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许多使人衰弱的神经和精神疾病的基础，包括自闭症、阿尔茨海默病、智力迟钝和精神分裂症。因此，了解并潜在地改善这种疾病将取决于确定控制突触发育的分子。这项工作的长期目标是了解粘附分子如何促进突触连接的建立和特异性，目前的应用主要集中在这样的一个分子家族，y-原钙粘蛋白（Pcdhs），它是这种作用的主要候选者。初步研究表明，22个Pcdh-y基因家族成员被删除的小鼠在出生后不久就会死亡，并因神经元间突触丧失和脊髓神经退行性变而出现严重的神经异常。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，即使当神经变性被遗传阻断。然而，关于v-Pcdh蛋白家族所扮演的确切角色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态将通过以下方式确定：1)在完整CMS组织的多个发育阶段检测v-Pcdh蛋白在不同类型突触上的定位；2)利用延时共聚焦显微镜观察表达荧光标记的y-Pcdhs的海马切片培养物在成熟过程中定位到脊柱突触的动态。其次，v-Pcdh在突触形成、成熟和维持中的特定功能将通过以下方式确定：1)将组织特异性Cre小鼠系与携带两个条件Pcdh-y突变等位基因的小鼠杂交，以在先前无法解决的离散神经元群体（包括皮质、海马和感觉神经元）中发育破坏v-Pcdh功能；2)将他莫昔芬诱导的Cre小鼠系与条件Pcdh-y突变小鼠杂交，以便在成熟神经元形成突触后暂时控制y-Pcdh功能的破坏。总之，这些研究将定义不同的v-Pcdh蛋白家族的突触功能，并确定它们起作用的神经元回路，从而为研究单个v-Pcdh亚型作为指定适当的突触前和突触后伙伴的线索的令人兴奋的可能性提供关键工具。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，因此本文所述的研究将通过为未来新治疗方法的发展提供基础科学基础而有益于公众健康。

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.