Genetic Analysis of Synapse Formation and Function

描述（由申请人提供）：本研究项目探讨分泌信号和细胞外基质蛋白聚糖在神经肌肉连接（NMJ）突触发育中的相互作用。我们的长期策略是利用果蝇正向遗传筛选来鉴定新基因，并表征驱动胚胎突触发生和突触功能分化的新机制。在过去五年中发现的许多基因突变，通过强调细胞外糖基化和蛋白水解机制形成跨突触信号的重要性，为这一建议提供了基础。我们在提案中重点研究的多种相互作用蛋白包括：1)硫酸肝素（HS） 6- o -硫基转移酶Hs6st和2)HS 6- o -磺化酶Sulf1，这两种功能配对的酶作用于相同的n -磺基氨基葡萄糖C6碳；3)分泌的Jelly Belly （Jeb）突触反式信号与4)间变性淋巴瘤激酶（Alk）受体酪氨酸激酶结合，这两种信号均受5)GlcNAc转移酶1 （Mgat1）和6)分泌的Mind-the-Gap （Mtg）凝集素对n -乙酰氨基葡萄糖（GlcNAc）蛋白聚糖的调节；7)分泌基质金属蛋白酶（MMP）和组织金属蛋白酶抑制剂（TIMP），这两种酶与脆性X智力迟钝蛋白（FMRP）相互作用，调控突触发生。从这个丰富的基础上，我们建议集中研究驱动NMJ发展的细胞间相互作用的细胞外糖生物学。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种跨突触信号。我们提出三个具体目标来检验这一假设。在Aim I中，我们使用sulf1/hs6st突变体作为工具来研究硫酸肝素蛋白聚糖（HSPG）机制在WNT (Wingless, Wg)/TGFb/BMP （Glass Bottom Boat, Gbb）跨突触信号传导中的作用。在Aim II中，我们测试了细胞外n -聚糖调节新定义的Jeb-Alk突触转导信号的机制，以及与Wg和Gbb通路的相互作用。在Aim III中，我们测试了MMPs和TIMP在突触ECM重塑中的作用，并结合FMRP的突触生成需求。我们将测试这一机制如何调节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），脆性X综合征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.