Postnatal development, maturation and aging in the mouse cochlea and their effects on hair cell regeneration

Abstract

The organ of Corti in the mammalian inner ear is comprised of mechanosensory hair cells (HCs) and nonsensory supporting cells (SCs), both of which are believed to be terminally post-mitotic beyond late embryonic ages. Consequently, regeneration of HCs and SCs does not occur naturally in the adult mammalian cochlea, though recent evidence suggests that these cells may not be completely or irreversibly quiescent at earlier postnatal ages. Furthermore, regenerative processes can be induced by genetic and pharmacological manipulations, but, more and more reports suggest that regenerative potential declines as the organ of Corti continues to age. In numerous mammalian systems, such effects of aging on regenerative potential are well established. However, in the cochlea, the problem of regeneration has not been traditionally viewed as one of aging. This is an important consideration as current models are unable to elicit widespread regeneration or full recovery of function at adult ages yet regenerative therapies will need to be developed specifically for adult populations. Still, the advent of gene targeting and other genetic manipulations has established mice as critically important models for the study of cochlear development and HC regeneration and suggests that auditory HC regeneration in adult mammals may indeed be possible. Thus, this review will focus on the pursuit of regeneration in the postnatal and adult mouse cochlea and highlight processes that occur during postnatal development, maturation, and aging that could contribute to an age-related decline in regenerative potential. Second, we will draw upon the wealth of knowledge pertaining to age related senescence in tissues outside of the ear to synthesize new insights and potentially guide future research aimed at promoting HC regeneration in the adult cochlea.

Figure1. Expression of Proliferating Cell Nuclear Antigen (PCNA) persists in the neonatal mouse cochlea

PCNA is a cyclin protein that is a co-factor for DNA polymerase and thus critical for cell proliferation. Whole mount preparations from C57Bl/6 mouse cochleae at postnatal days 1 and 2 (P1 & P2 respectively) exhibit PCNA positive nuclei (green) in hair cells (purple) and supporting cells (red), labeled by hair cell specific myosin VI (Myo6) and the supporting cell marker SRY-box 2 (Sox2), respectively. By P5, no PCNA positive hair cells or supporting cells were detected. The persistence of PCNA suggests that postnatal cochlear hair cells and supporting cells may retain some of the factors necessary for cell cycle entry.

Figure 2. Several factors that may promote or inhibit auditory hair cell regeneration during postnatal development and aging of the rodent cochlea

Factors are grouped by predicted roles of either preventing regenerative processes (red and yellow, above the age line) or promoting regenerative processes (green, below the age line). Factors with expression that increases with age are shown in red, while those with decreasing expression are shown in green. Factors whose expression persists with postnatal age are shown in yellow. The age line runs from birth, i.e postnatal day zero (P0), to adulthood, with each hatch mark representing 7 days. In most cases, the lengths of the colored bars cover the distance between timepoints at which expression levels were measured. However, for ease of visual representation, both p53 and PTEN have been added in their current positions despite the fact that their levels of expression were not measured at 1 month of age but rather between ~3 months of age and 18 months of age for PTEN, and ~3 months of age and 30 months of age for p53. Abbreviations: p53 = tumor protein 53, PTEN = phosphatase and tensin homolog, p16INK4a = cyclin dependent kinase inhibitor 2a, ECM = components of the extracellular matrix such as laminins and integrins, Cx26 = connexin 26, miR15a = microRNA 15a, DFNA5 = deafness autosomal dominant gene 5, let 7 = the let 7 family of microRNAs, EGFR = epidermal growth factor receptor, Hmga2 = high mobility group AT-hook 2, Prox1 = prospero related homeobox 1, Cx43 = connexin 43, Notch = activated Notch (and its canonical mediator Hes5), FGFs = acidic fibroblast growth factor and fibroblast growth factor receptor 1, PCNA = proliferating cell nuclear antigen.

Figure 3. Putative effectors of senescence and their potential interactions in promoting or preventing cell proliferation

At the bottom of the figure, entry of cells into the synthesis phase (S-phase) of the cell cycle is inhibited by the interaction of the retinoblastoma protein (pRb) and members of the E2F family of transcription factors (E2F). When pRb is hyperphosphorylated (P) by cyclin/cyclin dependent kinase (CDK) complexes, it dissociates from E2F transcription factors, resulting in progression into the cell cycle. Several cyclin dependent kinase inhibitors (e.g. p16INK4a, p21Cip1, p27Kip1) are known to be upregulated with age in many cell types, and act to repress cell proliferation by preventing cyclin-CDK dependent phosphorylation of pRb. The histone-lysine N-methyltransferase Ezh2 may prevent proliferation by promoting increased expression of the cyclin dependent kinase inhibitor (CDKN) p16INK4a. Similarly, the tumor (suppressor) protein 53 also prevents cell cycle entry by upgregulating the CDKNs p16INK4a and p21Cip1. However, p53 itself may be inhibited by high expression or activity of the telomerase reverse transcriptase (TERT) enzyme, which thus promotes cell cycle entry. TERT itself can be upregulated by the high mobility group AT-hook 2 protein (Hmga2) which has also been shown to inhibit p16INK4a and the interaction between pRb proteins and E2F family members, thus promoting cell proliferation. Let7 microRNA family members can target and degrade Hmga2 thus preventing proliferation, while histone deacetylases (HDAC) have been shown to increase Hmga2 expression, thus promoting cell cycle entry. Seemingly separate from these factors, the forkhead box M1 protein isoform B, FoxM1B, promotes cell proliferation by multiple mechanisms. First, FoxM1B upregulates cyclins and CDKs while simultaneously preventing p21Cip1 and p27kip1 from inhibiting the cyclin/CDK complexes. Second, FoxM1B upregulates Aurora B kinase (ABK) and polo-like kinase 1 (PLK1) which are both necessary for progression into the mitosis phase (M-phase) of the cell cycle. Arrows denote upregulation or activation of one factor by another, while flat bars denote the downregulation or inhibition of one factor by another.