Gene transfer in inner ear cells: a challenging race.

Recent advances in human genomics led to the identification of numerous defective genes causing deafness, which represent novel putative therapeutic targets. Future gene-based treatment of deafness resulting from genetic or acquired sensorineural hearing loss may include strategies ranging from gene therapy to antisense delivery. For successful development of gene therapies, a minimal requirement involves the engineering of appropriate gene carrier systems. Transfer of exogenous genetic material into the mammalian inner ear using viral or non-viral vectors has been characterized over the last decade. The nature of inner ear cells targeted, as well as the transgene expression level and duration, are highly dependent on the vector type, the route of administration and the strength of the promoter driving expression. This review summarizes and discusses recent advances in inner ear gene-transfer technologies aimed at examining gene function or identifying new treatment for inner ear disorders.

Retinoic acid stimulates regeneration of mammalian auditory hair cells.

Sensorineural hearing loss resulting from the loss of auditory hair cells is thought to be irreversible in mammals. This study provides evidence that retinoic acid can stimulate the regeneration in vitro of mammalian auditory hair cells in ototoxic-poisoned organ of Corti explants in the rat. In contrast, treatment with retinoic acid does not stimulate the formation of extra hair cells in control cultures of Corti's organ. Retinoic acid-stimulated hair cell regeneration can be blocked by cytosine arabinoside, which suggests that a period of mitosis is required for the regeneration of auditory hair cells in this system. These results provide hope for a recovery of hearing function in mammals after auditory hair cell damage.

New insights into peripherin expression in cochlear neurons.

Peripherin is an intermediate filament protein that is expressed in peripheral and enteric neurons. In the cochlear nervous system, peripherin expression has been extensively used as a differentiation marker by preferentially labeling the type II neuronal population at adulthood, but yet without knowing its function. Since the expression of peripherin has been associated in time with the process of axonal extension and during regeneration of nerve fibers in other systems, it was of interest to determine whether peripherin expression in cochlear neurons was a static phenotypic trait or rather prone to modifications following nerve injury. In the present study, we first compared the expression pattern of peripherin and beta III-tubulin from late embryonic stages to the adult in rat cochlea. The staining for both proteins was seen before birth within all cochlear neurons. By birth, and for 2 or 3 days, peripherin expression was gradually restricted to the type II neuronal population and their projections. In contrast, from postnatal day (P) 10 onwards, while the expression of beta III-tubulin was still found in projections of all cochlear neurons, only the type I population had beta III-tubulin immunoreactivity in their cell bodies. We next investigated the expression of peripherin in axotomized cochlear neurons using an organotypic explant model. Peripherin expression was surprisingly re-expressed in a vast majority of neurons after axotomy. In parallel, the expression and localization of beta III-tubulin and peripherin in dissociated cultures of cochlear neurons were studied. Both proteins were distributed along the entire neuronal length but exhibited complementary distribution, especially within the projections. Moreover, peripherin immunoreactivity was still abundant in the growth cone, whereas that of beta III-tubulin was decreasing at this compartment. Our findings are consistent with a model in which peripherin plays an important structural role in cochlear neurons and their projections during both development and regenerative processes and which is compatible with the assumption that frequently developmentally regulated factors are reactivated during neuronal regeneration.

Molecular pathways involved in apoptotic cell death in the injured cochlea: cues to novel therapeutic strategies.

Most hearing loss results from lesions of the sensory cells and/or neurons of the auditory portion of the inner ear. To date, only the cochlear implantation offers long-term hearing-aid benefit, but still with limited performance and expensive cost. While the underlying causes of deafness are not clear, the death or hair cells and/or neurons and the loss of neuronal contacts are key pathological features. Pinpointing molecular events that control cell death in the cochlea is critical for the development of new strategies to prevent and treat deafness, whether in combination or not with cochlear implant therapy.

[Differentiation, protection and regeneration of hair cells and auditory neurons in mammals]. / Différenciation, protection et régéneration des cellules ciliées et des neurones auditifs de mammifères.

Perception deafness, as opposed to transmission deafness, results from a lesion of the sensory cells and/or of the neurons of the auditory part of inner ear. There is currently no treatment able to stop the progression of a hearing loss or to restore a lost auditory function. We review here the progress which has been made with respect to the regeneration and the protection of neurosensory structures in the inner ear. A spontaneous post-lesional regeneration of hair cells in the sensory epithelium has been observed in amphibians and birds as well as in the vestibular part of the mammalian inner ear. In contrast, hair cells regeneration in the auditory portion of the inner ear of mammals appears to be limited to the developmental stages. The objective of this first part of our work is to try to identify progenitor cells in the cochlea and to characterize factors that are able to induce their proliferation and differentiation. Otoprotection of the sensory epithelium is another therapeutical strategy aimed at preventing further degradation of the auditory function. The prevention of the deleterious effect on hair cells of several drugs such as aminoglycosides and platin derivatives has been obtained using free radical scavengers or growth factors. Studies of the development of the innervation of the inner ear suggest the potential therapeutic interest of various growth factors, mostly the neurotrophins, to maintain the innervation of the adult Corti's organ.

In vitro and in vivo modulation of 5-hydroxytryptamine-, thyrotropin-releasing hormone- and calcitonin-gene related peptide-like immunoreactivities in adult rat sensory neurons.

In a previous work we have shown that culturing adult rat dorsal root ganglia neurons modifies their neurotransmitter phenotype in such a way that cultured neurons synthesize transmitters that are not found in situ, while several other transmitters are expressed in a much higher percentage of neurons in culture than in situ [Schoenen J. et al. (1989) J. Neurosci. Res. 22, 473-487]. The aim of the present study was to investigate the origin and the nature of the relevant environmental signals that allow this plasticity to be expressed, focusing on three neurotransmitters: 5-hydroxytryptamine, thyrotropin-releasing hormone and calcitonin-gene related peptide. The main results can be summarized as follows: (1) culturing cells in fetal calf serum or on feeder layers of astrocytes, Schwann cells or fibroblasts partially inhibits the serotoninergic phenotype of dorsal root ganglia neurons; (2) in vivo disconnection of dorsal root ganglia from their spinal targets but not from their peripheral or supraspinal targets induces a significant increase of the percentage of 5-hydroxytryptamine- and thyrotropin-releasing hormone-positive neurons in disconnected ganglia; (3) growth factors such as ciliary neuronotrophic factor or basic fibroblast growth factor but not nerve growth factor repress 5-hydroxytryptamine and calcitonin gene-related peptide immunoreactivity in cultured sensory neurons. In conclusion, neurotransmitter gene expression of adult dorsal root ganglia neurons is controlled by complex influences. Our data suggest that thyrotropin-releasing hormone and 5-hydroxytryptamine gene expression are tonically repressed in vivo by factors originating from the spinal segmental level and that growth factors such as ciliary neurotrophic factor or basic fibroblast growth factor could be potential vectors of this repressing effect.

The anti-epileptic drug levetiracetam reverses the inhibition by negative allosteric modulators of neuronal GABA- and glycine-gated currents.

1. In this study in vitro and in vivo approaches were combined in order to investigate if the anti-epileptic mechanism(s) of action of levetiracetam (LEV; Keppra) may involve modulation of inhibitory neurotransmission. 2. GABA- and glycine-gated currents were studied in vitro using whole-cell patch-clamp techniques applied on cultured cerebellar granule, hippocampal and spinal neurons. Protection against clonic convulsions was assessed in vivo in sound-susceptible mice. The effect of LEV was compared with reference anti-epileptic drugs (AEDs): carbamazepine, phenytoin, valproate, clonazepam, phenobarbital and ethosuximide. 3. LEV contrasted the reference AEDs by an absence of any direct effect on glycine-gated currents. At high concentrations, beyond therapeutic relevance, it induced a small reduction in the peak amplitude and a prolongation of the decay phase of GABA-gated currents. A similar action on GABA-elicited currents was observed with the reference AEDs, except ethosuximide. 4. These minor direct effects contrasted with a potent ability of LEV (EC(50)=1 - 10 microM) to reverse the inhibitory effects of the negative allosteric modulators zinc and beta-carbolines on both GABA(A) and glycine receptor-mediated responses. 5. Clonazepam, phenobarbital and valproate showed a similar ability to reverse the inhibition of beta-carbolines on GABA-gated currents. Blockade of zinc inhibition of GABA responses was observed with clonazepam and ethosuximide. Phenytoin was the only AED together with LEV that inhibited the antagonism of zinc on glycine-gated currents and only clonazepam and phenobarbital inhibited the action of DMCM. 6. LEV (17 mg kg(-1)) produced a potent suppression of sound-induced clonic convulsions in mice. This protective effect was significantly abolished by co-administration of the beta-carboline FG 7142, from a dose of 5 mg kg(-1). In contrast, the benzodiazepine receptor antagonist flumazenil (up to 10 mg kg(-1)) was without any effect on the protection afforded by LEV. 7. The results of the present study suggest that a novel ability to oppose the action of negative modulators on the two main inhibitory ionotropic receptors may be of relevance for the anti-epileptic mechanism(s) of action of LEV.

Astroglia-released factor with negative allosteric modulatory properties at the GABA A receptor.

We have previously shown, using whole-cell patch-clamp techniques, that astrocytes release a negative allosteric modulator of the gamma-aminobutyric acid type A receptor (GABAA receptor) with beta-carboline-like properties, thus, likely to act at the benzodiazepine site. Here, using patch-clamp and binding techniques, we confirm that the low-molecular-weight fraction of astroglia-conditioned medium (ACM lmf) contains a factor(s) that negatively modulates GABAA-receptor function. This factor, like beta-carbolines, enhances the specific binding of [35S]t-butyl bicyclophosphorothionate (TBPS) to adult rat cortical membranes in the presence of GABA. However, it fails to interact with various ligands of the benzodiazepine (BZD) site of the GABAA receptor ([3H]flunitrazepam, [3H]Ro 15-1788 and [3H]Ro 15-4513). The question of the actual binding site of the astroglia-derived factor on the GABAA receptor, thus, remains open and can be addressed only after the purification of the active molecule(s) of ACM Imf has been completed, and a labeled form of the endogenous ligand becomes available. Taken together, however, the data suggest that type 1 astrocytes are able to modulate the effects of the main inhibitory neurotransmission in the central nervous system.

Pharmacological modulation of the bystander effect in the herpes simplex virus thymidine kinase/ganciclovir gene therapy system: effects of dibutyryl adenosine 3',5'-cyclic monophosphate, alpha-glycyrrhetinic acid, and cytosine arabinoside.

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) suicide gene/ganciclovir system was first applied to the treatment of glioblastoma tumors, but was hampered by the low gene transfection yield. Fortunately, the gap junction-dependent diffusion of phosphorylated ganciclovir metabolites from transfected cells to their neighbors proved to enhance the overall benefit of this strategy. However, as tumor cells are often gap junction-deficient, we sought to restore this property pharmacologically and hence to improve the efficacy of the treatment. We demonstrated that this approach was feasible in glioblastoma cells using dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) (100 microM) as a pharmacological inducer of gap junctions. alpha-Glycyrrhetinic acid (25 microM), on the other hand, strongly inhibited both gap junction-mediated intercellular communication and the bystander effect, thus confirming the role of gap junctions in HSV-tk-mediated bystander killing. Using cytosine arabinoside as a growth inhibitor, we underlined the role of tumor cell proliferation in the sensitivity of HSV-tk-transfected cells to ganciclovir and demonstrated its correlation with the importance of the bystander effect.

Beta-carbolines induce apoptotic death of cerebellar granule neurones in culture.

Apart from its role in fast inhibitory transmission, only neurotrophic effects have been reported following activation of the GABAA receptor. Here, we show that n-butyl-beta-carboline-3-carboxylate and n-methyl-beta-carboline-3-carboxamide, which are negative allosteric modulators of the GABAA receptor acting at the benzodiazepine site, are neurotoxic for cerebellar granule neurones in culture. The beta-carboline-induced neuronal death is apoptotic since DNA internucleosomal fragmentation was induced and the neurotoxicity could be prevented by inhibitors of mRNA or protein synthesis. As GABA and benzodiazepine ligands (diazepam and Ro 15-1788) protect cerebellar granule cells against beta-carboline-induced toxicity, these data raise the possibility that the interaction between the beta-carbolines and the GABAA receptor is the triggering event leading to neuronal apoptosis.

NT-3 has a tropic effect on process outgrowth by postnatal auditory neurones in vitro.

CONFOCAL analysis of early postnatal auditory neurones in a bicompartmental culture system was used to test for chemoattractant properties of NGF, BDNF and NT-3 on neuronal process outgrowth. NT-3 exerted a strong tropic effect on neuritic outgrowth from auditory neurones in this system. BDNF and NGF did not have any tropic activity that directed processes outgrowth from auditory neurones. However, BDNF was important for the support of neuronal survival in NGF-treated cultures and for neuritogenesis in NT-3-treated cultures. Since NT-3 has been identified as both a survival factor and a chemotropic agent for auditory neurones, it is likely that this neurotrophin will be a useful therapeutic agent in the treatment of damaged cochleae for the recovery of hearing.

Neurotrophins affect survival and neuritogenesis by adult injured auditory neurons in vitro.

This study evaluates the trophic effects of three neurotrophins on traumatized adult auditory neurons in culture, and the presence of these neurotrophins in cochlear nucleus tissue. BDNF and NT-3 promoted survival but very limited neuritogenesis by adult auditory neurons in vitro, while NGF, although without a survival effect, evoked a robust neuritic outgrowth response when combined with BDNF. Messenger RNAs that encode for NGF, BDNF and NT-3 were detected by RT-PCR in RNA extracts from adult cochlear nuclei tissue. Based on these in vitro and in vivo findings, we propose NT-3 as the agent of the peripheral target-derived survival promoting effect and NGF, BDNF, and NT-3 as mediators of trophic influences originating from the central target (i.e. cochlear nucleus).

Caspase inhibitors prevent cisplatin-induced apoptosis of auditory sensory cells.

In vitro studies tested the efficacy of three caspase inhibitors, Ac-VAD-cmk (caspase-1 inhibitor), z-DEVD-fmk (caspase-3 inhibitor) and B-D-fmk (BOCDFK, a general inhibitor), for protecting auditory sensory cells from cisplatin-damage induced loss. Treatment of 3-day-old rat organ of Corti explants with these caspase inhibitors protected > 80% of the auditory hair cells from cisplatin-damage initiated apoptosis. Dissociated cell cultures of 3-day-old rat spinal ganglia treated with any of these three caspase inhibitors in addition to exogenous neurotrophin have highly significant increases in neuronal survival following cisplatin exposure. These results indicate that loss of auditory sensory cells as a result of cisplatin-induced damage involves apoptosis and that blocking of this cell death pathway at the caspase level effectively rescues both hair cells and neurons.

Developmental regulation of neuroligand-induced responses in cultured oligodendroglia.

Using whole-cell patch-clamp techniques, we show that oligosphere-derived oligodendrocyte progenitor cells (OP) display GABA-, glutamate-, 5-HT-, glycine- and acetylcholine-gated inward currents. When OP differentiate into oligodendrocytes (ODC), the amplitude of peak currents elicited by saturating concentrations of these transmitters decreases except for 5-HT. Intracellular Ca2+ concentration changes induced by microperfusion of glutamate, 5-HT, TRH, met-enkephalin and substance P were monitored using a fluo-3-based calcium imaging system. When OP cells differentiate into ODC, a global decrease of the proportion of responding cells is observed. During type-2 astrocytes commitment, this proportion decreases for 5-HT, TRH- and metenkephalin stimulations whereas it remains constant for substance P and glutamate. These data demonstrate a development regulation of neurotransmitter- and neuropeptide-induced responses within the oligodendroglial lineage.

NT-3 and/or BDNF therapy prevents loss of auditory neurons following loss of hair cells.

Destruction of auditory hair cells results in a subsequent loss of auditory neurons. In situ hybridization and neuronal cell culture studies as well as analyses of the inner ears of neurotrophin and neurotrophin receptor gene knockout mice have shown that NT-3 and BDNF mediate both the development and survival of auditory neurons. In this study guinea pigs were exposed to the ototoxic combination of an aminoglycoside antibiotic and a loop diuretic and then received 8 weeks of intracochlear infusion of either NT-3, BDNF or NT-3 + BDNF to determine whether site-specific application of these neurotrophins could prevent the loss of auditory neurons that follows a loss of auditory hair cells. Infusion of either NT-3 or NT-3 + BDNF into the scala tympani resulted in a > 90% survival of auditory neurons while BDNF infusion yielded a 78% survival rate, compared with a 14-24% neuronal survival rate in untreated ototoxin-exposed cochleae. These results show that loss of auditory neurons that occurs subsequent to a loss of auditory hair cells can be prevented by in vivo neurotrophin therapy with either NT-3 or BDNF.

Effects of neurotrophins on early auditory neurones in cell culture.

During the first week of postnatal development, the innervation of the organ of Corti changes from an immature to an adult pattern. Dissociated cell cultures of early postnatal spiral ganglia were used to investigate the effects of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on maturing auditory neurones. BDNF was the most potent stimulator of neuritogenesis, NT-3 provided the strongest support for neuronal survival, while NGF supported limited neuritogenesis, and only at pharmacological levels. These findings suggest that both BDNF and NT-3 participate in the postnatal maturation of cochlear innervation and that NGF is most probably not involved in this process.

NT-3 combined with CNTF promotes survival of neurons in modiolus-spiral ganglion explants.

Auditory neurons depend upon the integrity of both their peripheral (auditory hair cells) and central (cochlear nucleus) targets for survival. One proposed trophic mechanism is the production of neurotrophin-3 (NT-3) by auditory hair cells. Modiolus-spiral ganglion explants from adult rats that closely mirror cell-cell interactions and in vivo tissue relationships within this ganglion provide a model for testing trophic factors. Brain derived neurotrophic factor (BDNF), NT-3 and ciliary neurotrophic factor (CNTF) were tested for their ability, both individually and in combination, to support neuronal survival. NT-3 was the strongest individual promoter of survival, while CNTF (a cytokine) with NT-3 (a neurotrophin) was the most effective combination for promoting the survival of auditory neurons.

Diazepam-insensitive GABAA receptors on postnatal spiral ganglion neurones in culture.

Using dissociated spiral ganglion cell cultures obtained from 3-day-old rat cochlea, we investigated the response of auditory neurones to gamma-aminobutyric acid (GABA) using patch-clamp techniques. In our recording conditions, GABA elicited inward currents in > 95% of the neurones which reversed around 0 mV. Similar inward currents were measured using isoguvacin, a specific agonist of GABAA receptors. GABA-gated currents were reversibly inhibited by the channel blocker picrotoxin and the GABA competitive antagonist bicuculline. These functional GABAA receptors are characterized by an insensitivity to benzodiazepines and a relatively high sensitivity to beta-carbolines and barbiturates. These results show that the GABAA receptor pharmacological properties of spiral ganglion neurones are close to those of cerebellar granule cells.

AAV2 vectors mediate efficient and sustained transduction of rat embryonic ventral mesencephalon.

The success of transplantation of human embryonic mesencephalic tissue to treat parkinsonian patients is limited by the poor survival of the transplant. We show that an AAV2 vector mediates efficient expression of the egfp reporter gene in organotypic cultures of freshly explanted solid fragments of rat embryonic ventral mesencephalon (VM). We observed early and sustained transgene expression (4 days to > or = 6 weeks). Furthermore, rAAV-infected rat embryonic VM transplanted in the adult striatum continued to express EGFP for > or = 3 months. More than 95% of the transduced cells were neurons. Dopaminergic neurons were transduced at low frequency at earlier time points. This method of gene delivery could prove useful to achieve local, continuous secretion of neurotrophic factors at physiologically relevant doses to treat Parkinson's disease.

Cytokine production by human thymic epithelial cells: control by the immune recognition of the neurohypophysial self-antigen.

Oxytocin (OT) has been shown to be the dominant peptide of the neurohypophysial family expressed by thymic epithelial and nurse cells (TEC/TNC) in various species. Thymic OT is not secreted but, after translocation of a hybrid neurophysin/MHC class I protein, is integrated within the plasma membrane of TEC, thus allowing its presentation to pre-T cells. In order to further demonstrate that thymic OT behaves like a membrane antigen, we assessed the effect of mAbs to OT on cytokine productions by cultures enriched in human TEC. 75-85% pure TEC cultures were prepared from human thymic fragments. Using immunofluorescence and confocal microscopy, ir-OT, ir-interleukin-1 beta (IL-1 beta), ir-interleukin-6 (IL-6) and ir-leukemia inhibitory factor (LIF) could be detected in these TEC cultures. ir-OT was restricted to TEC, while some ir-IL-6 and ir-LIF were also seen in occasional fibroblasts. In basal conditions, ir-IL-6 and ir-LIF (but not ir-OT and ir-IL-1 beta) were detected in the supernatants of human TEC cultures. MAbs to OT induced a marked increase of ir-IL-6 and ir-LIF secretion in TEC cultures. No significant effect was observed using mAbs against vasopressin, mouse immunoglobulins, or control ascitic fluid controls. These data show that OT is fully processed and recognized by specific mAbs at the outer surface of TEC plasma membrane. They further support that thymic OT behaves as the self-antigen of the neurohypophysial family.