Role of semaphorin signaling in neuronal recovery from dendritic injury: a comparative case study in-vitro and in-vivo - Both axons and dendrites are damaged during traumatic brain injury and Spinal Cord Injury, causing a loss of synaptic connectivity and neural network breakdown. Yet, most studies of neural regeneration focus on axons, leaving dendritic responses to injury vastly unexplored. It is unknown whether and how dendrites reestablish themselves, influence axonal regeneration, or even promote circuit reconnection. The proper patterning of axons and dendrites during development, and regeneration, rely heavily on the precise morphogenesis of these processes, which in turn relies on guidance molecule signaling. Here we focus on semaphorins and their receptors, the plexins and neuropilins. Evolutionary conserved, the semaphorin signaling pathways are crucial to the establishment of neural circuits from invertebrates to vertebrates during development but their involvement in response from injury is unknown. Our long-term goal is to determine the network, cellular, and molecular mechanisms that affect recovery from injury. Our central hypotheses are that soluble semaphorins promote dendritic proliferation after injury, similar to their role during development, while membrane-bound semaphorins are limiting factors that need to be overcome to allow dendritic proliferation as well as synaptic formation. Here we set out to determine the shared fundamental mechanisms in dendritic response to injury by leveraging the in-vivo approach using the invertebrate C. elegans, with in-vitro methods based on mouse primary neuronal culture. We will use a femtosecond-pulse laser to precisely disconnect individual dendrites of neurons in live, behaving nematode and in a primary culture of mouse cortical neurons. For each of these complimentary model systems we adapted a microfluidic device that will improve control of ligand application and survivability. We will then test the roles of different semaphorins and plexins in response to dendritic transection by using C. elegans knockout strains for their genes, as well as transgenic overexpression and siRNA to regulate the protein levels, and application of synthetic Sema3A ligand and siRNA on mouse neurons. We will assay the time course of morphological changes, synaptogenesis, neuronal activity in individual cells and the corresponding circuit, as well as functional recovery of connections. Together, these aims will examine, both in-vitro and in-vivo, roles of semaphorin signaling in neuronal response after dendritic injury. Our anticipated results will uncover cellular and network mechanisms that share a molecular signaling pathway. The proposed research will be carried out by a talented PhD candidate and several mentored undergraduate students at the New Jersey Institute of Technology. We will recruit motivated, diverse, and capable students, and encourage their curiosity and supporting their career goals. The skills and methods that the participating young scientists will acquire will increase their desirability for the next steps in their career.
