Secreted serine proteases are abundant in the intact CNS and become deregulated by injury and disease, yet we lack knowledge regarding their physiological functions and contributions to pathology. Several years ago, the discovery of a set of enzyme-activated G protein-coupled receptors, the Protease Activated Receptors (PARs), led to a new conceptual framework for understanding the physiological impact of proteases. PARs permit activating enzymes to signal in a hormone-like fashion to modulate key cellular functions, but when overactivated can contribute to pathology. The PI?s team recently discovered that mice with global PAR1 gene knockout exhibit significant improvements in locomotor recovery after spinal cord injury (SCI). Functional improvements were accompanied by reductions in inflammation and astrogliosis and improvements in the appearance of myelin and axons, all integral substrates to support restoration of function. We also documented that CNS injury relevant proteases, such as thrombin and kallikrein 6 elicit Ca2+, MAPK and STAT3 signaling linked to neuroinflammation and pro-injury responses across neurons and neuroglia in a PAR1-dependent manner. Together, these studies highlight the likely multifactorial roles played by PAR1 in key cellular and molecular events positioned to govern outcomes after SCI. These findings also highlight the potential to target PAR1 for neural protection and repair. Despite these encouraging findings the cellular mechanisms by which blocking PAR1 improves recovery after SCI have not been defined and this knowledge gap hampers progress towards translation of existing FDA approved and orally bioavailable PAR1 small molecule inhibitors. Additionally, whether blocking PAR1 therapeutically at acute or chronic time points after SCI are both capable of improving neural recovery is unknown. Based on recently published findings, taken with new preliminary results, we propose 3 integrated Aims to test the Central Hypothesis that PAR1 is an essential regulator of reactivity across the microglial- astrocyte compartments and can be selectively blocked to improve glial-neuronal trophic coupling, neuroprotection and repair after SCI.
In Aim 1, we will determine the impact of pharmacologic PAR1 inhibition initiated at acute or chronic time points after injury on signs of neuroprotection, neural repair and recovery of sensorimotor function and use ribosomal mRNA capture techniques to document cellular and molecular mechanisms engaged across the astroglial and microglial/monocyte compartments.
In Aim 2, we will determine whether conditional deletion of PAR1 selectively in astrocytes, microglia or peripheral monocytes is sufficient to enhance recovery.
In Aim 3, we will use glial-neuron co-cultures as bioassays to establish PAR1-regulated glial- neural trophic coupling mechanisms relevant to neuroprotection and repair. The studies proposed address key mechanistic questions regarding the functional roles of PAR1 in neural injury and will provide new information needed to optimize therapeutic targeting strategies for recovery of function.
There are more than 300,000 individuals living with disabilities associated with traumatic spinal cord injury (SCI) in the United States, with approximately 17,000 new cases each year, yet there are no clinically approved treatments to significantly improve outcomes. We have determined that a highly druggable cell surface receptor, the thrombin receptor, can be blocked to improve functional outcomes in experimental murine SCI models. The proposed experiments will move these findings closer to clinical translation by providing essential insights into the mechanisms involved and when after SCI receptor targeting is likely to be most effective.
