Estela Carnicero

Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca2+ transients that modulate secretion.

Activation of calcium-ion (Ca2+) channels on the plasma membrane and on intracellular Ca2+ stores, such as the endoplasmic reticulum, generates local transient increases in the cytosolic Ca2+ concentration that induce Ca2+ uptake by neighbouring mitochondria. Here, by using mitochondrially targeted aequorin proteins with different Ca2+ affinities, we show that half of the chromaffin-cell mitochondria exhibit surprisingly rapid millimolar Ca2+ transients upon stimulation of cells with acetylcholine, caffeine or high concentrations of potassium ions. Our results show a tight functional coupling of voltage-dependent Ca2+ channels on the plasma membrane, ryanodine receptors on the endoplasmic reticulum, and mitochondria. Cell stimulation generates localized Ca2+ transients, with Ca2+ concentrations above 20–40 µM, at these functional units. Protonophores abolish mitochondrial Ca2+ uptake and increase stimulated secretion of catecholamines by three- to fivefold. These results indicate that mitochondria modulate secretion by controlling the availability of Ca2+ for exocytosis.

Functional measurements of [Ca2+] in the endoplasmic reticulum using a herpes virus to deliver targeted aequorin

Changes in the free calcium concentration of the endoplasmic reticulum ([Ca2+]er) play a central role controlling cellular functions like contraction, secretion or neuronal signaling. We recently reported that recombinant aequorin targeted to the endoplasmic reticulum (ER) [Montero M., Brini M., Marsault R. et al. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J 1995; 14: 5467-5475, Montero M., Barrero M.J., Alvarez J. [Ca2+] microdomains control agonist-induced Ca2+ release in intact cells. FASEB J 1997; 11: 881-886] can be used to monitor selectively [Ca2+]er in intact HeLa cells. Here we have used a herpes simplex virus type 1 (HSV-1) based system to deliver targeted aequorin into a number of different cell types including both postmitotic primary cells (anterior pituitary cells, chromaffin cells and cerebellar neurons) and cell lines (HeLa, NIH3T3, GH3 and PC12 cells). Functional studies showed that the steady state lumenal [Ca2+]er ranged from around 300 microM in granule cells to 800 microM in GH3 cells. InsP3-coupled receptor stimulation with agonists like histamine (in HeLa, NIH3T3 and chromaffin cells), UTP and bradykinin (in PC12 cells) or thyrotropin-releasing hormone (TRH, in GH3 cells) produced a very rapid decrease in lumenal [Ca2+]er. Caffeine caused a rapid Ca2+ depletion of the ER in chromaffin cells, but not in the other cell types. Depolarization by high K+ produced an immediate and reversible increase of [Ca2+]er in all the excitable cells (anterior pituitary, GH3, chromaffin cells and granule neurons). We conclude that delivery of recombinant aequorin to the ER using HSV amplicon provides the first direct quantitative and dynamic measurements of [Ca2+]er in several primary non-dividing cells.

A novel vestibular approach for gene transfer into the inner ear

The aim of gene transfer to the cochlea and vestibular organ is to protect the inner ear from different disorders. Although various vectors for gene delivery have been used with some success, there remains a need for a reliable transfer of genes into the inner ear without damaging cochlear function. Here, we have tested a novel application method for gene transfer into the rat inner ear in vivo using herpes simplex virus type-1(HSV-1)-based amplicon vectors. Our goal was to find an entry route into the inner ear that leaves its function intact. Besides other non-invasive and invasive application techniques, we applied the viral vector via injection through a small opening of the utriculus. Using this method, efficient β-galactosidase reporter gene expression was achieved in nearly all neurons in the vestibulum and cochlea, without functional hearing deficits. At the time point of maximal expression 5 days after injection, β-galactosidase activity was also observed in axonal fibres and synaptic endings close to inner and outer hair cells. Our results thus describe an efficient and reliable protocol for short-term expression of potential therapeutic genes in the neuronal compartment of the inner ear.

Defining Responsiveness of Avian Cochlear Neurons to Brain‐Derived Neurotrophic Factor and Nerve Growth Factor by HSV‐1‐Mediated Gene Transfer

Abstract: The importance of individual members of the neurotrophin gene family for avian inner ear development is not clearly defined. Here we address the role of two neurotrophins, brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF), for innervation of the chicken cochlea. We have used defective herpes simplex virus type 1 (HSV‐1) vectors, or amplicons, to express these neurotrophins in dissociated cultures of cochlear neurons. HSV‐1‐mediated expression of BDNF promotes neuronal survival similar to the maximal level seen by exogenously added BDNF and exceeds its potency to produce neurite outgrowth. In contrast, cochlear neurons transduced with an amplicon producing bioactive NGF show no response. These results confirm BDNF as an important mediator of neurotrophin signaling inside avian cochlear neurons. However, these neurons can be rendered NGF‐responsive by transducing them with the high‐affinity receptor for NGF, TrkA. This study underlines the usefulness of amplicons to study and modify neurotrophin signaling inside neurons.

Generation of inner ear sensory cells from bone marrow-derived human mesenchymal stem cells

AIM Hearing loss is the most common sensory disorder in humans, its main cause being the loss of cochlear hair cells. We studied the potential of human mesenchymal stem cells (hMSCs) to differentiate towards hair cells and auditory neurons. MATERIALS & METHODS hMSCs were first differentiated to neural progenitors and subsequently to hair cell- or auditory neuron-like cells using in vitro culture methods. RESULTS Differentiation of hMSCs to an intermediate neural progenitor stage was critical for obtaining inner ear sensory lineages. hMSCs generated hair cell-like cells only when neural progenitors derived from nonadherent hMSC cultures grown in serum-free medium were exposed to EGF and retinoic acid. Auditory neuron-like cells were obtained when treated with retinoic acid, and in the presence of defined growth factor combinations containing Sonic Hedgehog. CONCLUSION The results show the potential of hMSCs to give rise to inner ear sensory cells.

Differential roles of fibroblast growth factor-2 during development and maintenance of auditory sensory epithelia

Fibroblast growth factor‐2 (FGF2) has been postulated to be a key regulator involved in the proliferation, differentiation, and regeneration of sensory hair cells. Here we have addressed the potential functions of FGF2 during the formation and regeneration of the auditory epithelium in chicken and mice. By using viral gene transfer, based on herpes simplex type 1 virus (HSV‐1), we show that ectopically applied FGF2 drastically increases the number of cells expressing early hair cell markers during embryonic development in avians. Intriguingly, FGF2 does not stimulate cell division during this process. These data suggest that FGF2 plays a role during differentiation of sensory hair cells in avians. To address the potential functions of FGF2 during murine inner ear development, we analyzed FGF2 mouse mutants. Mice lacking FGF2 showed normal formation of the inner ear, and no abnormalities were observed at the adult stage. Moreover, FGF2 mouse mutants showed similar hearing thresholds compared with those observed in control mice before and after noise damage. Therefore, endogenous FGF2 appears not to be essential for the development or functional maintenance of the auditory organ in mammals. In light of these results, the differential roles of FGF2 in the vertebrate inner ear are discussed with respect to its previously postulated functions.

Herpes simplex virus type 1-mediated transfer of neurotrophin-3 stimulates survival of chicken auditory sensory neurons.

Neurotrophin-3 (NT-3) is one of the most potent stimulators for survival of auditory sensory neurons. Viral transfer of neurotrophins into auditory neurons may offer a route to provide a permanent supply of the growth factor and guarantee their long-term survival. Herpes simplex virus type 1 (HSV-1)-based vectors have demonstrated their effectiveness to transfer genes into peripheral sensory neurons. In the present report, we have produced a HSV-1-based amplicon vector expressing NT-3. This vector efficiently infects isolated auditory neurons and stimulates their survival during distinct developmental stages of the inner ear. Therefore, this vector may present a unique entry point to develop therapies preventing or treating hearing impairment caused by the degeneration of auditory neurons.

Roles of fibroblast growth factor 2 during innervation of the avian inner ear

The importance of individual members of the fibroblast growth factor gene family during innervation of the vertebrate inner ear is not clearly defined. Here we address the role of fibroblast growth factor 2 (FGF‐2 or basic FGF) during development of the chicken inner ear. We found that FGF‐2 stimulated survival of isolated cochlear and vestibular neurons during distinct phases of inner ear innervation. The potential neurotrophic role of FGF‐2 was confirmed by its expression in the corresponding sensory epithelia and the detection of one of its high‐affinity receptors in inner ear neurons. Finally, we have analysed the potential of the amplicon system based on defective herpes simplex virus type 1 (HSV‐1) vectors to express FGF‐2 in cochlear neurons. Overexpression of FGF‐2 in cochlear neurons resulted in neuronal differentiation demonstrating the presence of biologically active growth factor. This study underlines the potential of FGF‐2 to control innervation and development of sensory epithelia in the avian inner ear. Furthermore, amplicon vectors may provide a useful tool to analyse gene function in isolated neurons of the vertebrate inner ear.

Differential effects on the survival of neuronal and non-neuronal cells after infection by herpes simplex virus type 1 mutants

Replication-defective mutants of herpes simplex virus type 1 (HSV-1) are powerful tools to transfer genes into postmitotic neurons and show promise for gene therapy protocols in vivo. To evaluate the efficacy and safety of these vectors for the treatment of deafness we infected dissociated cochlear ganglia with HSV mutants defective in the immediate early genes IE 2 (5dl1.2) or IE 3 (d120). Our results reveal striking differences in the survival of neuronal and non-neuronal cells caused by these mutants. Surprisingly, cochlear neurons infected with 5dl1.2 at various concentrations show a significant increase in survival after 2 days in culture. In contrast, many non-neuronal cells undergo apoptosis reducing cell number to less than 50%. In both neuronal and non-neuronal cell types we also observe a population of cells with important changes in morphology. Analysis of dissociated cochlear ganglia infected with d120 reveals a decrease of neuronal survival, whereas non-neuronal cells were almost unaffected. To further characterize and compare the effects of 5dl1.2 and d120 we transduced central nervous system-derived cell types including cortical neurons and astrocytes. Similarly, as observed for cochlear neurons, infection with 5dl1.2 results in increased survival of cortical neurons, whereas d120 shows cytotoxic effects. Survival of astrocytes is equally reduced by both HSV deletion mutants. We conclude that HSV-1 mutants defective in immediate early genes cause very distinct cytopathic phenotypes depending on the cellular context. Possible reasons for these differences, like various patterns of cellular and viral gene expression, and the implications for the use of HSV-1 vectors for gene transfer are discussed.