Author ORCID Identifier
https://orcid.org/0009-0001-6250-105X
Defense Date
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmacology & Toxicology
First Advisor
Dana E. Selley
Abstract
Neuropathic pain is a prevalent health problem, yet current treatments have limited efficacy and can result in deleterious side-effects, highlighting need for novel treatment options. The sphingosine-1-phosphate (S1P) axis is a promising target for neuropathic pain treatment. Specifically, the immunomodulatory pro-drug FTY720 has been shown to alleviate neuropathic pain across several preclinical models, likely through S1P-receptor-type-1 (S1PR1) on astrocytes. However, the mechanism of FTY720-mediated antinociception is unclear as conflicting evidence implicates either agonism or functional antagonism via recruitment of negative regulation of S1PR1. This dissertation aimed to further investigate the mechanism of S1PR1-mediated antinociception in neuropathic pain using mouse models of traumatic nerve injury and chemotherapy-induced peripheral neuropathy (CIPN). We first examined the effects of S1PR1 conditional knockout (cKO) from various cell types in the central nervous system (CNS) on FTY720-induced alleviation of mechanical allodynia after acute or 14-day treatment using a chronic constriction sciatic nerve injury (CCI) model of neuropathy in mice. In parallel, we determined the effect of S1PR1 cKO, nerve injury, and drug treatment on S1PR1-mediated G-protein activation using agonist-stimulated [35S]GTP𝛾S binding in CNS tissues. Our results showed that S1PR1-mediated G-protein activity in the lumbar spinal cord and throughout the brain is mainly localized in astrocytes, and that S1PR1 expression in this cell type is required for sustained reversal of mechanical allodynia by FTY720. The acute effects of FTY720, however, appear to be mediated by another cell type as S1PR1 cKO from neurons, astrocytes, and oligodendrocytes, but not deletion from astrocytes only, blocked FTY720-induced reversal of CCI-induced mechanical allodynia. Furthermore, both acute and repeated FTY720 treatment desensitized S1PR1 activity in the spinal cord, with 14-day treatment approaching levels of S1PR1 cKO. Moreover, deletion of S1PR1 in any of the targeted cell types did not abrogate the development of CCI-induced allodynia, arguing against a pronociceptive role of S1PR1. To address findings that S1PR1 appears to be required in select CNS cell types for the anti-allodynic effects of FTY720 despite significant FTY720-induced desensitization of S1PR1 activity, we examined the role of the regulatory protein 𝛽-arrestin 2 in the CNS on the anti-allodynic effects of FTY720 in the CCI model after acute and 14-day treatment. 𝛽-arrestin 2 cKO from neurons, astrocytes, and oligodendrocytes attenuated sustained reversal of allodynia by FTY720, similar to results in the S1PR1 cKO mice. However, 𝛽-arrestin 2 cKO from astrocytes alone had no effect on FTY720-induced anti-allodynia after acute or 14-day treatment, indicating that 𝛽-arrestin 2 in astrocytes does not play a role in the anti-allodynic effects of FTY720. Interestingly, 𝛽-arrestin 2 cKO from any of these cell types did not decrease FTY720-induced desensitization of S1PR1 activity in the lumbar spinal cord, suggesting that 𝛽-arrestin 2 alone does not mediate S1PR1 desensitization. Lastly, to approach the question of whether S1PR1-mediated antinociception in neuropathic pain is mediated by S1PR1 agonism or functional antagonism using a pharmacological approach, the antinociceptive effects of novel, non-lymphopenic S1PR1-selective agonists that may not cause S1PR1 adaptation, was assessed using a paclitaxel-induced CIPN model. Our results showed that TRV045 and TRV6589 appear to act as full agonists for G-protein activation but are less potent than the active form of FTY720, FTY720-phosphate. Moreover, TRV045 and TRV6589 doses that acutely reversed paclitaxel-induced mechanical and cold allodynia in mice did not produce significant S1PR1 desensitization or downregulation of S1PR1 protein in the lumbar spinal cord or periaqueductal gray after a single administration. Repeated administration of TRV045 continued to reverse allodynia. However, no desensitization or downregulation of S1PR1 by TRV045 was found in either CNS region after repeated treatment, while repeated treatment with FTY720 significantly desensitized and downregulated S1PR1. In summary, our results suggests that direct agonism of S1PR1 is capable of alleviating neuropathic pain signs in two distinct mouse pain models irrespective of S1PR1 adaptation in CNS regions involved in pain processing.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-5-2024