surgical-method-for-virally-mediated-gene-delivery-to-mouse-inner-ear

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

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.

Progressive deafness and altered cochlear innervation in knock-out mice lacking prosaposin.

Nachdem ein Hefe-zwei-Hybrid-Bildschirm mit der nikotinische Acetylcholin-Rezeptor (nAChR) Untereinheit alpha10 Prosaposin als potenzielle zusammenwirkendes Protein identifiziert, wurden Studien durchgef&uuml;hrt, um Prosaposin im normalen Nagetier Innenohr zu charakterisieren. Prosaposin veranschaulicht diffuse Organ von Corti Ausdruck bei der Geburt mit schrittweisen Lokalisierung und der inneren Haarzellen (IHCs) und seine unterst&uuml;tzenden Zellen, innere S&auml;ule Zellen und synaptischen Region der &auml;u&szlig;eren Haarzellen (elektrische) und Deiters Zellen (DCs) durch postnatale 21.Tag (P21). Microdissected OHC und DC quantitative Reverse Transkriptase-PCR und Immunhistologisch lokalisiert Prosaposin mRNA DCs und elektrische Protein &uuml;berwiegend an der Spitze des DCs. nachtr&auml;gliche Studies in Prosaposin Knock Out (KO) (- / -) Maus zeigte intakt aber leicht reduziert durch P19, aber Taubheit P25 und geringere Verzerrung Produkt Otoakustische Emissionen von P15 weiter h&ouml;ren. Beginnend bei P12, die Prosaposin KO-M&auml;usen zeigten histologische Organ von Corti &Auml;nderungen einschlie&szlig;lich zellul&auml;rer Hypertrophie in der Region der IHC und gr&ouml;&szlig;ere epitheliale Ridge, ein Verlust von elektrische von cochlear Spitze und Vakuolisierung der elektrische. Immunfluoreszenz ergab &uuml;berschw&auml;nglich &Uuml;berwuchterung des auditorischen afferente Neurites in der Region der IHCs und Vermehrung von auditiven ableitendes Neurites in der Region des Tunnels von Corti. IHC-Mitschnitte dieser KO-M&auml;usen zeigten normale-V-Kurven und Reaktionen auf angewandte Acetylcholin. Diese Ergebnisse empfehlen zusammen, dass Prosaposin hilft normale Innervation Muster auf das Organ von Corti zu pflegen. Dar&uuml;ber hinaus empfehlen Prosaposin der &uuml;berlappende Entwicklungsst&ouml;rungen Ausdrucksmuster und verbindliche Kapazit&auml;t gegen&uuml;ber der nAChR alpha10, dass alpha10 in dieser Funktion auch eine Rolle spielen kann.

Progrediente Schwerh&ouml;rigkeit und ver&auml;nderte cochlear Innervation im Knock-out-M&auml;use Prosaposin fehlt.

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.

Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3.

Der Ausdruck des unkonventionellen vesikul&auml;ren Glutamat-Transporters VGLUT3 von Neuronen bekannt, einen anderen klassischen Sender ver&ouml;ffentlichen neue Rollen f&uuml;r die Signalisierung durch Glutamat, aber diese Verteilung vorgeschlagen hat hat ausgel&ouml;st, Fragen, ob das Protein tats&auml;chlich zu Glutamat Release beitr&auml;gt. Wir berichten nun, dass M&auml;usen fehlt VGLUT3 aufgrund des Fehlens von Glutamat-Freisetzung aus Haarzellen bei der ersten Synapse in der auditiven Pathway profund taub sind. Die fr&uuml;he Degeneration von einigen cochlear Ganglion-Neuronen im Knock-out-M&auml;use gibt auch Entwicklungsst&ouml;rungen ma&szlig;geblich f&uuml;r das Glutamat von Haarzellen vor Beginn der Anh&ouml;rung ver&ouml;ffentlicht. Dar&uuml;ber hinaus zeigen die M&auml;use, prim&auml;re Generalisierte Epilepsie, die mit einem bemerkenswert geringe &Auml;nderung im laufenden motor Verhalten einhergeht. Die Glutamat-Freisetzung aus der Ausdruck der VGLUT3 hat somit eine wesentliche Rolle, Funktion und Entwicklung des auditiven Signalweges sowie bei der Kontrolle der kortikale Erregbarkeit.

Schallempfindungs-Schwerh&ouml;rigkeit und Ergreifungen in M&auml;usen vesikul&auml;ren Glutamat Transporter 3 fehlt.

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.

Muscle-like nicotinic receptor accessory molecules in sensory hair cells of the inner ear.

The goal of this study was to determine the role of an influx copper transporter, CTR1, in the ototoxicity induced by cisplatin, a potent anticancer platinum analog used in the treatment of a variety of solid tumors. As determined through reverse transcriptase-PCR (RT-PCR), quantitative RT-PCR, Western blot, and immunohistochemistry, mouse CTR1 (Ctr1) was found to be abundantly expressed and highly localized at the primary sites of cisplatin toxicity in the inner ear, mainly outer hair cells (OHCs), inner hair cells, stria vascularis, spiral ganglia, and surrounding nerves in the mouse cochlea. A CTR1 substrate, copper sulfate, decreased the uptake and cytotoxicity of cisplatin in HEI-OC1, a cell line that expresses many molecular markers reminiscent of OHCs. Small interfering RNA-mediated knockdown of Ctr1 in this cell line caused a corresponding decrease in cisplatin uptake. In mice, intratympanic administration of copper sulfate 30 min before intraperitoneal administration of cisplatin was found to prevent hearing loss at click stimulus and 8, 16, and 32 kHz frequencies. To date, the utility of cisplatin remains severely limited because of its ototoxic effects. The studies described in this report suggest that cisplatin-induced ototoxicity and cochlear uptake can be modulated by administration of a CTR1 inhibitor, copper sulfate. The possibility of local administration of CTR1 inhibitors during cisplatin therapy as a means of otoprotection is thereby raised.

Role of the copper transporter, CTR1, in platinum-induced ototoxicity.

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.

Tissue-specific calibration of extracellular matrix material properties by transforming growth factor-β and Runx2 in bone is required for hearing.

This review highlights recent advances in cochlear gene therapy over the past several years. Cochlear gene therapy has undergone tremendous advances over the past decade. Beginning with some groundbreaking work in 2005 documenting hair cell regeneration using virally mediated delivery of the mouse atonal 1 gene, gene therapy is now being explored as a possible treatment for a variety of causes of hearing loss.

Cochlear gene therapy.

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.

Severe vestibular dysfunction and altered vestibular innervation in mice lacking prosaposin.

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.

Comparison of functional and morphologic characteristics of mice models of noise-induced hearing loss.

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.

Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy.

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.

Disrupted bone remodeling leads to cochlear overgrowth and hearing loss in a mouse model of fibrous dysplasia.

Saposin B (Sap B) is an essential activator protein for arylsulfatase A in the hydrolysis of sulfatide, a lipid component of myelin. To study Sap B's role in hearing and balance, a Sap B-deficient (B(-/-)) mouse was evaluated. At both light and electron microscopy (EM) levels, inclusion body accumulation was seen in satellite cells surrounding spiral ganglion (SG) neurons from postnatal month 1 onward, progressing into large vacuoles preceding satellite cell degeneration, and followed by SG degeneration. EM also revealed reduced or absent myelin sheaths in SG neurons from postnatal month 8 onwards. Hearing loss was initially seen at postnatal month 6 and progressed thereafter for frequency-specific stimuli, whereas click responses became abnormal from postnatal month 13 onward. The progressive hearing loss correlated with the accumulation of inclusion bodies in the satellite cells and their subsequent degeneration. Outer hair cell numbers and efferent function measures (distortion product otoacoustic emissions and contralateral suppression) were normal in the B(-/-) mice throughout this period. Alcian blue staining of SGs demonstrated that these inclusion bodies corresponded to sulfatide accumulation. In contrast, changes in the vestibular system were much milder, but caused severe physiologic deficits. These results demonstrate that loss of Sap B function leads to progressive sulfatide accumulation in satellite cells surrounding the SG neurons, leading to satellite cell degeneration and subsequent SG degeneration with a resultant loss of hearing. Relative sparing of the efferent auditory and vestibular neurons suggests that alternate glycosphingolipid metabolic pathways predominate in these other systems.

Article	Year
Progressive deafness and altered cochlear innervation in knock-out mice lacking prosaposin. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 17167097	2006
Sensorineural deafness and seizures in mice lacking vesicular glutamate transporter 3. Neuron\| PubMed ID: 18215623	2008
Muscle-like nicotinic receptor accessory molecules in sensory hair cells of the inner ear. Molecular and cellular neurosciences\| PubMed ID: 18420419	2008
Role of the copper transporter, CTR1, in platinum-induced ototoxicity. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 20631178	2010
Tissue-specific calibration of extracellular matrix material properties by transforming growth factor-β and Runx2 in bone is required for hearing. EMBO reports\| PubMed ID: 20847738	2010
Cochlear gene therapy. Current opinion in neurology\| PubMed ID: 22157110	2012
Severe vestibular dysfunction and altered vestibular innervation in mice lacking prosaposin. Neuroscience research\| PubMed ID: 22326583	2012
Comparison of functional and morphologic characteristics of mice models of noise-induced hearing loss. Auris, nasus, larynx\| PubMed ID: 22364846	2013
Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy. Neuron\| PubMed ID: 22841313	2012
Disrupted bone remodeling leads to cochlear overgrowth and hearing loss in a mouse model of fibrous dysplasia. PloS one\| PubMed ID: 24788917	2014
Spiral ganglion degeneration and hearing loss as a consequence of satellite cell death in saposin B-deficient mice. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 25698761	2015

Omar Akil

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