Elucidating Functions of the Gamma-Protocadherins in CNS Synapse Development

描述（由申请人提供）：突触的形成、模式化、成熟和维持中的缺陷被认为是广泛的使人衰弱的神经和精神障碍的基础，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，理解和潜在地改善这些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理解粘附分子如何促进突触连接的建立和特异性，并且本申请集中于这样的分子的一个家族，y-原钙粘蛋白（Pcdhs），其是这样的作用的主要候选者。初步研究表明，22个成员的Pcdh-y基因家族被删除的小鼠在出生后不久就死于严重的神经异常，这是由于脊髓中的神经元间突触丢失和神经变性。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，即使神经变性被基因阻断。然而，关于v-Pcdh蛋白质家族所起的确切作用的关键问题仍然没有答案，并且将通过本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通过以下来确定：1）在多个发育阶段检查v-Pcdh蛋白在完整CMS组织中不同类型的突触的定位;和2）使用表达荧光标记的γ-Pcdh的海马切片培养物的延时共聚焦显微镜来跟踪它们在成熟期间定位到棘突触的动力学。1）将组织特异性Cre小鼠系与携带两个条件性Pcdh-y突变等位基因的小鼠杂交，以发育性地破坏先前不可寻址的离散神经元群体（包括皮质、海马和感觉神经元）中的v-Pcdh功能;和2）将他莫昔芬诱导的Cre小鼠系与条件性Pcdh-y突变小鼠杂交，以允许在成熟神经元形成突触后暂时控制γ-Pcdh功能的破坏。总之，这些研究将定义不同的v-Pcdh蛋白质家族的突触功能，并确定它们作用的神经元回路，从而提供必要的关键工具，以调查令人兴奋的可能性，个别v-Pcdh亚型作为线索，以指定适当的突触前和突触后的合作伙伴。由于突触连接的异常模式化可能是人类许多认知、情感和行为缺陷的基础，因此诸如本文所述的那些研究将通过有助于未来开发新治疗方法所需的基础科学基础而有益于公共健康。

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.