The role of calcium, Akt and ERK signaling in cadmium-induced hair cell death.

Exposure to heavy metals has been shown to cause damage to a variety of different tissues and cell types including hair cells, the sensory cells of our inner ears responsible for hearing and balance. Elevated levels of one such metal, cadmium, have been associated with hearing loss and shown to cause hair cell death in multiple experimental models. While the mechanisms of cadmium-induced cell death have been extensively studied in other cell types they remain relatively unknown in hair cells. We have found that calcium signaling, which is known to play a role in cadmium-induced cell death in other cell types through calmodulin and CaMKII activation as well as IP3 receptor and mitochondrial calcium uniporter mediated calcium flow, does not appear to play a significant role in cadmium-induced hair cell death. While calmodulin inhibition can partially protect hair cells this may be due to impacts on mechanotransduction activity. Removal of extracellular calcium, and inhibiting CaMKII, the IP3 receptor and the mitochondrial calcium uniporter all failed to protect against cadmium-induced hair cell death. We also found cadmium treatment increased pAkt levels in hair cells and pERK levels in supporting cells. This activation may be protective as inhibiting these pathways enhances cadmium-induced hair cell death rather than protecting cells. Thus cadmium-induced hair cell death appears distinct from cadmium-induced cell death in other cell types where calcium, Akt and ERK signaling all promote cell death.

Mechanotransduction Activity Facilitates Hair Cell Toxicity Caused by the Heavy Metal Cadmium.

Hair cells are sensitive to many insults including environmental toxins such as heavy metals. We show here that cadmium can consistently kill hair cells of the zebrafish lateral line. Disrupting hair cell mechanotransduction genetically or pharmacologically significantly reduces the amount of hair cell death seen in response to cadmium, suggesting a role for mechanotransduction in this cell death process, possibly as a means for cadmium uptake into the cells. Likewise, when looking at multiple cilia-associated gene mutants that have previously been shown to be resistant to aminoglycoside-induced hair cell death, resistance to cadmium-induced hair cell death is only seen in those with mechanotransduction defects. In contrast to what was seen with mechanotransduction, significant protection was not consistently seen from other ions previously shown to compete for cadmium uptake into cells or tissue including zinc and copper. These results show that functional mechanotransduction activity is playing a significant role in cadmium-induced hair cell death.

The zebrafish merovingian mutant reveals a role for pH regulation in hair cell toxicity and function.

Control of the extracellular environment of inner ear hair cells by ionic transporters is crucial for hair cell function. In addition to inner ear hair cells, aquatic vertebrates have hair cells on the surface of their body in the lateral line system. The ionic environment of these cells also appears to be regulated, although the mechanisms of this regulation are less understood than those of the mammalian inner ear. We identified the merovingian mutant through genetic screening in zebrafish for genes involved in drug-induced hair cell death. Mutants show complete resistance to neomycin-induced hair cell death and partial resistance to cisplatin-induced hair cell death. This resistance is probably due to impaired drug uptake as a result of reduced mechanotransduction ability, suggesting that the mutants have defects in hair cell function independent of drug treatment. Through genetic mapping we found that merovingian mutants contain a mutation in the transcription factor gcm2. This gene is important for the production of ionocytes, which are cells crucial for whole body pH regulation in fish. We found that merovingian mutants showed an acidified extracellular environment in the vicinity of both inner ear and lateral line hair cells. We believe that this acidified extracellular environment is responsible for the defects seen in hair cells of merovingian mutants, and that these mutants would serve as a valuable model for further study of the role of pH in hair cell function.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line.

Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.

EphB receptors co-distribute with a nicotinic receptor subtype and regulate nicotinic downstream signaling in neurons.

Activation of nicotinic acetylcholine receptors (nAChRs) on neurons engages calcium-dependent signaling pathways regulating numerous events. Receptors containing alpha7 subunits (alpha7-nAChRs) are prominent in this because of their abundance and high relative calcium permeability. We show here that EphB2 receptors are co-localized with postsynaptic alpha7-nAChRs on chick ciliary ganglion neurons and that treatment of the cells with an ephrinB1 construct to activate the EphB receptors exerts physical restraints on both classes of receptors, diminishing their dispersal after spine retraction or lipid raft disruption. Moreover, the ephrinB1/EphB receptor complex specifically enhances the ability of alpha7-nAChRs to activate the transcription factor CREB, acting through a pathway including a receptor tyrosine kinase, a Src family member, PI3 kinase, and protein kinase A most distally. The enhancement does not appear to result from a change in the alpha7-nAChR current amplitude, suggesting a downstream target. The results demonstrate a role for ephrin/EphB action in nicotinic signaling.