Kevin K. Ohlemiller

Early elevation of cochlear reactive oxygen species following noise exposure.

Reactive oxygen species (ROS) have been implicated in a growing number of neurological disease states, from acute traumatic injury to neurodegenerative conditions such as Alzheimer’s disease. Considerable evidence suggests that ROS also mediate ototoxicant- and noise-induced cochlear injury, although most of this evidence is indirect. To obtain real-time assessment of noise-induced cochlear ROS production in vivo, we adapted a technique which uses the oxidation of salicylate to 2,3-dihydroxybenzoic acid as a probe for the generation of hydroxyl radical. In a companion paper we described the development and characterization of this method in cochlear ischemia-reperfusion. In the present paper we use this method to demonstrate early elevations in ROS production following acute noise exposure. C57BL/6J mice were exposed for 1 h to intense broad-band noise sufficient to cause permanent threshold shift (PTS), as verified by auditory brainstem responses. Comparison of noise-exposed animals with unexposed controls indicated that ROS levels increase nearly 4-fold in the period 1–2 h following exposure and do not decline over that time. Our ROS measures extend previous results indicating that noise-induced PTS is associated with elevated cochlear ROS production and ROS-mediated injury. Persistent cochlear ROS elevation following noise exposure suggests a sustained process of oxidative stress which might be amenable to intervention with chronic antioxidant therapy.

Targeted deletion of the cytosolic Cu/Zn-superoxide dismutase gene (Sod1) increases susceptibility to noise-induced hearing loss.

Reactive oxygen species (ROS) such as superoxide, peroxide and hydroxyl radicals are generated during normal cellular metabolism and are increased in acute injury and in many chronic disease states. When their production is inadequately regulated, ROS accumulate and irreversibly damage cell components, causing impaired cellular function and death. Antioxidant enzymes such as superoxide dismutase (SOD) play a vital role in minimizing ROS levels and ROS-mediated damage. The cytosolic form of Cu/Zn-SOD appears specialized to remove superoxide produced as a result of injury. ‘Knockout’ mice with targeted deletion of Sod1, the gene that codes for Cu/Zn-SOD, develop normally but show enhanced susceptibility to central nervous system injury. Since loud noise is injurious to the cochlea and is associated with elevated cochlear ROS, we hypothesized that Sod1 knockout mice would be more susceptible to noise-induced permanent threshold shifts (PTS) than wild-type and heterozygous control mice. Fifty-nine mice (15 knockout, 29 heterozygous and 15 wild type for Sod1) were exposed to broad-band noise (4.0–45.0 kHz) at 110 dB SPL for 1 h. Hearing sensitivity was evaluated at 5, 10, 20 and 40 kHz using auditory brainstem responses before exposure and 1, 14 and 28 days afterward. Cu/Zn-SOD deficiency led to minor (0–7 dB) threshold elevations prior to noise exposure, and about 10 dB of additional noise-induced PTS at all test frequencies, compared to controls. The distribution of thresholds at 10 and 20 kHz at 28 days following exposure contained three modes, each showing an effect of Cu/Zn-SOD deficiency. Thus another factor, possibly an additional unlinked gene, may account for the majority of the observed PTS. Our results indicate that genes involved in ROS regulation can impact the vulnerability of the cochlea to noise-induced hearing loss.

Contributions of mouse models to understanding of age- and noise-related hearing loss

Once an oddity, mice have become the most widely used hearing research model. Their value for research in noise-induced hearing loss (NIHL) stems from their high vulnerability to noise and reduced variance of results, made possible by genetic standardization. To research in age-related hearing loss (ARHL), they offer economies of small size and a short lifespan, both of which reduce housing costs. Inbred mouse strains show a wide range of noise sensitivities and rates of hearing loss with age. These can be studied using classical genetic analysis, as well as hypothesis-driven experiments utilizing genetic engineering. Through such investigations, presently 3 loci have been identified to date that contribute to NIHL, 10 that promote ARHL, and at least 6 loci that promote both. The types of genes involved implicate homeostatic and protective mechanisms as key to the appearance of either type of pathology and support a causal link between injury and some apparent ARHL. While the majority of mouse ARHL models examined most closely resemble sensory ARHL, recent work has identified mice possessing the essential characteristics of neural and strial ARHL. Using these models, it should be possible to identify genes and alleles that promote the major forms of ARHL and their combinations.

Activity-dependent transcription regulation of PSD-95 by neuregulin-1 and Eos

Neuregulin-1 (Nrg-1) contains an intracellular domain (Nrg-ICD) that translocates into the nucleus, where it may regulate gene expression upon neuronal depolarization. However, the identity of its target promoters and the mechanisms by which it regulates transcription have been elusive. Here we report that, in the mouse cochlea, synaptic activity increases the level of nuclear Nrg-ICD and upregulates postsynaptic density protein-95 (PSD-95), a scaffolding protein that is enriched in post-synaptic structures. Nrg-ICD enhances the transcriptional activity of the PSD-95 promoter by binding to a zinc-finger transcription factor, Eos. The Nrg-ICD–Eos complex induces endogenous PSD-95 expression in vivo through a signaling pathway that is mostly independent of γ-secretase regulation. This upregulation of PSD-95 expression by the Nrg-ICD–Eos complex provides a molecular basis for activity-dependent synaptic plasticity.

Prevention of systemic clinical disease in MPS VII mice following AAV-mediated neonatal gene transfer.

For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have previously shown that systemic treatment of neonatal mucopolysaccharidosis type VII (MPS VII) mice with recombinant adeno-associated virus (AAV) vectors results in relatively long-term expression of β-glucuronidase (GUSB) in multiple tissues, and a reduction in lysosomal storage. Here, we demonstrate that therapeutic levels of enzyme persist for at least 1 year following a single intravenous injection of virus in neonatal MPS VII mice. The level and distribution of GUSB expression achieved is sufficient to prevent the development of many aspects of clinical disease over the life of the animal. Following treatment, bone lengths, weights and retinal function were maintained at nearly normal levels throughout the life of the animal. In addition, significant improvements in survival and auditory function were seen in AAV-treated MPS VII mice when compared with untreated mutant siblings. These data suggest that AAV-mediated gene transfer in the neonatal period can lead to prevention of many of the clinical symptoms associated with MPS VII in the murine model of this disease.

Mechanisms and Genes in Human Strial Presbycusis from Animal Models

Schuknecht proposed a discrete form of presbycusis in which hearing loss results principally from degeneration of cochlear stria vascularis and decline of the endocochlear potential (EP). This form was asserted to be genetically linked, and to arise independently from age-related pathology of either the organ of Corti or cochlear neurons. Although extensive strial degeneration in humans coincides with hearing loss, EPs have never been measured in humans, and age-related EP reduction has never been verified. No human genes that promote strial presbycusis have been identified, nor is its pathophysiology well understood. Effective application of animal models to this issue requires models demonstrating EP decline, and preferably, genetically distinct strains that vary in patterns of EP decline and its cellular correlates. Until recently, only two models, Mongolian gerbils and Tyrp1(B-lt) mice, were known to undergo age-associated EP reduction. Detailed studies of seven inbred mouse strains have now revealed three strains (C57BL/6J, B6.CAST-Cdh23(CAST), CBA/J) showing essentially no EP decline with age, and four strains ranging from modest to severe EP reduction (C57BL/6-Tyr(c-2J), BALB/cJ, CBA/CaJ, NOD.NON-H2(nbl)/LtJ). Collectively, animal models support five basic principles regarding a strial form of presbycusis: 1) Progressive EP decline from initially normal levels as a defining characteristic; 2) Non-universality, not all age-associated hearing loss involves EP decline; 3) A clear genetic basis; 4) Modulation by environment or stochastic events; and 5) Independent strial, organ of Corti, and neural pathology. Shared features between human strial presbycusis, gerbils, and BALB/cJ and C57BL/6-Tyr(c-2J) mice further suggest this condition frequently begins with strial marginal cell dysfunction and loss. By contrast, NOD.NON-H2(nbl) mice may model a sequence more closely associated with strial microvascular disease. Additional studies of these and other inbred mouse and rat models should reveal candidate processes and genes that promote EP decline in humans.

Cochlear and retinal degeneration in the tubby mouse.

A number of autosomal recessive syndromes feature both sensorineural hearing loss and retinal degeneration. The mouse mutant tubby also combines hearing loss with progressive retinal degeneration, and thus may constitute a useful model of one form of human sensorineural deafness/retinal dystrophic syndrome. It has not been directly demonstrated that the hearing loss in this mouse involves the cochlea, however. We have examined the cochleas of adult tubby mice using light microscopy. The tubby cochlea shows pronounced degeneration of the organ of Corti and loss of afferent neurons in the base, with relative sparing of the apex. Our findings support the tubby mouse as a model of human sensorineural deafness/retinal dystrophic syndrome. Possible human counterparts include Usher’s, Alstrom’s, and Bardet–Biedl syndromes.

Age-related hearing loss: the status of Schuknecht's typology.

Purpose of reviewRecent developments in age-related hearing loss (ARHL) are reviewed with an emphasis on their relation to the framework advocated by Schuknecht. More than a classification scheme, Schuknecht’s typology incorporates testable hypotheses about the bases of ARHL. Since there is presently no widely accepted competing framework, research in this area should be aimed at supporting, modifying, or replacing Schuknecht scheme. Only recently has our understanding of cellular changes and gene/environment interactions in ARHL achieved the level needed for hypothesis-driven experiments in this area. Recent findingsNew findings largely support or amplify aspects of Schuknecht’s framework. Consideration of the kinds of cells involved in ARHL has broadened to include more nonsensory and supporting cells. This should provide more complete criteria for comparing models, and for diagnosing particular forms of ARHL. Newly discovered genetic effects and more detailed comparisons have imparted mechanistic significance to the often-noted similarity between sensory ARHL and noise injury. Recent comparative studies, and studies of cell replacement in the cochlear lateral wall, suggest variations in the relation between strial and ligament pathology, and indicate why cell loss occurs during aging. Mouse models carrying mutations affecting processes that may give rise to ARHL are receiving increased attention, even as detailed studies bolster support for mice as valid ARHL models. SummaryUsing Schuknecht’s framework as a guide, basic research can now seek to model specific forms of ARHL by combining genetic defects and appropriate environmental conditions. Identification of distinct risk factors for age-related degeneration of organ of Corti, afferent neurons, and stria would verify a key tenet of Schuknecht’s scheme, and point the way to interventions.

Biodistribution, Kinetics, and Efficacy of Highly Phosphorylated and Non-phosphorylated β-Glucuronidase in the Murine Model of Mucopolysaccharidosis VII

Enzyme replacement therapy (ERT) has been shown to be effective at reducing the accumulation of undegraded substrates in lysosomal storage diseases. Most ERT studies have been performed with recombinant proteins that are mixtures of phosphorylated and non-phosphorylated enzyme. Because different cell types use different receptors to take up phosphorylated or non-phosphorylated enzyme, it is difficult to determine which form of enzyme contributed to the clinical response. Here we compare the uptake, distribution, and efficacy of highly phosphorylated and non-phosphorylated β-glucuronidase (GUSB) in the MPS VII mouse. Highly phosphorylated murine GUSB was efficiently taken up by a wide range of tissues. In contrast, non-phosphorylated murine GUSB was taken up primarily by tissues of the reticuloendothelial (RE) system. Although the tissue distribution was different, the half-lives of both enzymes in any particular tissue were similar. Both preparations of enzyme were capable of preventing the accumulation of lysosomal storage in cell types they targeted. An important difference in clinical efficacy emerged in that phosphorylated GUSB was more efficient than non-phosphorylated enzyme at preventing the hearing loss associated with this disease. These data suggest that both forms of enzyme contribute to the clinical responses of ERT in MPS VII mice but that enzyme preparations containing phosphorylated GUSB are more broadly effective than non-phosphorylated enzyme.

Lentiviral-transduced human mesenchymal stem cells persistently express therapeutic levels of enzyme in a xenotransplantation model of human disease

Bone marrow‐derived mesenchymal stem cells (MSCs) are a promising platform for cell‐ and gene‐based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD‐SCID MPSVII]). Primary human bone marrow‐derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme β‐glucuronidase (MSCs‐GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD‐SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs‐GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs‐GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease.