Joshua A. Weiner - US grants

neurogenetics; developmental neurobiology; neurogenesis; developmental genetics; gene expression; embryonic stem cell; genetic mapping; cell differentiation; vertebrate embryology; retinoate; gene targeting; transfection; laboratory mouse; genetic library; cell line;

Synaptic patterning in several regions of the CNS depends on laminar specificity, in which afferent populations confine their synapses to defined subsets of layers, or laminae, within their target region. Studies have implicated the cadherin family of cell adhesion molecules as regulators of selective synaptogenesis. This proposal will test the hypothesis that cadherins are critical for the establishment of lamina-specific connectivity. Experiments will employ the chick optic tectum as a model system, because of its experimental accessibility and well-defined patterns of laminar afferent projections. The first aim is to examine the expression of cadherins in specific tectal laminae, and in their afferent populations, to provide support for a "cadherin code" guiding laminar connectivity. The second aim is to test the role of cadherins in the establishment of laminar-specific connectivity by using gene transfer to achieve loss- or gain-of-function in the tectum in vivo. The third aim is to more precisely examine the role of cadherins in the regulation of retinotectal synapse formation and maturation by using similar gene transfer strategies in vitro. The final aim is to extend these analyses to the protocadherins, a large family of related adhesion molecules about whose function little is known. Together, these studies will advance our understanding of how molecular cues guide the establishment of specific synaptic connections, a process that is critical for the development and normal function of the brain.

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.

DESCRIPTION (provided by applicant): Current understanding of the interactions between neurons, and between neurons and glia, required for the formation of neural circuits is incomplete. Fundamental gaps include identifying cell adhesion molecules that can generate the diversity needed to promote specific recognition between cells of the developing mammalian CNS, and elucidating associated signaling pathways that regulate several key steps, including elaboration of dendritic arbors and synaptogenesis. The long-term goal is to identify the molecular mechanisms that control the proper formation of neural circuits during development. The objective of this renewal application is to identify the molecular mechanisms by which the gamma-Pcdhs, a family of 22 cadherin superfamily adhesion molecules, regulate cortical dendrite arborization. The central hypothesis is that homophilic interactions between gamma-Pcdh tetramers on cortical neurons and astrocytes promote dendrite arborization by inhibiting a PKC signaling pathway. This hypothesis is based on extensive preliminary data generated by the applicant's laboratory during the prior funding period, and will be tested by pursuing 3 Specific Aims: 1) Determine the extent to which homophilic gamma -Pcdh interactions promote dendrite arborization in cortical neurons; 2) Identify roles for astrocytic gamma -Pcdhs in cortica neuron dendrite arborization; and 3) Identify intracellular signaling mech- anisms regulating the gamma -Pcdhs' role in arborization. Under Aim 1, a model resulting from preliminary in vitro assays--combinatorially diverse gamma -Pcdh cis-tetramers interact homophilically in trans--will be applied to cortical development. Neuronal gamma -Pcdh tetramer composition will be manipulated using transfection and several novel Pcdh- gamma knock-in transgenic mouse lines to directly address the role of interaction specificity in dendrite arborization. Aim 2 build on preliminary data establishing astrocytic gamma -Pcdhs as key regulators of circuit form- ation in the spinal cord. Using astrocyte-restricted Cre transgenics with conditional Pcdh- gamma mutants and knock- ins in vivo, the role of astrocytic gamma -Pcdhs in dendrite arborization of cortical neurons will be delineated. Aim 3 expands on preliminary data showing that a PKC signaling pathway is inhibited by the gamma -Pcdhs to promote dendrite arborization. A C-terminal residue shared by all gamma -Pcdhs has been identified that can be phosphorylated by PKC. We hypothesize that this disrupts the gamma -Pcdhs' ability to inhibit PKC signaling via inhibition of FAK, providing a signaling feedback mechanism. This will be tested using a series of point mutant and truncation Pcdh- gamma constructs in biochemical assays and neuronal cultures. The proposed research is significant, because it will identify molecular mechanisms that can account for diverse cell-cell interactions driving a key step in neural circuit formation, filling an important gap in current knowledge in the field. Such information will be critical to understanding, and eventually ameliorating, the many neurodevelopmental disorders that involve defective dendrite development and synaptogenesis.