Age-related hearing loss is experienced by one-third of individuals aged 65 years and older and can be socially debilitating. Historically, there has been poor correlation between age-related threshold changes, loss of speech understanding, and loss of cochlear hair cells. We examined changes in ribbon synapse number at four different ages in Fisher Brown Norway rats, an extensively studied rat model of aging. In contrast to previous work in mice/Wistar rats, we found minimal ribbon synapse loss before 20 months, with significant differences in 24- and 28-month-old rats at 4 kHz. Significant outer HC loss was observed at 24 and 28 months in low- to mid-frequency regions. Age-related reductions in auditory brainstem response wave I amplitude and increases in threshold were strongly correlated with ribbon synapse loss. Wave V/I ratios increased across age for click, 2, 4, and 24 kHz. Together, we find that ribbon synapses in the Fisher Brown Norway rat cochlea show resistance to aging until ∼60% of their life span, suggesting species/strain differences may underpin decreased peripheral input into the aging central processor.

Supporting cells (SCs) are known to spontaneously regenerate hair cells (HCs) in the neonatal mouse cochlea, yet little is known about the relative contribution of distinct SC subtypes which differ in morphology and function. We have previously shown that HC regeneration is linked to Notch signaling, and some SC subtypes, but not others, lose expression of the Notch effector Hes5. Other work has demonstrated that Lgr5-positive SCs have an increased capacity to regenerate HCs; however, several SC subtypes express Lgr5. To further investigate the source for spontaneous HC regeneration, we used three CreER lines to fate-map distinct groups of SCs during regeneration. Fate-mapping either alone or combined with a mitotic tracer showed that pillar and Deiters' cells contributed more regenerated HCs overall. However, when normalized to the total fate-mapped population, pillar, Deiters', inner phalangeal and border cells had equal capacity to regenerate HCs, and all SC subtypes could divide after HC damage. Investigating the mechanisms that allow individual SC subtypes to regenerate HCs and the postnatal changes that occur in each group during maturation could lead to therapies for hearing loss.

During cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important role in the specification of the prosensory region, which gives rise to sound-sensing hair cells and neighboring supporting cells (SCs). While JAG1's expression is maintained in SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we demonstrate that JAG1 is essential for the formation and maintenance of Hensen's cells, a highly specialized SC subtype located at the edge of the auditory epithelium. Using Sox2 CreERT2/1 ::Jag1 loxP/loxP mice of both genders, we show that Jag1 deletion at the onset of differentiation, at embryonic day 14.5, disrupted Hensen's cell formation. Similar loss of Hensen's cells was observed when Jag1 was deleted after Hensen's cell formation at postnatal day (P) 0/P1 and fate-mapping analysis revealed that in the absence of Jag1, some Hensen's cells die, but others convert into neighboring Claudius cells. In support of a role for JAG1 in cell survival, genes involved in mitochondrial function and protein synthesis were downregulated in the sensory epithelium of P0 cochlea lacking Jag1. Finally, using Fgfr3-iCreER T2 ::Jag1 loxP/loxP mice to delete Jag1 at P0, we observed a similar loss of Hensen's cells and found that adult Jag1 mutant mice have hearing deficits at the low-frequency range.

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 CAG-eGFP reporter consistently labeled fewer cells, both reporter lines are valuable 44 depending on the goal of the study.

22During cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important 23 role in the specification of the prosensory region, which gives rise to sound-sensing hair 24 cells and neighboring supporting cells (SCs). While JAG1's expression is maintained in 25 SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we 26 demonstrate that JAG1 is essential for the formation and maintenance of Hensen cells 27 (HeCs), a highly specialized SC-subtype located at the edge of the auditory epithelium. 28Deletion of Jag1 at the onset of differentiation, at stage E14.5, disrupted HeC formation. 29Similar loss of HeCs was observed when Jag1 was deleted at P0/P1 and fate-mapping 30 analysis revealed that in the absence of Jag1 some HeCs die, but others convert into 31 neighboring Claudius cells. In support of a role for JAG1 in cell survival, genes involved 32 in mitochondrial function and protein synthesis were downregulated in P0 cochlea lacking 33 Jag1. 34 35 106 deletion disrupted the formation or patterning of SCs and/or HCs. To label HCs, we used 107 immuno-staining against myosin VIIa, which labels both inner HCs (IHCs) and outer HCs 108 (OHCs) (Hasson et al., 1995). To visualize SCs, we immuno-stained against SOX2, which 109 at E18.5/P0 is highly expressed in the nucleus of all SC subtypes, including HeCs, and is 110 expressed at a lower level in the nucleus of IHCs and OHCs (Kempfle et al., 2016). We 111 found that Jag1 deletion had no effect on the number (density) of IHCs or OHCs (Fig. 1E, 112 F, I). Our analysis of the SC phenotype, however revealed a significant reduction in the 113 number of HeCs in Jag1 CKO mice compared to all three controls ( Fig. 1E-I). In cochlear 114 tissue of control animals, SOX2 + HeCs were located between the 3 rd row of DCs and 115 Claudius cells (CCs), with their nuclei residing in both the HC and SC layers, and with 2-116 3 HeCs sitting on top of each other (Fig. 1A, E, G). By contrast, Jag1 CKO cochlear tissue 117 contained either no or only a few scattered HeCs within the HC and SC layers (Fig. 1F, 118 H). 119In addition, DCs in Jag1 CKO mice had enlarged nuclei compared to control mice, 120 and their arrangement appeared to be disorganized, suggesting defects in DC 121 differentiation ( Fig. 1G, H). Decreased numbers of DCs in the 2 nd and 3 rd row (DC2 and 122 DC3) was observed in Jag1 CKO samples compared to the Jag1 fx/fx treated and/or 123 Jag1 fx/fx untreated control groups, but not when compared to Sox2 CreERT2/+ ::Jag1 fx/fx 124 untreated controls (Fig. 1I). A similar result was observed for IPCs and OPCs (Fig. 1I), 125 suggesting that Jag1 deficiency combined with Sox2 haploinsufficiency negatively affects 126 the differentiation of DCs and PCs. Unfortunately, we were unable to address how Jag1 127 deficiency combined with Sox2 haploinsufficiency may impact DC and PC maturation as 128 conditional deletion of Jag1 at E14.5 resulted in early postnatal lethality. In summary, our 129 analysis demonstrates that JAG1's function is ...