Investigate The Effectiveness Of Salicylate In Preventing Hpβcd-Induced Ototoxicity In The NPC1 Mouse Model

HPCD is a cholesterol-chelating agent currently undergoing clinical trial for the treatment of NPC1 disease. Although studies in NPC1 animal models established the effectiveness of HPCD in alleviating neurological phenotypes, the compound is unfortunately ototoxic in both animal models and in NPC1 patients. Previous studies identified outer hair cells (OHCs), sensory cells in the hearing organ, as being particularly vulnerable to cholesterol extraction by HPCD, resulting in cell death (Crumling et al., PLoS One, 2013). Recently, we found that this HPCD-induced ototoxicity is dependent on prestin, an OHC-specific molecular motor (Takahashi et al., Sci. Rep., 2016). This new discovery identified prestin as one of the determinants of HPCD-induced ototoxicity, and implicates prestin as a novel therapeutic target to prevent hearing loss in NPC1 patients responding to HPCD treatment. OHCs are required for cochlear amplification, a process that is responsible for the sensitivity and sharp frequency selectivity of mammalian hearing. Prestin is a voltage-dependent motor protein that powers OHC amplification. Although this process is influenced by cholesterol, the detailed mechanisms were not fully understood. Recently, we found that prestin and cholesterol can directly interact. As prestin is essential for OHC electromotility, structural integrity, and survival, disruption of this close interaction between cholesterol and prestin may be detrimental and could result in rapid cell death. Although factors other than prestin may be involved, it is likely that prestin’s structural and/or electromotile functions contribute to HPCD-induced OHC death. Salicylate, a commonly used anti-inflammatory drug, is known to reversibly inhibit OHC electromotility. Several lines of evidence warrant evaluation of salicylate as a potential therapeutic agent to minimize ototoxicity in the presence of HPCD: 1) salycilate is thought to directly interact with prestin to confer its inhibitory effect (Golbunov et al., Nat. comm. 2014). Thus, it is conceivable that prestin-cholesterol interaction may be modulated in the presence of salicylate; 2) long-term, low-does administration of salicylate increases prestin expression and OHC electromotility (Yu et al., Cell Mol. Life Sci. 2008); 3) salicylate, in combination with -cyclodextrin, increases the mobility of prestin at the PM of OHCs in vitro (Yamashita et al., PLoS Gen., 2015); 4) salicylate treatment alone did not have any adverse effects on NPC1 knockout (KO) mice. In fact, salicylate treatment exhibited a tendency to lengthen the life span of the animals though not reaching statistical significance (Smith et al., Neurobiol. Dis. 2009). These observations support the possibility of salicylate having an effect when administered in conjunction with HPCD, which will uncover not only a mechanism for HPCD-induced OHC death but also a potential target with a known inhibitor already on market. In this study, we propose to investigate the effects of salicylate in the context of a NPC1 disease model in the presence of HPCD. First, we will examine prestin expression and OHC function in the NPC1-KO mouse model to understand the cochlea physiology in the NPC1 disease state. Cholesterol has significant impact on prestin function, and various degrees of hearing impairment are reported for NPC1 patients without treatment (King et al., Ear Hear. 2014). Previous studies on NPC1-KOs described an early-onset hearing loss that progressed over time (King et al., J. Assoc. Res. Otolaryngol., 2014). We will further eva