Novel mechanisms in pain processing - involvement of alternative RNA splicing

The extent and complexity of the proteome in higher eukaryotes derives from alternative pre-mRNA splicing, through which multiple mRNAs and hence proteins can be generated from a single gene. Alternative pre-mRNA splicing and its control are implicated in numerous processes from development to premature ageing. Alternative mRNA splicing can generate completely different proteins from a single gene (e.g. calcitonin and calcitonin-gene-related peptide), or structurally and functionally related families, for example vascular endothelial growth factor). Alternative mRNA splicing is a rapid dynamic process, now recognised to have importance in physiological processes, such as synaptic function, and importantly for pain, responses to neuronal depolarisation/signalling (1).

We have developed specific small molecule inhibitors of Serine aRginine Protein Kinase 1 (SRPK1), a kinase that controls the activity of a downstream RNA binding protein/splicing factor, the SR protein SRSF1. SRSF1 is known to control the alternative splicing of fewer than 20 genes, most of which have been implicated in neoplastic processes. Our first identified SRSF1 target is the VEGF-A gene, which is alternatively spliced into two families, that have opposing actions on both angiogenesis (2) and pain (3). Contributions of alternative pre-mRNA splicing have not been previously investigated in pain processing, but our data show that either local depot, or intrathecal administration of SRPK1 inhibitor completely prevents the onset of neuropathic pain in animal models. This project will determine the efficacy and site and mechanism(s) of action of inhibitors of SRPK1/SRSF1-mediated alternative pre-mRNA splicing in models of acute and chronic pain.

The project has the following aims:

1. To determine the cellular and molecular targets of the inhibitor drugs in modulating pain. We will study the effects of SRPK1 inhibitors on alternative splicing in a) neurons, astrocytes, microglia and Schwann cells in culture, and b) nervous tissues from animals with acute pain. Cells will be cultured in the presence and absence of inhibitor, and the effect on differential SRPK1 binding to specific pre-mRNA splice variants of potential targets will be identified using iCLIP.

2. To determine the effect of alternative splicing inhibition in acute and chronic inflammatory pain and compare to effects on chronic osteoarthritic and diabetic neuropathic pain. In all of these models, we will determine the effect of peripheral and central (intrathecal) SRPK1 inhibition on pain, and any associated tissue damage. We will study the effect of SRPK1 inhibitors on SRSF1 activation in specific cell types in vivo in models of pain, and also the effects on known SRSF1-controlled splicing targets, such as VEGF, as well as key additional targets identified in aim 1.
This project will train a student in molecular and in vivo approaches to drug discovery, and will elucidate the contribution of a novel mechanism to pain signalling in both peripheral and central nervous systems, in multiple pain models.