Azel Zine

Blocking c-Jun-N-terminal kinase signaling can prevent hearing loss induced by both electrode insertion trauma and neomycin ototoxicity.

Neomycin ototoxicity and electrode insertion trauma both involve activation of the mitogen activated protein kinase (MAPK)/c-Jun-N-terminal kinase (JNK) cell death signal cascade. This article discusses mechanisms of cell death on a cell biology level (e.g. necrosis and apoptosis) and proposes the blocking of JNK signaling as a therapeutic approach for preventing the development of a permanent hearing loss that can be initiated by either neomycin ototoxicity or electrode insertion trauma. Blocking of JNK molecules incorporates the use of a peptide inhibitor (i.e. D-JNKI-1), which is specific for all three isoforms of JNK and has been demonstrated to prevent loss of hearing following either electrode insertion trauma or loss of both hearing and hair cells following exposure to an ototoxic level of neomycin. We present previously unpublished results that control for the effect of perfusate washout of aminoglycoside antibiotic by perfusion of the scala tympani with an inactive form of D-JNKI-1 peptide, i.e. JNKI-1(mut) peptide, which was not presented in the original J. Neurosci. article that tested locally delivered D-JNKI-1 peptide against both noise- and neomycin-induced hearing loss (i.e. Wang, J., Van De Water, T.R., Bonny, C., de Ribaupierre, F., Puel, J.L., Zine, A. 2003a. A peptide inhibitor of c-Jun N-terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss. J. Neurosci. 23, 8596-8607). D-JNKI-1 is a cell permeable peptide that blocks JNK signaling at the level of the three JNK molecular isoforms, which when blocked prevents the increases in hearing thresholds and the loss of auditory hair cells. This unique therapeutic approach may have clinical application for preventing: (1) hearing loss caused by neomycin ototoxicity; and (2) the progressive component of electrode insertion trauma-induced hearing loss.

The Injured Cochlea as a Target for Inflammatory Processes, Initiation of Cell Death Pathways and Application of Related Otoprotective Strategies

One of the causes of sensorineural hearing loss is the loss of auditory hair cells following exposure to environmental stresses. Auditory hair cell death in response to cochlear trauma occurs via both necrosis and apoptosis. Apoptosis of hair cells involves the caspase and MAPK/JNK pathways which are activated by oxidative stress and secretion of inflammatory cytokines in response to trauma. Identification of the pathways that lead to apoptosis provides therapeutic targets for the conservation of hearing. Antioxidants reduce the level of reactive oxygen species and reactive nitrogen species generated by oxidative stress in response to acoustic trauma, aminoglycoside and platinum-based drugs. Caspase inhibitors affect both the extrinsic and intrinsic apoptotic pathways thereby reducing cisplatin, aminoglycoside, hydraulic trauma and ischemia-induced hearing losses. Corticosteroid therapy reduces inflammation and inhibits apoptosis while activating pro-survival pathways in the organ of Corti following exposure to noise, vibration, cisplatin, aminoglycoside, ischemia/reperfusion injury, bacterial meningitis and electrode insertion trauma. Inhibitors of JNK signaling pathway prevent apoptosis of auditory hair cells following electrode insertion trauma, acute labyrinthitis, acoustic trauma and aminoglycoside ototoxicity. This review provides an overview of the different pathways involved in auditory hair cell death following an environmental stress and both traditional and newly developed drugs that are currently being studied or used for the treatment of acute hearing loss. Recent patents related to otoprotective strategies to conserve hearing and auditory hair cells are also discussed in this review.

D-JNKI-1 treatment prevents the progression of hearing loss in a model of cochlear implantation trauma.

Hypotheses: 1) Hearing loss caused by electrode insertion trauma has both acute and delayed components; and 2) the delayed component of trauma-initiated hearing loss can be prevented by a direct delivery of a peptide inhibitor of the c-Jun N-terminal kinase cell death signal cascade, that is, D-JNKI-1, immediately after the electrode insertion within the cochlea. Background: Acute trauma to the macroscopic elements of the cochlea from electrode insertion is well known. The impact of trauma-induced oxidative stress within injured cochlear tissues and the efficacy of drugs (e.g., D-JNKI-1) to prevent apoptosis of damaged hair cells is not well defined. Methods: Hearing function was tested by pure-tone evoked auditory brainstem responses (ABRs) and distortion products of otoacoustic emissions (DPOAEs). D-JNKI-1 in artificial perilymph (AP) or AP alone was delivered into the scala tympani immediately after electrode trauma and for 7 days. Controls were nontreated contralateral and D-JNKI-1-treated ears without electrode insertion trauma. Results: There was no increase in the hearing thresholds of either the contralateral control ears or in the D-JNKI-1 without trauma animals. There was a progressive increase in ABR thresholds and decrease in DPOAE amplitudes after electrode insertion trauma in untreated and in AP-treated cochleae. Treatment with D-JNKI-1 prevented the progressive increase in ABR thresholds and decrease in DPOAE amplitudes that occur after electrode insertion trauma. Conclusion: Hearing loss caused by cochlear implant electrode insertion trauma in guinea pigs has both acute and delayed components. The delayed component can be prevented by treating the cochlea with D-JNKI-1.

Targeting the JNK pathway as a therapeutic protective strategy for nervous system diseases.

The c-Jun N-terminal kinases (JNKs) are members of the family of mitogen activated protein kinases (MAPKs). While the functions of the JNKs under physiological conditions are diverse and not completely understood, there is increasing evidence that JNKs are potent effectors of apoptosis in both the brain and the mammalian inner ear following a variety of injuries. The activation of the inducible transcription factor c-Jun by N-terminal phosphorylation is a central event in JNK-mediated neural and inner ear hair cell death. A cell permeable peptide designed specifically to inhibit JNK signaling has proven successful in in vivo models of both neuronal degeneration following cerebral ischemia and auditory hair cell degeneration following exposure to either acoustic trauma or a toxic level of an aminoglycoside antibiotic. Here we discuss the evidence supporting the application of JNK inhibitors to prevent cellular degeneration in several central nervous system (CNS) and peripheral nervous system (PNS) diseases with an emphasis on traumatic ischemic damage to the CNS and acquired deafness in the PNS receptors.

Cochlear stem/progenitor cells from a postnatal cochlea respond to Jagged1 and demonstrate that notch signaling promotes sphere formation and sensory potential

Hair cells and supporting cells of the mammalian cochlea terminally differentiate during development. Recent in vitro evidence suggests the presence of hair cell progenitors in the postnatal cochlea. Phenotypic properties of these cells and factors that promote their ability to generate spheres in aggregate cultures have not been reported. We define an in vitro system that allows stem/progenitor cells harvested from the early postnatal cochlea to develop into spheres. These spheres contain Abcg2, Jagged1 and Notch1 positive progenitor cells that can divide and generate new hair cell-like cells, i.e. immunopositive for specific hair cell markers, including Myosin VI, Myosin VIIa, Math1 and ability to uptake FM1-43. We demonstrate that reducing Notch signaling with a gamma secretase inhibitor decreases the number of spheres generated following treatment of the stem/progenitor cell cultures. Additionally, activation of Notch by an exogenous soluble form of a Notch ligand, i.e. Jagged1 protein, promotes sphere formation and the sensory potential of cochlear stem/progenitor cells. Our findings suggest that Notch1/Jagged1 signaling plays a role in maintaining a population of Abcg2 sensory stem/progenitor cells in the postnatal cochlea.

Disorganized Innervation and Neuronal Loss in the Inner Ear of Slitrk6-Deficient Mice

Slitrks are type I transmembrane proteins that share conserved leucine-rich repeat domains similar to those in the secreted axonal guidance molecule Slit. They also show similarities to Ntrk neurotrophin receptors in their carboxy-termini, sharing a conserved tyrosine residue. Among 6 Slitrk family genes in mammals, Slitrk6 has a unique expression pattern, with strong expression in the sensory epithelia of the inner ear. We generated Slitrk6-knockout mice and investigated the development of their auditory and vestibular sensory organs. Slitrk6-deficient mice showed pronounced reduction in the cochlear innervation. In the vestibule, the innervation to the posterior crista was often lost, reduced, or sometimes misguided. These defects were accompanied by the loss of neurons in the spiral and vestibular ganglia. Cochlear sensory epithelia from Slitrk6-knockout mice have reduced ability in promoting neurite outgrowth of spiral ganglion neurons. Indeed the Slitrk6-deficient inner ear showed a mild but significant decrease in the expression of Bdnf and Ntf3, both of which are essential for the innervation and survival of sensory neurons. In addition, the expression of Ntrk receptors, including their phosphorylated forms was decreased in Slitrk6-knockout cochlea. These results suggest that Slitrk6 promotes innervation and survival of inner ear sensory neurons by regulating the expression of trophic and/or tropic factors including neurotrophins from sensory epithelia.

Molecular mechanisms that regulate auditory hair-cell differentiation in the mammalian cochlea

Mechanosensory hair cells of the vertebrate cochlea offer an excellent developmental system to study cell-fate specification, and to gain insight into the many human neurological deficits which result in a hearing loss, by affecting primarily the hair cells. Therefore, there is great interest in studying the molecular mechanisms that regulate their specification and differentiation.Recent studies, based mostly on loss-of-function experiments that target the role of Notch signaling and basic helix-loop-helix genes in inner-ear development have indicated that they can regulate mechanosensory hair cell-fate specification and their initial differentiation.

Expression of candidate markers for stem/progenitor cells in the inner ears of developing and adult GFAP and nestin promoter-GFP transgenic mice.

Loss of hair cells in the mammalian cochlea leads to permanent sensori-neural hearing loss. Hair cells degenerate and their places are taken by phalangeal scars formed by non-sensory supporting cells. Current data indicate that early postnatal post-mitotic supporting cells can proliferate and differentiate into hair cell-like cells in culture. In this study, we used GFAP and nestin promoter-GFP transgenic mice in combination with other stem cell markers to characterize supporting cell subtypes in the postnatal day-3 (P3) and adult organs of Corti with potential stem/progenitor cell phenotype. In P3 organ of Corti, we show GFAP-GFP signal in all the supporting cell subtypes while the nestin-GFP was restricted to the supporting cells in the inner hair cell area. At this stage, GFAP and selected stem/progenitor markers displayed overlapping expression pattern in the supporting cell population. In the adult, GFAP expression is down-regulated from the supporting cells in the outer hair cell area and nestin expression is down-regulated in the supporting cells of the inner hair cell area. Sox2 and Jagged1 expression is maintained in the mature supporting cells, while Abcg2 was down-regulated in these cells. In contrast, GFAP and Abcg2 expression was up-regulated in the inner sulcus limbal cells outside the mature organ of Cortis area. Using quantitative reverse transcription-PCR, we found a decrease in transcripts for Jagged1 and Sox2 in adult cochleae. Our findings suggest that the loss of regenerative capacity of the adult organ of Corti is related to down-regulation of stem/progenitor key-markers from the mature supporting cells.

Molecular regulation of auditory hair cell death and approaches to protect sensory receptor cells and/or stimulate repair following acoustic trauma

Loss of auditory sensory hair cells (HCs) is the most common cause of hearing loss. This review addresses the signaling pathways that are involved in the programmed and necrotic cell death of auditory HCs that occur in response to ototoxic and traumatic stressor events. The roles of inflammatory processes, oxidative stress, mitochondrial damage, cell death receptors, members of the mitogen-activated protein kinase (MAPK) signal pathway and pro- and anti-cell death members of the Bcl-2 family are explored. The molecular interaction of these signal pathways that initiates the loss of auditory HCs following acoustic trauma is covered and possible therapeutic interventions that may protect these sensory HCs from loss via apoptotic or non-apoptotic cell death are explored.

Culture conditions have an impact on the maturation of traceable, transplantable mouse embryonic stem cell-derived otic progenitor cells.

The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1‐green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two‐step short‐term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF‐β inhibitors and insulin‐like growth factor IGF‐1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin‐injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright