Reactive astrogliosis after spinal cord injury (SCI) serves as a critical defense mechanism, but it also can be detrimental, causing swelling that leads to ischemia, and formation of a glial scar that inhibits axonal regeneration. Astrogliosis can be prevented by deleting aquaporin (Aqp) 4, but this too can be detrimental, since a glial scar is essential for restricting inflammation and lesion size. Therefore, down-modulation of astrogliosis and of Aqp4, but not total inhibition, are important therapeutic goals. Aqp channels passively transport H2O across membranes, with the amount of H2O transported determined by the osmotic gradient. The molecular mechanisms by which osmotic gradients are regulated in order to drive the flux of H2O are poorly understood, but likely involve ion channels. In resting astrocytes, Aqp4 physically co-associates with transient receptor potential (Trp) V4, a non-selective cation channel, with the assembled molecular complex being required for normal cell volume control. We discovered that the molecular mechanism by which H2O is handled undergoes a fundamental change when astrocytes become activated. Following SCI, the partner for Aqp4 co-association switches from TrpV4 to TrpM4, with molecular experiments showing that TrpM4 co-associates preferentially with Aqp4-M23, the isoform that assembles into 'orthogonal arrays of particles'that are associated with high rates of H2O transport. This switch provides an opportunity to down-modulate Aqp4-M23 preferentially by inhibiting TrpM4, and thereby favorably influence astrogliosis without directly or completely inhibiting Aqp4. In this competitive renewal, we will use a unique model involving fibrinogen injection into the spinal cord to study reactive astrogliosis in situ. We propose 3 Specific Aims (SA) to expand on our novel preliminary data on the role of TrpM4 in regulating Aqp4 and reactive astrogliosis. DESCRIPTION:
In Specific Aim (SA) 1, we will test the hypothesis that TrpM4 deletion will favorably influence astrogliosis and astrocyte swelling. In SA2, we will test the hypothesis that Aqp4 and TrpM4 physically co-associate to form an integral molecular complex for the control of H2O transport in reactive astrocytes. In SA3, we will test the hypothesis that TrpM4 activation osmotically drives the flux of water via Aqp4 in reactive astrocytes. Each hypothesis in each aim is supported by robust preliminary data.
Each year, SCI devastates the lives of thousands of people worldwide. Short of actual prevention, the best hope for reducing the impact of SCI rests with decreasing secondary injury suffered after trauma. Tremendous progress has been made in cell replacement therapies and rehabilitation, but these treatments work best when administered in the context of the smallest possible lesion. Spinal cord swelling and astrogliosis are responsible for ischemia and impaired axonal growth, but effective treatments are lacking. The purpose of this proposal is to demonstrate that inhibition of TrpM4 can exert a significant salutary effect in SCI due to inhibition of astrogliosis and astrocyte swelling.
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