ABSTRACT: The underlying molecular mechanisms of age-related hearing loss (ARHL) in humans and many strains of mice have not been fully characterized. This common age-related disorder is assumed to be closely associated with oxidative stress. Here, we demonstrate that mTORC1 signaling is highly and specifically activated in the cochlear neurosensory epithelium (NSE) in aging mice, and rapamycin injection prevents ARHL. To further examine the specific role of mTORC1 signaling in ARHL, we generated murine models with NSE-specific deletions of Raptor or Tsc1, regulators of mTORC1 signaling. Raptor-cKO mice developed hearing loss considerably more slowly than WT littermates. Conversely, Tsc1 loss led to the early-onset death of cochlear hair cells and consequently accelerated hearing loss. Tsc1-cKO cochleae showed features of oxidative stress and impaired antioxidant defenses. Treatment with rapamycin and the antioxidant N-acetylcysteine rescued Tsc1-cKO hair cells from injury in vivo. In addition, we identified the peroxisome as the initial signaling organelle involved in the regulation of mTORC1 signaling in cochlear hair cells. In summary, our findings identify overactive mTORC1 signaling as one of the critical causes of ARHL and suggest that reduction of mTORC1 activity in cochlear hair cells may be a potential strategy to prevent ARHL.
Project description:Neuropilin-1 (Nrp1) encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific Nrp1 conditional knockout (CKO) mouse line because Nrp1 constitutive knockouts are embryonic lethal. In situ hybridization demonstrated weak Nrp1 mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, Nrp1 CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old Nrp1 CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old Nrp1 CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of Nrp1 in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of Nrp1 show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons in vitro. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that Nrp1 may play a role in ARHL.
Project description:Presbycusis, or age-related hearing loss (ARHL), occurs in most mammals with variations in the age of onset, rate of decline, and magnitude of degeneration in the central nervous system and inner ear. The affected cochlear structures include the stria vascularis and its vasculature, spiral ligament, sensory hair cells and auditory neurons. Dysfunction of the stria vascularis results in a reduced endocochlear potential. Without this potential, the cochlear amplification provided by the electro-motility of the outer hair cells is insufficient, and a high-frequency hearing-loss results. Degeneration of the sensory cells, especially the outer hair cells also leads to hearing loss due to lack of amplification. Neuronal degeneration, another hallmark of ARHL, most likely underlies difficulties with speech discrimination, especially in noisy environments. Noise exposure is a major cause of ARHL. It is well-known to cause sensory cell degeneration, especially the outer hair cells at the high frequency end of the cochlea. Even loud, but not uncomfortable, sound levels can lead to synaptopathy and ultimately neuronal degeneration. Even in the absence of a noisy environment, aged cells degenerate. This pathology most likely results from damage to mitochondria and contributes to degenerative changes in the stria vascularis, hair cells, and neurons. The genetic underpinnings of ARHL are still unknown and most likely involve various combinations of genes. At present, the only effective strategy for reducing ARHL is prevention of noise exposure. If future strategies can improve mitochondrial activity and reduce oxidative damage in old age, these should also bring relief.
Project description:Tendons transmit contractile forces between musculoskeletal tissues. Whereas the biomechanical properties of tendons have been studied extensively, the molecular mechanisms regulating postnatal tendon development are not well understood. Here we examine the role of mTORC1 signaling in postnatal tendon development using mouse genetic approaches. Loss of mTORC1 signaling by removal of Raptor in tendons caused severe tendon defects postnatally, including decreased tendon thickness, indicating that mTORC1 is necessary for postnatal tendon development. By contrast, activation of mTORC1 signaling in tendons increased tendon cell numbers and proliferation. In addition, Tsc1 conditional knockout mice presented severely disorganized collagen fibers and neovascularization in the tendon midsubstance. Interestingly, collagen fibril diameter was significantly reduced in both Raptor and Tsc1 conditional knockout mice, albeit with variations in severity. We performed RNA-seq analysis using Achilles tendons to investigate the molecular changes underlying these tendon phenotypes. Raptor conditional knockout mice showed decreased extracellular matrix (ECM) structure-related gene expression, whereas Tsc1 conditional knockout mice exhibited changes in genes regulating TGF-?/BMP/FGF signaling, as well as in genes controlling ECM structure and disassembly. Collectively, our studies suggest that maintaining physiological levels of mTORC1 signaling is essential for postnatal tendon development and maturation.
Project description:Age-related hearing loss (ARHL) is the progressive, bilateral loss of high-frequency hearing in elderly people. Mutations in GJB2, encoding the cochlear gap junction protein connexin26 (Cx26), are the most frequent cause of hereditary deafness; however, a common molecular pathology between ARHL and GJB2-related hearing loss has not been reported. Here, we investigated the quantitative change in expression and molecular pathology of Cx26 in ARHL. We used C57BL/6J mice as a model of ARHL. Hearing levels that were evaluated by auditory brainstem response thresholds increased gradually between 4 and 32 weeks of age and increased sharply at 36 weeks. Gap junctions in the cochleae of 4-week-old mice had linear plaques along cell-cell junction sites. In contrast, the cochleae from 32-week-old mice had significantly shorter gap junctions. Severe hair cell loss was not observed during this period. Based on western blotting, Cx26 and connexin30 (Cx30) levels were significantly decreased at 32 weeks compared with 4 weeks.Moreover, Cx26 was more significantly enriched in the hydrophilic fraction at 4 weeks but was more significantly enriched in the hydrophobic fraction at 32 weeks, indicating an age-related conversion of this biochemical property. Thus, the hydrophobic conversion of Cx26 and disruption of gap junction proteins and plaques may be involved in the pathogenesis of ARHL and may occur before severe hair cell degeneration.
Project description:Age-related hearing loss (ARHL) is one of the most common disorders affecting elderly individuals. There is an urgent need for effective preventive measures for ARHL because none are currently available. Cockayne syndrome (CS) is a premature aging disease that presents with progressive hearing loss at a young age, but is otherwise similar to ARHL. There are two human genetic complementation groups of CS, A and B. While the clinical phenotypes in patients are similar, the proteins have very diverse functions, and insight into their convergence is of great interest. Here, we use mouse models for CS (CSA -/- and CSB m/m ) that recapitulate the hearing loss in human CS patients. We previously showed that NAD+, a key metabolite with various essential functions, is reduced in CS and associated with multiple CS phenotypes. In this study, we report that NAD+ levels are reduced in the cochlea of CSB m/m mice and that short-term treatment (10 days) with the NAD+ precursor nicotinamide riboside (NR), prevents hearing loss, restores outer hair cell loss, and improves cochlear health in CSB m/m mice. Similar, but more modest effects were observed in CSA -/- mice. Remarkably, we observed a reduction in synaptic ribbon counts in the presynaptic zones of inner hair cells in both CSA -/- and CSB m/m mice, pointing to a converging mechanism for cochlear defects in CS. Ribbon synapses facilitate rapid and sustained synaptic transmission over long periods of time. Ribeye, a core protein of synaptic ribbons, possesses an NAD(H) binding pocket which regulates its activity. Intriguingly, NAD+ supplementation rescues reduced synaptic ribbon formation in both CSA -/- and CSB m/m mutant cochleae. These findings provide valuable insight into the mechanism of CS- and ARHL-associated hearing loss, and suggest a possible intervention.
Project description:Age-related hearing loss (ARHL) negatively impacts quality of life in the elderly population. The prevalent cause of ARHL is loss of mechanosensitive cochlear hair cells (HCs). The molecular and cellular mechanisms of HC degeneration remain poorly understood. Using RNA-seq transcriptomic analyses of inner and outer HCs isolated from young and aged mice, we show that HC aging is associated with changes in key molecular processes, including transcription, DNA damage, autophagy, and oxidative stress, as well as genes related to HC specialization. At the cellular level, HC aging is characterized by loss of stereocilia, shrinkage of HC soma, and reduction in outer HC mechanical properties, suggesting that functional decline in mechanotransduction and cochlear amplification precedes HC loss and contributes to ARHL. Our study reveals molecular and cytological profiles of aging HCs and identifies genes such as Sod1, Sirt6, Jund, and Cbx3 as biomarkers and potential therapeutic targets for ameliorating ARHL.
Project description:Age-associated decline in retina function is largely responsible for the irreversible vision deterioration in the elderly population. It is also an important risk factor for the development of degenerative and angiogenic diseases. However, the molecular mechanisms involved in the process of aging in the retina remain largely elusive. This study investigated the role of mTORC1 signaling in aging of the retina. We showed that mTORC1 was activated in old-aged retina, particularly in the ganglion cells. The role of mTORC1 activation was further investigated in Chx10-Cre;Tsc1fx/fx mouse (Tsc1-cKO). Activation of mTORC1 was found in bipolar and some of the ganglion and amacrine cells in the adult Tsc1-cKO retina. Bipolar cell hypertrophy and Müller gliosis were observed in Tsc1-cKO since 6 weeks of age. The abnormal endings of bipolar cell dendritic tips at the outer nuclear layer resembled that of the old-aged mice. Microglial cell activation became evident in 6-week-old Tsc1-cKO. At 5 months, the Tsc1-cKO mice exhibited advanced features of old-aged retina, including the expression of p16Ink4a and p21, expression of SA-β-gal in ganglion cells, decreased photoreceptor cell numbers, decreased electroretinogram responses, increased oxidative stress, microglial cell activation, and increased expression of immune and inflammatory genes. Inhibition of microglial cells by minocycline partially prevented photoreceptor cell loss and restored the electroretinogram responses. Collectively, our study showed that the activation of mTORC1 signaling accelerated aging of the retina by both cell autonomous and nonautonomous mechanisms. Our study also highlighted the role of microglia cells in driving the decline in retina function.
Project description:Age-related hearing (ARHL) loss affects a large part of the human population with a major impact on our aging societies. Yet, underlying mechanisms are not understood, and no validated therapy or prevention exists. NADPH oxidases (NOX), are important sources of reactive oxygen species (ROS) in the cochlea and might therefore be involved in the pathogenesis of ARHL. Here we investigate ARHL in a mouse model. Wild type mice showed early loss of hearing and cochlear integrity, while animals deficient in the NOX subunit p22phox remained unaffected up to six months. Genes of the excitatory pathway were down-regulated in p22phox-deficient auditory neurons. Our results demonstrate that NOX activity leads to upregulation of genes of the excitatory pathway, to excitotoxic cochlear damage, and ultimately to ARHL. In the absence of functional NOXs, aging mice conserve hearing and cochlear morphology. Our study offers new insights into pathomechanisms and future therapeutic targets of ARHL.
Project description:<h4>Background</h4>Age-related hearing loss (ARHL), also known as presbycusis, is the most common sensory impairment seen in elderly people. However, the cochlear aging process does not affect people uniformly, suggesting that both genetic and environmental (e.g., noise, ototoxic drugs) factors and their interaction may influence the onset and severity of ARHL. Considering the potential links between thyroid hormone, mitochondrial activity, and hearing, here, we probed the role of p43, a N-terminally truncated and ligand-binding form of the nuclear receptor TR?1, in hearing function and in the maintenance of hearing during aging in p43<sup>-/-</sup> mice through complementary approaches, including in vivo electrophysiological recording, ultrastructural assessments, biochemistry, and molecular biology.<h4>Results</h4>We found that the p43<sup>-/-</sup> mice exhibit no obvious hearing loss in juvenile stages, but that these mice developed a premature, and more severe, ARHL resulting from the loss of cochlear sensory outer and inner hair cells and degeneration of spiral ganglion neurons. Exacerbated ARHL in p43<sup>-/-</sup> mice was associated with the early occurrence of a drastic fall of SIRT1 expression, together with an imbalance between pro-apoptotic Bax, p53 expression, and anti-apoptotic Bcl2 expression, as well as an increase in mitochondrial dysfunction, oxidative stress, and inflammatory process. Finally, p43<sup>-/-</sup> mice were also more vulnerable to noise-induced hearing loss.<h4>Conclusions</h4>These results demonstrate for the first time a requirement for p43 in the maintenance of hearing during aging and highlight the need to probe the potential link between human THRA gene polymorphisms and/or mutations and accelerated age-related deafness or some adult-onset syndromic deafness.
Project description:AIMS:Previous study suggests that mTOR signaling pathway may play an important role in epileptogenesis. The present work was designed to explore the contribution of raptor protein to the development of epilepsy and comorbidities. METHODS:Mice with conditional knockout of raptor protein were generated by cross-bred Rptorflox/flox mice with nestin-CRE mice. The expression of raptor protein was analyzed by Western blotting in brain tissue samples. Neuronal death and mossy fiber sprouting were detected by FJB staining and Timm staining, respectively. Spontaneous seizures were recorded by EEG-video system. Morris water maze, open field test, and excitability test were used to study the behaviors of Rptor CKO mice. RESULTS:As the consequence of deleting Rptor, downstream proteins of raptor in mTORC1 signaling were partly blocked. Rptor CKO mice exhibited decrease in body and brain weight under 7 weeks old and accordingly, cortical layer thickness. After kainic acid (KA)-induced status epilepticus, overactivation of mTORC1 signaling was markedly reversed in Rptor CKO mice. Although low frequency of spontaneous seizure and seldom neuronal cell death were observed in both Rptor CKO and control littermates, KA seizure-induced mossy fiber spouting were attenuated in Rptor CKO mice. Additionally, cognitive-deficit and anxiety-like behavior after KA-induced seizures were partly reversed in Rptor CKO mice. CONCLUSION:Loss of the Rptor gene in mice neural progenitor cells affects normal development in young age and may contribute to alleviate KA seizure-induced behavioral abnormalities, suggesting that raptor protein plays an important role in seizure comorbidities.