Genetic Analysis of Synapse Formation and Function

描述(申请人提供)：这项研究计划调查分泌信号和细胞外基质蛋白多糖在神经肌肉接头(NMJ)突触发育中的交互作用。我们的长期策略是使用果蝇正向遗传筛选来识别新的基因，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大量基因突变通过强调细胞外糖基化和蛋白分解机制塑造跨突触信号的重要性，为这一提议提供了基础。我们在提案中关注的多种相互作用的蛋白质包括：1)硫酸乙酰肝素(HS)6-O-磺酸转移酶Hs6st和2)HS 6-O-硫酸盐酶Sulf1，两个作用于相同N-磺基葡萄糖C6碳的功能配对的酶；3)分泌的Jelly Belly(JEB)反式突触信号与4)间变性淋巴瘤激酶(ALK)受体酪氨酸激酶(TK)结合，两者均受5)GlcNAc转移酶(MGAT1)和6)分泌的Mind-the-Gap(Mtg)凝集素的N-乙酰氨基葡萄糖(Glcc)-蛋白多糖的调节；7)分泌基质金属蛋白酶及其组织抑制物(TIMP)，两者在基因上与脆性X智力低下蛋白(FMRP)相互作用，调节突触发生。从这一肥沃的基础上，我们建议集中关注驱动NMJ发育的细胞间相互作用的细胞外糖生物学。我们的核心假设是，细胞外多聚糖作为阶段性平台，结合并组合调制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。在目的I中，我们以Sul1/hs6st突变体为工具，研究了硫酸肝素蛋白多糖(HSPG)机制在WNT(Wingless，Wg)/TGFb/BMP(Glass Bottom Boat，GBB)跨突触信号转导中的作用。在AIM II中，我们测试了细胞外N-糖链调节新定义的JEB-ALK跨突触信号的机制，以及与Wg和GBB通路的相互作用。在目标III中，我们结合FMRP的突触形成要求，测试了MMPs和TIMP在突触ECM重塑中的作用。我们将测试这一机制如何调节HSPG来控制JEB、Wg和GBB配体的组合跨突触信号。这项工作与许多神经肌肉和神经疾病直接相关，包括先天性糖基化紊乱(CDG)和脆性X综合征(FXS)，后者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.