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Mehmet Asoglu,1 Mehmet Aslan,2 Okan Imre,1 Yuksel Kivrak,3 Oznur Akil,1 Emin Savik,4 Hasan Buyukaslan,5 Ulker Fedai,1 Abdurrahman Altındag6 1Department of Psychiatry, Faculty of Medicine, Harran University, Sanliurfa, 2Department of Internal Medicine, Faculty of Medicine, Yuzuncu Yil University, Van, 3Department of Psychiatry, Faculty of Medicine, Kafkas University, Kars, 4Department of Clinical Biochemistry, Faculty of Medicine, Harran University, 5Department of Emergency Medicine, Faculty of Medicine, Harran University, Sanliurfa, 6Department of Psychiatry, Faculty of Medicine, Gaziantep University, Gaziantep, Turkey Background: As the relationship between psychological stress and platelet activation has been widely studied in recent years, activated platelets lead to certain biochemical changes, which occur in the brain in patients with mental disorders. However, data relating to the mean platelet volume (MPV) in patients with panic disorder (PD) are both limited and controversial. Herein, we aimed to evaluate, for the first time, the red cell distribution width (RDW) levels combined with MPV levels in patients with PD.Patients and methods: Between January 2012 and June 2015, data of 30 treatment-naïve patients (16 females, 14 males; mean age: 37±10 years; range: 18–59 years) who were diagnosed with PD and 25 age- and sex-matched healthy volunteers (10 females, 15 males; mean age: 36±13 years; range: 18–59 years) (control group) were retrospectively analyzed. The white blood cell count (WBC), MPV, and RDW levels were measured in both groups.Results: The mean WBC, MPV, and RDW levels were 9,173.03±2,400.31/mm3, 8.19±1.13 fl, and 12.47±1.14%, respectively, in the PD group. These values were found to be 7,090.24±1,032.61, 6.85±0.67, and 11.63±0.85, respectively, in the healthy controls. The WBC, MPV, and RDW levels were significantly higher in the patients with PD compared to the healthy controls (P=0.001, P=0.001, and P=0.003, respectively). However, there was no significant difference in the platelet number between the patients with PD and healthy controls (P>0.05).Conclusion: Our study results are the first to demonstrate that the RDW levels combined with MPV levels significantly increase among patients with PD. We believe that increased RDW and MPV levels can be used as a novel marker for PD. Keywords: panic disorder, mean platelet volume, red cell distribution width, white blood cell count, platelet number

Normal hearing requires exquisite cooperation between bony and sensorineural structures within the cochlea. For example, the inner ear secretes proteins such as osteoprotegrin (OPG) that can prevent cochlear bone remodeling. Accordingly, diseases that affe

Adeno-associated virus (AAV)-mediated gene therapy has evolved from the bench to the bedside, and is now considered the therapy of choice for certain inherited diseases. AAVs are attractive vectors for several reasons: they are nonpathogenic, result in long-term transgene expression, have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms allowing the targeting of specific cell types. However, one of the greatest limitations of AAVs is the limited genome-packaging capacity of ∼4.7 kb. Given that numerous diseases are caused by mutations in genes with coding sequences exceeding this capacity, packaging into a single AAV capsid is currently unfeasible for larger genes. Taking advantage of the AAV genome's ability to concatemerize, multiple strategies have been explored to overcome the size limit of AAV vectors. One strategy is to split large transgenes into two or three parts, generating dual or triple AAV vectors. Coinfection of a cell with these two or three AAVs will then, through a variety of mechanisms, result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector. This review: 1) documents AAV dual and triple vector strategies currently employed in a variety of tissues, and highlights the advantages and disadvantages of each method; 2) describes the first successful studies using the dual vector approach to restore hearing and prevent deafness in a mouse model of non-syndromic deafness due to absence of the otoferlin protein function, and the implications of these findings for the future of gene therapy in the human inner ear; and 3) highlights additional different deafness genes that could be potential future targets for gene therapy using the dual vector approach., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hearing impairment is a major health and economic concern worldwide. Currently, the cochlear implant (CI) is the standard of care for remediation of severe to profound hearing loss, and in general, contemporary CIs are highly successful. But there is great variability in outcomes among individuals, especially in children, with many CI users deriving much less or even marginal benefit. Much of this variability is related to differences in auditory nerve survival, and there has been substantial interest in recent years in exploring potential therapies to improve survival of the cochlear spiral ganglion neurons (SGN) after deafness. Preclinical studies using osmotic pumps and other approaches in deafened animal models to deliver neurotrophic factors (NTs) directly to the cochlea have shown promising results, especially with Brain-Derived Neurotrophic Factor (BDNF). More recent studies have focused on the use of NT gene therapy to force expression of NTs by target cells within the cochlea. This could provide the means for a one-time treatment to promote long-term NT expression and improve neural survival after deafness. This review summarizes the evidence for the efficacy of exogenous NTs in preventing SGN degeneration after hearing loss and reviews the animal research to date suggesting that NT gene therapy can elicit long-term NT expression in the cochlea, resulting in significantly improved SGN and radial nerve fiber survival after deafness. In addition, we discuss NT gene therapy in other non-auditory applications and consider some of the remaining issues with regard to selecting optimal vectors, timing of treatment, and place/method of delivery, etc. that must be resolved prior to considering clinical application., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The consequences of neonatal white matter injury are devastating and represent a major societal problem as currently there is no cure. Prematurity, low weight birth and maternal pre-natal infection are the most frequent causes of acquired myelin deficiency in the human neonate leading to cerebral palsy and cognitive impairment. In the developing brain, oligodendrocyte (OL) maturation occurs perinatally, and immature OLs are particularly vulnerable. Cell replacement therapy is often considered a viable option to replace progenitors that die due to glutamate excitotoxicity. We previously reported directed specification and mobilization of endogenous committed and uncommitted neural progenitors by the combination of transferrin and insulin growth factor 1 (TSC1). Here, considering cell replacement and integration as therapeutic goals, we examined if OL progenitors (OLPs) grafted into the brain parenchyma of mice that were subjected to an excitotoxic insult could rescue white matter injury. For that purpose, we used a well-established model of glutamate excitotoxic injury. Four-day-old mice received a single intraparenchymal injection of the glutamate receptor agonist N-methyl-D-aspartate alone or in conjunction with TSC1 in the presence or absence of OLPs grafted into the brain parenchyma. Energetics and expression of stress proteins and OL developmental specific markers were examined. A comparison of the proteomic profile per treatment was also ascertained. We found that OLPs did not survive in the excitotoxic environment when grafted alone. In contrast, when combined with TSC1, survival and integration of grafted OLPs was observed. Further, energy metabolism in OLPs was significantly increased by N-methyl-D-aspartate and modulated by TSC1. The proteomic profile after the various treatments showed elevated ubiquitination and stress/heat shock protein 90 in response to N-methyl-D-aspartate. These changes were reversed in the presence of TSC1 and ubiquitination was decreased. The results obtained in this pre-clinical study indicate that the use of a combinatorial intervention including both trophic support and healthy OLPs constitutes a promising approach for long-term survival and successful graft integration. We established optimal conditioning of the host brain environment to promote long-term survival and integration of grafted OLPs into an inflamed neonate host brain. Experimental procedures were performed under the United States Public Health Service Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee at (UCLA) (ARC #1992-034-61) on July 1, 2010., Competing Interests: None

Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin-1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT-PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single-cell RNA-sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock-in mouse with a hemagglutinin (HA) epitope-tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1-specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Over 450 million people worldwide suffer from hearing loss, leading to an estimated economic burden of ∼$750 billion. The past decade has seen significant advances in the understanding of the molecular mechanisms that contribute to hearing, and the environmental and genetic factors that can go awry and lead to hearing loss. This in turn has sparked enormous interest in developing gene therapy approaches to treat this disorder. This review documents the most recent advances in cochlear gene therapy to restore hearing loss, and will cover viral vectors and construct designs, potential routes of delivery into the inner ear, and, lastly, the most promising genes of interest., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)

Outcomes with contemporary cochlear implants (CI) depend partly upon the survival and condition of the cochlear spiral ganglion (SG) neurons. Previous studies indicate that CI stimulation can ameliorate SG neural degeneration after deafness, and brain-derived neurotrophic factor (BDNF) delivered by an osmotic pump can further improve neural survival. However, direct infusion of BDNF elicits undesirable side effects, and osmotic pumps are impractical for clinical application. In this study, we explored the potential for two adeno-associated viral vectors (AAV) to elicit targeted neurotrophic factor expression in the cochlea and promote improved SG and radial nerve fiber survival. Juvenile cats were deafened prior to hearing onset by systemic aminoglycoside injections. Auditory brainstem responses showed profound hearing loss by 16-18 days postnatal. At ~ 4 weeks of age, AAV2-GFP (green fluorescent protein), AAV5-GFP, AAV2-hBDNF, or AAV5-hGDNF (glial-derived neurotrophic factor) was injected through the round window unilaterally. For GFP immunofluorescence, animals were studied ~ 4 weeks post-injection to assess cell types transfected and their distributions. AAV2-GFP immunofluorescence demonstrated strong expression of the GFP reporter gene in residual inner (IHCs), outer hair cells (OHCs), inner pillar cells, and in some SG neurons throughout the cochlea. AAV5-GFP elicited robust transduction of IHCs and some SG neurons, but few OHCs and supporting cells. After AAV-neurotrophic factor injections, animals were studied ~ 3 months post-injection to evaluate neural survival. AAV5-hGDNF elicited a modest neurotrophic effect, with 6 % higher SG density, but had no trophic effect on radial nerve fiber survival, and undesirable ectopic fiber sprouting occurred. AAV2-hBDNF elicited a similar 6 % increase in SG survival, but also resulted in greatly improved radial nerve fiber survival, with no ectopic fiber sprouting. A further study assessed whether AAV2-hBDNF neurotrophic effects would persist over longer post-injection periods. Animals examined 6 months after virus injection showed substantial neurotrophic effects, with 14 % higher SG density and greatly improved radial nerve fiber survival. Our results suggest that AAV-neurotrophin gene therapy can elicit expression of physiological concentrations of neurotrophins in the cochlea, supporting improved SG neuronal and radial nerve fiber survival while avoiding undesirable side effects. These studies also demonstrate the potential for application of cochlear gene therapy in a large mammalian cochlea comparable to the human cochlea and in an animal model of congenital/early acquired deafness.

Autosomal recessive genetic forms (DFNB) account for most cases of profound congenital deafness. Adeno-associated virus (AAV)-based gene therapy is a promising therapeutic option, but is limited by a potentially short therapeutic window and the constrained packaging capacity of the vector. We focus here on the otoferlin gene underlying DFNB9, one of the most frequent genetic forms of congenital deafness. We adopted a dual AAV approach using two different recombinant vectors, one containing the 5' and the other the 3' portions of otoferlin cDNA, which exceed the packaging capacity of the AAV when combined. A single delivery of the vector pair into the mature cochlea of Otof -/- mutant mice reconstituted the otoferlin cDNA coding sequence through recombination of the 5' and 3' cDNAs, leading to the durable restoration of otoferlin expression in transduced cells and a reversal of the deafness phenotype, raising hopes for future gene therapy trials in DFNB9 patients., Competing Interests: Conflict of interest statement: W.W.H. and the University of Florida (UF) have a financial interest in the use of AAV therapies, and own equity in a company, Applied Genetic Technologies Corp. (AGTC), that might, in the future, commercialize some aspects of this work, and a joint international patent application (International Patent Application No.: PCT/US2018/031009 - WGS “Whole Genome Sequencing” Ref. U1197.70110WO00) has been submitted by UF and University of California, San Francisco (F.D., W.W.H., O.A.)., (Copyright © 2019 the Author(s). Published by PNAS.)

Aim: To better elucidate the cellular dynamics by which perforations in the tympanic membrane (TM) are healed., Background: Under normal conditions, epidermal cells are born and then migrate radially outward from the malleus in the TM. It is unknown what the relative contribution of newly proliferated cells from different lineages is in the healing of TM perforations., Methods: Thirty-six female mice were used in this study. Ethynyl deoxyuridine, a thymidine analogue that labels newly proliferated cells, was injected intraperitoneally into each mouse and then subsequently supplied in the drinking water. Acute perforations were performed on the right TM and the left TM served as the control and remained intact. The animals were sacrificed at six time points between 2 hours and 6 days. We stained for proliferative, epithelial, mesenchymal markers, and ethynyl deoxyuridine and analyzed the distribution of cells., Results: In control TMs, newly proliferated cells were detected around the malleus handle and then migrated radially outward. Perforated TMs had a significantly higher number of newly proliferated cells throughout the tympanic membrane with a marked proliferative response of epithelial, mesenchymal, and mucosal cells in the region of the malleus and perforation. The majority of cells in the healed perforation were newly proliferated. In the anterior TM opposite the perforation, an increased turnover of keratinocytes was noted, but not mesenchymal cells., Conclusions: Perforation of the TM alters the cellular dynamics throughout the entire TM, rather than simply adjacent to the perforation. This argues for long distance signaling occurring in the perforated TM.

Contemporary cochlear implants (CI) are generally very effective for remediation of severe to profound sensorineural hearing loss, but outcomes are still highly variable. Auditory nerve survival is likely one of the major factors underlying this variability. Neurotrophin therapy therefore has been proposed for CI recipients, with the goal of improving outcomes by promoting improved survival of cochlear spiral ganglion neurons (SGN) and/or residual hair cells. Previous studies have shown that glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor, and neurotrophin-3 can rescue SGNs following insult. The current study was designed to determine whether adeno-associated virus vector serotype 5 (AAV-5) encoding either green fluorescent protein or GDNF can transduce cells in the mouse cochlea to express useful levels of neurotrophin and to approximate the optimum therapeutic dose(s) for transducing hair cells and SGN. The findings demonstrate that AAV-5 is a potentially useful gene therapy vector for the cochlea, resulting in extremely high levels of transgene expression in the cochlear inner hair cells and SGN. However, overexpression of human GDNF in newborn mice caused severe neurological symptoms and hearing loss, likely due to Purkinje cell loss and cochlear nucleus pathology. Thus, extremely high levels of transgene protein expression should be avoided, particularly for proteins that have neurological function in neonatal subjects.

Cochlear gene therapy has made tremendous strides over the past 5 years. The first study documenting successful restoration of congenital hearing loss using AAV-mediated gene therapy occurred in a mouse model of deafness lacking vesicular glutamate transporter 3 (VGLUT 3). This study utilized a trans-bulla round window membrane (RWM) delivery approach. Since this study, these methodologies have been applied to a number of other mouse models of genetic deafness with varying degrees of success, lending promise for future clinical application of this burgeoning technology. Here we describe a method of virally mediated gene delivery into the cochlear scala tympani through the RWM. This method involves negligible damage to essential structures of the middle and inner ear while preserving hearing. The efficacy of this surgical technique will be demonstrated by the restoration of hearing to the VGLUT3 knockout mice (a mouse model of congenital deafness) after delivery of VGLUT3 gene to the inner ear using an adeno-associated virus as a vector.

Hearing loss is a significant public health concern, affecting over 250 million people worldwide. Both genetic and environmental etiologies are linked to hearing loss, but in many cases the underlying cellular pathophysiology is not well understood, highlighting the importance of further discovery. We found that inactivation of the gene Tmtc4 (transmembrane and tetratricopeptide repeat 4), which was broadly expressed in the mouse cochlea, caused acquired hearing loss in mice. Our data showed Tmtc4 enriched in the endoplasmic reticulum, and that it functioned by regulating Ca2+ dynamics and the unfolded protein response (UPR). Given this genetic linkage of the UPR to hearing loss, we demonstrated a direct link between the more common noise-induced hearing loss (NIHL) and the UPR. These experiments suggested a novel approach to treatment. We demonstrated that the small-molecule UPR and stress response modulator ISRIB (integrated stress response inhibitor), which activates eIF2B, prevented NIHL in a mouse model. Moreover, in an inverse genetic complementation approach, we demonstrated that mice with homozygous inactivation of both Tmtc4 and Chop had less hearing loss than knockout of Tmtc4 alone. This study implicated a novel mechanism for hearing impairment, highlighting a potential treatment approach for a broad range of human hearing loss disorders.

Clarin-1, a tetraspan-like membrane protein defective in Usher syndrome type IIIA (USH3A), is essential for hair bundle morphogenesis in auditory hair cells. We report a new synaptic role for clarin-1 in mouse auditory hair cells elucidated by characterization of Clrn1 total (Clrn1ex4-/-) and postnatal hair cell-specific conditional (Clrn1ex4fl/fl Myo15-Cre+/-) knockout mice. Clrn1ex4-/- mice were profoundly deaf, whereas Clrn1ex4fl/fl Myo15-Cre+/- mice displayed progressive increases in hearing thresholds, with, initially, normal otoacoustic emissions and hair bundle morphology. Inner hair cell (IHC) patch-clamp recordings for the 2 mutant mice revealed defective exocytosis and a disorganization of synaptic F-actin and CaV1.3 Ca2+ channels, indicative of a synaptopathy. Postsynaptic defects were also observed, with an abnormally broad distribution of AMPA receptors associated with a loss of afferent dendrites and defective electrically evoked auditory brainstem responses. Protein-protein interaction assays revealed interactions between clarin-1 and the synaptic CaV1.3 Ca2+ channel complex via the Cavβ2 auxiliary subunit and the PDZ domain-containing protein harmonin (defective in Usher syndrome type IC). Cochlear gene therapy in vivo, through adeno-associated virus-mediated Clrn1 transfer into hair cells, prevented the synaptic defects and durably improved hearing in Clrn1ex4fl/fl Myo15-Cre+/- mice. Our results identify clarin-1 as a key organizer of IHC ribbon synapses, and suggest new treatment possibilities for USH3A patients.

Neuronal synapse formation and remodeling are essential to central nervous system (CNS) development and are dysfunctional in neurodevelopmental diseases. Innate immune signals regulate tissue remodeling in the periphery, but how this affects CNS synapses is largely unknown. Here, we show that the interleukin-1 family cytokine interleukin-33 (IL-33) is produced by developing astrocytes and is developmentally required for normal synapse numbers and neural circuit function in the spinal cord and thalamus. We find that IL-33 signals primarily to microglia under physiologic conditions, that it promotes microglial synapse engulfment, and that it can drive microglial-dependent synapse depletion in vivo. These data reveal a cytokine-mediated mechanism required to maintain synapse homeostasis during CNS development., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Our understanding of the mechanisms underlying inherited forms of inner ear deficits has considerably improved during the past 20 y, but we are still far from curative treatments. We investigated gene replacement as a strategy for restoring inner ear functions in a mouse model of Usher syndrome type 1G, characterized by congenital profound deafness and balance disorders. These mice lack the scaffold protein sans, which is involved both in the morphogenesis of the stereociliary bundle, the sensory antenna of inner ear hair cells, and in the mechanoelectrical transduction process. We show that a single delivery of the sans cDNA by the adenoassociated virus 8 to the inner ear of newborn mutant mice reestablishes the expression and targeting of the protein to the tips of stereocilia. The therapeutic gene restores the architecture and mechanosensitivity of stereociliary bundles, improves hearing thresholds, and durably rescues these mice from the balance defects. Our results open up new perspectives for efficient gene therapy of cochlear and vestibular disorders by showing that even severe dysmorphogenesis of stereociliary bundles can be corrected., Competing Interests: Conflict of interest statement: A patent involving A.E., C.P., and S.S. (PCT/EP2016/053613) has been deposited by the Institut Pasteur, INSERM, and CNRS.

Bone remodeling, a combination of bone resorption and formation, requires precise regulation of cellular and molecular signaling to maintain proper bone quality. Whereas osteoblasts deposit and osteoclasts resorb bone matrix, osteocytes both dynamically resorb and replace perilacunar bone matrix. Osteocytes secrete proteases like matrix metalloproteinase-13 (MMP13) to maintain the material quality of bone matrix through perilacunar remodeling (PLR). Deregulated bone remodeling impairs bone quality and can compromise hearing since the auditory transduction mechanism is within bone. Understanding the mechanisms regulating cochlear bone provides unique ways to assess bone quality independent of other aspects that contribute to bone mechanical behavior. Cochlear bone is singular in its regulation of remodeling by expressing high levels of osteoprotegerin. Since cochlear bone expresses a key PLR enzyme, MMP13, we examined whether cochlear bone relies on, or is protected from, osteocyte-mediated PLR to maintain hearing and bone quality using a mouse model lacking MMP13 (MMP13(-/-)). We investigated the canalicular network, collagen organization, lacunar volume via micro-computed tomography, and dynamic histomorphometry. Despite finding defects in these hallmarks of PLR in MMP13(-/-) long bones, cochlear bone revealed no differences in these markers, nor hearing loss as measured by auditory brainstem response (ABR) or distortion product oto-acoustic emissions (DPOAEs), between wild type and MMP13(-/-) mice. Dynamic histomorphometry revealed abundant PLR by tibial osteocytes, but near absence in cochlear bone. Cochlear suppression of PLR corresponds to repression of several key PLR genes in the cochlea relative to long bones. These data suggest that cochlear bone uniquely maintains bone quality and hearing independent of MMP13-mediated osteocytic PLR. Furthermore, the cochlea employs parallel mechanisms to inhibit remodeling by osteoclasts and osteoblasts, and by osteocytes, to protect hearing. Understanding the cellular and molecular mechanisms that confer site-specific control of bone remodeling has the potential to elucidate new pathways that are deregulated in skeletal disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The auditory brainstem response (ABR) test provides information about the inner ear (cochlea) and the central pathways for hearing. The ABR reflects the electrical responses of both the cochlear ganglion neurons and the nuclei of the central auditory pathway to sound stimulation (Zhou et al. , 2006; Burkard et al. , 2007). The ABR contains 5 identifiable wave forms, labeled as I-V. Wave I represents the summated response from the spiral ganglion and auditory nerve while waves II-V represent responses from the ascending auditory pathway. The ABR is recorded via electrodes placed on the scalp of an anesthetized animal. ABR thresholds refer to the lowest sound pressure level (SPL) that can generate identifiable electrical response waves. This protocol describes the process of measuring the ABR of small rodents (mouse, rat, guinea pig, etc. ), including anesthetizing the mouse, placing the electrodes on the scalp, recording click and tone burst stimuli and reading the obtained waveforms for ABR threshold values. As technology continues to evolve, ABR will likely provide more qualitative and quantitative information regarding the function of the auditory nerve and brainstem pathways involved in hearing.

Objectives/hypothesis: Saposins are small proteins derived from a precursor protein, prosaposin. Each of the four saposins (A-D) is necessary for the activity of lysosomal glycosphingolipid hydrolases. Individual saposin mutations lead to lysosomal storage diseases, some of which are associated with hearing loss. Here we evaluate the effects of the loss of saposins C and D on auditory and vestibular function in transgenic mice., Methods: Transgenic mice with either loss of saposin C function or a combined loss of saposin C + D function were studied. Light microscopy and immunofluorescence were used to evaluate histologic and morphologic changes in the auditory and vestibular organs. Acoustic brainstem response thresholds and distortion product otoacoustic emissions were used to study the auditory phenotype., Results: A null mutation of saposin C did not result in any identifiable histologic changes or loss of hearing through postnatal day 55. Combined losses of saposins C and D similarly did not result in any changes in organ of Corti histology or loss of hearing. However, inclusions within the vestibular end organs was noted, consistent with afferent and efferent neuronal sprouting, although to a much milder degree than seen in the previously studied prosaposin knockout mouse., Conclusions: Loss of saposin C and D function, although causing mild phenotypic changes in the vestibular end organs, otherwise results in minimal functional impairment and no changes in the auditory system. It is more likely that the auditory and vestibular effects of the loss of prosaposin are mediated through the actions of saposin A and/or B., Level of Evidence: NA., (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

Normal hearing requires exquisite cooperation between bony and sensorineural structures within the cochlea. For example, the inner ear secretes proteins such as osteoprotegrin (OPG) that can prevent cochlear bone remodeling. Accordingly, diseases that affect bone regulation can also result in hearing loss. Patients with fibrous dysplasia develop trabecular bone overgrowth resulting in hearing loss if the lesions affect the temporal bones. Unfortunately, the mechanisms responsible for this hearing loss, which could be sensorineural and/or conductive, remain unclear. In this study, we used a unique transgenic mouse model of increased Gs G-protein coupled receptor (GPCR) signaling induced by expression of an engineered receptor, Rs1, in osteoblastic cells. These ColI(2.3)+/Rs1+ mice showed dramatic bone lesions that histologically and radiologically resembled fibrous dysplasia. We found that ColI(2.3)+/Rs1+ mice showed progressive and severe conductive hearing loss. Ossicular chain impingement increased with the size and number of dysplastic lesions. While sensorineural structures were unaffected, ColI(2.3)+/Rs1+ cochleae had abnormally high osteoclast activity, together with elevated tartrate resistant acid phosphatase (TRAP) activity and receptor activator of nuclear factor kappa-B ligand (Rankl) mRNA expression. ColI(2.3)+/Rs1+ cochleae also showed decreased expression of Sclerostin (Sost), an antagonist of the Wnt signaling pathway that normally increases bone formation. The osteocyte canalicular networks of ColI(2.3)+/Rs1+ cochleae were disrupted and showed abnormal osteocyte morphology. The osteocytes in the ColI(2.3)+/Rs1+ cochleae showed increased expression of matrix metalloproteinase 13 (MMP-13) and TRAP, both of which can support osteocyte-mediated peri-lacunar remodeling. Thus, while the ossicular chain impingement is sufficient to account for the progressive hearing loss in fibrous dysplasia, the deregulation of bone remodeling extends to the cochlea as well. Our findings suggest that factors regulating bone remodeling, including peri-lacunar remodeling by osteocytes, may be useful targets for treating the bony overgrowths and hearing changes of fibrous dysplasia and other bony pathologies.

This protocol comprises the entire process of immunofluorescence staining on mouse cochlea whole mount, starting from tissue preparation to the mounting of the tissue. This technique provides "three-dimensional" views of the stained components in order to determine the localization of a protein of interest in the tissue in its natural state and environment.

Objectives: This study was conducted to compare morphologic and audiologic changes after noise exposure in two different strains of mice (CBA and C57) and to create morphologically proven models of noise-induced hearing loss., Methods: Mice were exposed to white noise at 110-dB sound-pressure level for 60 minutes at the age of 1 month. Hearing thresholds and outer hair cell functions were evaluated by auditory brainstem response recordings and distortion product otoacoustic emission immediately and 22 days after noise exposure. Cochlear pathology was observed and compared by light and electron microscopic studies., Results: Both mice strains showed hearing threshold shifts with decreased outer hair cell function immediately and 22 days after noise exposure. More severe auditory brainstem response threshold shifts were observed in C57 mice compared with CBA mice at click, 8-, 16-, and 32-kHz tone-burst stimuli. A cochlear morphologic study demonstrated predominant outer hair cell degeneration at all turns of the cochlea; degeneration was most severe at the basal turn in both mice strains. A scanning electron microscopic study revealed more severe ultrastructural damage of outer hair cells at each turn of the cochlea in C57 mice. The lateral wall of the cochlea was more severely degenerated in CBA mice., Conclusion: Both mice strains showed consistent, permanent noise-induced hearing loss with different susceptibilities and site vulnerabilities. Further studies to investigate the mechanism of the different degree and cochlear site vulnerability to noise exposure between two mice strains are necessary., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Mice lacking the vesicular glutamate transporter-3 (VGLUT3) are congenitally deaf due to loss of glutamate release at the inner hair cell afferent synapse. Cochlear delivery of VGLUT3 using adeno-associated virus type 1 (AAV1) leads to transgene expression in only inner hair cells (IHCs), despite broader viral uptake. Within 2 weeks of AAV1-VGLUT3 delivery, auditory brainstem response (ABR) thresholds normalize, along with partial rescue of the startle response. Lastly, we demonstrate partial reversal of the morphologic changes seen within the afferent IHC ribbon synapse. These findings represent a successful restoration of hearing by gene replacement in mice, which is a significant advance toward gene therapy of human deafness., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Prosaposin, a precursor of four glycoprotein activators (saposin A, B, C and D) for lysosomal hydrolases, has previously been shown to be important for normal adult cochlear innervation and the maintenance of normal hearing. In these studies, we now investigate prosaposin in normal vestibular epithelium and the functional impairment of balance caused by prosaposin ablation. In normal mice, prosaposin is localized to all 3 vestibular end-organs (ampullae, saccule, and utricle) and Scarpa's ganglion by RT-PCR, Western blot analysis and immunofluorescence. Ablation of prosaposin function caused severe vestibular dysfunction on a battery of behavioral tasks. Histologically, the KO mice demonstrated an exuberant cellular proliferation below the vestibular hair cells with disruption of the supporting cells. Electron microscopy further demonstrated inclusion bodies and cellular proliferation disturbing the normal neuroepithelial structure of the vestibular end-organs. Lastly, immunofluorescence (neurofilament 200 and synaptophysin) staining suggests that this cellular proliferation corresponds to afferent and efferent neuronal overgrowth. These data suggest that prosaposin plays a role not only in the maintenance of normal hearing but also an important role in the neuronal maturation processes of the vestibular sensory epithelium and the maintenance of normal vestibular system function., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Objectives/hypothesis: Efferent nerves under the outer hair cells (OHCs) play a role in the protection of these cells from loud stimuli. Previously, we showed that cochlear α-synuclein expression is localized to efferent auditory synapses at the base of the OHCs. To prove our hypothesis that α-synuclein deficiency and efferent auditory deficit might be a cause of hearing loss, we compared the morphology of efferent nerve endings and α-synuclein expression within the cochleae of two mouse models of presbycusis., Study Design: Comparative animal study of presbycusis., Methods: The C57BL/6J(C57) mouse strain, a well-known model of early-onset hearing loss, and the CBA mouse strain, a model of relatively late-onset hearing loss, were examined. Auditory brainstem responses and distortion product otoacoustic emissions were recorded, and cochlear morphology with efferent nerve ending was compared. Western blotting was used to examine α-synuclein expression in the cochlea., Results: Compared with CBA mice, C57 mice showed earlier onset high-frequency hearing loss and decreased function in OHCs, especially within high-frequency regions. C57 mice demonstrated more severe pathologic changes within the cochlea, particularly within the basal turn, than CBA mice of the same age. Weaker α-synuclein and synaptophysin expression in the efferent nerve endings and cochlear homogenates in C57 mice was observed., Conclusions: Our results support the hypothesis that efferent nerve degeneration, possibly due to differential α-synuclein expression, is a potential cause of early-onset presbycusis. Further studies at the cellular level are necessary to verify our results., (Copyright © 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Physical cues, such as extracellular matrix stiffness, direct cell differentiation and support tissue-specific function. Perturbation of these cues underlies diverse pathologies, including osteoarthritis, cardiovascular disease and cancer. However, the molecular mechanisms that establish tissue-specific material properties and link them to healthy tissue function are unknown. We show that Runx2, a key lineage-specific transcription factor, regulates the material properties of bone matrix through the same transforming growth factor-β (TGFβ)-responsive pathway that controls osteoblast differentiation. Deregulated TGFβ or Runx2 function compromises the distinctly hard cochlear bone matrix and causes hearing loss, as seen in human cleidocranial dysplasia. In Runx2+/⁻ mice, inhibition of TGFβ signalling rescues both the material properties of the defective matrix, and hearing. This study elucidates the unknown cause of hearing loss in cleidocranial dysplasia, and demonstrates that a molecular pathway controlling cell differentiation also defines material properties of extracellular matrix. Furthermore, our results suggest that the careful regulation of these properties is essential for healthy tissue function.

Nothing is known about the regulation of nicotinic acetylcholine receptors (nAChRs) in hair cells of the inner ear. MuSK, rapsyn and RIC-3 are accessory molecules associated with muscle and brain nAChR function. We demonstrate that these accessory molecules are expressed in the inner ear raising the possibility of a muscle-like mechanism for clustering and assembly of nAChRs in hair cells. We focused our investigations on rapsyn and RIC-3. Rapsyn interacts with the cytoplasmic loop of nAChR alpha9 subunits but not nAChR alpha10 subunits. Although rapsyn and RIC-3 increase nAChR alpha9 expression, rapsyn plays a greater role in receptor clustering while RIC-3 is important for acetylcholine-induced calcium responses. Our data suggest that RIC-3 facilitates receptor function, while rapsyn enhances receptor clustering at the cell surface.

The expression of unconventional vesicular glutamate transporter VGLUT3 by neurons known to release a different classical transmitter has suggested novel roles for signaling by glutamate, but this distribution has raised questions about whether the protein actually contributes to glutamate release. We now report that mice lacking VGLUT3 are profoundly deaf due to the absence of glutamate release from hair cells at the first synapse in the auditory pathway. The early degeneration of some cochlear ganglion neurons in knockout mice also indicates an important developmental role for the glutamate released by hair cells before the onset of hearing. In addition, the mice exhibit primary, generalized epilepsy that is accompanied by remarkably little change in ongoing motor behavior. The glutamate release conferred by expression of VGLUT3 thus has an essential role in both function and development of the auditory pathway, as well as in the control of cortical excitability.

After a yeast two-hybrid screen identified prosaposin as a potential interacting protein with the nicotinic acetylcholine receptor (nAChR) subunit alpha10, studies were performed to characterize prosaposin in the normal rodent inner ear. Prosaposin demonstrates diffuse organ of Corti expression at birth, with gradual localization to the inner hair cells (IHCs) and its supporting cells, inner pillar cells, and synaptic region of the outer hair cells (OHCs) and Deiters' cells (DCs) by postnatal day 21 (P21). Microdissected OHC and DC quantitative reverse transcriptase-PCR and immunohistology localizes prosaposin mRNA to DCs and OHCs, and protein predominantly to the apex of the DCs. Subsequent studies in a prosaposin knock-out (KO) (-/-) mouse showed intact but slightly reduced hearing through P19, but deafness by P25 and reduced distortion product otoacoustic emissions from P15 onward. Beginning at P12, the prosaposin KO mice showed histologic organ of Corti changes including cellular hypertrophy in the region of the IHC and greater epithelial ridge, a loss of OHCs from cochlear apex, and vacuolization of OHCs. Immunofluorescence revealed exuberant overgrowth of auditory afferent neurites in the region of the IHCs and proliferation of auditory efferent neurites in the region of the tunnel of Corti. IHC recordings from these KO mice showed normal I-V curves and responses to applied acetylcholine. Together, these results suggest that prosaposin helps maintain normal innervation patterns to the organ of Corti. Furthermore, prosaposin's overlapping developmental expression pattern and binding capacity toward the nAChR alpha10 suggest that alpha10 may also play a role in this function.