Browsing by Subject "hearing loss"

Our hearing perception is based on the ability to discriminate mechanical sound waves and to amplify and transduce them into electrical stimuli.This function is based on the complex cellular organization of the cochlea, the hearing organ. The sensory epithelium in the organ of Corti spirals along the cochlear duct in a tonotopic arrangement: every sound frequency elicits the strongest response at allocation along this duct. Sound stimulus is detected by three rows of outer hair cells (OHCs) which amplify- and tone-discriminate the sound stimulus, and by one row of inner hair cells (IHCs), which transduce the mechanical stimulus into electric impulses. Basal regions of the cochlea detect high- frequency sounds and apical regions detect low- frequency sounds. The complexity and sensitivity of the cochlea is linked with its vulnerability to various traumas. Most kinds of damage to the mammalian hair cells is irreversible, because these cells are not capable of regeneration. Hearing impairment has many etiologies. Common to them is that damage is permanent and no pharmacotherapy is available. Hearing impairment is often a disabling condition and it has vast societal consequences. The number of hearing impaired people is constantly increasing and the WHO has estimated that 10% of the world`s population will suffer from disabling hearing loss in 2050. Mesencephalic astrocyte- derived neurotrophic factor (MANF) is an unconventional, ER-resident protein that promotes ER- homeostasis. It has been associated with cytoprotective functions in many neurodegenerative disease- models and shown to promote recovery after ischemic trauma. MANF expression has been previously found in many cell-types in the cochlea, including OHCs and IHCs. Its deficiency in a mouse model led to upregulation of ER-stress markers and a robust, tonotopic base –to apex gradient loss of outer hair cells and severe hearing loss. This study examines the role of MANF in noise-induced trauma in the hair cells of the cochlea. In a conditionally inactivated (Manf -/- cKO) mouse model in the C57BL/6J – background, where Manf has been inactivated from most of the cochlear cells, I studied, if Manf -deficiency sensitizes the cells to noise-induced cell death in two age-groups. I also examined the basic and noise- induced MANF expression, using two mouse- strains, C57BL/6J and CBA/Ca. I also examined OHC stereociliary bundle morphology to find out if noise induces morphological changes in Manf cKO-mice that differ from noise-exposed C57BL/6j wild type mice. This study found that OHCs have a low MANF- expression, whereas in IHCs the expression is strong. MANF is expressed in a base- to apex gradient in the OHCs of the two mouse-strains examined, in a uniform pattern, that correlates with vulnerability, implicating that low levels of MANF predispose basal OHCs to vulnerability. MANF expression in the IHCs was non-gradiental. Noise did not induce upregulation, as was expected, but instead noise induced downregulation of MANF in the basal region of the OHCs by an unknown mechanism in both mouse-strains.This suggests that noise-induced trauma induces ER dyshomeostasis, possibly independent of ER stress response pathways ,unfold protein response (UPR). This study also demonstrates that MANF deficiency sensitizes the OHCs to noise- induced trauma, resulting in more elevated OHC loss and hearing thresholds. This sensitization is mainly caused by a progressive degenerative changes seen in the OHC stereociliary bundles of Manf cKO-mice, and is associated with more severe noise-induced hearing loss. The results of my study suggest that MANF has an important, yet unknown, protective role in noise-induced trauma in OHCs. These results support the possible role of MANF as a therapeutic agent in a noise-induced trauma.

Recent studies have associated ER stress with various types of hearing loss, such as drug- and noise-induced, age-related, and hereditary hearing loss. However, the research has mostly focused on auditory sensory cell (hair cell) death, and it is not well understood if other molecular mechanisms can drive ER stress-dependent hearing loss. We used Manfflox/flox;Pax2-Cre conditional knockout (cKO) mice under the C57BL/6J (B6) mouse strain to study the effects of genetically-induced chronic ER stress on hearing function. In these mice, the gene coding for mesencephalic astrocyte-derived neurotrophic factor (Manf) has been silenced specifically in the cochlea. Manf is thought to act as an ER homeostasis regulator, and it has shown cytoprotective properties in different disease models both in vitro and in vivo. However, Manf’s mode of action is still poorly understood and even less is known about its function in the inner ear. Previously, cKO mice were found to upregulate ER stress markers in the cochlear hair cells. These mice develop progressive high-frequency hearing loss characterized by high-frequency outer hair cell (OHC) death. However, they have elevated hearing thresholds already at postnatal day 22 (P22) before any OHC death takes place and have elevated hearing thresholds in hearing frequencies where OHCs are retained. Therefore, there has to be another pathological mechanism besides OHC death accounting for the elevations in their hearing thresholds. Hence, we wanted to study the effect of ER stress on the outer hair cell hair bundle structure. The hair bundle is located at the apical pole of the hair cells, and it consists of filamentous actin (F-actin)-filled stereocilia. In mechanotransduction (MET), sound stimuli-induced motions of cochlear fluids cause stereocilia to deflect towards the tallest stereocilia row, allowing for depolarization of hair cells and transformation of mechanical force into electrical signal. Therefore, hair bundle is an essential structure for the hearing function. We used scanning electron microscopy (SEM) and fluorescent microscopy to study OHC hair bundles of cKO mice. We saw disorganization of the bundle structure already at P22. It progressed with age and advanced to strong stereocilia fusion by P56. At this age, all of the high-frequency OHCs of cKO mice displayed stereocilia fusion. We used cochlear whole mounts and immunostainings to study the protein composition of OHC stereocilia of Manf-deficient mice. The base of the stereocilia, termed as the tapering region, contains proteins that link the plasma membrane of stereocilia to their F-actin core, ensuring the cohesion of individual stereocilia. Mutations in these proteins have been associated with stereocilia fusion and hair bundle disorganization. At P56, we saw that stereocilia tapering region proteins radixin (RDX) and myosin 6 (Myo6) were mislocalized from the tapering region towards the apical tips of stereocilia in the high-frequency OHCs of cKO mice. Additionally, we saw that PTPRQ – a tapering region protein that is under normal conditions expressed only in the IHCs of mature cochlea – was upregulated in OHCs of cKO mice, yielding an expression pattern similar to RDX and Myo6. In addition, we used the F-actin probe phalloidin to quantitatively compare F-actin densities in the cuticular plates of cKO and WT mice. Cuticular plate is a structure responsible for attaching stereocilia to hair cell body. It consists of a dense F-actin network and prior studies have associated defects in the cuticular plate composition with hearing loss and stereocilia bundle abnormalities. We found a significant decrease in phalloidin staining intensity in the cuticular plates of high-frequency OHCs of cKO mice, indicating that their cuticular plate F-actin rigidity had been reduced. Together our data shows that Manf deficiency promotes diverse impairments in the OHC hair bundles, consequently inducing hearing loss. To conclude, our study presents novel insights into the complexity of ER stress-induced cochlear pathology. We show that ER stress impairs MET by inducing structural changes in the OHC hair bundle. It appears to be the major reason for hearing loss in the cKO mice, rather than hair cell death. In the future, the impact of Manf deficiency to the inner ear should be further studied. For example, younger and aged cKO mice could be studied to better characterize the progression of Manf deficiency-induced cochlear pathology and hearing loss. Similarly, Manf’s effect on hearing should be studied in other ER stress models to determine its role in the hearing function.