The role of SOCE in microglia and secondary degeneration after SCI - Abstract: Neurological outcome after spinal cord injury (SCI) is dependent on the extent of primary injury and the delayed secondary degeneration of spared white matter (WM) and grey matter. Although Ca2+ overload is established as a key mediator of secondary injury, therapeutically targeting external sources of Ca2+ to date have failed to improve neurological recovery in pre-clinical studies and following human SCI. This proposal will assess the role of store-operated Ca2+ entry (SOCE) directly in spinal cord neurons and microglia after SCI using cell type specific knockout of key SOCE mediators. Although, SOCE plays a vital role in maintaining Ca2+ homeostasis and is necessary to sustain intracellular Ca2+ at critical levels for immune cell function, we propose that aberrant and excessive SOCE causes secondary degeneration of WM. The role of SOCE in WM injury, microglial activation, and function remains poorly understood. Furthermore, the precise role of the essential SOCE components including stromal interaction molecule (stim 1 and -2), and Orai 1-3 (form the Ca2+ channel pore) in microglial function and neuronal injury remains unclear. Our preliminary data support an important role for SOCE in mediating secondary degeneration, microglial activation and proinflammatory cytokine release, and worsening neurological recovery. We hypothesize that dysregulation of SOCE mediates “bystander” secondary axonal degeneration following SCI by increasing Ca2+ permeability through Stim and/or Orai channels causing Ca2+ overload in axons. In addition, we hypothesize that SOCE regulates microglial activation and release of proinflammatory factors that negatively impacts neurological recovery after SCI. Our specific Aims are to, 1. Determine the role of SOCE in secondary degeneration and neurological recovery following SCI. 2. Determine the role of microglial SOCE in secondary degeneration, wound healing, and neurological recovery after SCI. To accomplish these aims, we will use two photon excitation intravital imaging to visualize the dynamic response of spinal cord axons and microglia after SCI in real time. We will use both pharmacological SOCE inhibitors and cell type specific knockout of key SOCE mediators directly in spinal neurons and microglia. The most efficacious approach will then be assessed using behavioral testing, amount of spared WM and grey matter, and wound healing. The technology and approach may help advance the field as live imaging of axons over time allows unequivocal determination of the fate of injured axons and whether they can be rescued in real-time with treatment. Furthermore, it allows direct visualization of microglia simultaneously with axons and their interactions as these events are unfolding in the injured spinal cord. This proposal is innovative and uses advanced imaging techniques to explore overlooked areas of SCI research. The approach taken may also unveil novel drug targets and repurpose FDA approved drugs found to inhibit SOCE (e.g., teriflunomide). The underlying mechanisms of WM injury may also be relevant to other neurological diseases of the nervous system such as multiple sclerosis, stroke, and TBI.
