Synapse formation is a critical step in brain development that requires precise regulation to prevent miswiring that can manifest as neurological dysfunction. In recent years, it has come to light that astrocytes induce excitatory synaptogenesis in the central nervous system by secreting multiple factors that regulate synaptic formation and maturation. However, the question of why astrocytes release a variety of signals affecting synaptogenesis represents a significant gap in knowledge in the field of neurodevelopment. The primary focus of our lab is to understand the contributions of three of these astrocyte-released signals, the matricellular proteins thrombospondin (TSP), hevin and SPARC, to the initial formation and maturation of excitatory synapses. The first goal in this proposed study is to determine the role of TSP in forming the initial synaptic contacts between presynaptic axons and postsynaptic dendrites. Intriguingly, TSP binds to the neuronal calcium channel subunit a2d-1 to stimulate the formation and target-seeking behavior of dendritic filopodia in cultured retinal ganglion cells (RGCs). Further preliminary findings indicate that TSP/a2d-1 may induce downstream Rho GTPase signaling pathways in order to exert effects on actin dynamics in filopodia, representing a potential molecular mechanism for TSP/a2d-1-induced synapse formation. Like TSP, the matricellular protein hevin is prosynaptogenic both in cultured neurons and in vivo. SPARC, which shares a high degree of homology with hevin, is specifically antagonistic to hevin's synaptogenic effects. The second goal of the proposed study is to determine how the balance between hevin and SPARC affects the maturation of CNS synapses. Preliminary results from serial section electron microscopy (EM) revealed a dramatic phenotype in hevin knockout (KO) mice whereby postsynaptic spines were largely absent from dendrites, replaced instead by immature filopodia-like protrusions. Furthermore, excitatory synapses were primarily made onto the dendritic shaft rather than at the tips of protrusions. Based on these findings, here I will test the hypotheses that a2d-1 promotes the initiation of excitatory synaptic contacts during the dendritic filopodial stage of development, while precise regulation of the levels of hevin and SPARC controls the stabilization and maturation of synapses. To achieve this objective, I propose two specific aims: 1) how do TSP and a2d-1 regulate the initial phase of excitatory synapse formation? 2) How do hevin and SPARC regulate the morphological and functional maturation of excitatory synapses? Through a combination of live confocal imaging, serial section EM and electrophysiology in both purified cortical neurons and slice preparations from visual cortex, the proposed work should provide new molecular insight into the role of astrocytes in excitatory synaptogenesis during development. Better understanding of TSP, a2d-1, hevin and SPARC may lead to improvements in the treatment of neurological diseases such as schizophrenia and autism which are characterized by aberrant synaptic connectivity.
Many neurological disease states, including but not limited to epilepsy, schizophrenia and autism, are characterized by aberrant synapse formation. The proposed work should provide new molecular insight into excitatory synapse formation during development both in vitro and in vivo. Better understanding of the roles of the astrocyte-secreted proteins thrombospondin, hevin and SPARC in synapse formation may lead to improvements in the treatment of a wide variety of brain disorders.
