Synapse Maturation by Activity-Dependent Ectodomain Shedding of SIRP

描述(申请人提供)：功能性突触连接的形成对大脑的正常运作至关重要。在最初的突触分化后，突触在神经活动的作用下成熟和稳定，以建立适当的突触连接。在成熟的突触前束扩大的过程中，更多的突触小泡聚集到突触前终末，活动区的数量和突触后的密度增加，棘突的形状随着突触的活动而改变。然而，依赖活性的突触成熟的分子机制仍有待阐明。使用在培养的神经元中聚集突触小泡的能力作为生物测试，我们已经纯化了可以组织发育中的大脑的突触前终末的分子。这种纯化揭示了诱导突触囊泡聚集的两个活性高峰。其中一个峰含有FGF22，我们已经证明FGFs促进小脑、神经肌肉和海马区突触的分化1-3。我们在这里关注的另一个峰包含信号调节蛋白1(SIRP1)的胞外结构域，SIRP1是一个跨膜免疫球蛋白超家族成员4。在突触成熟前后，SIRP1在海马区高表达。它定位于树突，集中在突触。有趣的是，SIRP1的胞外区在细胞激活时被裂解和脱落。将SIRP1的胞外区应用于培养的海马神经元，可促进突触囊泡聚集。条件性SIRP1基因敲除小鼠在突触前成熟方面存在缺陷。根据这些初步结果，我们提出了以下海马区突触活性依赖的成熟模型：在轴突-树突接触形成突触后，突触前终末释放的神经递质诱导突触后SIRP1的裂解，SIRP1的外区脱落反过来促进突触前终末的成熟。为了验证这一假说，我们建议：7目标1：确定SIRP1突触前效应是否需要胞外脱落7目标2：研究神经活动在SIRP1依赖的突触前成熟中的作用7目标3：检查SIRP1胞外脱落对体内突触前成熟的重要性我们将使用分子和细胞生物学、生化、成像和电生理方法。通过这些研究，我们可以了解通过神经活动在海马区建立功能性突触的分子机制。许多形式的神经疾病，包括自闭症、精神分裂症和阿尔茨海默病，都与海马体突触的异常变化有关。此外，SIRP1受体CD475与学习、记忆、阿尔茨海默病和精神分裂症有关。因此，我们的研究还将有助于设计预防和治疗此类神经疾病的策略。在未来的研究中，我们将使用条件性SIRP1基因敲除小鼠来研究SIRP1及其胞外结构域脱落在学习、记忆形成和神经功能障碍中的体内作用。 与公共健康相关：为了在大脑中建立适当的突触连接，突触必须在发育过程中通过神经活动成熟。然而，依赖活性的突触成熟的分子机制尚不清楚。在这里，我们将确定SIRP1的活性依赖性切割在海马区突触成熟中的作用，SIRP1是一种突触细胞黏附分子，海马区的结构对长期记忆的形成至关重要。由于SIRP1信号的缺陷与阿尔茨海默病和精神分裂症有关，我们的研究应该会对这种破坏性神经疾病的病理生理学和治疗产生新的见解。

DESCRIPTION (provided by applicant): Formation of functional synaptic connections is critical for proper functioning of the brain. After initial synaptic differentiation, synapses are maturated and stabilized by neural activity to establish appropriate synaptic connections. During maturation presynaptic boutons enlarge, more synaptic vesicles accumulate to the presynaptic terminal, the number of active zones and postsynaptic densities increases, and the shape of spines changes in response to synaptic activity. However, the molecular mechanisms underlying activity- dependent synapse maturation remain to be elucidated. Using the ability to cluster synaptic vesicles in cultured neurons as a bioassay, we have purified molecules that can organize presynaptic terminals from developing brains. This purification revealed two peaks of activity that induced synaptic vesicle clustering. One peak contains FGF22, and we have shown that FGFs promote differentiation of cerebellar, neuromuscular and hippocampal synapses1-3. The other peak, on which we focus here, contains the extracellular domain of signal regulatory protein 1 (SIRP1), a transmembrane immunoglobulin superfamily member4. SIRP1 is highly expressed in the hippocampus around the time of synapse maturation. It is localized in dendrites and concentrated at synapses. Interestingly, the extracellular domain of SIRP1 is cleaved and shed in response to cellular activation. The application of the extracellular domain of SIRP1 to cultured hippocampal neurons promotes synaptic vesicle clustering. Conditional SIRP1 knockout mice show defects in presynaptic maturation. From these preliminary results, we propose the following model for activity-dependent maturation of hippocampal synapses: After initial synapse formation by axon-dendrite contacts, neurotransmitter release from the presynaptic terminal induces the cleavage of postsynaptic SIRP1, and the shed ectodomain of SIRP1 in turn promotes the maturation of the presynaptic terminal. To test this hypothesis, we propose to: 7 Aim 1: Determine whether ectodomain shedding is required for the presynaptic effect of SIRP1 7 Aim 2: Investigate the role of neural activity for SIRP1-dependent presynaptic maturation 7 Aim 3: Examine the importance of ectodomain shedding of SIRP1 for presynaptic maturation in vivo We will use molecular and cellular biological, biochemical, imaging and electrophysiological approaches. Through these studies we should understand the molecular mechanisms underlying functional synapse establishment in the hippocampus by neural activity. Many forms of neurological disorders including autism, schizophrenia, and Alzheimer's disease are associated with abnormal alterations of synapses in the hippocampus. Furthermore, a SIRP1 receptor, CD475 is implicated in learning, memory, Alzheimer's disease and schizophrenia6-9. Thus, our studies will also help design strategies to prevent and treat such neurological disorders. In future studies, we will use conditional SIRP1 knockout mice to investigate the in vivo role of SIRP1 and its ectodomain shedding in learning, memory formation and neurological disorders. PUBLIC HEALTH RELEVANCE: To establish appropriate synaptic connections in the brain, synapses must be maturated by neural activity during development. However, the molecular mechanisms underlying activity-dependent synapse maturation are not known. Here, we will determine the role of activity-dependent cleavage of SIRP1, a synaptic cell adhesion molecule, in synapse maturation in the hippocampus, the structure known to be critical for long-term memory formation. Because defects in SIRP1 signaling are implicated in Alzheimer's disease and schizophrenia, our studies should yield novel insights into the pathophysiology and treatment of such devastating neurological disorders.