Elucidating Functions of the Gamma-Protocadherins in CNS Synapse Development

描述(由申请人提供)：突触的形成、模式、成熟和维持方面的缺陷被认为是一系列衰弱的神经和精神疾病的基础，包括自闭症、阿尔茨海默病、精神发育迟滞和精神分裂症。因此，理解并潜在地改善这种疾病将取决于识别控制突触发育的分子。这项工作的长期目标是了解黏附分子如何促进突触连接的建立和特异性，目前的应用集中在这类分子中的一个家族，即γ-原钙粘附素(Pcdhs)，它们是这种作用的主要候选者。初步研究表明，由22个成员组成的Pcdh-y基因家族缺失的小鼠在出生后不久就会死于严重的神经异常，原因是神经元间突触丢失和脊髓神经变性。Pcdh-y突变的脊髓中间神经元表现出更少的和生理上更弱的突触连接，即使在神经退变被遗传阻止的情况下。然而，关于v-Pcdh蛋白家族扮演的确切角色的关键问题仍然没有得到回答，这将通过这项提案的两个具体目标来解决。首先，v-Pcdh突触定位的特异性和动力学将由以下方面确定：1)检测v-Pcdh蛋白在多个发育阶段完整的CMS组织中对不同类型突触的定位；以及2)使用时间推移共聚焦显微镜对表达荧光标记的y-Pcdhs的海马片培养物进行跟踪，以跟踪它们在成熟过程中对脊髓突触的定位动态。其次，v-Pcdhs在突触形成、成熟和维持中的特定功能将由以下方面确定：1)将组织特异的Cre小鼠品系与含有两个条件性Pcdh-y突变等位基因的小鼠杂交，以破坏先前无法寻址的离散神经元群体的v-Pcdh功能，包括皮质、海马神经元和感觉神经元；2)将他莫昔芬可诱导的Cre小鼠品系与条件性Pcdh-y突变小鼠杂交，使成熟神经元形成突触后，能够暂时地控制干扰他们的y-Pcdh功能。总之，这些研究将定义不同的v-Pcdh蛋白家族的突触功能，并确定它们在其中发挥作用的神经元电路，从而提供必要的关键工具来研究单个v-Pcdh亚型作为线索来指定适当的突触前和突触后伙伴的令人兴奋的可能性。由于突触连接的异常模式可能是人类许多认知、情感和行为缺陷的基础，本文所述的研究将为未来开发新的治疗方法所需的基础科学基础做出贡献，从而使公众健康受益。

DESCRIPTION (provided by applicant): Defects in the formation, patterning, maturation, and maintenance of synapses are believed to underlie a wide range of debilitating neurological and psychiatric disorders, including autism, Alzheimer's disease, mental retardation, and schizophrenia. Understanding and, potentially, ameliorating such disorders will thus depend on identifying the molecules that control synapse development. The long term goal of this work is to understand how adhesion molecules promote the establishment and specificity of synaptic connections, and the present application is focused on one family of such molecules, the y-protocadherins (Pcdhs), that are prime candidates for such roles. Preliminary studies have shown that mice in which the 22-member Pcdh-y gene family is deleted die shortly after birth with severe neurological abnormalities due to interneuron synapse loss and neurodegeneration in the spinal cord. Pcdh-y mutant spinal interneurons exhibit fewer and physiologically weaker synaptic connections even when neurodegeneration is blocked genetically. Critical questions about the precise roles played by the v-Pcdh family of proteins remain unanswered, however, and will be addressed by the two specific aims of this proposal. First, the specificity and dynamics of v-Pcdh synaptic localization will be determined by: 1) examining the localization of v-Pcdh proteins to distinct types of synapses in intact CMS tissues at multiple developmental stages; and 2) using time-lapse confocal microscopy of hippocampal slice cultures expressing fluorescently-tagged y-Pcdhs to follow the dynamics of their localization to spine synapses during maturation. Second, specific functions of the v-Pcdhs in synapse formation, maturation, and maintenance will be determined by: 1) crossing tissue-specific Cre mouse lines with mice harboring two conditional Pcdh-y mutant alleles to developmental^ disrupt v-Pcdh function in discrete neuronal populations not addressable previously, including cortical, hippocampal, and sensory neurons; and 2) crossing a tamoxifen-inducible Cre mouse line with conditional Pcdh-y mutant mice to allow temporally controlled disruption of y-Pcdh function in mature neurons after they have formed synapses. Together, these studies will define synaptic functions for the diverse v-Pcdh family of proteins and identify the neuronal circuits in which they act, thus providing critical tools needed to investigate the exciting possibility that individual v-Pcdh isoforms serve as cues to specify appropriate pre- and post-synaptic partners. As aberrant patterning of synaptic connections is likely to underlie many cognitive, emotional, and behavioral deficits in humans, studies such as those described herein will benefit public health by contributing to the basic science foundation needed for the development of new therapeutic approaches in the future.