Date of Award

12-1-2017

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Ramkumar, Vickram

Second Advisor

Rybak, Leonard

Abstract

Endocannabinoid (eCB) system is composed of endogenous CB ligands including anandamide (AEA) and 2-Arachidonyl glycerol (2-AG), enzymes involved in their biosynthesis and degradation such as diacylglycerol lipase-α (DAGL- α), and CB receptors. Primarily, there are three types of CB receptors - CB receptor 1(CB1), CB receptor 2 (CB2) and non CB1 non CB2 types of CB receptor (e.g. GPR, TRPV1) and they belong to G-protein (Gi/o) coupled receptors (GPCR) family.CB1 receptors are abundant in the brain where they modulate neuronal activities. On the other hand, CB2 receptors are predominantly expressed in the immune cells and regulate the growth and proliferation of different immune cells and modulate the activities of cytokines network and anti-oxidant machinery in stress conditions. Inflammation plays a central role in hearing loss (HL) caused by different ototoxic insults including anti-neoplastic agents such as cisplatin, aminoglycosides and acoustic trauma. These insults can trigger chronic production of reactive oxygen species (ROS) in regions of cochlea such as organ of Corti, stria vascularis (SVA), spiral ligament (SL) and spiral ganglion neurons (SG). This leads to increased synthesis of pro-inflammatory cytokines, disruption of mitochondrial membrane integrity, activation of DNA damage/repair pathways and activation of pro-apoptotic enzymes. Jeong et al. (2007) have shown that CB2 receptor specific agonist (JWH-015) protects the HEI-OC1 hair cell cultures against cisplatin-induced cytotoxicity in-vitro. The goal of the current study was to examine the distribution and function of CB receptors (mainly CB2) in the cochlea and determine whether activation of these receptors could protect the cochlea by altering the expression of ROS generating proteins, along with pro-inflammatory and pro-apoptotic proteins. This study also investigated whether inhibition of eCB synthesis can causes HL. Aim 1 of the current study investigated the expression of CB receptors in the cochlea using different in-vivo models such as male Wistar rat and knock-in mice with GFP-tagged CB2 receptors, in-vitro models such as organotypic culture of neonatal mouse (C57BL/6) cochlea and University of Bristol organ of Corti (UB/OC1) cells. We show that both CB1 and CB2 receptors are expressed in the outer and the inner hair cells (OHCs and IHCs), SV, SG and supporting cells (SCs) included outer and inner pillar cells. The distribution of DAGL- α was also examined in the male Wistar rats and we found the similar distribution pattern of this enzymes as CB2. DAGL- α catalyzes the hydrolysis of DAG to synthesize 2-AG, which acts as a chief endogenous CB2 ligand. Our initial studies suggested a role of CB2 and not CB1 in protection, leading us to focus on CB2 receptors for subsequent studies. Aim 2 examined the otoprotective role of trans-tympanic application of CB2 specific agonist (JWH-015) against cisplatin-induced hearing loss in male Wistar rats. Activation of CB2 receptors restored cisplatin-induced elevations in ABR thresholds which was significantly reversed by CB2 antagonist AM-630. Pre-treatment with JWH-015 protected against cisplatin-induced loss of hair cell and synaptic ribbons. In-vitro studies in UB/OC-1 cells demonstrated that pre-treatment of JWH-015 modulates the activities of signal transducer and activator of transcription 1 and 3 (STAT1 and STAT3), increases the expression of anti-apoptotic protein Bcl-xL, indicating its role in regulating the apoptosis Activation of CB2 also abrogated cisplatin-induced decrease in Na+/K+ATPase- α in the SV and SL fibrocytes and ameliorated the expression of different pro-inflammatory genes including TRPV1, COX2, NOX3, KIM1, iNOS and TNF- α. We also found that blocking of CB2 by AM630 itself resulted in hearing loss and loss in CB2 receptors, indicating eCB system is tonically active and could be important for physiological function of the cochlea. Indeed, we observed that inhibition of DAGL- α by RHC80267 results in HL. Aim 3 of this current study investigated whether pre-treatment of CB2 agonist will interfere with anti-cancer efficacy of cisplatin against various cancer cell lines head and neck cancer cells (UMSCC10B), and colon cancer cells (HCT116). Our data indicate that JWH-015 did not interfere with cisplatin-induced apoptosis in these cells. Overall, this study provides novel insights into the essential role eCBs plays in protection the cochlea under non-stressed conditions and following exposure to ototoxic agents. It also demonstrates that application of exogenous CB2 agonist (JWH-015) could serve as an effective protective agent against cisplatin ototoxicity These data suggest that localized delivery of CB2 agonists should be studied in human for protection against hearing loss.

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