially by decreasing the culture temperature and was associated with concomitant decrease in Aβ secretion. Correcting SorLA maturation in the context of maturation-defective missense variants could thus be a relevant strategy to improve SorLA protective function against AD

Both cytoplasmic and nuclear accumulations of the protein are neurotoxic in Drosophila models of TDP-43 proteinopathies

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Wingless Capture by Frizzled and Frizzled2 in Drosophila Embryos

International audienceA variety of factors could influence how far developmental signals spread. For example, the Patched receptor limits the range of its ligand Hedgehog. Somehow, the Frizzled2 receptor has the opposite effect on its ligand. Increasing the level of Frizzled2 stabilizes Wingless and thus extends the Wingless gradient in Drosophila wing imaginal disks. Here we ask whether Frizzled or Frizzled2 affects the spread of Wingless in Drosophila embryos. We show that in the embryonic epidermis, the combined expression of both receptors is lowest in the engrailed domain. This is because expression of Frizzled is repressed by the Engrailed transcription factor, whereas that of Frizzled2 is repressed by Wingless signaling. Receptor downregulation correlates with an early asymmetry in Wingless distribution, characterized by the loss of Wingless staining in the engrailed domain. Raising the expression of either Frizzled or Frizzled2 in this domain prevents the early disappearance of Wingless-containing vesicles. Apparently, Wingless is captured, stabilized, and quickly internalized by either receptor. As far as we can tell, captured Wingless is not passed on to further cells and does not contribute to the spread of Wingless. Receptor downregulation in the posterior compartment may contribute to dampening the signal at the time when cuticular fates are specified

Drosophila models of human tauopathies indicate that Tau protein toxicity in vivo is mediated by soluble cytosolic phosphorylated forms of the protein

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A Connected Network of Interacting Proteins Is Involved in Human-Tau Toxicity in Drosophila

International audienceTauopathies are neurodegenerative diseases characterized by the presence of aggregates of abnormally phosphorylated Tau. Deciphering the pathophysiological mechanisms that lead from the alteration of Tau biology to neuronal death depends on the identification of Tau cellular partners. Combining genetic and transcriptomic analyses in Drosophila, we identified 77 new modulators of human Tau-induced toxicity, bringing to 301 the number of Tau genetic interactors identified so far in flies. Network analysis showed that 229 of these genetic modulators constitute a connected network. The addition of 77 new genes strengthened the network structure, increased the intergenic connectivity and brought up key hubs with high connectivities, namely Src64B/FYN, Src42A/FRK, kuz/ADAM10, heph/PTBP1, scrib/SCRIB, and Cam/CALM3. Interestingly, we established for the first time a genetic link between Tau-induced toxicity and ADAM10, a recognized Alzheimer Disease protective factor. In addition, our data support the importance of the presynaptic compartment in mediating Tau toxicity

Tau is not normally degraded by the proteasome

International audienceTau-positive inclusions in neurons are consistent neuropathologic features of the most common causes of dementias such Alzheimer's disease and frontotemporal dementia. Ubiquitinated tau-positive inclusions have been reported in brains of Alzheimer's disease patients, but involvement of the ubiquitin-dependent proteasomal system in tau degradation remains controversial. Before considering the tau degradation in pathologic conditions, it is important to determine whether or not endogenous tau is normally degraded by the proteasome pathway. We therefore investigated this question using two complementary approaches in vitro and in vivo. Firstly, SH-SY5Y human neuroblastoma cells were treated with different proteasome inhibitors, MG132, lactacystin, and epoxomicin. Under these conditions, neither total nor phosphorylated endogenous tau protein levels were increased. Instead, an unexpected decrease of tau protein was observed. Secondly, we took advantage of a temperature-sensitive mutant allele of the 20S proteasome in Drosophila. Genetic inactivation of the proteasome also resulted in a decrease of tau levels in Drosophila. These results obtained in vitro and in vivo demonstrate that endogenous tau is not normally degraded by the proteasome