Genetic Analysis of Synapse Formation and Function

描述（由申请人提供）：本研究项目研究神经肌肉接头（NMJ）突触发育中分泌信号和细胞外基质蛋白聚糖的相互作用。我们的长期策略是使用果蝇正向遗传筛选来识别新基因，并描述驱动胚胎突触发生和突触功能分化的新机制。过去五年中发现的许多遗传突变体通过突出细胞外糖基化和蛋白水解机制塑造跨突触信号传导的重要性为这一提议提供了基础。本文重点研究的多个相互作用蛋白包括：1）硫酸乙酰肝素（HS）6-O-磺基转移酶Hs 6st和2）HS 6-O-硫酸酯酶Sulf 1，两个功能配对的酶作用于相同的N-磺基葡萄糖胺C6碳; 3）分泌的Jelly Belly（Jeb）跨突触信号与4）间变性淋巴瘤激酶（Alk）受体酪氨酸激酶结合，两者都受到5）GlcNAc转移酶1（Mgat 1）和6）分泌的N-乙酰葡萄糖胺（GlcNAc）-蛋白聚糖的Mind-the-Gap（Mtg）凝集素的调节;和7）分泌的基质金属蛋白酶（MMP）和MMP的组织抑制剂（TIMP），这两种基因都与脆性X智力迟钝蛋白（FMRP）在调节突触发生中相互作用。从这个肥沃的基础上，我们建议集中在细胞间相互作用驱动NMJ发展的胞外糖生物学。我们的核心假设是细胞外聚糖作为分期平台，结合并组合调节驱动突触发生的多个跨突触信号。我们提出了三个具体目标来检验这一假设。目的一：以sulf 1/hs 6st突变体为工具，研究硫酸乙酰肝素蛋白聚糖（HSPG）在WNT（Wingless，Wg）/TGFb/BMP（Glass Bottom Boat，Gbb）跨突触信号转导中的作用。在目标II中，我们测试了细胞外N-聚糖机制在新定义的Jeb-Alk跨突触信号传导的调节中，以及与Wg和Gbb通路的相互作用。在目的III中，我们测试MMPs和TIMP在突触ECM重塑中的作用，以及FMRP突触发生的要求。我们将测试这种机制如何调节HSPGs来控制Jeb，Wg和Gbb配体的组合跨突触信号传导。这项工作与许多神经肌肉和神经系统疾病直接相关，包括先天性糖基化障碍（CDG）和脆性X综合征（FXS），这是认知障碍和自闭症谱系障碍的主要遗传原因。

DESCRIPTION (provided by applicant): This research program investigates interactive roles of secreted signals and extracellular matrix proteoglycans in the development of neuromuscular junction (NMJ) synapses. Our long-term strategy has been to use Drosophila forward genetic screens to identify new genes and characterize novel mechanisms driving embryonic synaptogenesis and the differentiation of synaptic function. Numerous genetic mutants discovered over the last five years provide the foundation of this proposal, by highlighting the importance of extracellular glycosylation and proteolytic mechanisms shaping trans-synaptic signaling. The multiple interacting proteins we focus on in the proposal include: 1) Heparan Sulfate (HS) 6-O-sulfotransferase Hs6st and 2) HS 6-O-sulfatase Sulf1, two functionally-paired enzymes acting on the same N-sulfoglucosamine C6 carbon; 3) the secreted Jelly Belly (Jeb) trans-synaptic signal binding to the 4) Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase, both of which are regulated by 5) GlcNAc Transferase 1 (Mgat1) and 6) secreted Mind-the-Gap (Mtg) lectin for N-acetylglucosamine (GlcNAc)-proteoglycans; and 7) secreted matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP), both of which genetically interact with 8) Fragile X Mental Retardation Protein (FMRP) in regulating synaptogenesis. From this fertile foundation, we propose to focus intensively on the extracellular glycobiology of intercellular interactions driving NMJ development. Our core hypothesis is that extracellular glycans function as staging platforms that bind and combinatorially modulate multiple trans-synaptic signals driving synaptogenesis. We propose three specific aims to test this hypothesis. In Aim I, we use sulf1/hs6st mutants as a tool to investigate the roles of Heparan Sulfate Proteoglycan (HSPG) mechanisms in WNT (Wingless, Wg)/TGFb/BMP (Glass Bottom Boat, Gbb) trans-synaptic signaling. In Aim II, we test extracellular N-glycan mechanisms in regulation of the newly-defined Jeb-Alk trans-synaptic signaling, as well as interactions with the Wg and Gbb pathways. In Aim III, we test roles of MMPs and TIMP in synaptic ECM remodeling, in conjunction with FMRP synaptogenic requirements. We will test how this mechanism regulates HSPGs to control the combinatorial trans-synaptic signaling of Jeb, Wg and Gbb ligands. This work has direct relevance for numerous neuromuscular and neurological diseases, including Congenital Disorders of Glycosylation (CDG) and Fragile X syndrome (FXS), the leading heritable cause of cognitive impairment and autism spectrum disorders.