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.

MicroRNAs tune cerebral cortical neurogenesis.

MicroRNAs (miRNAs) are non-coding RNAs that promote post-transcriptional silencing of genes involved in a wide range of developmental and pathological processes. It is estimated that most protein-coding genes harbor miRNA recognition sequences in their 3' untranslated region and are thus putative targets. While functions of miRNAs have been extensively characterized in various tissues, their multiple contributions to cerebral cortical development are just beginning to be unveiled. This review aims to outline the evidence collected to date demonstrating a role for miRNAs in cerebral corticogenesis with a particular emphasis on pathways that control the birth and maturation of functional excitatory projection neurons.

Oxidative stress in the cochlea: an update.

This paper will focus on understanding the role and action of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the molecular and biochemical pathways responsible for the regulation of the survival of hair cells and spiral ganglion neurons in the auditory portion of the inner ear. The pivotal role of ROS/RNS in ototoxicity makes them potentially valuable candidates for effective otoprotective strategies. In this review, we describe the major characteristics of ROS/RNS and the different oxidative processes observed during ototoxic cascades. At each step, we discuss their potential as therapeutic targets because an increasing number of compounds that modulate ROS/RNS processing or targets are being identified.

Ototoxic drugs: difference in sensitivity between mice and guinea pigs.

The development of experimental animal models has played an invaluable role in understanding the mechanisms of neurosensory deafness and in devising effective treatments. The purpose of this study was to develop an adult mouse model of ototoxic drug-induced hearing loss and to compare the ototoxicity in the adult mouse to that in the well-described guinea pig model. Mice are a powerful model organism, especially due to the large availability of antibodies, probes and genetic mutants. In this study, mice (n=114) and guinea pigs (n=35) underwent systemic treatment with either kanamycin or cisplatin. Auditory brainstem responses showed a significant threshold shift in guinea pigs 2 weeks after the beginning of the ototoxic treatment, while there was no significant hearing impairment recorded in mice. Hair cells and neuronal loss were correlated with hearing function in both guinea pigs and mice. These results indicate that the mouse is not a good model for ototoxicity, which should be taken into consideration in all further investigations concerning ototoxicity-induced hearing loss.

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.

Caspases, the enemy within, and their role in oxidative stress-induced apoptosis of inner ear sensory cells.

This review covers the general roles of members of the cysteine protease family of caspases in the process of apoptosis (programmed cell death) looking at their participation in both the "extrinsic" cell death receptor and the "intrinsic" mitochondrial cell death pathways. It defines the difference between initiator and effector caspases and shows the progression of caspase activations that ends up in the apoptotic cell death and elimination of a damaged cell. The review then presents what is currently know about the participation of caspases in the programmed cell death of inner ear sensory cells during the process of normal development and maturation of the inner ear and their importance in this process as illustrated by the results of caspase-3 gene knockout experiments. The participation of specific caspases and the sequence of their activation in the elimination (apoptosis) of damaged sensory cells from adult inner ears after an injury that generates oxidative stress are reviewed. Both the possibility and the potential efficacy of caspase inhibition with a broad-spectrum pancaspase inhibitor as an interventional therapy to treat and rescue oxidative stress-damaged inner ear sensory cells from apoptosis are presented and discussed.

Substance P protects spiral ganglion neurons from apoptosis via PKC-Ca2+-MAPK/ERK pathways.

In the current study, we have investigated the ability of substance P (SP) to protect 3-day-old (P3) rat spiral ganglion neurons (SGNs) from trophic factor deprivation (TFD)-induced cell death. The presence of SP high affinity neurokinin-1 receptor (NK1) transcripts was detected in the spiral ganglion and the NK1 protein localized to SGNs both ex vivo and in vitro. Treatment with SP increased cytoplasmic Ca2+ in SGNs, further arguing for the presence of functional NK1 on these neurons. Both SP and the agonist [Sar9,Met(O2)11]-SP significantly decreased SGN cell death induced by TFD, with no effect on neurite outgrowth. The survival promoting effect of SP was blocked by the NK1 antagonist, WIN51708. Both pan-caspase inhibitor BOC-D-FMK and SP treatments markedly reduced activation of caspases and DNA fragmentation in trophic factor deprived-neurons. The neuroprotective action of SP was antagonised by specific inhibitors of second messengers, including 1.2-bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, the protein kinase C (PKC) inhibitors bisindolylmaleimide I, Gö6976 and LY333531 and the MAPK/ERK inhibitor U0126. In contrast, nifedipine, a specific inhibitor of l-type Ca2+ channel, and LY294002, a phosphatidylinositol-3-OH kinase (PI3K) inhibitor, had no effect on SP trophic support of SGNs. Moreover, activation of endogenous PKC by 4 beta-phorbol 12-myristate 13-acetate (PMA) also reduced the loss of trophic factor-deprived SGNs. Thus, NK1 expressed by SGNs transmit a survival-promoting regulatory signal during TFD-induced SGN cell death via pathways involving PKC activation, Ca2+ signalling and MAPK/ERK activation, which can be accounted for by an inhibition of caspase activation.

Epithelial supporting cells can differentiate into outer hair cells and Deiters' cells in the cultured organ of Corti.

The organ of Corti is a complex structure containing a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs), supported respectively by one row of inner phalangeal cells and three rows of Deiters' cells. When fetal rat organ of Corti explants are cultured, supernumerary OHCs and supernumerary Deiters' cells are produced, without any additional cell proliferation. Analysis of semi- and ultrathin sections revealed that supernumerary OHCs are produced at the distal edge of the organ of Corti. Quantitative analysis of cell types present in the organ of Corti demonstrates that when the number of OHCs increases: (i) the total number of cells remains constant; (ii) the number of Deiters' cells increases; (iii) the number of tectal cells decreases and of Hensen's cells decreases. Using specific HC markers, i.e. jagged2 (Jag2) and Math1, we showed that in addition to existing OHCs, supernumerary OHCs, tectal cells and Hensen's cells expressed these markers in embryonic day 19 organ of Corti explants after 5 days in vitro. The results of this study suggest that Hensen's cells retain the capacity to differentiate into either tectal cells, which differentiate into OHCs, or into undertectal cells which differentiate into Deiters' 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.

Effect of systemic nitroglycerin on CGRP and 5-HT afferents to rat caudal spinal trigeminal nucleus and its modulation by estrogen.

Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraine patients a delayed attack of unknown mechanism. After puberty migraine is more prevalent in women. Attacks can be triggered by abrupt falls in plasma estrogen levels, which accounts in part for sexual dimorphism, but lacks an established neurobiological explanation. We studied the effect of nitroglycerin on the innervated area of calcitonin gene-related peptide (CGRP) and serotonin-immunoreactive afferents to the superficial laminae of the spinal portion of trigeminal nucleus caudalis, and its modulation by estrogen. In male rats, nitroglycerin produced after 4 h a significant decrease of the area innervated by CGRP-immunoreactive afferents and an increase of that covered by serotonin-immunoreactive fibres. These effects were not observed in the superficial laminae of thoracic dorsal horns. The effect of nitroglycerin was similar in ovariectomized females. In estradiol-treated ovariectomized females the area in the spinal portion of trigeminal nucleus caudalis laminae I-II covered by CGRP-immunoreactive fibres was lower and that of serotonin-immunoreactive fibres was higher than in males and for both transmitters not significantly changed after nitroglycerin. The bouton size of CGRP profiles was smaller in estradiol-treated ovariectomized females, whereas after nitroglycerin it decreased significantly but only in males and ovariectomized females. Nitroglycerin, i.e. nitric oxide, is thus able to differentially influence afferent fibres in the superficial laminae of rat spinal trigeminal nucleus caudalis. Estradiol modulates the basal expression of these transmitters and blocks the nitroglycerin effect. These data may contribute to understanding the mechanisms by which estrogens influence migraine severity and the triggering of attacks by nitric oxide.

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.

Pharmacologic treatment of inner ear: from basic science to the patient.

Most of the deafness are of sensorineural origin and are characterized by a loss of hair cells and of spiral ganglion neurons. At the present time, hearing aids are the only treatment. However, in some diseases of the inner ear, pharmacological treatment have been proposed and used successfully. In this paper, we will review some basic science aspects of the biology of the neurosensory structures of the inner ear, in particular of the auditory neurons, that lead to the rationale of some treatments for the inner ear diseases. Developmental studies, neuronal cell culture experiments, and analyses of gene knockout animals reveal a number of growth factors which are important for the rescue and repair of injured auditory neurons in the inner ear. These factors rescue the injured auditory neurons in vivo. Furthermore, perfusion of antioxydant to the cochlea prevented the hearing loss induced by cisplatin. These in vitro and in vivo experiments demonstrate that it is possible to manipulate the neurosensory structures of the inner ear and provide an effective treatment to prevent the degeneration of the neurons. The molecules or drugs can be administered locally to the inner ear through a direct perilymphatic perfusion or through the round window membrane. As an example, we will discuss the treatment of patients suffering from idiopathic sensorineural hearing loss which can be treated successfully by a perfusion through the round window membrane, improving their hearing threshold and their speech discrimination.

Neurotransmitters as early signals for central nervous system development.

During brain ontogenesis, the temporal and spatial generation of the different types of neuronal and glial cells from precursors occurs as a sequence of successive progenitor stages whose proliferation, survival and cell-fate choice are controlled by environmental and cellular regulatory molecules. Neurotransmitters belong to the chemical microenvironment of neural cells, even at the earliest stages of brain development. It is now established that specific neurotransmitter receptors are present on progenitor cells of the developing central nervous system and could play, during neural development, a role that has remained unsuspected until recently. The present review focuses on the occurrence of neurotransmitters and their corresponding ligand-gated ion channel receptors in immature cells, including neural stem cells of specific embryonic and neonatal brain regions. We summarize in vitro and in vivo data arguing that neurotransmitters could regulate morphogenetic events such as proliferation, growth, migration, differentiation and survival of neural precursor cells. The understanding of neurotransmitter function during early neural maturation could lead to the development of pharmacological tools aimed at improving adult brain repair strategies.

Supernumerary outer hair cells arise external to the last row of sensory cells in the organ of corti.

During the development of the mammalian inner ear, the number of hair cells produced is highly regulated and remains constant throughout life. The mechanism underlying this regulation is beginning to be understood although many aspects still remain obscure. When late embryonic or early postnatal rat organs of Corti were cultured, the production of supernumerary hair cells was observed. This overproduction of sensory cells could be modulated by the addition of several growth factors. In this study, we examined explants of rat organs of Corti that produced supernumerary hair cells. In the supernumerary hair cell region, up to two rows of inner hair cells and five rows of outer hair cells were observed. Morphological evaluation of these specimens revealed that less mature hair cells were located in the most external rows of these sensory cells. When a supernumerary hair cell was produced, a supporting cell (i.e. Deiters' cell) was also produced, strongly suggesting that the conversion of a Deiters' cell into a hair cell was not the mechanism that produced these extra hair cells. Based on these results, we propose that prosensory cells located at the external edge of the organ of Corti retain a capacity to form hair cells and that it is these prosensory cells that differentiate into supernumerary hair cells and Deiters' cells.

Peripheral nerve regeneration using bioresorbable macroporous polylactide scaffolds.

The ability of DRG-derived neurons to survive and attach onto macroporous polylactide (PLA) foams was assessed in vitro. The foams were fabricated using a thermally induced polymer-solvent phase separation. Two types of pore structures, namely oriented or interconnected pores, can be produced, depending on the mechanism of phase separation, which in turn can be predicted by the thermodynamics of the polymer-solvent pair. Coating of the porous foams with polyvinylalcohol (PVA) considerably improved the wettability of the foams and allowed for cell culture. The in vitro biocompatibility of the PVA-coated supports was demonstrated by measuring cell viability and neuritogenesis. Microscopic observations of the cells seeded onto the polymer foams showed that the interconnected pore networks were more favorable to cell attachment than the anisotropic ones. The capacity of highly oriented foams to support in vivo peripheral nerve regeneration was studied in rats. A sciatic nerve gap of 5-mm length was bridged with a polymer implant showing macrotubes of 100 microm diameter. At 4 weeks postoperatively, the polymer implant was still present. It was well integrated and had restored an anatomic continuity. An abundant cell migration was observed at the outer surface of the polymer implant, but not within the macrotubes. This dense cellular microenvironment was found to be favorable for axogenesis.

Glycine triggers an intracellular calcium influx in oligodendrocyte progenitor cells which is mediated by the activation of both the ionotropic glycine receptor and Na+-dependent transporters.

Using fluo-3 calcium imaging, we demonstrate that glycine induces an increase in intracellular calcium concentration ([Ca2+]i) in cortical oligodendrocyte progenitor (OP) cells. This effect results from a calcium entry through voltage-gated calcium channels (VGCC), as it is observed only in OP cells expressing such channels, and it is abolished either by removal of calcium from the extracellular medium or by application of an L-type VGCC blocker. Glycine-triggered Ca2+ influx in OP cells actually results from an initial depolarization that is the consequence of the activation of both the ionotropic glycine receptor (GlyR) and Na+-dependent transporters, most probably the glycine transporters 1 (GLYT1) and/or 2 (GLYT2) which are colocalized in these cells. Through this GlyR- and transporter-mediated effect on OP intrcellular calcium concentration [Ca2+]i, glycine released by neurons may, as well as other neurotransmitters, serve as a signal between neurons and OP during development.

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.

Oxidative stress-induced apoptosis of cochlear sensory cells: otoprotective strategies.

Apoptosis is an important process, both for normal development of the inner ear and for removal of oxidative-stress damaged sensory cells from the cochlea. Oxidative-stressors of auditory sensory cells include: loss of trophic factor support, ischemia-reperfusion, and ototoxins. Loss of trophic factor support and cisplatin ototoxicity, both initiate the intracellular production of reactive oxygen species and free radicals. The interaction of reactive oxygen species and free radicals with membrane phospholipids of auditory sensory cells creates aldehydic lipid peroxidation products. One of these aldehydes, 4-hydroxynonenal, functions as a mediator of apoptosis for both auditory neurons and hair cells. We present several approaches for the prevention of auditory sensory loss from reactive oxygen species-induced apoptosis: 1) preventing the formation of reactive oxygen species; (2) neutralizing the toxic products of membrane lipid peroxidation; and 3) blocking the damaged sensory cells' apoptotic pathway.