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Materials and methods
Results
Discussion Prior studies established that Notch signaling regulates the differentiation of hair cells and supporting cells during inner ear development (reviewed in Kiernan (2013)). Notch signaling is a multistep process that requires the interaction between a Notch ligand and receptor, followed by sequential proteolytic processing of the receptor by the ADAM10 metalloprotease and γ-secretase, and culminating in intercellular signaling to the nucleus and changes in gene expression (reviewed in Kopan (2012)). The present study examined the roles of ADAM10 and γ-secretase in regulating hair cell regeneration in the mature avian utricle. In agreement with prior studies conducted on other hair cell epithelia (described below), we found that inhibition of these proteases led to an increase in the numbers of replacement hair cells. Unexpectedly, we also found that inhibition of γ-secretase or ADAM10 following hair cell damage led to a significant increase in the proliferation of hair cell progenitors (supporting cells). More surprisingly, inhibition of either protease in the undamaged chicken utricle (which normally contains a small number of proliferating supporting cells) led to large-scale flecainide acetate sale entry among supporting cells. These results demonstrate that, in addition to controlling cell fate, γ-secretase and ADAM10 also play an essential role in the regulation of supporting cell division. In order to help identify the molecular pathways affected by blocking these proteases, we characterized gene expression in the sensory epithelium of the utricle in response to γ-secretase inhibition. Our data indicate that inhibition of γ-secretase caused statistically significant changes in the expression levels of 253 genes. We also found that the sensory epithelium contains mRNAs for 45 proteins that are known γ-secretase substrates, including genes in the Notch signaling pathway (e.g., Notch1 and Dll1). While these expression data are consistent with the hypothesis that Notch signaling regulates supporting cell division, we cannot rule out parallel contributions from other biochemical pathways that also require γ-secretase and/or ADAM10 (see below).
Introduction Abnormalities of the endolysosomal and autophagy systems are both found in Alzheimer’s disease (AD) (Nixon, 2017, Lie and Nixon, 2018). Prominent enlargement of endosomal compartments precedes the appearance of neurofibrillary tangles and neuritic plaques in mouse models of AD (Cataldo et al., 2000). Similarly, selective accumulation of lysosomal dense bodies and autophagic vacuoles in dystrophic neurites has been observed in post-mortem brain samples of people with AD (Nixon et al., 2005), suggesting that autolysosomal proteolysis is impaired in AD (Nixon, 2017). Genome-wide association studies have identified SNPs in several genes with roles in the endolysosomal and autophagy systems as impacting disease risk, including PICALM, CLU, SORL1, ABCA7, and BIN1 (Karch and Goate, 2015). These underscore the significance of the endolysosomal and autophagy systems as a genetic risk hotspot in AD (Nixon, 2017). However, it is not clear whether defects in lysosomal and autophagic systems are a cause or consequence of degenerative processes in AD. Collectively, genetic data place APP processing by γ-secretase as central to disease initiation in monogenic forms of AD, including familial AD due to PSEN1 and APP mutations (Selkoe, 2002). The majority of known familial AD mutations are autosomal dominant and affect the amyloid precursor protein (APP) gene or the catalytic components of the γ-secretase APP-processing complex, presenilin (PSEN) 1 and 2 (Bertram and Tanzi, 2008). As well as missense mutations, increased APP gene dosage due either to trisomy of chromosome 21 (Ts21) or duplication of the APP locus (APP (dup)) also leads to early onset AD (Rovelet-Lecrux et al., 2006). Conversely, a protective mutation near the BACE-cleavage site in APP has been identified that reduces the risk of developing dementia (Jonsson et al., 2012).