Differential heparan sulfate dependency of the Drosophila glypicans.

Heparan sulfate proteoglycans (HSPGs) are composed of a core protein and glycosaminoglycan (GAG) chains and serve as coreceptors for many growth factors and morphogens. To understand the molecular mechanisms by which HSPGs regulate morphogen gradient formation and signaling, it is important to determine the relative contributions of the carbohydrate and protein moieties to the proteoglycan function. To address this question, we generated ΔGAG alleles for dally and dally-like protein (dlp), two Drosophila HSPGs of the glypican family, in which all GAG-attachment serine residues are substituted to alanine residues using CRISPR/Cas9 mutagenesis. In these alleles, the glypican core proteins are expressed from the endogenous loci with no GAG modification. Analyses of the dallyΔGAG allele defined Dally functions that do not require heparan sulfate (HS) chains and that need both core protein and HS chains. We found a new, dallyΔGAG-specific phenotype, the formation of a posterior ectopic vein, which we have never seen in the null mutants. Unlike dallyΔGAG, dlpΔGAG mutants do not show most of the dlp null mutant phenotypes, suggesting that HS chains are dispensable for these dlp functions. As an exception, HS is essentially required for Dlp's activity at the neuromuscular junction. Thus, Drosophila glypicans show strikingly different levels of HS dependency. The ΔGAG mutant alleles of the glypicans serve as new molecular genetic toolsets highly useful to address important biological questions, such as molecular mechanisms of morphogen gradient formation.

Heparan sulfate proteoglycan molecules, syndecan and perlecan, have distinct roles in the maintenance of Drosophila germline stem cells.

The Drosophila female germline stem cell (GSC) niche provides an excellent model for understanding the stem cell niche in vivo. The GSC niche is composed of stromal cells that provide growth factors for the maintenance of GSCs and the associated extracellular matrix (ECM). Although the function of stromal cells/growth factors has been well studied, the function of the ECM in the GSC niche is largely unknown. In this study, we investigated the function of syndecan and perlecan, molecules of the heparan sulfate proteoglycan (HSPG) family, as the main constituents of the ECM. We found that both of these genes were expressed in niche stromal cells, and knockdown of them in stromal cells decreased GSC number, indicating that these genes are important niche components. Interestingly, our genetic analysis revealed that the effects of syndecan and perlecan on the maintenance of GSC were distinct. While the knockdown of perlecan in the GSC niche increased the number of cystoblasts, a phenotype suggestive of delayed differentiation of GSCs, the same was not true in the context of syndecan. Notably, the overexpression of syndecan and perlecan did not cause an expansion of the GSC niche, opposing the results reported in the context of glypican, another HSPG gene. Altogether, our data suggest that HSPG genes contribute to the maintenance of GSCs through multiple mechanisms, such as the control of signal transduction, and ligand distribution/stabilization. Therefore, our study paves the way for a deeper understanding of the ECM functions in the stem cell niche.

Glypicans and Heparan Sulfate in Synaptic Development, Neural Plasticity, and Neurological Disorders.

Heparan sulfate proteoglycans (HSPGs) are components of the cell surface and extracellular matrix, which bear long polysaccharides called heparan sulfate (HS) attached to the core proteins. HSPGs interact with a variety of ligand proteins through the HS chains, and mutations in HSPG-related genes influence many biological processes and cause various diseases. In particular, recent findings from vertebrate and invertebrate studies have raised the importance of glycosylphosphatidylinositol-anchored HSPGs, glypicans, as central players in the development and functions of synapses. Glypicans are important components of the synapse-organizing protein complexes and serve as ligands for leucine-rich repeat transmembrane neuronal proteins (LRRTMs), leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (RPTPs), and G-protein-coupled receptor 158 (GPR158), regulating synapse formation. Many of these interactions are mediated by the HS chains of glypicans. Neurexins (Nrxs) are also synthesized as HSPGs and bind to some ligands in common with glypicans through HS chains. Therefore, glypicans and Nrxs may act competitively at the synapses. Furthermore, glypicans regulate the postsynaptic expression levels of ionotropic glutamate receptors, controlling the electrophysiological properties and non-canonical BMP signaling of synapses. Dysfunctions of glypicans lead to failures in neuronal network formation, malfunction of synapses, and abnormal behaviors that are characteristic of neurodevelopmental disorders. Recent human genetics revealed that glypicans and HS are associated with autism spectrum disorder, neuroticism, and schizophrenia. In this review, we introduce the studies showing the roles of glypicans and HS in synapse formation, neural plasticity, and neurological disorders, especially focusing on the mouse and Drosophila as potential models for human diseases.

The HSPG Glypican Regulates Experience-Dependent Synaptic and Behavioral Plasticity by Modulating the Non-Canonical BMP Pathway.

Under food deprivation conditions, Drosophila larvae exhibit increases in locomotor speed and synaptic bouton numbers at neuromuscular junctions (NMJs). Octopamine, the invertebrate counterpart of noradrenaline, plays critical roles in this process; however, the underlying mechanisms remain unclear. We show here that a glypican (Dlp) negatively regulates type I synaptic bouton formation, postsynaptic expression of GluRIIA, and larval locomotor speed. Starvation-induced octopaminergic signaling decreases Dlp expression, leading to increases in synapse formation and locomotion. Dlp is expressed by postsynaptic muscle cells and suppresses the non-canonical BMP pathway, which is composed of the presynaptic BMP receptor Wit and postsynaptic GluRIIA-containing ionotropic glutamate receptor. We find that during starvation, decreases in Dlp increase non-canonical BMP signaling, leading to increases in GluRIIA expression, type I bouton number, and locomotor speed. Our results demonstrate that octopamine controls starvation-induced neural plasticity by regulating Dlp and provides insights into how proteoglycans can influence behavioral and synaptic plasticity.

Suppression of the synaptic localization of a subset of proteins including APP partially ameliorates phenotypes of the Drosophila Alzheimer's disease model.

APP (amyloid precursor protein), the causative molecule of Alzheimer's disease, is synthesized in neuronal cell bodies and subsequently transported to synapses. We previously showed that the yata gene is required for the synaptic transport of the APP orthologue in Drosophila melanogaster. In this study, we examined the effect of a reduction in yata expression in the Drosophila Alzheimer's disease model, in which expression of human mutant APP was induced. The synaptic localization of APP and other synaptic proteins was differentially inhibited by yata knockdown and null mutation. Expression of APP resulted in abnormal synaptic morphology and the premature death of animals. These phenotypes were partially but significantly rescued by yata knockdown, whereas yata knockdown itself caused no abnormality. Moreover, we observed that synaptic transmission accuracy was impaired in our model, and this phenotype was improved by yata knockdown. Thus, our data suggested that the phenotypes caused by APP can be partially prevented by inhibition of the synaptic localization of a subset of synaptic proteins including APP.

Heparan sulfate proteoglycans in Drosophila neuromuscular development.

Heparan sulfate proteoglycans (HSPGs) are glycoconjugates bearing heparan sulfate (HS) chains covalently attached to core proteins, which are ubiquitously distributed on the cell surface and in the extracellular matrix. HSPGs interact with a number of molecules mainly through HS chains, which play critical roles in diverse physiological and disease processes. Among these, recent vertebrate studies showed that HSPGs are closely involved in synapse development and function. However, the detailed molecular mechanisms remain elusive. Genetic studies from fruit flies, Drosophila melanogaster, have begun to reveal the molecular mechanisms by which HSPGs regulate synapse formation at neuromuscular junctions (NMJs). In this review, we introduce Drosophila studies showing how HSPGs regulate various signaling pathways in developing NMJs. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.

The Strip-Hippo Pathway Regulates Synaptic Terminal Formation by Modulating Actin Organization at the Drosophila Neuromuscular Synapses.

Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. Here, we demonstrate that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo) pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. Our study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization.

Perlecan regulates bidirectional Wnt signaling at the Drosophila neuromuscular junction.

Heparan sulfate proteoglycans (HSPGs) play pivotal roles in the regulation of Wnt signaling activity in several tissues. At the Drosophila melanogaster neuromuscular junction (NMJ), Wnt/Wingless (Wg) regulates the formation of both pre- and postsynaptic structures; however, the mechanism balancing such bidirectional signaling remains elusive. In this paper, we demonstrate that mutations in the gene of a secreted HSPG, perlecan/trol, resulted in diverse postsynaptic defects and overproduction of synaptic boutons at NMJ. The postsynaptic defects, such as reduction in subsynaptic reticulum (SSR), were rescued by the postsynaptic activation of the Frizzled nuclear import Wg pathway. In contrast, overproduction of synaptic boutons was suppressed by the presynaptic down-regulation of the canonical Wg pathway. We also show that Trol was localized in the SSR and promoted postsynaptic accumulation of extracellular Wg proteins. These results suggest that Trol bidirectionally regulates both pre- and postsynaptic activities of Wg by precisely distributing Wg at the NMJ.

In vivo manipulation of heparan sulfate structure and its effect on Drosophila development.

Heparan sulfate proteoglycans (HSPGs) participate in a wide range of biological processes through interactions with a number of ligand proteins. The nature of these interactions largely depends on the heparan sulfate (HS) moiety of HSPGs, which undergoes a series of modifications by various HS-modifying enzymes (HSMEs). Although the effects of alterations in a single HSME on physiological processes have started to be studied, it remains elusive how a combination of these molecules control the structure and function of HS. Here we systematically manipulated the HS structures and analyzed their effect on morphogenesis and signaling, using the genetically tractable model organism, Drosophila. We generated transgenic fly strains overexpressing HSMEs alone or in combination. Unsaturated disaccharide analyses of HS showed that expression of various HSMEs generates distinct HS structures, and the enzymatic activities of HSMEs are influenced by coexpression of other HSMEs. Furthermore, these transgenic HSME animals showed a different extent of lethality, and a subset of HSMEs caused specific morphological defects due to defective activities of Wnt and bone morphogenetic protein signaling. There is no obvious relationship between HS unsaturated disaccharide composition and developmental defects in HSME animals, suggesting that other structural factors, such as domain organization or sulfation sequence, might regulate the function of HS.

Drosophila heparan sulfate 6-O endosulfatase regulates Wingless morphogen gradient formation.

Heparan sulfate proteoglycans (HSPGs) play critical roles in the distribution and signaling of growth factors, but the molecular mechanisms regulating HSPG function are poorly understood. Here, we characterized Sulf1, which is a Drosophila member of the HS 6-O endosulfatase class of HS modifying enzymes. Our genetic and biochemical analyses show that Sulf1 acts as a novel regulator of the Wg morphogen gradient by modulating the sulfation status of HS on the cell surface in the developing wing. Sulf1 affects gradient formation by influencing the stability and distribution of Wg. We also demonstrate that expression of Sulf1 is induced by Wg signaling itself. Thus, Sulf1 participates in a feedback loop, potentially stabilizing the shape of the Wg gradient. Our study shows that the modification of HS fine structure provides a novel mechanism for the regulation of morphogen gradients.

Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface.

Decapentaplegic (Dpp), a Drosophila homologue of bone morphogenetic proteins, acts as a morphogen to regulate patterning along the anterior-posterior axis of the developing wing. Previous studies showed that Dally, a heparan sulfate proteoglycan, regulates both the distribution of Dpp morphogen and cellular responses to Dpp. However, the molecular mechanism by which Dally affects the Dpp morphogen gradient remains to be elucidated. Here, we characterized activity, stability, and gradient formation of a truncated form of Dpp (Dpp(Delta N)), which lacks a short domain at the N-terminus essential for its interaction with Dally. Dpp(Delta N) shows the same signaling activity and protein stability as wild-type Dpp in vitro but has a shorter half-life in vivo, suggesting that Dally stabilizes Dpp in the extracellular matrix. Furthermore, genetic interaction experiments revealed that Dally antagonizes the effect of Thickveins (Tkv; a Dpp type I receptor) on Dpp signaling. Given that Tkv can downregulate Dpp signaling by receptor-mediated endocytosis of Dpp, the ability of dally to antagonize tkv suggests that Dally inhibits this process. Based on these observations, we propose a model in which Dally regulates Dpp distribution and signaling by disrupting receptor-mediated internalization and degradation of the Dpp-receptor complex.

Specific and flexible roles of heparan sulfate modifications in Drosophila FGF signaling.

Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, we characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. We found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts.

Characterization of Drosophila aspartic proteases that induce the secretion of a Golgi-resident transferase, heparan sulfate 6-O-sulfotransferase.

Alzheimer's beta-secretase (BACE1), an aspartic protease, cleaves amyloid precursor protein to produce a neurotoxic peptide, amyloid-beta, which plays a role in triggering Alzheimer's disease. We previously found that BACE1 also cleaves a glycosyltransferase, alpha2,6-sialyltransferase, as a physiological substrate. In the present study, we performed a BLAST homology search, identified two Drosophila aspartic proteases that are homologous to human BACE1, and isolated their cDNAs. The proteins encoded by the cDNAs were designated as DASP1 and DASP2, which exhibited 59% and 50% similarity to human BACE1, respectively. Each protein contained a pair of active site motifs (Asp/Thr or Ser/Gly), which is a common characteristic of aspartic proteases including BACE1. Although DASP1 and DASP2 did not contain an apparent transmenbrane domain, the proteases overexpressed in COS cells were localized in the Golgi area. Some of the DASP1 overexpressed in S2 cells was secreted, but none of the DASP2 was. DASP1 transcripts were expressed in the head of fruitflies, whereas DASP2 transcripts were mainly expressed in the body. When either DASP1 or DASP2 was coexpressed together with a Golgi-resident transferase, Drosophila heparan sulfate 6-O-sulfotransferase, the protease enhanced the secretion of the transferase from the cells, indicating that both DASP1 and DASP2 can induce the secretion of the 6-O-sulfotransferase.

Regulation of Notch signaling by Drosophila heparan sulfate 3-O sulfotransferase.

Heparan sulfate (HS) regulates the activity of various ligands and is involved in molecular recognition events on the cell surface and in the extracellular matrix. Specific binding of HS to different ligand proteins depends on the sulfation pattern of HS. For example, the interaction between antithrombin and a particular 3-O sulfated HS motif is thought to modulate blood coagulation. However, a recent study of mice defective for this modification suggested that 3-O sulfation plays other biological roles. Here, we show that Drosophila melanogaster HS 3-O sulfotransferase-b (Hs3st-B), which catalyzes HS 3-O sulfation, is a novel component of the Notch pathway. Reduction of Hs3st-B function by transgenic RNA interference compromised Notch signaling, producing neurogenic phenotypes. We also show that levels of Notch protein on the cell surface were markedly decreased by loss of Hs3st-B. These findings suggest that Hs3st-B is involved in Notch signaling by affecting stability or intracellular trafficking of Notch protein.

Dally regulates Dpp morphogen gradient formation in the Drosophila wing.

Decapentaplegic (Dpp), a Drosophila TGF beta/bone morphogenetic protein homolog, functions as a morphogen to specify cell fate along the anteroposterior axis of the wing. Dpp is a heparin-binding protein and Dpp signal transduction is potentiated by Dally, a cell-surface heparan sulfate proteoglycan, during assembly of several adult tissues. However, the molecular mechanism by which the Dpp morphogen gradient is established and maintained is poorly understood. We show evidence that Dally regulates both cellular responses to Dpp and the distribution of Dpp morphogen in tissues. In the developing wing, dally expression in the wing disc is controlled by the same molecular pathways that regulate expression of thick veins, which encodes a Dpp type I receptor. Elevated levels of Dally increase the sensitivity of cells to Dpp in a cell autonomous fashion. In addition, dally affects the shape of the Dpp ligand gradient as well as its activity gradient. We propose that Dally serves as a co-receptor for Dpp and contributes to shaping the Dpp morphogen gradient.