Published On: Mon, Jan 14th, 2019

The role of retrograde intraflagellar transport genes in aminoglycoside-induced hair cell death [RESEARCH ARTICLE]

INTRODUCTION

During development, hair cells – the sensory cells of the auditory and vestibular systems – contain a single primary cilium on their apical surface known as the kinocilium. Primary cilia are microtubule-based structures that are believed to be important for cellular signaling in other cell types (Satir and Christensen, 2007). The kinocilium is lost in auditory hair cells of many species but always maintained in vestibular hair cells (Ernstson and Smith, 1986; Lim and Anniko, 1985; Tanaka and Smith, 1978). While mutations in cilia-associated genes have been found in multiple human patients with hearing loss (Delmaghani et al., 2016; Grati et al., 2015; Hearn et al., 2005; Ross et al., 2005), how the kinocilium affects mature hair cells has remained largely a mystery. In addition to kinocilia, hair cells also have actin-based protrusions on their apical surface known as stereocilia. Mechanotransduction, the process by which hair cells respond to stimuli, is carried out through these stereocilia in mature hair cells (Hudspeth and Jacobs, 1979). The kinocilium and cilia genes have been shown to be important for determining stereocilia polarity in mammalian auditory hair cells (Jones et al., 2008; Ross et al., 2005), however, do not appear to have this function in vestibular hair cells (Sipe and Lu, 2011) or hair cells of the zebrafish lateral line (Kindt et al., 2012; Stawicki et al., 2016). Hearing loss found in animal models with mutations in cilia-associated genes can be seen in the absence of stereocilia polarity defects (Imtiaz et al., 2018) or can develop after stereocilia defects are observed (Jagger et al., 2011) suggesting these genes have roles in hair cells independent of functions determining stereocilia polarity.

There are a number of genes required for the proper development, maintenance and function of cilia. We have previously found a role for a subset of these cilia genes in aminoglycoside-induced hair cell death (Stawicki et al., 2016). Aminoglycoside antibiotics are known to kill hair cells across a range of species and can cause hearing loss and vestibular dysfunction in human patients (Lerner et al., 1986; Moore et al., 1984). Genes important for ciliary intraflagellar transport (IFT) show a particularly large reduction in neomycin-induced hair cell death when mutated (Stawicki et al., 2016). The majority of these IFT gene mutations, including mutations of the retrograde IFT motor protein gene dync2h1, also lead to reductions in the amount of neomycin and FM1-43 entering hair cells. There was one exception to this: wdr35, a component of the IFT-A complex. Mutations of this gene showed a similar reduction in neomycin-induced hair cell death as other IFT mutants, but not as large of a change in neomycin or FM1-43 uptake (Stawicki et al., 2016).

Intraflagellar transport is the process by which proteins are trafficked along cilia and is crucial for cilia maintenance. Anterograde IFT, transport from the cell body to the ciliary tip, depends on the kinesin-2 motor and the IFT-B complex of adaptor proteins. Whereas, retrograde IFT, transport from the tip back to the base, depends on the dynein-2 motor and the IFT-A complex of adaptor proteins (Pedersen et al., 2006; Scholey, 2003). While both dynein-2 and the IFT-A complex are required for retrograde IFT it has previously been shown that mutations in genes of these two different complexes can lead to different phenotypes. For example, while Dync2h1 mutant mice show a loss of sonic hedgehog (Shh) signaling (Huangfu and Anderson, 2005; May et al., 2005), mouse mutants in IFT-A complex genes can show excess Shh signaling (Ashe et al., 2012; Qin et al., 2011; Tran et al., 2008). Individual IFT-A gene mutants and Dync2h1 also show different defects in cilia morphology (Cortellino et al., 2009; Liem et al., 2012; Mill et al., 2011; Ocbina et al., 2011; Tran et al., 2008) and for some cilia localized genes, transport is only affected by a subset of IFT-A gene mutations (Hirano et al., 2017; Mukhopadhyay et al., 2010). Reductions in IFT-A gene products can actually partially rescue Dync2h1 mutant phenotypes (Liem et al., 2012; Ocbina et al., 2011).

Given these observations we wanted to further investigate whether phenotypic differences in hair cells of dync2h1 and wdr35 mutants were generalized to other dynein motor complex and IFT-A complex genes by looking at mutants in the dynein motor complex gene dync2li1 and the IFT-A adaptor complex genes ift122, ift140 and wdr19. Here we report that all these genetic mutants show comparable resistance to neomycin-induced hair cell death, and defects in neomycin and FM1-43 loading into hair cells, comparable to what was previously shown for dync2h1 mutants. We also show that wdr35 and dync2h1 mutants show similar resistance to a second aminoglycoside, gentamicin. We find that that wdr35 mutants fail to show any genetic interaction effects when combined with other IFT mutants, suggesting wdr35 may function via a similar mechanism as other IFT genes. Lastly, we show that unlike those in anterograde IFT genes, retrograde IFT gene mutations do not lead to alterations in the localization of Usher complex genes. Overall, these results suggest that disruption of either the dynein motor or IFT-A adaptor complex will limit aminoglycoside uptake into hair cells and subsequent hair cell toxicity.

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