Project 2: Endocannabinoid Signaling in Development of Inner Ear

Project Summary The endocannabinoid signaling is composed of ligands (e.g. anandamide, 2 arachidonoyl glycerol), receptors to which the ligands bind (e.g. CNR1, CNR2), and enzymes that synthesize or degrade the ligands (e.g. NAPE- PLD, DAGL α/β, FAAH, MGLL). The ECS regulates neurogenesis, axonal pathfinding and modulates synaptic transmission in the central nervous system. Our prior studies have shown distribution of cannabinoid receptors (CNR) in the adult cochlea and support roles for the ECS in protection against ototoxic drugs like cisplatin. However, the expression and function of ECS components during inner ear development remains unelucidated. The major goals of this study are - 1. Map the distribution of various endocannabinoid signaling components in the otic vesicle during embryogenesis and in the sensory and non-sensory cells of the early postnatal inner ear; and 2. Investigate the role of CNR1-mediated cannabinoid signaling in development of spiral ganglion neurons (SGNs). Aim1 of this study will test the hypothesis that cannabinoid receptors (CNR1 and CNR2) and endocannabinoid metabolizing (both biosynthesis and degradation) enzymes are robustly and dynamically expressed in the sensory (hair cells and neurons) and non-sensory cells (supporting cells and glial cells) of embryonic and early postnatal inner ear. I will investigate expression of Cnr1, Cnr2, Dagla/b, Napepld, Faah and Mgll at embryonic (E) days E8.5, E10.5, E16.5 and postnatal days (P) P0, P10 and P32. Aim2 of the study will investigate the role of CNR1 mediated cannabinoid signaling in SGN development. I will test the hypothesis that the germline deletion of Cnr1 (Cnr1-/-) will interfere with axonal pathfinding of SGNs and disrupt the normal pattern of hair cell innervation. I further predict that Cnr1 deletion will adversely affect inner hair cell synapse development and resulting hearing function in the adult mice. To further delineate whether CNR1 signaling works in a cell or non-cell autonomous fashion, I will conditionally delete Cnr1 from SGNs or cochlear duct via Bhlhb5-cre or Emx2-cre, respectively, and investigate if either deletion can recapitulate the phenotypes of germline knockout model. Aim3 of the study will test the hypothesis that germline deletion of Cnr1 will downregulate the expression of axon guidance and /or planar cell polarity genes in the cochlea. I will conduct single cell RNASeq of both SGNs and cochlear duct at E16.5 and P10. Unbiased gene analysis will also reveal the target genes regulated by Cnr1 in the inner ear.