A Frameshift Mutation in GRXCR 2 Causes Recessively Inherited Hearing Loss

More than 360 million humans are affected with some degree of hearing loss, either early or later in life. A genetic cause for the disorder is present in a majority of the cases. We mapped a locus ( DFNB 101) for hearing loss in humans to chromosome 5q in a consanguineous P akistani family. Exome sequencing revealed an insertion mutation in GRXCR 2 as the cause of moderate‐to‐severe and likely progressive hearing loss in the affected individuals of the family. The frameshift mutation is predicted to affect a conserved, cysteine‐rich region of GRXCR 2, and to result in an abnormal extension of the C ‐terminus. Functional studies by cell transfections demonstrated that the mutant protein is unstable and mislocalized relative to wild‐type GRXCR 2, consistent with a loss‐of‐function mutation. Targeted disruption of G rxcr2 is concurrently reported to cause hearing loss in mice. The structural abnormalities in this animal model suggest a role for GRXCR 2 in the development of stereocilia bundles, specialized structures on the apical surface of sensory cells in the cochlea that are critical for sound detection. Our results indicate that GRXCR 2 should be considered in differential genetic diagnosis for individuals with early onset, moderate‐to‐severe and progressive hearing loss. We mapped a new deafness locus DFNB101 and discovered an insertion mutation in GRXCR2 as the cause of moderate‐to‐severe hearing loss in humans. The frameshift mutation was predicted to affect the conserved, cysteine‐rich domain of GRXCR2, and to result in an abnormal extension of the C‐terminus. Cell transfection experiments demonstrated that the mutant protein is unstable and mislocalized relative to the wild type GRXCR2, consistent with a loss of function mutation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106992/1/humu22545.pd

An SV40 transformation revertant due to a host mutation: Isolation and complementation analysis

We have isolated an SV40 transformation revertant cell line, CLi L, by selection for normal cells whose growth is inhibited under low serum conditions. This line expresses a single, wild-type copy of large T antigen, yet is not transformed. It is not retransformable by transfection of SV40 DNA or infection with a recombinant retrovirus encoding large T antigen. Resistance to transformation therefore appears to be due to a cellular mutation. Fusion of CL1 L cells to normal 3T3 cells or SV40-transformed cells results in somatic cell hybrids that are transformed, indicating that resistance is recessive. In addition, fusion of CL1L cells to another SV40 transformation-resistant line, A27, results in transformed hybrids, indicating the existence of discrete complementation groups with respect to SV40 transformation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30140/1/0000517.pd

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 ( sh2 ) and pirouette ( pi ) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions “cytocauds.” The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47461/1/11068_2004_Article_278960.pd

Allelic mutations of the sodium channel SCN8A reveal multiple cellular and physiological functions

Allelic mutations of Scn8a in the mouse have revealed the range of neurological disorders that can result from alternations of one neuronal sodium channel. Null mutations produce the most severe phenotype, with motor neuron failure leading to paralysis and juvenile lethality. Two less severe mutations cause ataxia, tremor, muscle weakness, and dystonia. The electrophysiological effects have been studied at the cellular level by recording from neurons from the mutant mice. The data demonstrate that Scn8a is required for the complex spiking of cerebellar Purkinje cells and for persistent sodium current in several classes of neurons, including some with pacemaker roles. The mouse mutations of Scn8a have also provided insight into the mode of inheritance of channelopathies, and led to the identification of a modifier gene that affects transcript splicing. These mutations demonstrate the value of mouse models to elucidate the pathophysiology of human disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42795/1/10709_2004_Article_5381441.pd

Visualizing the metazoan proliferation-quiescence decision in vivo

Cell proliferation and quiescence are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these key events of the cell cycle in Caenorhabditis elegans and zebrafish through live-cell imaging. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from quiescent cells in G0, revealing a possible commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. Finally, we derive a predictive model of future proliferation behavior in C. elegans based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitate in vivo studies of cell-cycle control in a wide-range of developmental contexts. </div

Axon–glia interactions in the ascending auditory system

The auditory system detects and encodes sound information with high precision to provide a high‐fidelity representation of the environment and communication. In mammals, detection occurs in the peripheral sensory organ (the cochlea) containing specialized mechanosensory cells (hair cells) that initiate the conversion of sound‐generated vibrations into action potentials in the auditory nerve. Neural activity in the auditory nerve encodes information regarding the intensity and frequency of sound stimuli, which is transmitted to the auditory cortex through the ascending neural pathways. Glial cells are critical for precise control of neural conduction and synaptic transmission throughout the pathway, allowing for the precise detection of the timing, frequency, and intensity of sound signals, including the sub‐millisecond temporal fidelity is necessary for tasks such as sound localization, and in humans, for processing complex sounds including speech and music. In this review, we focus on glia and glia‐like cells that interact with hair cells and neurons in the ascending auditory pathway and contribute to the development, maintenance, and modulation of neural circuits and transmission in the auditory system. We also discuss the molecular mechanisms of these interactions, their impact on hearing and on auditory dysfunction associated with pathologies of each cell type.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/169355/1/dneu22813_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/169355/2/dneu22813.pd

\u3ci\u3eGrxcr2\u3c/i\u3e is required for stereocilia morphogenesis in the cochlea

Hearing and balance depend upon the precise morphogenesis and mechanosensory function of stereocilia, the specialized structures on the apical surface of sensory hair cells in the inner ear. Previous studies of Grxcr1 mutant mice indicated a critical role for this gene in control of stereocilia dimensions during development. In this study, we analyzed expression of the paralog Grxcr2 in the mouse and evaluated auditory and vestibular function of strains carrying targeted mutations of the gene. Peak expression of Grxcr2 occurs during early postnatal development of the inner ear and GRXCR2 is localized to stereocilia in both the cochlea and in vestibular organs. Homozygous Grxcr2 deletion mutants exhibit significant hearing loss by 3 weeks of age that is associated with developmental defects in stereocilia bundle orientation and organization. Despite these bundle defects, the mechanotransduction apparatus assembles in relatively normal fashion as determined by whole cell electrophysiological evaluation and FM1-43 uptake. Although Grxcr2 mutants do not exhibit overt vestibular dysfunction, evaluation of vestibular evoked potentials revealed subtle defects of the mutants in response to linear accelerations. In addition, reduced Grxcr2 expression in a hypomorphic mutant strain is associated with progressive hearing loss and bundle defects. The stereocilia localization of GRXCR2, together with the bundle pathologies observed in the mutants, indicate that GRXCR2 plays an intrinsic role in bundle orientation, organization, and sensory function in the inner ear during development and at maturity

Cochlear Neurotrophin-3 overexpression at mid-life prevents age-related inner hair cell synaptopathy and slows age-related hearing loss

Age-related hearing loss (ARHL) is the most prevalent sensory deficit in the elderly. This progressive pathology often has psychological and medical comorbidities, including social isolation, depression, and cognitive decline. Despite ARHL’s enormous societal and economic impact, no therapies to prevent or slow its progression exist. Loss of synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs), a.k.a. IHC synaptopathy, is an early event in cochlear aging, preceding neuronal and hair cell loss. To determine if age-related IHC synaptopathy can be prevented, and if this impacts the time-course of ARHL, we tested the effects of cochlear overexpression of neurotrophin-3 (Ntf3) starting at middle age. We chose Ntf3 because this neurotrophin regulates the formation of IHC-SGN synapses in the neonatal period. We now show that triggering Ntf3 overexpression by IHC supporting cells starting in middle age rapidly increases the amplitude of sound-evoked neural potentials compared with age-matched controls, indicating that Ntf3 produces a positive effect on cochlear function when the pathology is minimal. Furthermore, near the end of their lifespan, Ntf3-overexpressing mice have milder ARHL, with larger sound-evoked potentials along the ascending auditory pathway and reduced IHC synaptopathy compared with age-matched controls. Our results also provide evidence that an age-related decrease in cochlear Ntf3 expression contributes to ARHL and that Ntf3 supplementation could serve as a therapeutic for this prevalent disorder. Furthermore, these findings suggest that factors that regulate synaptogenesis during development could prevent age-related synaptopathy in the brain, a process involved in several central nervous system degenerative disorders.We show that triggering cochlear Ntf3 overexpression starting in middle age rapidly increases the amplitude of sound-evoked neural potentials and reduces age-related inner hair cells synapse loss. Thus, near the end of their lifespan, Ntf3-overexpressing mice have milder age-related hearing loss and maintain larger sound-evoked potentials along the ascending auditory pathway. These findings provide evidence that Ntf3 supplementation could serve as a therapeutic for this prevalent disorder.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/175054/1/acel13708_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/175054/2/acel13708.pd

Characterization of two transgene insertional mutations at pirouette, a mouse deafness locus

The mouse mutant ‘pirouette’ (pi) exhibits profound hearing loss and vestibular defects due to inheritance of a recessive mutation on chromosome 5. Dysfunction has been correlated with defects during maturation of sensory cells in the inner ear. As an initial step in characterizing pirouette at the genetic level, we have localized the candidate interval to a small region on central chromosome 5 by analysis of a congenic strain of pirouette mice. This region exhibits conserved synteny with human chromosome 4 and suggests that pirouette may be a genetic model of the human nonsyndromic deafness disorder DFNB25, which has been localized to 4p15.3–q12. In addition to the original spontaneous pirouette strain, we have identified and characterized 2 additional mouse strains with allelic mutations at the same locus. Analysis of the morphology in each of the 3 pirouette alleles indicated very similar early postnatal alterations in maturation of stereocilia and suggests that the gene affected in pirouette normally plays a role in building or maintaining these structures that are critical for sensory mechanotransduction

Localization of the Homolog of a Mouse Craniofacial Mutant to Human Chromosome 18q11 and Evaluation of Linkage to Human CLP and CPO

The transgene-induced mutation 9257 and the spontaneous mutation twirler cause craniofacial and inner ear malformations and are located on mouse chromosome 18 near the ataxia locusax.To map the human homolog of 9257, a probe from the transgene insertion site was used to screen a human genomic library. Analysis of a cross-hybridizing human clone identified a 3-kb conserved sequence block that does not appear to contain protein coding sequence. Analysis of somatic cell hybrid panels assigned the human locus to 18q11. The polymorphic microsatellite markers D18S1001 and D18S1002 were isolated from the human locus and mapped by linkage analysis using the CEPH pedigrees. The 9257 locus maps close to the centromeres of human chromosome 18q and mouse chromosome 18 at the proximal end of a conserved linkage group. To evaluate the role of this locus in human craniofacial disorders, linkage to D18S1002 was tested in 11 families with autosomal dominant nonsyndromic cleft lip and palate and 3 families with autosomal dominant cleft palate only. Obligatory recombinants were observed in 8 of the families, and negative lod scores from the other families indicated that these disorders are not linked to the chromosome 18 loci