Year: 2006
Type: article
Abstract: Public Health Relevance: The United States has 80 million employees with chronic pain resulting in annual losses of 61.2 billion dollars due to pain-related productive time lost. In addition, pain-related depression and inactivity reduce the quality of life. The development of effective analgesics is therefore important from a public health perspective. In this dissertation, the natural properties of herpes simplex virus (HSV-1) vectors are exploited to (i) develop an HSV-1 vector-based selection system that can potentially identify natural or chemical inhibitors of chronic pain and (ii) to test HSV-1 vector-expressed dominant negative PKCε (DNP) as a strategy to treat chronic pain. The vanilloid/capsaicin receptor (TRPV1) is a pro-nociceptive calcium ion channel that is upregulated in chronic pain. This occurs partly due to protein kinase C epsilon (PKCε)−mediated receptor phosphorylation. An HSV-1 vector expressing TRPV1 (vTT) was engineered and vTT-expressed TRPV1 functionality was confirmed.Treatment of vTT-infected cells with capsaicin or resiniferatoxin caused concentration-dependent Ca+2 influx, leading to cell-death and a dramatic reduction in infectious particle yield. TRPV1 antagonists, ruthenium red and SB-366791 reversed agonist-induced cell-death and rescued vTT growth, providing a basis for selection. Selection for antagonists was modeled using a mixed infection of vTT and vHG (capsaicin resistant control vector) and virus passage in the presence capsaicin. These experiments demonstrated that a single control vector particle was readily isolated from a population of 10^5 vTT particles. This approach can be used to identify antagonists from chemical or gene libraries and offers advantages of (i) a platform assay applicable to other ion channels and (ii) adaptability to high throughput formats. Dominant negative PKCε (DNP) was engineered into HSV-1 to create the vector, vHDNP. Following functional confirmation of vHDNP in U2OS, Vero cells and neurons, cobalt uptake showed a reduction of capsaicin sensitive vHDNP-transduced neurons. Electrophysiology confirmed this and also demonstrated a knockdown of TRPV1-PKCε coupling in nociceptive neurons. In-vivo studies of noxious heat-induced nocisponsive behavior in vHDNP-inoculated rats showed a subtle inhibition of withdrawal responses when compared with controls. In conclusion, HSV-1 expressed dominant negative PKCε is a viable strategy to specifically inhibit TRPV1 function in order to treat chronic pain.
Author: Rahul Srinivasan
Language: English
Subfield: Physiology
Field: Medicine
Domain: Health Sciences
Open Access status: green
Awards: G1144762115, G1347292224, Early Adversity Shapes Adolescent Risk Behavior Trajectories in Mexican Americans, Molecular Mechanisms of Obesity in Children Exposed to Phthalates in Utero, G3709884776, Maintaining and Expanding the CHAMACOS Epidemiology Cohort Infrastructure for Future Generations, Early life influences on Epigenetic Aging in Mexican-American children, DDT & PBDE exposure, puberty onset and neurodevelopment in Mexican-American girls, PRENATAL AND POSTNATAL EXPOSURE TO ENVIRONMENTAL MIXTURES: NEURODEVELOPMENT AND DNA METHYLATION BIOMARKERS, Effect of early life exposure to social adversity and pesticides on risk-taking behavior of 16-18 year olds: the CHAMACOS study, Center for Children's Environmental Health Research, G9000013131, Common and Distinct Influences of Prenatal and Postnatal Early-Life Adversity on Epigenomic Trajectories in Mexican American Children, Fostering Scientific and Workforce Development by Maintaining and Enriching the CHAMACOS Birth Cohort
OpenAlex ID: W35912433