Biomedical subjects

Matthew C Holley

Publications and source records attributed to Matthew C Holley.

11 recordsLinked to original sources

PubMed2006

Stem cell therapy for hearing loss: Math1 overexpression in VOT-E36 cells.

HYPOTHESIS: VOT-E36 cells acquire mechanosensitivity after mammalian atonal homolog 1 (Math1) overexpression. BACKGROUND: VOT-E36 cells are derived from a population of epithelial cells in the ventral region of the otocyst at embryonic Day 10.5, before hair cell differentiation. These cells express a number of specific molecular markers for hair cells under both proliferation and differentiation states. Overexpression of Math1 can convert nonsensory epithelial cells into hair cells in the cochlea. Based on this information, we tested whether VOT-E36 cells can be converted into hair cells by Math1 overexpression. METHODS: Using reverse transcriptase-polymerase chain reaction-based analysis, we first compared the expression patterns of various molecular markers for hair cell development in VOT-E36 cells between proliferation and differentiation states, and also before and after overexpression of Math1. Subsequently, with a standard calcium imaging method, we examined whether VOT-E36 cells overexpressing Math1 could detect mechanical vibrations and activate spiral ganglion neurons in a coculture model. In addition, using confocal and scanning electron microscopes, we examined morphologic changes of VOT-E36 cells after Math1 overexpression. RESULTS: Consistent with previous reports, this study has shown that VOT-E36 cells express a number of specific molecular markers for hair cells in both proliferation and differentiation states. Under appropriate culture conditions, Math1 is transiently expressed in this cell line during conditional differentiation. In VOT-E36 cells overexpressing Math1, the normal expression pattern of certain molecular markers for mature hair cells is partially restored. Interestingly, after coculture with spiral ganglion neurons, VOT-E36 cells overexpressing Math1 are able to respond to mechanical vibrations and activate spiral ganglion neurons. Possible molecular mechanisms underlying this novel finding have been explored. CONCLUSION: Math1 overexpression can partially restore presumably downstream signaling cascades for normal hair cell differentiation in VOT-E36 cells, which are able to detect mechanical vibrations after being cocultured with spiral ganglion neurons.

Animals↗

PubMed2005

Keynote review: The auditory system, hearing loss and potential targets for drug development.

There is a huge potential market for the treatment of hearing loss. Drugs are already available to ameliorate predictable, damaging effects of excessive noise and ototoxic drugs. The biggest challenge now is to develop drug-based treatments for regeneration of sensory cells following noise-induced and age-related hearing loss. This requires careful consideration of the physiological mechanisms of hearing loss and identification of key cellular and molecular targets. There are many molecular cues for the discovery of suitable drug targets and a full range of experimental resources are available for initial screening through to functional analysis in vivo. There is now an unparalleled opportunity for translational research.

Animals↗

PubMed2004

Development of outward potassium currents in inner and outer hair cells from the embryonic mouse cochlea.

We recorded K(+) currents in inner (IHCs) and outer (OHCs) hair cells from mice at embryonic days 16 and 18 and on the day of birth (PO) to characterize their early physiological differentiation. In both cell types, outward currents increased in size during late embryonic development, in cells situated in both the apical and basal coils of the cochlea. Currents increased up to six-fold, with current density increasing four-fold. Currents in basal cells were generally larger than in the apex, and currents in IHCs were larger than in OHCs at any given stage. In OHCs, they were initially non-inactivating but gained the partial inactivation characteristic of the K(+) current of neonatal mouse cochlear hair cells, I(K,neo), by day 18 in the base and by P0 in the apex. In IHCs, there was little change, other than in amplitude, with partial inactivation already evident in the base by embryonic day 16. These results suggest that changes in the channel complement of OHCs occur within a few days of terminal mitosis, whereas in IHCs any such development would occur earlier. The progressive development of K(+) currents correlates with a developmental delay of around 2 days from the base to the apex of the cochlea.

Animals↗

PubMed2004

GATA3 and NeuroD distinguish auditory and vestibular neurons during development of the mammalian inner ear.

The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expressed GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD and that migrate from the antero-ventral, otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3.

Animals↗

PubMed2003

Developmental changes in the expression of potassium currents of embryonic, neonatal and mature mouse inner hair cells.

Developmental changes in electrophysiological membrane properties of mouse cochlear inner hair cells (IHCs) were studied from just after terminal differentiation up to functional maturity. As early as embryonic day 14.5 (E14.5) newly differentiated IHCs express a very small outward K+ current that is largely insensitive to 4-aminopyridine (4-AP). One day later the inward rectifier, IK1, is first observed. These immature cells initially exhibit only slow graded voltage responses under current clamp. From E17.5 spontaneous action potentials occur. During the first week of postnatal development, the outward K+ current steadily increases in size and a progressively larger fraction of the current is sensitive to 4-AP. During the second postnatal week, the activation of the 4-AP-sensitive current, by now contributing about half of the outward K+ current, shifts in the hyperpolarizing direction. Together with an increase in size of IK1, this hyperpolarizes the cell, thus inhibiting the spontaneous spike activity, although spikes could still be evoked upon depolarizing current injection. Starting at about the onset of hearing (postnatal day 12, P12) immature IHCs make the final steps towards fully functional sensory receptors with fast graded voltage responses. This is achieved mainly by the expression of the large-conductance Ca2+-activated K+ current IK,f, but also of a current indistinguishable from the negatively activating IK,n previously described in mature outer hair cells (OHCs). The 4-AP-sensitive current continues to increase after the onset of hearing to form the major part of the mature delayed rectifier, IK,s. By P20 IHCs appear mature in terms of their complement of K+ conductances.

4-Aminopyridine↗

PubMed2003

Hair cell re-growth.

Hair cell loss is usually a function of age, noise, ototoxic drugs and genetics. Therapeutic strategies fall into two categories: protection and regeneration. Protective methods include targeted application of growth factors and other agents to promote cell survival, and systemic application of drugs to prevent activation of programmed cell death. These strategies are related to treatments that cause predictable damage, such as the use of aminoglycoside antibiotics. The challenge of hair cell regeneration is more difficult. Instead of preventing cell loss, we must consider methods of stimulating cell division and hair cell differentiation from existing cells. We need to know much more about the molecular mechanisms that govern these processes so that we can identify potential targets for specific drugs or gene therapies. One method of approaching the issue is to combine in vitro models of developing hair cells with genomic and proteomic technologies. The benefits of hair cell re-growth may not be limited to full replacement of pre-existing cells. Surrogate hair cells may help to maintain cochlear innervation, even if they do not detect sound, and this property could be harnessed to improve the performance of cochlear implants.

Aminoglycosides↗

PubMed2002

Cell lines in inner ear research.

Cell lines have provided important experimental tools that have enhanced our understanding of neural and sensory function. They are particularly valuable in inner ear research because the auditory and vestibular systems are small, complex, and encased in several layers of bone. Organotypic cultures provide an invaluable experimental resource but require repeated microdissection and culture, and remain complex in terms of cell types and states of differentiation. A number of laboratories have established cell lines that offer a range of potential applications to hearing research. This review describes the advances that have already been made with these lines and the potential applications that they offer in the future. The majority of the cell lines are immortalized with a conditionally expressed, temperature sensitive variant of the SV40 tumor antigen. We discuss the value of these cells in developmental studies.

Animals↗

PubMed2002

Differential expression of alpha 3 and alpha 6 integrins in the developing mouse inner ear.

The development of the mammalian inner ear involves a complex series of cell-cell and cell-extracellular matrix interactions. These interactions are likely to be mediated by families of adhesion molecules, including the integrins. We have studied the expression of three integrin subunits known to be expressed on epithelia in a number of tissues (namely, alpha3, alpha6, and beta4) during the development of the murine inner ear. At E10.5, both alpha3 and alpha6 were expressed in the epithelial layers of the otocyst. The expression of alpha6 was concentrated in an anterioventral region of the epithelium and in a proportion of the cells forming the cochlear-vestibular and facial ganglia. By E12.5, alpha6 showed a more restricted expression, confined mainly to the pro-sensory epithelia and the neural processes from the cochlear-vestibular ganglion. In contrast, alpha3 was expressed in epithelia adjacent to the pro-sensory areas. This reciprocal expression pattern was maintained until birth. Between birth and P6, a switch in expression occurred such that alpha3 was upregulated and alpha6 was downregulated in the sensory epithelia of both the auditory and vestibular systems. At this stage, alpha3 was expressed in all the epithelia lining the scala media, thus defining the endolymph compartment. The expression of beta4 was restricted to epithelial/mesenchymal borders throughout the developmental stages studied, suggesting that alpha6 expression observed within the epithelium and neuronal tissue was alpha6beta1. The early expression and changing pattern of alpha3 and alpha6 integrins during development of the mammalian inner ear suggests that they may be involved in the molecular processes that define epithelial boundaries and guide sensory innervation.

Animals↗

PubMed2002

Asymmetric segregation of mitochondria and mortalin correlates with the multi-lineage potential of inner ear sensory cell progenitors in vitro.

The sensory epithelia of the inner ear include hair cells and supporting cells that share a common precursor. One possible mechanism involved in the genesis of these cell types is through asymmetric cell division. In this work we have studied asymmetric division of inner ear sensory cell progenitors in vitro in an attempt to understand how the different cell phenotypes are generated. In the search for molecules that will segregate asymmetrically we have found that mitochondria in general, and a mitochondrial protein named mortalin in particular, are asymmetrically segregated during certain cell divisions. In one conditionally immortal cell line (UB/OC-1), which represents a population of committed hair cell precursors, mortalin is uniformly distributed in the cytoplasm and shared equally between sibling cells during division. In another cell line (UB/UE-1), which represents a bipotent, vestibular supporting cell that can produce both neonatal hair cells as well as supporting cells, mortalin segregates asymmetrically. In UB/UE-1, approximately 12% of the cells display an asymmetric distribution of mortalin and mitochondria. The proportion of asymmetric cells increases immediately after the release of the immortalizing gene and before the onset of differentiation. The asymmetric segregation of mortalin in the bipotent cell line and its uniform distribution in a committed, lineage-restricted cell line raises the possibility that it may play a role in cell fate determination.

Animals↗

PubMed2002

Application of new biological approaches to stimulate sensory repair and protection.

The inner ear governs hearing and balance via six sense organs, each composed of a few thousand mechanosensory hair cells. Most inner ear disorders involve irreversible loss of hair cells and their associated nerves. They are a function of age, genetic abnormalities and environmental factors such as noise and the use of ototoxic drugs. The genetics and cell biology of the inner ear have revealed some key molecular mechanisms of development and sensory degeneration that raise hopes for new therapeutic approaches to the regeneration of sensory function. This review highlights these advances and the approaches that might be taken to effect protection and repair. It concludes with the suggestion that we can expect tangible, practical progress towards the clinic over the next 5-10 years and that, to provide the training and skills required to take full advantage of emerging technologies, we should forge much closer links between specialist clinicians and basic scientists.

Animals↗

PubMed2002

Transcript profiling of functionally related groups of genes during conditional differentiation of a mammalian cochlear hair cell line.

We have used Affymetrix high-density gene arrays to generate a temporal profile of gene expression during differentiation of UB/OC-1, a conditionally immortal cell line derived from the mouse cochlea. Gene expression was assessed daily for 14 days under differentiating conditions. The experiment was replicated in two separate populations of cells. Profiles for selected genes were correlated with those obtained by RT-PCR, TaqMan analysis, immunoblotting, and immunofluorescence. The results suggest that UB/OC-1 is derived from a population of nonsensory epithelial cells in the greater epithelial ridge that have the potential to differentiate into a hair-cell-like phenotype, without the intervention of Math1. Elements of the Notch signaling cascade were identified, including the receptor Notch3, with a transient up-regulation that suggests a role in hair cell differentiation. Several genes showed a profile similar to Notch3, including the transcriptional co-repressor Groucho1. UB/OC-1 also expressed Me1, a putative partner of Math1 that may confer competence to differentiate into hair cells. Cluster analysis revealed expression profiles for neural guidance genes associated with Gata3. The temporal dimension of this analysis provides a powerful tool to study genetic mechanisms that underlie the conversion of nonsensory epithelial cells into hair cells.

Animals↗