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Do adult mammalian cochlear inner hair cells regenerate?

Do adult mammalian cochlear inner hair cells regenerate?


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The consensus seems to be no, but I see conflicting evidence.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5361427/

Supernumerary human hair cells-signs of regeneration or impaired development? A field emission scanning electron microscopy study

The combination of scarring and proximity to the supernumerary cells suggests it is regeneration and not just misplacement.

Do adult mammalian cochlear inner hair cells regenerate?


Short answer
The current consensus is that hair cells in the cochlea of humans do not regenerate spontaneously.

Background
I took the liberty to show the linked paper to a colleague of mine. This guy has been doing histology on the inner ear for his entire professional career.

He pointed out that the consensus is that in mammals, cochlear hair cells do not regenerate. This as opposed to birds and fish where they can regenerate. In mammals, vestibular hair cells can also regenerate (Santaolalla et al., 2013).

Where do these 'supernumerary inner hair cells' (sIHCs) the authors of the paper show in their (quite stunning) EM photos come from? I wish to point you to the last lines of the Discussion of your linked paper (Rask-Anderson et al., 2017):

Taken together, it cannot be settled if the sIHC represent renewed or redundant accessory IHCs. Further molecular studies are needed to verify if the regenerative capacity of the human auditory periphery might have been underestimated.

In other words, the extra hair cells may have appeared during development in utero, or perhaps due to regenerative processes later on. Note that the inner ear in mammals is fully developed and doesn't grow anymore in size. The EM photos in the article don't allow for the identification of functionality either. Also note that sIHCs have been identified in the 1800s in other animals and that the beauty of this paper lies more in the fact that it shows that sIHCs exist in humans, rather than showing that IHCs regenerate in man (that would likely not appear in the Upsula journal of medical sciences, but in Nature or the likes). On a side note - the authors of the paper are giants in the field.

Literature
- Rask-Andersen, Ups J Med Sci (2017); 122(1): 1-19
- Santaolalla et al., Neural Regen Res (2013); 8(24): 2284-9


Regeneration of Hair Cells

To date, research shows that mammalian cochlear hair cells do not regenerate, either spontaneously or after damage. However, lower vertebrates (fishes, amphibians, reptiles, and birds) can spontaneously regrow hair cells, under normal conditions and/or after damage. Hair cell regeneration allows birds to hear again. These findings provide hope that, if hair cell regeneration were to be stimulated in mammals, the new hair cells would be sufficient to restore hearing function.


Towards local and specific pharmacology.

We have seen that the large majority of hearing problems are caused by environmental trauma (loud noise, ototoxic drugs. ). To achieve good results with hearing aids and implants, it is crucial that cells survive. Research is already well under way.

We now know more and more about the biological and molecular mechanisms involved in cell death in the inner ear. We can therefore target protection treatments at sensory cells or cochlear neurons or increase their resistance to damage.

Currently, the primary obstacle to human clinical trials is the potential side effects of these therapeutic molecules if they are administered non-specifically.

Local administration of these effective molecules at the round window is possible (as in the drawing below) !

Mini-pump implant

The commercialisation of a number of delivery systems (catheter, syringe, etc. ) allows us to conceptualise numerous promising therapies which could become acute treatments. Such devices are already used to treat dizziness!

More sophisticated methods such as the implantation of a pump that can be recharged through the skin can now be imagined. These pumps could be coupled with cochlear implants, with an aim to keep as many neurons as possible alive, improving the performance of the implant.

Showing that cell degeneration can be stopped in patients with cochlear implants will allow this type of pharmacological approach to spread to other pathologies, such as presbycusis, .


Could hair cell regrowth restore lost hearing?

You are free to share this article under the Attribution 4.0 International license.

New research marks an important step toward what may become a new approach to restore hearing loss.

Scientists have been able to regrow the sensory hair cells found in the cochlea—a part of the inner ear—that converts sound vibrations into electrical signals and can be permanently lost due to age or noise damage.

An estimated 30 million Americans suffer from some degree of hearing loss—and people have long been accepted it as a fact of life for the aging population. But animals, including birds, frogs, and fish, can regenerate lost sensory hair cells.

“It’s funny, but mammals are the oddballs in the animal kingdom when it comes to cochlear regeneration,” says study coauthor Jingyuan Zhang of the biology department at the University of Rochester. “We’re the only vertebrates that can’t do it.”

Pathway switch

In 2012, researchers identified a family of receptors called epidermal growth factor (EGF) that are responsible for activating support cells in the auditory organs of birds. When triggered, these cells proliferate and foster the generation of new sensory hair cells. Scientists speculated that they could potentially manipulate this signaling pathway to produce a similar result in mammals.

“In mice, the cochlea expresses EGF receptors throughout the animal’s life, but they apparently never drive regeneration of hair cells,” says lead author Patricia White, research associate professor in the University of Rochester Medical Center (URMC) Del Monte Institute for Neuroscience.

“Perhaps during mammalian evolution, there have been changes in the expression of intracellular regulators of EGF receptor family signaling. Those regulators could have altered the outcome of signaling, blocking regeneration,” White says.

“Our research is focused on finding a way switch the pathway temporarily, in order to promote both regeneration of hair cells and their integration with nerve cells, both of which are critical for hearing,” she explains.

“This research… could represent a new approach to cochlear regeneration and, ultimately, restoration of hearing.”

In the new study, the team tested the theory that signaling from the EGF family of receptors could play a role in cochlear regeneration in mammals. The researchers focused on a specific receptor called ERBB2 in cochlear support cells.

They investigated a number of different methods to activate the EGF signaling pathway. One set of experiments involved using a virus to target ERBB2 receptors. Another, involved mice genetically modified to overexpress an activated ERBB2. A third experiment involved testing two drugs, originally developed to stimulate stem cell activity in the eyes and pancreas, that are known activate ERBB2 signaling.

Complex problem

The researchers found that activating the ERBB2 pathway triggered a cascading series of cellular events by which cochlear support cells began to proliferate and start the process of activating other neighboring stem cells to become new sensory hair cells.

Further, it appears that this process not only could affect the regeneration of sensory hair cells, but also support their integration with nerve cells.

“The process of repairing hearing is a complex problem and requires a series of cellular events,” White says. “You have to regenerate sensory hair cells and these cells have to function properly and connect with the necessary network of neurons.

“This research demonstrates a signaling pathway that can be activated by different methods and could represent a new approach to cochlear regeneration and, ultimately, restoration of hearing,” she says.

Additional researchers are from URMC and the Massachusetts Ear and Eye Infirmary, which is part of Harvard Medical School. The National Institute for Deafness and Communication Disorders supported the work.


Hearing loss is one of the most common disabilities in the United States, with approximately half of all adults suffering from some degree of hearing loss by the time they reach retirement age. Hearing loss can be due to the effects of aging, or to specific environmental insults such as industrial or recreational noise or exposure to aminoglycoside antibiotics and platinum-based chemotherapy drugs. Noise-induced hearing loss has also been a huge problem in combat veterans returning from Iraq and Afghanistan.

The most common form of hearing loss is caused by the death of cochlear hair cells in the organ of Corti, and once lost, hair cells in humans and other mammals do not regenerate. In contrast, non-mammalian vertebrates can functionally recover from deafening injury by mobilizing supporting cells in the cochlea to divide and differentiate to replace lost hair cells. Since the discovery of hair cell regeneration in birds in the 1980s, research has focused on trying to understand the cellular and molecular mechanisms underlying regeneration and why these processes do not occur in mammals.

In the past 10 years, a number of labs including ours have shown that mammalian supporting cells from new born mice have a limited and transient ability to re-enter the cell cycle and produce hair cells. However, by the time mice are able to hear, at two weeks of age, this capacity for proliferation has been largely lost. Since no new supporting cells are formed after birth, this suggests that the maturation of supporting cells must in some way lead to a loss of their regenerative potential.

The Notch signaling pathway is activated as hair cells and supporting cells differentiate, and has been recently shown to be necessary to maintain the differentiated state of hair cells and supporting cells in early postnatal life. Notch signaling is also activated during hair cell regeneration in birds, and we and others have shown that disruption of Notch signaling in the mammalian cochlea causes supporting cells to trans-differentiate into hair cells. Once again, the ability of supporting cells to do this declines with age – they show a robust response to a blockade of the Notch signaling pathway at the time of birth, but a complete lack of response to such blockade just a few days later. We recently used RNA-seq to identify transcriptional changes in supporting cells in the first postnatal week, and we compared these transcriptomes to those of supporting cells cultured in the presence of Notch pathway inhibitors. Strikingly, we found that the transcriptional response to Notch blockade disappears almost completely in the first postnatal week.

Finally, attention has focused on several pathways and molecules implicated in hair cell development and regeneration. The basic helix-loop-helix transcription factor Atoh1 is one of the first transcription factors to be switched on in hair cells as they differentiate. Atoh1 has been demonstrated to be both necessary and sufficient for the development of hair cells, but its ability to induce hair cells declines rapidly after birth. We have collaborated with Huda Zoghbi’s lab at Baylor to identify direct targets of Atoh1 in cochlear and vestibular hair cells, and we are now testing whether epigenetic silencing contributes to the loss of regenerative ability in the mammalian cochlea.


Cochlear Hair Cell Regeneration

Paths of regeneration may be different from those of development. However lower vertebrates fishes amphibians reptiles and birds can spontaneously regrow hair cells under normal conditions andor after damage.

Cochlear Hair Cell Regeneration After Noise Induced Hearing Loss Does Regeneration Follow Development Sciencedirect

Mature mammalian cochlear hair cells HCs do not spontaneously regenerate once lost leading to life-long hearing deficits.

Cochlear hair cell regeneration. Hearing is an amazing process and its all thanks to the 15000 or so tiny hair cells inside our cochlea the small snail-shaped organ for hearing in the inner ear. Thus hearing loss due to hair cell damage is permanent. Youm I1 Li W2.

Cochlear hair cells are mechanoreceptors of the auditory system and cannot spontaneously regenerate in adult mammals. Advancing science for the future A group of researchers have come together and started a company Frequency Therapeutics which has licensed this technology and said last year that it would begin initial trial testing on human subjects within as short a time as eighteen months. This symposium will bring together experts of the auditory system and hair cell regeneration to give an overview of the recent advancements in the field identify knowledge gaps and outline future directions towards a cure for age-related hearing loss.

New research marks an important step toward what may become a new approach to restore hearing loss. In contrast hair cells in nonmammalian vertebrates such as birds and in the zebrafish lateral line have the ability to regenerate after hair cell loss. However in the mid 1980s several researchers discovered that certain species of birds and sharks could actually regenerate cochlear hair cells that had been damaged.

This would then stimulate the cells causing hair to regenerate and with the hair restored hearing. Currently many researchers have foc Cochlear hair cell regeneration. There are two kinds of HC regeneration mechanisms in vertebrates namely direct transdifferentiation in which SCs directly differentiate into HCs without cell division 5 6 and mitotic regeneration in which SCprogenitor cells reenter the cell cycle to proliferate first and then several days later switch fates to become HCs.

To date research shows that mammalian cochlear hair cells do not regenerate either spontaneously or after damage. Drug discovery holds promise in clinical therapy for hearing loss. Attempts to induce HC regeneration in adult mammals have used over.

The discovery of avian cochlear hair cell regeneration in the late 1980s and the concurrent development of new techniques in molecular and developmental biology generated a renewed interest in understanding the genetic mechanisms that regulate hair cell development in the embryonic avian and mammalian cochlea and regeneration in the mature avian cochlea. Once cochlear hair cells are lost they are not replaced. Repeat cell counting gave a test variation of.

Transient MYC and NOTCH activities enable adult supporting cells to respond to transcription factor Atoh1 and efficiently transdifferentiate into hair cell-like cells. The cell number was determined from DAPI- or Hoechst-positive nuclei. But that may not always be the case.

2Hough Ear Institute 3400 NW 56th Street Oklahoma City OK 73112 USA. When sounds are too loud for too long these bundles are damaged. 7 8 In the mouse cochlea HCs are interdigitated by SCs and.

Co-activation of cell cycle activator Myc and inner ear progenitor gene Notch1 induces robust proliferation of diverse adult cochlear sensory epithelial cell types. This has led to a flurry of research into the field of cochlear hair cell regeneration. Cochlear hair cells are sensitive to acoustic trauma drug insults aging and environmental or genetic influences that can cause permanent hearing loss.

Spontaneous and forced hair cell regeneration occurs in mammalian cochleae in vivo. Their findings show that activating ERBB2 started a process that led to the production of cochlear support cells. Hair cells were identified with myosin VIIa antibodies or endogenous GFP in Atoh1.

The term regeneration is commonly used for describing generation of new hair cells. Scientists have been able to regrow the sensory hair cells found in the cochleaa part of the. This then resulted in stem cells transforming into the sensory hair cells.

Hair cell regeneration allows birds to hear again. Cochlear hair cell regeneration. The cells are called hair cells because tiny bundles of stereocilia which look like hairs under a microscope sit on top of each hair cell.

For explants inner hair cells outer hair cells and supporting cells in the outer hair cell region were counted on cochlear whole mounts. Multiple factors can enhance hair cell regeneration. An emerging opportunity to cure noise-induced sensorineural hearing loss Drug Discov Today.

Using a sustained-release formulation of small interfering RNAs delivered via nanoparticles into the cochlea of noise-injured guinea pigs. In the scientific and medical disciplines dealing with neural repair and re-growth. 1Hough Ear Institute 3400 NW 56th Street Oklahoma City OK 73112 USA.

2018 Aug2381564 -1569. Epigenetic memory is a key to hair cell regeneration. An emerging opportunity to cure noise-induced sensorineural hearing loss.

The cochlear hair cells of humans and other mammalsunlike some animal species like birds for examplecannot be restored after damageRecent research has indicated however that manipulation of signaling pathways could lead to hair cell regeneration. The cochlear hair cells dont regenerate so right now damage to them is permanent and common among people with some types of hearing loss.

Figure 3 From Sensory Hair Cell Development And Regeneration Similarities And Differences Semantic Scholar

Sensory Hair Cells Regenerated Hearing Restored In Mammal Ear

Mammalian Cochlear Hair Cell Regeneration And Ribbon Synapse Reformation

The Proposed Role Of Wnt Catenin Signaling In Hair Cell Regeneration Download Scientific Diagram

Cochlear Architecture Critical Part Of Hair Cell Regeneration

Sensory Hair Cell Development And Regeneration Similarities And Differences Development

New Method Enables Systematic Study Of Hair Cell Loss And Regeneration In Chickens Hearing Health Foundation

5 Hair Cell Regeneration Neurowiki 2013

Frontiers Recent Advancements In The Regeneration Of Auditory Hair Cells And Hearing Restoration Frontiers In Molecular Neuroscience

Cochlear Hair Cell Regeneration An Emerging Opportunity To Cure Noise Induced Sensorineural Hearing Loss Sciencedirect

Study Shows Hair Cell Regrowth With New Drug

Model Of Hair Cell Regeneration In The Neonatal Mouse Utricle Striolar Download Scientific Diagram

Sensory Hair Cell Development And Regeneration Similarities And Differences Development


Mammalian Cochlear Hair Cell Regeneration and Ribbon Synapse Reformation.

Mammalian HCs loss by noise trauma, ototoxic drugs, or infection is a major cause of deafness [1]. HCs in mammalian inner ear, unlike invertebrate animals such as birds and fish, do not undergo spontaneous regeneration, even though vestibular supporting cells (SCs) retain a limited capacity to divide [2, 3]. There are two approaches of HC regeneration: (1) direct transdifferentiation of surrounding SCs that directly change cell fate and become HCs and (2) induction of a proliferative response in the SCs which mitotically divide and further differentiate to replace damaged HCs [4-6]. There are various numbers of genes and cell signaling pathways involved in these two mechanisms that remain challenging to understand the molecular mechanism underneath hair cell regeneration. Several studies showed reinnervation of the regenerated HCs after HC regeneration [6-8]. However, innervation of new regenerated HCs still needs to be determined in all kinds of hearing loss.

2. The Anatomy and Function of the Organ of Corti

The organ of Corti, also called the spiral organ, is the spiral structure on the basement membrane of the cochlear duct. The sensory epithelium of the organ of Corti is made up of HCs and SCs. HCs, which can be divided into inner HCs and outer HCs, are sensory receptor cells whose mechanically sensitive hair bundles convert mechanical force produced by sound waves into neural impulses. HCs are surrounded by SCs and connected with cochlear nerve fibers by forming synaptic connection. There are several types of SCs, such as pillar cells and phalangeal cells. Pillar cells can be divided into inner and outer pillar cells found in the middle of the inner and outer HCs separately. The top and bottom of the inner and outer pillar cells are combined, but the middle of them is separated, forming the two edge sides of the triangular tunnel. In the lateral of inner and outer HCs rows, inner and outer phalangeal cells (also called the Deiters' cells) reside, respectively. The finger like projection of Deiters' cells are tightly connected with the apical of outer pillar cells forming a thin, hard reticular membrane, also called reticular layer. The stereocilium of outer HCs is tightly bounded trough the mesh of reticular layer. The reticular layer constitutes fiber and matrix and is found below the tectorial membrane. HCs are sensory cells, and they do not contain axons and dendrites. Instead, the basolateral surface of HCs form afferent synaptic contacts with the axonal terminals of the eighth nerve and receive efferent contacts from neurons in the brainstem. There are about 25,000 to 30,000 auditory nerve fibers connected with HCs. These fibers originate from bipolar spiral ganglion neurons in the modiolus, whose axonal terminals form synaptic connections with the ribbons at HCs and the dendrite forms connection with cochlear nucleus neuron (Figure 1).

The organ of Corti acts as an auditory receptor. Acoustic wave passes through the external auditory canal and reaches the tympanic membrane the tympanic membrane transmitted these vibrations to the oval window by auditory ossicles, causing the perilymph in scala vestibuli to further pass these vibrations to the vestibular membrane and endolymph in cochlear duct. At the same time, the vibration of perilymph in scala vestibuli can be transmitted to the scala tympani through helicotrema, causing the basement membrane to resonance. Due to the different length and diameter of hearing fiber in different parts of the basement membrane results in the different frequency of acoustic wave resonance in the different parts of the basement membrane. The vibration of corresponding parts causes the HCs to contact with the tectorial membrane, the stereocilia bends, and HCs become excited to translocate the mechanical vibration into electrical excitation, which further transmit to the central auditory nerve to eventually producing the sense of hearing.

The organ of Corti harbors HCs, which are vulnerable to infections and many pharmaceutical drugs such as aminoglycoside antibodies, for example, streptomycin and neomycin, and the chemotherapeutic agent cisplatin. Most importantly, HCs can be damaged by acoustic trauma. In nonmammalian vertebrates such as birds, after ototoxic drugs or damaged by noise, the inner ear sensory HCs can regenerate spontaneously and eventually replace the damaged HCs, thus maintaining and restoring the function of sensory epithelium [5, 9]. However, in mammals, spontaneous HC regeneration in vivo has only been identified in neonatal cochleae and also the number of regenerated HCs is quite low as a result the hearing loss is permanent in mammals [10, 11]. It is thought that the mammalian inner ear HCs and SCs originate from the common precursor cells and some of the reported studies suggested that some SCs become HCs when the microenvironment changes, such as damage to HCs and activation of particular genes SCs can continue to differentiate to form HCs [12, 13]. Thus, currently some of the SCs are more commonly recognized as progenitor cells in regenerating HCs. At present, in view of the origin and regeneration of mammalian HCs, there are mainly two mechanisms of HCs regeneration from SCs, one is mitotic division of SC and the other is transdifferentiation [4-6]. In mitotic division, the SCs can divide and then their daughter cells undergo differentiating into HCs in some portions. In transdifferentiation, the SCs directly undergo phenotypic conversion and thus transdifferentiate into a HC without mitosis. Many studies have been done that illustrated the important factors, which are involved in the process of HC differentiation, such as Atoh1, p27Kip1, and Rb. Also, the cell signaling pathways, such as Notch, Wnt, and FGF signaling pathway, play important roles in HC regeneration (Figure 2).

Atoh1, the bHLH differentiation factor, was relating to the formation of mechanoreceptor and photoreceptor in Drosophila [14, 15]. During the embryonic development of mice cochlea, the upregulation of Atoh1 causes an increase number of HCs [7, 16]. In the neonatal cochlea of mice, the upregulation of Atoh1 can activate the SCs to differentiate to form more HCs [17-19]. However, in the undamaged and mature cochleae, the differentiation capacity of SCs is significantly decreased when assessed in transgenic mice or via direct viral inoculation [20]. In consideration of the crucial role of Atoh1 gene during the development of HCs, various studies focused on the regulation of Atoh1 to produce HCs in the damaged and mature cochlear. It is reported that, after ototoxic injury in guinea pigs, immature HCs were regenerated through regulating the ectopic expression of Atoh1, and the hearing function was rescued to a certain extent [21]. However, other studies also found that the efficacy of this approach to regenerate HCs might be limited, and the regulation time of Atoh1 expression after damage is dependent [22, 23]. Moreover, it has been revealed that the H3K4me3/H3K27me3 bivalent chromatin structure is crucial for the function of Atoh1, which is observed at the Atoh1 locus of SCs, and might give an explanation for why these cells can keep the capacity to transdifferentiate into HCs [24].

Several cyclin/cyclin-dependent-kinases (CDKs), including p27Kip1, are dynamically expressed in the sensory epithelial [25, 26]. During the embryonic development of mammalian cochlea, the prosensory cells begin to express p27Kip1 from the apex to the base [25, 26]. Disruption of p27Kip1 gene in the mouse cochlea results in ongoing cell proliferation in the postnatal and adult mouse organ of Corti [25, 27]. Although this approach partially keep the capacity of prosensory cells to proliferate, the cell overproduction will cause dysfunction in the organ of Corti, which results in hearing loss [25]. These studies indicated that the proper expression level of p27Kip1 is necessary for maintaining the normal quantity of HCs and SCs. In contrast to the nonmammals, the mammalian organ of Corti completely lacks the phenomenon through which SCs reenter cell cycle [28-30]. One reason why the mature mammalian organ of Corti cannot reproduce HCs is because the SCs are mitotically quiescent after birth. When p27Kip1 is genetically deleted in the SCs in the neonatal cochlear, these SCs proliferate but do not differentiate into HCs [31-33]. The number of mitotic cells significantly decreased in the mature cochlea when compared to that in the neonatal cochlea [31-33]. When p27Kip is deleted in the HCs of neonatal cochlea, these HCs autonomously reenter into the cell cycle and regenerate new HCs also these newly generated HCs survived till adult age without compromising hearing function [34]. These findings revealed a new route to directly induce regeneration by renewing the proliferation capacity of surviving HCs in mammalian organ of Corti.

pRb is a retinoblastoma protein, which is encoded by the retinoblastoma gene Rb1. It plays a role in cell cycle exit, differentiation, and survival [35, 36]. It has been shown that the targeted deletion of Rb1 allowed them to undergo cell cycle and become highly differentiated and functional indicating that the differentiation of the sensory epithelia and cell division are not mutually exclusive [6, 37]. However, the proliferation due to Rb1 deletion is age dependent and eventually the cochlear HCs undergo apoptosis [38, 39]. Moreover, the transient downregulation of Rb1 is necessary to induce proliferation in adult cochlea, also together with Rb1 deletion some other strategies such as epigenetic modifications and reprogramming need to be further studied in order to regenerate HCs in mature cochlea.

The Notch signaling pathway plays multiple roles during the development of mammalian cochlea. The precise formation of mosaic structure of the HC and SC is mediated by lateral inhibition through dynamic expression of the Notch signaling pathway [40-42]. As the process of HCs differentiation begins, a prosensory cell chooses to become a HC or a SC under the precise regulation of lateral inhibition through Notch pathway. The HCs undergoing the differentiation express the Notch ligands and activate Notch signaling pathway in the neighboring SCs, thus preventing them from obtaining a HC fate. Eventually, the mosaic structure of HC and SC is formed. Moreover, in the germline deletion of the Notch ligand Jag2 or Delta-like 1 (Dll1), the HC number is increased at the cost of SCs [43, 44]. In a similar manner, when Notch/Jag2 and Dll1 are suppressed during early embryonic development, the prosensory cells proliferation becomes prolonged comparing with the normal control in the inner ear [43, 44]. On the contrary, the formation of prosensory domain is prevented when the Notch receptor Notch1 is conditionally knocked out meanwhile, there is increased number of HCs and a concomitant deceased number of SCs [43]. These findings demonstrated that the Notch pathway plays important roles in the specification of normal prosensory domain and regulates the differentiation of HCs in different levels through different combination of Notch ligands and receptors. Furthermore, the effects of Notch inhibition have also been explored on the regeneration process of HC. It is reported that the SCs can transdifferentiate into HCs when treated with Notch inhibitor in the undamaged neonatal mammalian cochlea [45-47]. Coincidently, this pharmacological approach produces significantly less number of HCs in the damaged and mature cochlea of mammals [48, 49] and these newly regenerated HCs are acquired through direct transdifferentiation of SCs [45-49]. Taken together, these findings suggests that both the proliferation of SCs and HC differentiation including their coordination might require the regeneration and function recovery of the organ of Corti.

Wnt are widely expressed and evolutionary conserved in the vertebrates and invertebrates animal tissues. Wnt play important roles in several biological processes, such as development, proliferation, metabolism, and regulation of stem cells. The activation of Wnt signaling pathway through beta-catenin overexpression protects HCs against neomycin insult [50]. When cochleae are cultured in vitro, the addition of Wnt inhibitors prevents the proliferation of prosensory cells and also the differentiation into HCs [51]. On the contrary, when supplied with Wnt signaling activators result in increased proliferation of prosensory cells and HCs [51]. These studies revealed that the canonical Wnt signaling pathway plays important roles in regulating the proliferation of prosensory cells and differentiation of HCs during cochlea development. Furthermore, when beta-catenin is ablated during cochlear development, which is a key gene of canonical Wnt signaling pathway, the proliferation of prosensory cells is significantly decreased and the large number of HCs was diminished [52]. Recent studies have found that Lgr5 positive SCs are the precursor cells with the capacity to regenerate HCs under certain conditions [13, 53]. In the neonatal cochleae of mammals, the Wnt target gene Lgr5 is expressed in a subset of SCs (the pillar cells, inner phalangeal cells, and Deiters' cells) [54], and these endogenous Lgr5+ cells maintain mitotic quiescence. The expression level of Wnt signaling pathway including Lgr5 regulated via the expression of Bmi1 [55]. When isolated as single cells using flow cytometry and cultured in vitro, they become proliferative and converted into HC-like cells [13]. In addition, the isolated Lgr5+ SCs significantly increase the Atoh1 expression and the number of HC-like cells after the addition of Wnt signaling pathway agonist [53]. Moreover, it is reported that the proliferation capacity of the Lgr5 positive cells in the apical turn is higher than the basal turn [56]. The conditional overexpression of beta-catenin in the neonatal transgenic mouse cochlea significantly increased the percentage of proliferative supporting cells [13, 53]. Prior study reveals that the inner pillar cells are more sensitive to the beta-catenin overexpression and can also upregulate the expression level of Atoh1 [57]. These studies suggested that the Wnt/beta-catenin signaling pathway participated in the proliferative response in the SCs of neonatal mammals and the interaction between Wnt and Notch signaling pathway is important in the inner ear [46, 58]. More excitingly, extensive SCs proliferation followed by mitotic HCs generation can be achieved through a genetic reprogramming process involving beta-catenin activation, Notch1 deletion, and Atoh1 overexpression. [59].

The FGF signaling pathway is important during inner ear development and morphogenesis. It is related to the induction of otic placode and the development of otic vesile [60-62]. When the FGF receptor 1 (Fgfr1) is genetically deleted in the inner ear, the number of proliferative prosensory cells decreases resulting in decreased number of HCs and SCs [63, 64]. It is reported that Fgf20, which is the candidate ligand for Fgfr1, might be the downstream target of Notch signaling pathway [42]. The addition of Ffg20 rescues the abnormal prosensory specification caused by Notch inhibition [42]. Moreover, downregulation of Fgf20 expression does not cause vestibular dysfunction, which indicates that the Fgf20 might be related to HCs specification in the cochlea. Moreover, it is identified that Fgf8 and Fgf3 are necessary for the development of pillar cells [65, 66]. So far, the function of FGF signaling pathway on HC regeneration is explored in the utricle of chicken and lateral line of zebrafish. When SCs robustly proliferate, the expression level of Fgf20 and Fgfr3 decreases [67]. It is found that the expression level of Fgfr3 is decreased in the cochlea of chicken and the lateral line of zebrafish [68, 69]. However, in the damaged and undamaged mammalian cochlea increased Fgfr3 expression was observed [70]. Taken together, these studies indicated that FGF signaling pathway plays an important role in the specification of prosensory cells and differentiation of HCs and SCs during development, but the function of FGF signaling on HC regeneration is still remain unknown.

4. Ribbon Synapse Reforming and Reinnervation in Regenerated Hair Cells

It is true that the regeneration of HCs is predominantly important and the pivotal issue for restoring hearing and balance function. The regeneration of synaptic connection between newly generated HCs and spiral ganglion neurons is also required. It is reported that when exposed to excessive noise, both HCs and spiral ganglion neuron are sensitive. In mammals, spiral ganglion neurons are hardly recovered from injury [71, 72] and the auditory nerve fibers often degenerate after ototoxic insult, including noise damage and ototoxic drugs. The process of degeneration has been revealed. At first, the unmyelinated terminal dendrites within the organ of Corti disappear (within hours to days), followed by the slow degeneration of peripheral axons in the osseous spiral lamina (within days to weeks). Then, the cell bodies in the spiral ganglion and their central axons that compose the cochlear nerve (over weeks to months and longer) degenerate in the last. Thus the regeneration of ribbon synapses and spiral ganglion neurons in combination with HCs are important for treating hearing loss.

The innervation of HCs is complex process. In the mammalian cochlea, the inner HCs are key component in the sound perception. The inner HC transmits signal to the nerve fibers of spiral ganglion neuron through transforming the mechanical signals induced by sound into electrochemical signal. On the other hand, the outer HC is related to the amplification of audible signals. In the auditory nervous system, there are two kinds of functional neuron population that works differently to convey sound information. In the adult mouse cochlea, there are approximately 800 inner HCs, which are exclusively innervated by 5-30 type I spiral ganglion neuron fibers. These type I spiral ganglion neurons are the main encoders of the auditory signal, which constitute almost 95 percent of the total neuron population [73-75]. In contrast, the type II spiral ganglion neurons constituting approximately 5 percent of the total neuron population innervate the approximately 2,600 outer HCs (almost 1-2 outer HCs per fiber) [76] (Figure 3). The innervation of type II spiral ganglion neuron to the outer HCs is likely to give sensory feedback as a component of the neural control loop, which includes the inhibitory olivocochlear efferent innervation of both outer HC and the postsynaptic region of the type I spiral ganglion neuron at the inner HC region. The mature organ of Corti receives extensive efferent innervation via the lateral olivocochlear (LOC) input to the boutons and dendrites of type I spiral ganglion neurons in the inner spiral plexus region and via the medial olivocochlear (MOC) bundle projection to the outer HCs [76] (Figure 3). This reorganization occurs just before the onset of hearing during the first postnatal week. There are three distinct stages in the formation and development of the afferent nerve fiber innervation to the inner and outer HCs [77]. From embryonic day 18 to postnatal day 0, two kinds of afferent nerve fibers begin to extend and the neurite grows towards HCs. From postnatal day 0 to day 3, the neurite of these afferent fibers begins to refine to form outer spiral bundles, which innervate outer HCs. From postnatal day 3 to day 6, the neurite and synapse structure of type I spiral ganglion neuron retract towards outer HCs and prune to eliminate the innervation between outer HCs and type I spiral ganglion neuron, while the innervation of inner HC is retained by type I spiral ganglion neuron. Also, multiple factors and signaling pathways have been studied in the development and regeneration processes in inner HC ribbon synapses, such as neurotrophins, hormonal signaling, thrombospondins, Gata3-mafb, and Foxo3 networks [78,79].

The neurotransmission between inner HCs and type I spiral ganglion neurons and outer HCs and type II spiral ganglion neurons are conveyed by the ribbon synapses, which are crucial for the accurate encoding of acoustic information [76, 80]. The key component of the ribbon synapse is the glutamatergic synaptic complexes, which are composed of presynaptic ribbons and postsynaptic densities. This kind of afferent ribbon synapse is capable of releasing neurotransmitter quickly and synchronously [81]. The presynaptic ribbons in the inner ear basolateral membrane was found in the opposite side of the postsynaptic glutamate receptors on the dendrite of afferent fibers. The presynaptic ribbons are settled in the active zone of HCs by electron-dense ribbon configuration. When responding to different acoustic signals, the presynaptic ribbons release multiple vesicles quickly and synchronously with high temporal resolution [82-84]. In the postsynaptic dendrite of afferent fibers, the excitatory neurotransmission is mediated by AMPA-type glutamate receptors [85].

In recent years, HC regeneration has made certain achievements thus, the reinnervation of newly generated HCs and reformation of ribbon synapses are urgently needed for restoring hearing and balance function. Cochlear ribbon synapses have limited intrinsic capacity to spontaneously regenerate [86-88]. Prior study reported that when cochleae is damaged in neonatal mice, the HCs spontaneously regenerate from the SCs, but the inner cell marker vesicular glutamate transporter VGlut3 was not detected in these newly regenerated HCs [10]. When the Notch1 signaling pathway overactivated to induce the ectopic HCs, the neural fiber marker Tuj1 was detected in the lateral edge of spiral ganglion neuron, while the synaptic marker synaptophysin was detected between the new HCs and neuronal cells in the spiral ganglion regions, but the synaptophysin signals adjacent to new HCs were weaker indicating that the synaptic structures among new HCs and neuronal cells were not fully mature [89]. The deletion of p27Kip1 induced regeneration of new HCs and these HCs were stained with espin (stereociliary bundles), C-terminal binding protein-2 (Ctbp2 ribbon synapses), and class III beta-tubulin (Tuj1 innervating nerve fibers). However, a portion of the postnatal derived inner HCs was negative for VGlut3 (synaptic transmission) marker [34]. Deletion of p27Kip1 reforms "synaptic structure" to some extent. Although hearing function was normal in adult mice, the functional reformation of synaptic contacts still remained unclear. The ectopic expression of Atoh1 induced HC regeneration, the synaptic markers, CSP, synaptophysin, and synaptotagmin 1 detected at the basal of the newly generated HCs. Although some synaptic markers were found at the site of newly regenerated HCs and neuron contacts, the normal synaptic ribbons were still absent [18].

To achieve a better innervation of the newly generated HCs, the regeneration of ribbon synapses is predominantly important. Recently, many factors and signaling pathways have been found to play a role in promoting axonal regeneration and synapse reformation [90,91]. The synaptotrophic factors are the most well-known factors. The members of the neutrophin family, such as the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT-4/5), are participated in the formation of ribbon synapse and promote the synaptic regeneration process [92-95]. BDNF and its congenital receptor TrkB and the NT-3 and its congenital receptor were detected in the cochlea [96]. It has been reported that BDNF and NT-3 are critical factors for the survival of sensory neurons and the initiation of nerve fibers extending towards the sensory epithelial in the cochlea and vestibule [97]. In the neonatal inner ear of mammals, the deletion of BDNF or NT-3 causes specific loss of ribbon synapses in the cochlea and vestibule, respectively, causing hearing loss and vestibular dysfunction [98, 99]. After ototoxic drug damage, the addition of BDNF and NT-3 promotes the reinnervation of spiral ganglion neurons in cultured cochleae and expresses the postsynaptic markers [100]. Moreover, it is likely that the NT-3 is more significant for ribbon synapses after noise exposure than BDNF [99]. Supporting cell-derived NT-3 promotes the regeneration of ribbon synapses and is helpful in the recovery of cochlear function [78,100] indicating that the neurotrophins are important for the formation of postsynaptic densities and ribbon synapse regeneration after injury. Glutamate is another important synaptotrophic factor [100]. In the deafferented organ of Corti, the number of newly generated synaptic contacts at the dendrite of spiral ganglion neurons was significantly decreased in the Vglut3 deletion mice when compared to normal controls, indicating that the proper releasing of glutamate transmitter is important for the regeneration of synaptic contacts in vitro [100]. However, the in vivo role of glutamate in synaptic regeneration still remains unclear. Furthermore, the contacts generated between cultured spiral ganglion neurons and denervated HCs were evaluated and found that the postsynaptic density protein PSD-95 was immunopositive and directly facing the HC ribbons [100]. The neurotrophins, BDNF and NT-3, significantly increase the number of new synapses. In consideration of the synapse formation activities, these neurotrophins reveal a potential to promote synapse regeneration in the newly regenerated HCs.

In the recent years, there is growing concern about the HC regeneration and synaptic plasticity around the globe and the great achievements have been made in revealing the mechanism and strategies to recover hearing function in mammals [10,13, 59,101]. Different levels of HC regeneration could be achieved through the regulation of factors and signaling pathways, which play important roles during the development of mammalian inner ear [23, 34, 48, 59]. Synapse and nerve fiber related markers are detected around the newly regenerated HCs [10, 34, 89]. However, we are still quite far from restoring the hearing function in the damaged inner ear. The maturation and survival of newly generated HCs are still challenging. Furthermore, the maturation of reinnervation of the regenerated HCs and the function of the reformed ribbon synapse remain open to question, such as the contact between stereocilium and tectorial membrane, reorganization of the innervation of afferent Type I and Type II spiral ganglion neuron, and the integral interplay of outer hair cell based cochlear amplification. To obtain a viable treatment option for future hair cell regeneration of patients suffering from hearing loss, the understanding of reinnervation of the regenerated hair cells and the function of the reformed ribbon synapse is essential and it remains to be explored and open to question.

The authors have declared that no competing interests exist.

Xiaoling Lu and Yilai Shu contributed equally to this work.

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Xiaoling Lu, (1) Yilai Shu, (1) Mingliang Tang, (2,3) and Huawei Li (1,4,5,6,7)

(1) Otorhinolaryngology Department of Affiliated Eye and ENT Hospital, State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200031, China

(2) Key Laboratory for Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing 210096, China

(3) Co-Innovation Center of Neuroregeneration, Nantong University, Nantong 226001, China

(4) Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China

(5) Key Laboratory of Hearing Medicine ofNHFPC, Shanghai 200031, China

(6) Shanghai Engineering Research Centre of Cochlear Implant, Shanghai 200031, China

(7) The Institutes of Brain Science and the Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China

Correspondence should be addressed to Mingliang Tang [email protected] and Huawei Li [email protected]

Received 9 September 2016 Revised 29 November 2016 Accepted 1 December 2016

Academic Editor: Genglin Li

Caption: Figure 1: Schematic model of the organ of Corti. IHC: inner hair cell OHCs: outer hair cells PCs: inner and outer pillar cells IPhC: inner phalangeal cell DCs: Deiters' cells IBC: inner border cell Hen: Hensen's cell GER: greater epithelial ridge LER: lesser epithelial ridge.

Caption: Figure 3: Schematic drawing of the innervation of hair cells. IHC: inner hair cell OHCs: outer hair cells AF: afferent fiber EF: efferent fiber LOC: lateral olivary complex MOC: medial olivary complex.


Mammalian Cochlear Hair Cell Regeneration and Ribbon Synapse Reformation

Hair cells (HCs) are the sensory preceptor cells in the inner ear, which play an important role in hearing and balance. The HCs of organ of Corti are susceptible to noise, ototoxic drugs, and infections, thus resulting in permanent hearing loss. Recent approaches of HCs regeneration provide new directions for finding the treatment of sensor neural deafness. To have normal hearing function, the regenerated HCs must be reinnervated by nerve fibers and reform ribbon synapse with the dendrite of spiral ganglion neuron through nerve regeneration. In this review, we discuss the research progress in HC regeneration, the synaptic plasticity, and the reinnervation of new regenerated HCs in mammalian inner ear.

1. Introduction

Mammalian HCs loss by noise trauma, ototoxic drugs, or infection is a major cause of deafness [1]. HCs in mammalian inner ear, unlike invertebrate animals such as birds and fish, do not undergo spontaneous regeneration, even though vestibular supporting cells (SCs) retain a limited capacity to divide [2, 3]. There are two approaches of HC regeneration: (1) direct transdifferentiation of surrounding SCs that directly change cell fate and become HCs and (2) induction of a proliferative response in the SCs which mitotically divide and further differentiate to replace damaged HCs [4–6]. There are various numbers of genes and cell signaling pathways involved in these two mechanisms that remain challenging to understand the molecular mechanism underneath hair cell regeneration. Several studies showed reinnervation of the regenerated HCs after HC regeneration [6–8]. However, innervation of new regenerated HCs still needs to be determined in all kinds of hearing loss.

2. The Anatomy and Function of the Organ of Corti

The organ of Corti, also called the spiral organ, is the spiral structure on the basement membrane of the cochlear duct. The sensory epithelium of the organ of Corti is made up of HCs and SCs. HCs, which can be divided into inner HCs and outer HCs, are sensory receptor cells whose mechanically sensitive hair bundles convert mechanical force produced by sound waves into neural impulses. HCs are surrounded by SCs and connected with cochlear nerve fibers by forming synaptic connection. There are several types of SCs, such as pillar cells and phalangeal cells. Pillar cells can be divided into inner and outer pillar cells found in the middle of the inner and outer HCs separately. The top and bottom of the inner and outer pillar cells are combined, but the middle of them is separated, forming the two edge sides of the triangular tunnel. In the lateral of inner and outer HCs rows, inner and outer phalangeal cells (also called the Deiters’ cells) reside, respectively. The finger like projection of Deiters’ cells are tightly connected with the apical of outer pillar cells forming a thin, hard reticular membrane, also called reticular layer. The stereocilium of outer HCs is tightly bounded trough the mesh of reticular layer. The reticular layer constitutes fiber and matrix and is found below the tectorial membrane. HCs are sensory cells, and they do not contain axons and dendrites. Instead, the basolateral surface of HCs form afferent synaptic contacts with the axonal terminals of the eighth nerve and receive efferent contacts from neurons in the brainstem. There are about 25,000 to 30,000 auditory nerve fibers connected with HCs. These fibers originate from bipolar spiral ganglion neurons in the modiolus, whose axonal terminals form synaptic connections with the ribbons at HCs and the dendrite forms connection with cochlear nucleus neuron (Figure 1).

The organ of Corti acts as an auditory receptor. Acoustic wave passes through the external auditory canal and reaches the tympanic membrane the tympanic membrane transmitted these vibrations to the oval window by auditory ossicles, causing the perilymph in scala vestibuli to further pass these vibrations to the vestibular membrane and endolymph in cochlear duct. At the same time, the vibration of perilymph in scala vestibuli can be transmitted to the scala tympani through helicotrema, causing the basement membrane to resonance. Due to the different length and diameter of hearing fiber in different parts of the basement membrane results in the different frequency of acoustic wave resonance in the different parts of the basement membrane. The vibration of corresponding parts causes the HCs to contact with the tectorial membrane, the stereocilia bends, and HCs become excited to translocate the mechanical vibration into electrical excitation, which further transmit to the central auditory nerve to eventually producing the sense of hearing.

3. Hair Cell Regeneration

The organ of Corti harbors HCs, which are vulnerable to infections and many pharmaceutical drugs such as aminoglycoside antibodies, for example, streptomycin and neomycin, and the chemotherapeutic agent cisplatin. Most importantly, HCs can be damaged by acoustic trauma. In nonmammalian vertebrates such as birds, after ototoxic drugs or damaged by noise, the inner ear sensory HCs can regenerate spontaneously and eventually replace the damaged HCs, thus maintaining and restoring the function of sensory epithelium [5, 9]. However, in mammals, spontaneous HC regeneration in vivo has only been identified in neonatal cochleae and also the number of regenerated HCs is quite low as a result the hearing loss is permanent in mammals [10, 11]. It is thought that the mammalian inner ear HCs and SCs originate from the common precursor cells and some of the reported studies suggested that some SCs become HCs when the microenvironment changes, such as damage to HCs and activation of particular genes SCs can continue to differentiate to form HCs [12, 13]. Thus, currently some of the SCs are more commonly recognized as progenitor cells in regenerating HCs. At present, in view of the origin and regeneration of mammalian HCs, there are mainly two mechanisms of HCs regeneration from SCs, one is mitotic division of SC and the other is transdifferentiation [4–6]. In mitotic division, the SCs can divide and then their daughter cells undergo differentiating into HCs in some portions. In transdifferentiation, the SCs directly undergo phenotypic conversion and thus transdifferentiate into a HC without mitosis. Many studies have been done that illustrated the important factors, which are involved in the process of HC differentiation, such as Atoh1, p27Kip1, and Rb. Also, the cell signaling pathways, such as Notch, Wnt, and FGF signaling pathway, play important roles in HC regeneration (Figure 2).

Atoh1, the bHLH differentiation factor, was relating to the formation of mechanoreceptor and photoreceptor in Drosophila [14, 15]. During the embryonic development of mice cochlea, the upregulation of Atoh1 causes an increase number of HCs [7, 16]. In the neonatal cochlea of mice, the upregulation of Atoh1 can activate the SCs to differentiate to form more HCs [17–19]. However, in the undamaged and mature cochleae, the differentiation capacity of SCs is significantly decreased when assessed in transgenic mice or via direct viral inoculation [20]. In consideration of the crucial role of Atoh1 gene during the development of HCs, various studies focused on the regulation of Atoh1 to produce HCs in the damaged and mature cochlear. It is reported that, after ototoxic injury in guinea pigs, immature HCs were regenerated through regulating the ectopic expression of Atoh1, and the hearing function was rescued to a certain extent [21]. However, other studies also found that the efficacy of this approach to regenerate HCs might be limited, and the regulation time of Atoh1 expression after damage is dependent [22, 23]. Moreover, it has been revealed that the H3K4me3/H3K27me3 bivalent chromatin structure is crucial for the function of Atoh1, which is observed at the Atoh1 locus of SCs, and might give an explanation for why these cells can keep the capacity to transdifferentiate into HCs [24].

Several cyclin/cyclin-dependent-kinases (CDKs), including p27Kip1, are dynamically expressed in the sensory epithelial [25, 26]. During the embryonic development of mammalian cochlea, the prosensory cells begin to express p27Kip1 from the apex to the base [25, 26]. Disruption of p27Kip1 gene in the mouse cochlea results in ongoing cell proliferation in the postnatal and adult mouse organ of Corti [25, 27]. Although this approach partially keep the capacity of prosensory cells to proliferate, the cell overproduction will cause dysfunction in the organ of Corti, which results in hearing loss [25]. These studies indicated that the proper expression level of p27Kip1 is necessary for maintaining the normal quantity of HCs and SCs. In contrast to the nonmammals, the mammalian organ of Corti completely lacks the phenomenon through which SCs reenter cell cycle [28–30]. One reason why the mature mammalian organ of Corti cannot reproduce HCs is because the SCs are mitotically quiescent after birth. When p27Kip1 is genetically deleted in the SCs in the neonatal cochlear, these SCs proliferate but do not differentiate into HCs [31–33]. The number of mitotic cells significantly decreased in the mature cochlea when compared to that in the neonatal cochlea [31–33]. When p27Kip is deleted in the HCs of neonatal cochlea, these HCs autonomously reenter into the cell cycle and regenerate new HCs also these newly generated HCs survived till adult age without compromising hearing function [34]. These findings revealed a new route to directly induce regeneration by renewing the proliferation capacity of surviving HCs in mammalian organ of Corti.

pRb is a retinoblastoma protein, which is encoded by the retinoblastoma gene Rb1. It plays a role in cell cycle exit, differentiation, and survival [35, 36]. It has been shown that the targeted deletion of Rb1 allowed them to undergo cell cycle and become highly differentiated and functional indicating that the differentiation of the sensory epithelia and cell division are not mutually exclusive [6, 37]. However, the proliferation due to Rb1 deletion is age dependent and eventually the cochlear HCs undergo apoptosis [38, 39]. Moreover, the transient downregulation of Rb1 is necessary to induce proliferation in adult cochlea, also together with Rb1 deletion some other strategies such as epigenetic modifications and reprogramming need to be further studied in order to regenerate HCs in mature cochlea.

The Notch signaling pathway plays multiple roles during the development of mammalian cochlea. The precise formation of mosaic structure of the HC and SC is mediated by lateral inhibition through dynamic expression of the Notch signaling pathway [40–42]. As the process of HCs differentiation begins, a prosensory cell chooses to become a HC or a SC under the precise regulation of lateral inhibition through Notch pathway. The HCs undergoing the differentiation express the Notch ligands and activate Notch signaling pathway in the neighboring SCs, thus preventing them from obtaining a HC fate. Eventually, the mosaic structure of HC and SC is formed. Moreover, in the germline deletion of the Notch ligand Jag2 or Delta-like 1 (Dll1), the HC number is increased at the cost of SCs [43, 44]. In a similar manner, when Notch/Jag2 and Dll1 are suppressed during early embryonic development, the prosensory cells proliferation becomes prolonged comparing with the normal control in the inner ear [43, 44]. On the contrary, the formation of prosensory domain is prevented when the Notch receptor Notch1 is conditionally knocked out meanwhile, there is increased number of HCs and a concomitant deceased number of SCs [43]. These findings demonstrated that the Notch pathway plays important roles in the specification of normal prosensory domain and regulates the differentiation of HCs in different levels through different combination of Notch ligands and receptors. Furthermore, the effects of Notch inhibition have also been explored on the regeneration process of HC. It is reported that the SCs can transdifferentiate into HCs when treated with Notch inhibitor in the undamaged neonatal mammalian cochlea [45–47]. Coincidently, this pharmacological approach produces significantly less number of HCs in the damaged and mature cochlea of mammals [48, 49] and these newly regenerated HCs are acquired through direct transdifferentiation of SCs [45–49]. Taken together, these findings suggests that both the proliferation of SCs and HC differentiation including their coordination might require the regeneration and function recovery of the organ of Corti.

Wnt are widely expressed and evolutionary conserved in the vertebrates and invertebrates animal tissues. Wnt play important roles in several biological processes, such as development, proliferation, metabolism, and regulation of stem cells. The activation of Wnt signaling pathway through beta-catenin overexpression protects HCs against neomycin insult [50]. When cochleae are cultured in vitro, the addition of Wnt inhibitors prevents the proliferation of prosensory cells and also the differentiation into HCs [51]. On the contrary, when supplied with Wnt signaling activators result in increased proliferation of prosensory cells and HCs [51]. These studies revealed that the canonical Wnt signaling pathway plays important roles in regulating the proliferation of prosensory cells and differentiation of HCs during cochlea development. Furthermore, when beta-catenin is ablated during cochlear development, which is a key gene of canonical Wnt signaling pathway, the proliferation of prosensory cells is significantly decreased and the large number of HCs was diminished [52]. Recent studies have found that Lgr5 positive SCs are the precursor cells with the capacity to regenerate HCs under certain conditions [13, 53]. In the neonatal cochleae of mammals, the Wnt target gene Lgr5 is expressed in a subset of SCs (the pillar cells, inner phalangeal cells, and Deiters’ cells) [54], and these endogenous Lgr5+ cells maintain mitotic quiescence. The expression level of Wnt signaling pathway including Lgr5 regulated via the expression of Bmi1 [55]. When isolated as single cells using flow cytometry and cultured in vitro, they become proliferative and converted into HC-like cells [13]. In addition, the isolated Lgr5+ SCs significantly increase the Atoh1 expression and the number of HC-like cells after the addition of Wnt signaling pathway agonist [53]. Moreover, it is reported that the proliferation capacity of the Lgr5 positive cells in the apical turn is higher than the basal turn [56]. The conditional overexpression of beta-catenin in the neonatal transgenic mouse cochlea significantly increased the percentage of proliferative supporting cells [13, 53]. Prior study reveals that the inner pillar cells are more sensitive to the beta-catenin overexpression and can also upregulate the expression level of Atoh1 [57]. These studies suggested that the Wnt/beta-catenin signaling pathway participated in the proliferative response in the SCs of neonatal mammals and the interaction between Wnt and Notch signaling pathway is important in the inner ear [46, 58]. More excitingly, extensive SCs proliferation followed by mitotic HCs generation can be achieved through a genetic reprogramming process involving beta-catenin activation, Notch1 deletion, and Atoh1 overexpression. [59].

The FGF signaling pathway is important during inner ear development and morphogenesis. It is related to the induction of otic placode and the development of otic vesile [60–62]. When the FGF receptor 1 (Fgfr1) is genetically deleted in the inner ear, the number of proliferative prosensory cells decreases resulting in decreased number of HCs and SCs [63, 64]. It is reported that Fgf20, which is the candidate ligand for Fgfr1, might be the downstream target of Notch signaling pathway [42]. The addition of Ffg20 rescues the abnormal prosensory specification caused by Notch inhibition [42]. Moreover, downregulation of Fgf20 expression does not cause vestibular dysfunction, which indicates that the Fgf20 might be related to HCs specification in the cochlea. Moreover, it is identified that Fgf8 and Fgf3 are necessary for the development of pillar cells [65, 66]. So far, the function of FGF signaling pathway on HC regeneration is explored in the utricle of chicken and lateral line of zebrafish. When SCs robustly proliferate, the expression level of Fgf20 and Fgfr3 decreases [67]. It is found that the expression level of Fgfr3 is decreased in the cochlea of chicken and the lateral line of zebrafish [68, 69]. However, in the damaged and undamaged mammalian cochlea increased Fgfr3 expression was observed [70]. Taken together, these studies indicated that FGF signaling pathway plays an important role in the specification of prosensory cells and differentiation of HCs and SCs during development, but the function of FGF signaling on HC regeneration is still remain unknown.

4. Ribbon Synapse Reforming and Reinnervation in Regenerated Hair Cells

It is true that the regeneration of HCs is predominantly important and the pivotal issue for restoring hearing and balance function. The regeneration of synaptic connection between newly generated HCs and spiral ganglion neurons is also required. It is reported that when exposed to excessive noise, both HCs and spiral ganglion neuron are sensitive. In mammals, spiral ganglion neurons are hardly recovered from injury [71, 72] and the auditory nerve fibers often degenerate after ototoxic insult, including noise damage and ototoxic drugs. The process of degeneration has been revealed. At first, the unmyelinated terminal dendrites within the organ of Corti disappear (within hours to days), followed by the slow degeneration of peripheral axons in the osseous spiral lamina (within days to weeks). Then, the cell bodies in the spiral ganglion and their central axons that compose the cochlear nerve (over weeks to months and longer) degenerate in the last. Thus the regeneration of ribbon synapses and spiral ganglion neurons in combination with HCs are important for treating hearing loss.

The innervation of HCs is complex process. In the mammalian cochlea, the inner HCs are key component in the sound perception. The inner HC transmits signal to the nerve fibers of spiral ganglion neuron through transforming the mechanical signals induced by sound into electrochemical signal. On the other hand, the outer HC is related to the amplification of audible signals. In the auditory nervous system, there are two kinds of functional neuron population that works differently to convey sound information. In the adult mouse cochlea, there are approximately 800 inner HCs, which are exclusively innervated by 5–30 type I spiral ganglion neuron fibers. These type I spiral ganglion neurons are the main encoders of the auditory signal, which constitute almost 95 percent of the total neuron population [73–75]. In contrast, the type II spiral ganglion neurons constituting approximately 5 percent of the total neuron population innervate the approximately 2,600 outer HCs (almost 1-2 outer HCs per fiber) [76] (Figure 3). The innervation of type II spiral ganglion neuron to the outer HCs is likely to give sensory feedback as a component of the neural control loop, which includes the inhibitory olivocochlear efferent innervation of both outer HC and the postsynaptic region of the type I spiral ganglion neuron at the inner HC region. The mature organ of Corti receives extensive efferent innervation via the lateral olivocochlear (LOC) input to the boutons and dendrites of type I spiral ganglion neurons in the inner spiral plexus region and via the medial olivocochlear (MOC) bundle projection to the outer HCs [76] (Figure 3). This reorganization occurs just before the onset of hearing during the first postnatal week. There are three distinct stages in the formation and development of the afferent nerve fiber innervation to the inner and outer HCs [77]. From embryonic day 18 to postnatal day 0, two kinds of afferent nerve fibers begin to extend and the neurite grows towards HCs. From postnatal day 0 to day 3, the neurite of these afferent fibers begins to refine to form outer spiral bundles, which innervate outer HCs. From postnatal day 3 to day 6, the neurite and synapse structure of type I spiral ganglion neuron retract towards outer HCs and prune to eliminate the innervation between outer HCs and type I spiral ganglion neuron, while the innervation of inner HC is retained by type I spiral ganglion neuron. Also, multiple factors and signaling pathways have been studied in the development and regeneration processes in inner HC ribbon synapses, such as neurotrophins, hormonal signaling, thrombospondins, Gata3-mafb, and Foxo3 networks [78, 79].

The neurotransmission between inner HCs and type I spiral ganglion neurons and outer HCs and type II spiral ganglion neurons are conveyed by the ribbon synapses, which are crucial for the accurate encoding of acoustic information [76, 80]. The key component of the ribbon synapse is the glutamatergic synaptic complexes, which are composed of presynaptic ribbons and postsynaptic densities. This kind of afferent ribbon synapse is capable of releasing neurotransmitter quickly and synchronously [81]. The presynaptic ribbons in the inner ear basolateral membrane was found in the opposite side of the postsynaptic glutamate receptors on the dendrite of afferent fibers. The presynaptic ribbons are settled in the active zone of HCs by electron-dense ribbon configuration. When responding to different acoustic signals, the presynaptic ribbons release multiple vesicles quickly and synchronously with high temporal resolution [82–84]. In the postsynaptic dendrite of afferent fibers, the excitatory neurotransmission is mediated by AMPA-type glutamate receptors [85].

In recent years, HC regeneration has made certain achievements thus, the reinnervation of newly generated HCs and reformation of ribbon synapses are urgently needed for restoring hearing and balance function. Cochlear ribbon synapses have limited intrinsic capacity to spontaneously regenerate [86–88]. Prior study reported that when cochleae is damaged in neonatal mice, the HCs spontaneously regenerate from the SCs, but the inner cell marker vesicular glutamate transporter VGlut3 was not detected in these newly regenerated HCs [10]. When the Notch1 signaling pathway overactivated to induce the ectopic HCs, the neural fiber marker Tuj1 was detected in the lateral edge of spiral ganglion neuron, while the synaptic marker synaptophysin was detected between the new HCs and neuronal cells in the spiral ganglion regions, but the synaptophysin signals adjacent to new HCs were weaker indicating that the synaptic structures among new HCs and neuronal cells were not fully mature [89]. The deletion of p27Kip1 induced regeneration of new HCs and these HCs were stained with espin (stereociliary bundles), C-terminal binding protein-2 (Ctbp2 ribbon synapses), and class III beta-tubulin (Tuj1 innervating nerve fibers). However, a portion of the postnatal derived inner HCs was negative for VGlut3 (synaptic transmission) marker [34]. Deletion of p27Kip1 reforms “synaptic structure” to some extent. Although hearing function was normal in adult mice, the functional reformation of synaptic contacts still remained unclear. The ectopic expression of Atoh1 induced HC regeneration, the synaptic markers, CSP, synaptophysin, and synaptotagmin 1 detected at the basal of the newly generated HCs. Although some synaptic markers were found at the site of newly regenerated HCs and neuron contacts, the normal synaptic ribbons were still absent [18].

To achieve a better innervation of the newly generated HCs, the regeneration of ribbon synapses is predominantly important. Recently, many factors and signaling pathways have been found to play a role in promoting axonal regeneration and synapse reformation [90, 91]. The synaptotrophic factors are the most well-known factors. The members of the neutrophin family, such as the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT-4/5), are participated in the formation of ribbon synapse and promote the synaptic regeneration process [92–95]. BDNF and its congenital receptor TrkB and the NT-3 and its congenital receptor were detected in the cochlea [96]. It has been reported that BDNF and NT-3 are critical factors for the survival of sensory neurons and the initiation of nerve fibers extending towards the sensory epithelial in the cochlea and vestibule [97]. In the neonatal inner ear of mammals, the deletion of BDNF or NT-3 causes specific loss of ribbon synapses in the cochlea and vestibule, respectively, causing hearing loss and vestibular dysfunction [98, 99]. After ototoxic drug damage, the addition of BDNF and NT-3 promotes the reinnervation of spiral ganglion neurons in cultured cochleae and expresses the postsynaptic markers [100]. Moreover, it is likely that the NT-3 is more significant for ribbon synapses after noise exposure than BDNF [99]. Supporting cell-derived NT-3 promotes the regeneration of ribbon synapses and is helpful in the recovery of cochlear function [78, 100] indicating that the neurotrophins are important for the formation of postsynaptic densities and ribbon synapse regeneration after injury. Glutamate is another important synaptotrophic factor [100]. In the deafferented organ of Corti, the number of newly generated synaptic contacts at the dendrite of spiral ganglion neurons was significantly decreased in the Vglut3 deletion mice when compared to normal controls, indicating that the proper releasing of glutamate transmitter is important for the regeneration of synaptic contacts in vitro [100]. However, the in vivo role of glutamate in synaptic regeneration still remains unclear. Furthermore, the contacts generated between cultured spiral ganglion neurons and denervated HCs were evaluated and found that the postsynaptic density protein PSD-95 was immunopositive and directly facing the HC ribbons [100]. The neurotrophins, BDNF and NT-3, significantly increase the number of new synapses. In consideration of the synapse formation activities, these neurotrophins reveal a potential to promote synapse regeneration in the newly regenerated HCs.

5. Conclusions

In the recent years, there is growing concern about the HC regeneration and synaptic plasticity around the globe and the great achievements have been made in revealing the mechanism and strategies to recover hearing function in mammals [10, 13, 59, 101]. Different levels of HC regeneration could be achieved through the regulation of factors and signaling pathways, which play important roles during the development of mammalian inner ear [23, 34, 48, 59]. Synapse and nerve fiber related markers are detected around the newly regenerated HCs [10, 34, 89]. However, we are still quite far from restoring the hearing function in the damaged inner ear. The maturation and survival of newly generated HCs are still challenging. Furthermore, the maturation of reinnervation of the regenerated HCs and the function of the reformed ribbon synapse remain open to question, such as the contact between stereocilium and tectorial membrane, reorganization of the innervation of afferent Type I and Type II spiral ganglion neuron, and the integral interplay of outer hair cell based cochlear amplification. To obtain a viable treatment option for future hair cell regeneration of patients suffering from hearing loss, the understanding of reinnervation of the regenerated hair cells and the function of the reformed ribbon synapse is essential and it remains to be explored and open to question.

Competing Interests

The authors have declared that no competing interests exist.

Authors’ Contributions

Xiaoling Lu and Yilai Shu contributed equally to this work.

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Copyright

Copyright © 2016 Xiaoling Lu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract

Supporting cells in the cochlea play critical roles in the development, maintenance, and function of sensory hair cells and auditory neurons. Although the loss of hair cells or auditory neurons results in sensorineural hearing loss, the consequence of supporting cell loss on auditory function is largely unknown. In this study, we specifically ablated inner border cells (IBCs) and inner phalangeal cells (IPhCs), the two types of supporting cells surrounding inner hair cells (IHCs) in mice in vivo. We demonstrate that the organ of Corti has the intrinsic capacity to replenish IBCs/IPhCs effectively during early postnatal development. Repopulation depends on the presence of hair cells and cells within the greater epithelial ridge and is independent of cell proliferation. This plastic response in the neonatal cochlea preserves neuronal survival, afferent innervation, and hearing sensitivity in adult mice. In contrast, the capacity for IBC/IPhC regeneration is lost in the mature organ of Corti, and consequently IHC survival and hearing sensitivity are impaired significantly, demonstrating that there is a critical period for the regeneration of cochlear supporting cells. Our findings indicate that the quiescent neonatal organ of Corti can replenish specific supporting cells completely after loss in vivo to guarantee mature hearing function.


Oncomodulin identifies different hair cell types in the mammalian inner ear

The tight regulation of Ca 2+ is essential for inner ear function, and yet the role of Ca 2+ binding proteins (CaBPs) remains elusive. By using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated the expression of oncomodulin (Ocm), a member of the parvalbumin family, relative to other EF-hand CaBPs in cochlear and vestibular organs in the mouse. In the mouse cochlea, Ocm is found only in outer hair cells and is localized preferentially to the basolateral outer hair cell membrane and to the base of the hair bundle. Developmentally, Ocm immunoreactivity begins as early as postnatal day (P) 2 and shows preferential localization to the basolateral membrane and hair bundle after P8. Unlike the cochlea, Ocm expression is substantially reduced in vestibular tissues at older adult ages. In vestibular organs, Ocm is found in type I striolar or central hair cells, and has a more diffuse subcellular localization throughout the hair cell body. Additionally, Ocm immunoreactivity in vestibular hair cells is present as early as E18 and is not obviously affected by mutations that cause a disruption of hair bundle polarity. We also find Ocm expression in striolar hair cells across mammalian species. These data suggest that Ocm may have distinct functional roles in cochlear and vestibular hair cells. J. Comp. Neurol. 518:3785–3802, 2010. © 2010 Wiley-Liss, Inc.

Additional Supporting Information may be found in the online version of this article.

Filename Description
CNE_22424_sm_suppinfoFig1.tif4.6 MB Supplemental Figure 1 Comparisons of Ocm expression in the β-Actin-CreOcm-/- homozygotes and wild type mouse at 4 weeks. Ocm (green) labels outer hair cells in the cochlea and striolar hair cells in the saculus. Calretinin (magenta) labels inner hair cells in cochlea and calyx endings in the sacculus. The scale bar represents 10 μm.
CNE_22424_sm_suppinfoFig2.tif2.8 MB Supplemental Figure 2 Same as Figure 2. Ocm immunoreactivity in the cochlea of the mouse. The scale bars in each panel represent 10 μm. a. A cross section through the organ of Corti of a P10 cochlear basal turn shows outer hair cells (OHCs) immunoreactive for Ocm (magenta). The inner hair cells (IHCs) did not show any Ocm immunoreactivity. Ocm labels the lateral wall of outer hair cells and the hair bundle (arrow). Phalloidin staining (green) labels the cuticular plate (arrow) and stereocilia. The Ocm labeling in the hair bundle is more intense than the phalloidin labeling. b. A confocal image projection of the organ of Corti in an adult mouse shows Ocm labeled (magenta) outer hair cells. The Ocm labeling in the hair bundle is predominately toward the outer portion of the apical surface of the outer hair cell (arrow). Ocm also labeled the basolateral wall of the outer hair cell. Punctuated Ocm labeling (asterisk) is found at the base of outer hair cells. Phalloidin staining (green) is shown in the cuticular plate and distal (apical) portion of the hair bundles. Phalloidin staining is also shown on the hair bundles of inner hair cells. The inset shows a single section where Ocm labeling is restricted to the hair bundle and the basal pole of outer hair cells. c. Colocalization of Ocm (magenta) and CaMK-IV (green) in cochlear outer hair cells in the P10 mouse. Ocm and CaMK-IV colocalized (yellow) to the lateral wall of outer hair cells (OHCs). A projection image shows punctuated regions of colocalization (arrows) as well as non-overlapping regions of labeling (asterisks). d. Colocalization of Ocm (magenta) and CaMK-IV (green) in cochlear hair cells in the P10 rat organ of Corti. A 3d reconstruction of a longitudinal view of three rows of outer hair cells (OHCs) shows Ocm and CaMK-IV concentrated at the peripheral edges of the outer hair cell lateral walls. The arrows identify regions of colocalization (yellow) between Ocm and CaMK-IV.
CNE_22424_sm_suppinfoFig3.tif2.5 MB Supplemental Figure 3 Same as Figure 3. Comparisons of Ca2+ binding protein immunoreactivity in the early postnatal cochlea. a. A confocal image shows a P3 mouse cochlear section from the basal turn. Calretinin (CR, green) and calbindin (CB, grey) label inner and outer hair cells. Ocm (magenta) labels outer hair cells. Afferent fibers below the inner hair cell are labeled by calretinin. Phalloidin (grey) staining is also present. The insets show labeling in the separate confocal channels. The scale bar represents 10 μm. b. A confocal image of a section taken from the basal turn of a P6 mouse cochlea labeled with phalloidin (green), Ocm (magenta), and calretinin (grey). The scale bar represents 10 μm. c. A confocal image shows a section from the midbasal turn of a P8 mouse cochlea. Parvalbumin (green) labels only the inner hair cells whereas Ocm (magenta) labels only the outer hair cells. The arrow identifies lateral wall labeling. The scale bar represents 15 μm.
CNE_22424_sm_suppinfoFig4.tif4.3 MB Supplemental Figure 4 Same as Figure 4. Patterns of Ocm labeling across vestibular organs. a. A confocal image taken from a 6-week-old mouse utricle labeled for Ocm (magenta) and phalloidin (green). The scale bar represents 100 μm. b. A confocal image taken from an 8-month-old mouse utricle labeled for Ocm (magenta) and phalloidin (green). The scale bar represents 50 μm. c. A 3d-reconstruction Ocm labeling in an adult mouse utricular striola shows Ocm labeling throughout the cytoplasm and hair bundle (arrows). The cells are predominately flask shaped. In contrast to the tight cluster at P3, the Ocm labeled hair cells are interspersed with non-Ocm labeled cells represented by their phalloidin-labeled hair bundles. d. A 3d reconstruction of the central zone of the cristae from an adult mouse shows Ocm (magenta) labeling and phalloidin (green) staining. Ocm labels throughout the cytoplasm and hair bundle (arrow). The scale bar represents 40 μm. e. Ocm and CaMK-IV immunoreactivity in hair cells of the utricular striola. In the P10 mouse utricle the CaMK-IV labeling was mostly concentrated to the narrowed neck region of the hair cell and Ocm labeling was diffuse throughout the cytoplasm. f. In the striola of the P10 rat utricle CaMK-IV labeling was punctate along the basolateral surface whereas Ocm was diffuse. Scale bar, 10 μm.
CNE_22424_sm_suppinfoFig5.tif3.9 MB Supplemental Figure 5 Same as Figure 5. a. A 3d reconstruction of an adult mouse utricle (Utr) and two cristae (Crst) shows labeling for phalloidin (green), Ocm (magenta), and calretinin (grey). The scale bars represent 100 μm. b, c, d. 3d reconstructions of the inset taken from the adult mouse utricle (a) identify the line of polarity reversal (solid white line). The scale bar represents 40 μm in b, c, and d. Using only the phalloidin (green) channel (b), the line of polarity reversal is shown. The arrows indicate hair bundle polarity vectors (i.e., direction of the kinocilium). The line divides the utricle into medial (M) and lateral (L) regions. The addition of the Ocm (magenta) channel (c) reveals the relation between Ocm-positive hair cells and the line of polarity reversal. The addition of the calretinin (grey) channel (d) reveals the calretinin positive calyces. e. Confocal image of an adult mouse cristae section. Calretinin (green) labels afferent calyces. Ocm (magenta) labels a central band of hair cells. Calbindin intensely labels calyx endings of hair cells in the adult mouse cristae. The hair bundles and cuticular plates are labeled with phalloidin (cyan). The scale bar represents 10 μm. f. A confocal image shows an adult mouse reconstruction of the utricular striola. Parvalbumin (green) labels calyx type endings and Ocm labels a center band of hair cells. The inset identifies two Ocm-positive cells that are contacted by a parvalbumin-labeled complex ending. The scale bar represents 20 μm.
CNE_22424_sm_suppinfoFig6.tif2.5 MB Supplemental Figure 6 Confocal reconstructions of Ocm labeling in the chinchilla inner ear. The scale bars represent 100 μm. An adult chinchilla utricle (a), sacculus (b), and cristae (c) show Ocm (magenta) labeling and phalloidin (green) staining.
CNE_22424_sm_suppinfoFig7.tif2.2 MB Supplemental Figure 7 Same as Figure 7. a. A flat mount of a normal E18 inner ear with utricle, saccule and cristae labeled for tubulin (grey), Ocm (magenta), and phalloidin staining (blue). Scale bar represents 100 μm. b. A flat mount of an E18 Looptail mutant inner ear labeled for tubulin (grey), Ocm (magenta), and phalloidin staining (blue) as above. Scale bar represents 40 μm. Ocm immunoreactivity is found in a subset of tightly clustered hair cells in an E18 utricle of the Looptail mutant. c. A 3d reconstruction of Ocm labeling (magenta) in the utricular striola is diffuse and extends throughout the cell body. Neurofilament-positive endings (grey) surround the Ocm-labeled hair cells. The scale bar represents 30 μm.

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