Nr2f1 heterozygous knockout mice recapitulate neurological phenotypes of Bosch-Boonstra-Schaaf optic atrophy syndrome and show impaired hippocampal synaptic plasticity.

Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) has been identified as an autosomal-dominant disorder characterized by a complex neurological phenotype, with high prevalence of intellectual disability and optic nerve atrophy/hypoplasia. The syndrome is caused by loss-of-function mutations in NR2F1, which encodes a highly conserved nuclear receptor that serves as a transcriptional regulator. Previous investigations to understand the protein's role in neurodevelopment have mostly used mouse models with constitutive and tissue-specific homozygous knockout of Nr2f1. In order to represent the human disease more accurately, which is caused by heterozygous NR2F1 mutations, we investigated a heterozygous knockout mouse model and found that this model recapitulates some of the neurological phenotypes of BBSOAS, including altered learning/memory, hearing defects, neonatal hypotonia and decreased hippocampal volume. The mice showed altered fear memory, and further electrophysiological investigation in hippocampal slices revealed significantly reduced long-term potentiation and long-term depression. These results suggest that a deficit or alteration in hippocampal synaptic plasticity may contribute to the intellectual disability frequently seen in BBSOAS. RNA-sequencing (RNA-Seq) analysis revealed significant differential gene expression in the adult Nr2f1+/- hippocampus, including the up-regulation of multiple matrix metalloproteases, which are known to be critical for the development and the plasticity of the nervous system. Taken together, our studies highlight the important role of Nr2f1 in neurodevelopment. The discovery of impaired hippocampal synaptic plasticity in the heterozygous mouse model sheds light on the pathophysiology of altered memory and cognitive function in BBSOAS.

Otud7a Knockout Mice Recapitulate Many Neurological Features of 15q13.3 Microdeletion Syndrome.

15q13.3 microdeletion syndrome is characterized by a wide spectrum of neurodevelopmental disorders, including developmental delay, intellectual disability, epilepsy, language impairment, abnormal behaviors, neuropsychiatric disorders, and hypotonia. This syndrome is caused by a deletion on chromosome 15q, which typically encompasses six genes. Here, through studies on OTU deubiquitinase 7A (Otud7a) knockout mice, we identify OTUD7A as a critical gene responsible for many of the cardinal phenotypes associated with 15q13.3 microdeletion syndrome. Otud7a-null mice show reduced body weight, developmental delay, abnormal electroencephalography patterns and seizures, reduced ultrasonic vocalizations, decreased grip strength, impaired motor learning/motor coordination, and reduced acoustic startle. We show that OTUD7A localizes to dendritic spines and that Otud7a-null mice have decreased dendritic spine density compared to their wild-type littermates. Furthermore, frequency of miniature excitatory postsynaptic currents (mEPSCs) is reduced in the frontal cortex of Otud7a-null mice, suggesting a role of Otud7a in regulation of dendritic spine density and glutamatergic synaptic transmission. Taken together, our results suggest decreased OTUD7A dosage as a major contributor to the neurodevelopmental phenotypes associated with 15q13.3 microdeletion syndrome, through the misregulation of dendritic spine density and activity.

Adenosine A2B receptor: A pathogenic factor and a therapeutic target for sensorineural hearing loss.

Over 466 million people worldwide are diagnosed with hearing loss (HL). About 90% of HL cases are sensorineural HL (SNHL) with treatments limited to hearing aids and cochlear implants with no FDA-approved drugs. Intriguingly, ADA-deficient patients have been reported to have bilateral SNHL, however, its underlying cellular and molecular basis remain unknown. We report that Ada-/- mice, phenocopying ADA-deficient humans, displayed SNHL. Ada-/- mice cochlea with elevated adenosine caused substantial nerve fiber demyelination and mild hair cell loss. ADA enzyme therapy in these mice normalized cochlear adenosine levels, attenuated SNHL, and prevented demyelination. Additionally, ADA enzyme therapy rescued SNHL by restoring nerve fiber structure in Ada-/- mice post two-week drug withdrawal. Moreover, elevated cochlear adenosine in untreated mice was associated with enhanced Adora2b gene expression. Preclinically, ADORA2B-specific antagonist treatment in Ada-/- mice significantly improved HL, nerve fiber density, and myelin compaction. We also provided genetic evidence that ADORA2B is detrimental for age-related SNHL by impairing cochlear myelination in WT aged mice. Overall, understanding purinergic molecular signaling in SNHL in Ada-/- mice allows us to further discover that ADORA2B is also a pathogenic factor underlying aged-related SNHL by impairing cochlear myelination and lowering cochlear adenosine levels or blocking ADORA2B signaling are effective therapies for SNHL.

An Atoh1-S193A Phospho-Mutant Allele Causes Hearing Deficits and Motor Impairment.

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix (bHLH) transcription factor that is essential for the genesis, survival, and maturation of a variety of neuronal and non-neuronal cell populations, including those involved in proprioception, interoception, balance, respiration, and hearing. Such diverse functions require fine regulation at the transcriptional and protein levels. Here, we show that serine 193 (S193) is phosphorylated in Atoh1's bHLH domain in vivo Knock-in mice of both sexes bearing a GFP-tagged phospho-dead S193A allele on a null background (Atoh1S193A/lacZ) exhibit mild cerebellar foliation defects, motor impairments, partial pontine nucleus migration defects, cochlear hair cell degeneration, and profound hearing loss. We also found that Atoh1 heterozygous mice of both sexes (Atoh1lacZ/+) have adult-onset deafness. These data indicate that different cell types have different degrees of vulnerability to loss of Atoh1 function and that hypomorphic Atoh1 alleles should be considered in human hearing loss.SIGNIFICANCE STATEMENT The discovery that Atonal homolog 1 (Atoh1) governs the development of the sensory hair cells in the inner ear led to therapeutic efforts to restore these cells in cases of human deafness. Because prior studies of Atoh1-heterozygous mice did not examine or report on hearing loss in mature animals, it has not been clinical practice to sequence ATOH1 in people with deafness. Here, in seeking to understand how phosphorylation of Atoh1 modulates its effects in vivo, we discovered that inner ear hair cells are much more vulnerable to loss of Atoh1 function than other Atoh1-positive cell types and that heterozygous mice actually develop hearing loss late in life. This opens up the possibility that missense mutations in ATOH1 could increase human vulnerability to loss of hair cells because of aging or trauma.

Survival of auditory hair cells.

The inability of mammals to regenerate auditory hair cells creates a pressing need to understand the means of enhancing hair cell survival following insult or injury. Hair cells are easily damaged by noise exposure, by ototoxic medications and as a consequence of aging processes, all of which lead to progressive and permanent hearing impairment as hair cells are lost. Significant efforts have been invested in designing strategies to prevent this damage from occurring since permanent hearing loss has a profound impact on communication and quality of life for patients. In this mini-review, we discuss recent progress in the use of antioxidants, anti-inflammatories and apoptosis inhibitors to enhance hair cell survival. We conclude by clarifying the distinction between protection and rescue strategies and by highlighting important areas of future research.

Conditional deletion of Atoh1 reveals distinct critical periods for survival and function of hair cells in the organ of Corti.

Atonal homolog1 (Atoh1) encodes a basic helix-loop-helix protein that is the first transcription factor to be expressed in differentiating hair cells. Previous work suggests that expression of Atoh1 in prosensory precursors is necessary for the differentiation and survival of hair cells, but it is not clear whether Atoh1 is required exclusively for these processes, or whether it regulates other functions later during hair cell maturation. We used EGFP-tagged Atoh1 knock-in mice to demonstrate for the first time that Atoh1 protein is expressed in hair cell precursors several days before the appearance of differentiated markers, but not in the broad pattern expected of a proneural gene. We conditionally deleted Atoh1 at different points in hair cell development and observe a rapid onset of hair cell defects, suggesting that the Atoh1 protein is unstable in differentiating hair cells and is necessary through an extended phase of their differentiation. Conditional deletion of Atoh1 reveals multiple functions in hair cell survival, maturation of stereociliary bundles, and auditory function. We show the presence of distinct critical periods for Atoh1 in each of these functions, suggesting that Atoh1 may be directly regulating many aspects of hair cell function. Finally, we show that the supporting cell death that accompanies loss of Atoh1 in hair cells is likely caused by the abortive trans-differentiation of supporting cells into hair cells. Together our data suggest that Atoh1 regulates multiple aspects of hair cell development and function.

Factors Associated With Adherence of Cervical Cancer Screening in Women With Systemic Lupus Erythematosus.

OBJECTIVE: The objective is to determine cervical cancer screening rates and factors associated with decreased cervical cancer screening in women with systemic lupus erythematosus (SLE). METHODS: We conducted a cross-sectional study that enrolled consecutive women (age 21-64 years) with SLE. We collected demographics, clinical characteristics, constructs of the Health Beliefs Model (HBM) (ie, susceptibility, severity, barriers, benefits, cues to action, and self-efficacy), and self-reported cervical cancer screening (confirmed with the electronic medical record). The primary outcome was adherence to cervical cancer screening according to current guidelines. Multivariable logistic regression models were used to examine the association between SLE disease activity and cervical cancer screening and explore mediation effects from HBM constructs. RESULTS: We enrolled 130 women with SLE. The median age was 42 years (interquartile range 32-52 years). The cervical cancer screening adherence rate was 61.5%. Women with high SLE disease activity were less likely to have cervical cancer screening versus those with low disease activity (odds ratio 0.59, 95% confidence interval [CI] 0.39-0.89; P = 0.01), which remained statistically significant after adjusting for baseline demographics and drug therapy in a multivariable model (odds ratio 0.25, 95% CI 0.08-0.79; P = 0.02). Regarding the HBM constructs, increased perceived barriers to cervical cancer screening (r = -0.30, P < 0.01) and decreased self-efficacy (r = -0.21, P = 0.02) correlated with decreased cervical cancer screening. CONCLUSION: Patients with SLE with high disease activity undergo cervical cancer screening less frequently than those with low disease activity. Perceived barriers to cervical cancer screening are moderately correlated with decreased screening. These data highlight the need to develop strategies to increase cervical cancer screening in this high-risk patient population.

Texas Developmental Center for AIDS Research CFAR Diversity, Equity, and Inclusion Pathway Initiative Program.

BACKGROUND: The Texas Developmental Center for AIDS Research (D-CFAR) diversity program, termed the CFAR Diversity, Equity, and Inclusion Pathway Initiative (CDEIPI), was created in 2021 to engage high school students and graduate students from Underrepresented Minorities/Black, Indigenous, and People of Color populations. SETTING: The Texas D-CFAR CDEIPI has partnered with 2 Texas high schools with predominantly economically disadvantaged and minority student populations-Michael E. DeBakey High School for Health Professions in Houston, TX, and the South Texas Independent School District Medical Professions High School in Olmito, TX in the Rio Grande Valley. METHODS: A total of 370 high school student learners at both partner schools participated in presentations of research and career paths related to HIV-1 and SARS-CoV-2 during the 2021-2022 academic year. Afterward, learners completed anonymous surveys to share their self-reported interest in research degrees and careers. RESULTS: Learners reported increased knowledge of related science content and interest in research careers, including HIV-1 research, after each of the sessions. CONCLUSIONS: The programming has been of interest to student learners, and future additions intend to build upon the Texas D-CFAR CDEIPI.

Centers for AIDS Research (CFAR) Diversity, Equity, and Inclusion Pathway Initiative (CDEIPI): Developing Career Pathways for Early-Stage Scholars From Racial and Ethnic Groups Underrepresented in HIV Science and Medicine.

BACKGROUND: There is an urgent need to increase diversity among scientific investigators in the HIV research field to be more reflective of communities highly affected by the HIV epidemic. Thus, it is critical to promote the inclusion and advancement of early-stage scholars from racial and ethnic groups underrepresented in HIV science and medicine. METHODS: To widen the HIV research career pathway for early-stage scholars from underrepresented minority groups, the National Institutes of Health supported the development of the Centers for AIDS Research (CFAR) Diversity, Equity, and Inclusion Pathway Initiative (CDEIPI). This program was created through partnerships between CFARs and Historically Black Colleges and Universities and other Minority Serving Institutions throughout the United States. RESULTS: Seventeen CFARs and more than 20 Historically Black Colleges and Universities and Minority Serving Institutions have participated in this initiative to date. Programs were designed for the high school (8), undergraduate (13), post baccalaureate (2), graduate (12), and postdoctoral (4) levels. Various pedagogical approaches were used including didactic seminar series, intensive multiday workshops, summer residential programs, and mentored research internship opportunities. During the first 18 months of the initiative, 257 student scholars participated in CDEIPI programs including 150 high school, 73 undergraduate, 3 post baccalaureate, 27 graduate, and 4 postdoctoral students. CONCLUSION: Numerous student scholars from a wide range of educational levels, geographic backgrounds, and racial and ethnic minority groups have engaged in CDEIPI programs. Timely and comprehensive program evaluation data will be critical to support a long-term commitment to this unique training initiative.

Ablation of ghrelin receptor in leptin-deficient ob/ob mice has paradoxical effects on glucose homeostasis when compared with ablation of ghrelin in ob/ob mice.

The orexigenic hormone ghrelin is important in diabetes because it has an inhibitory effect on insulin secretion. Ghrelin ablation in leptin-deficient ob/ob (Ghrelin(-/-):ob/ob) mice increases insulin secretion and improves hyperglycemia. The physiologically relevant ghrelin receptor is the growth hormone secretagogue receptor (GHS-R), and GHS-R antagonists are thought to be an effective strategy for treating diabetes. However, since some of ghrelin's effects are independent of GHS-R, we have utilized genetic approaches to determine whether ghrelin's effect on insulin secretion is mediated through GHS-R and whether GHS-R antagonism indeed inhibits insulin secretion. We investigated the effects of GHS-R on glucose homeostasis in Ghsr-ablated ob/ob mice (Ghsr(-/-):ob/ob). Ghsr ablation did not rescue the hyperphagia, obesity, or insulin resistance of ob/ob mice. Surprisingly, Ghsr ablation worsened the hyperglycemia, decreased insulin, and impaired glucose tolerance. Consistently, Ghsr ablation in ob/ob mice upregulated negative ß-cell regulators (such as UCP-2, SREBP-1c, ChREBP, and MIF-1) and downregulated positive ß-cell regulators (such as HIF-1α, FGF-21, and PDX-1) in whole pancreas; this suggests that Ghsr ablation impairs pancreatic ß-cell function in leptin deficiency. Of note, Ghsr ablation in ob/ob mice did not affect the islet size; the average islet size of Ghsr(-/-):ob/ob mice is similar to that of ob/ob mice. In summary, because Ghsr ablation in leptin deficiency impairs insulin secretion and worsens hyperglycemia, this suggests that GHS-R antagonists may actually aggravate diabetes under certain conditions. The paradoxical effects of ghrelin ablation and Ghsr ablation in ob/ob mice highlight the complexity of the ghrelin-signaling pathway.

Cysteine mutagenesis reveals transmembrane residues associated with charge translocation in prestin.

The solute carrier transmembrane protein prestin (SLC26A5) drives an active electromechanical transduction process in cochlear outer hair cells that increases hearing sensitivity and frequency discrimination in mammals. A large intramembraneous charge movement, the nonlinear capacitance (NLC), is the electrical signature of prestin function. The transmembrane domain (TMD) helices and residues involved in the intramembrane charge displacement remain unknown. We have performed cysteine-scanning mutagenesis with serine or valine replacement to investigate the importance of cysteine residues to prestin structure and function. The distribution of oligomeric states and membrane abundance of prestin was also probed to investigate whether cysteine residues participate in prestin oligomerization and/or NLC. Our results reveal that 1) Cys-196 (TMD 4) and Cys-415 (TMD 10) do not tolerate serine replacement, and thus maintaining hydrophobicity at these locations is important for the mechanism of charge movement; 2) Cys-260 (TMD 6) and Cys-381 (TMD 9) tolerate serine replacement and are probably water-exposed; and 3) if disulfide bonds are present, they do not serve a functional role as measured via NLC. These novel findings are consistent with a recent structural model, which proposes that prestin contains an occluded aqueous pore, and we posit that the orientations of transmembrane domain helices 4 and 10 are essential for proper prestin function.

Selective cell-surface labeling of the molecular motor protein prestin.

Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

Identification of functionally important residues/domains in membrane proteins using an evolutionary approach coupled with systematic mutational analysis.

Structure-function studies of membrane proteins present a unique challenge to researchers due to the numerous technical difficulties associated with their expression, purification and structural characterization. In the absence of structural information, rational identification of putative functionally important residues/regions is difficult. Phylogenetic relationships could provide valuable information about the functional significance of a particular residue or region of a membrane protein. Evolutionary Trace (ET) analysis is a method developed to utilize this phylogenetic information to predict functional sites in proteins. In this method, residues are ranked according to conservation or divergence through evolution, based on the hypothesis that mutations at key positions should coincide with functional evolutionary divergences. This information can be used as the basis for a systematic mutational analysis of identified residues, leading to the identification of functionally important residues and/or domains in membrane proteins, in the absence of structural information apart from the primary amino acid sequence. This approach is potentially useful in the context of the auditory system, as several key processes in audition involve the action of membrane proteins, many of which are novel and not well characterized structurally or functionally to date.

Functional prestin transduction of immature outer hair cells from normal and prestin-null mice.

Prestin is a membrane protein in the outer hair cell (OHC) that has been shown to be essential for electromotility. OHCs from prestin-null mice do not express prestin, do not have a nonlinear capacitance (the electrical signature of electromotility), and are smaller in size than wild-type OHCs. We sought to determine whether prestin-null OHCs can be transduced to incorporate functional prestin protein in a normal fashion. A recombinant helper-dependent adenovirus expressing prestin and green fluorescent protein (HDAd-prestin-GFP) was created and tested in human embryonic kidney cells (HEK cells). Transduced HEK cells demonstrated membrane expression of prestin and nonlinear capacitance. HDAd-prestin-GFP was then applied to cochlear sensory epithelium explants harvested from wild-type and prestin-null mice at postnatal days 2-3, the age at which native prestin is just beginning to become functional in wild-type mice. At postnatal days 4-5, we investigated transduced OHCs for (1) their prestin expression pattern as revealed by immunofluorescence; (2) their cell surface area as measured by linear capacitance; and (3) their prestin function as indicated by nonlinear capacitance. HDAd-prestin-GFP efficiently transduced OHCs of both genotypes and prestin protein localized to the plasma membrane. Whole-cell voltage clamp studies revealed a nonlinear capacitance in transduced wild-type and prestin-null OHCs, but not in non-transduced cells of either genotype. Prestin transduction did not increase the linear capacitance (cell surface area) for either genotype. In peak nonlinear capacitance, voltage at peak nonlinear capacitance, charge density of the nonlinear capacitance, and shape of the voltage-capacitance curves, the transduced cells of the two genotypes resembled each other and previously reported data from adult wild-type mouse OHCs. Thus, prestin introduced into prestin-deficient OHCs segregates normally to the cell membrane and generates a normal nonlinear capacitance, indicative of normal prestin function.

Trehalose reduces retinal degeneration, neuroinflammation and storage burden caused by a lysosomal hydrolase deficiency.

The accumulation of undegraded molecular material leads to progressive neurodegeneration in a number of lysosomal storage disorders (LSDs) that are caused by functional deficiencies of lysosomal hydrolases. To determine whether inducing macroautophagy/autophagy via small-molecule therapy would be effective for neuropathic LSDs due to enzyme deficiency, we treated a mouse model of mucopolysaccharidosis IIIB (MPS IIIB), a storage disorder caused by deficiency of the enzyme NAGLU (alpha-N-acetylglucosaminidase [Sanfilippo disease IIIB]), with the autophagy-inducing compound trehalose. Treated naglu-/ - mice lived longer, displayed less hyperactivity and anxiety, retained their vision (and retinal photoreceptors), and showed reduced inflammation in the brain and retina. Treated mice also showed improved clearance of autophagic vacuoles in neuronal and glial cells, accompanied by activation of the TFEB transcriptional network that controls lysosomal biogenesis and autophagic flux. Therefore, small-molecule-induced autophagy enhancement can improve the neurological symptoms associated with a lysosomal enzyme deficiency and could provide a viable therapeutic approach to neuropathic LSDs. ABBREVIATIONS: ANOVA: analysis of variance; Atg7: autophagy related 7; AV: autophagic vacuoles; CD68: cd68 antigen; ERG: electroretinogram; ERT: enzyme replacement therapy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GNAT2: guanine nucleotide binding protein, alpha transducing 2; HSCT: hematopoietic stem cell transplantation; INL: inner nuclear layer; LC3: microtubule-associated protein 1 light chain 3 alpha; MPS: mucopolysaccharidoses; NAGLU: alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); ONL: outer nuclear layer; PBS: phosphate-buffered saline; PRKCA/PKCα: protein kinase C, alpha; S1BF: somatosensory cortex; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; VMP/VPL: ventral posterior nuclei of the thalamus.

Essential helix interactions in the anion transporter domain of prestin revealed by evolutionary trace analysis.

Prestin, a member of the SLC26A family of anion transporters, is a polytopic membrane protein found in outer hair cells (OHCs) of the mammalian cochlea. Prestin is an essential component of the membrane-based motor that enhances electromotility of OHCs and contributes to frequency sensitivity and selectivity in mammalian hearing. Mammalian cells expressing prestin display a nonlinear capacitance (NLC), widely accepted as the electrical signature of electromotility. The associated charge movement requires intracellular anions reflecting the membership of prestin in the SLC26A family. We used the computational approach of evolutionary trace analysis to identify candidate functional (trace) residues in prestin for mutational studies. We created a panel of mutations at each trace residue and determined membrane expression and nonlinear capacitance associated with each mutant. We observe that several residue substitutions near the conserved sulfate transporter domain of prestin either greatly reduce or eliminate NLC, and the effect is dependent on the size of the substituted residue. These data suggest that packing of helices and interactions between residues surrounding the "sulfate transporter motif" is essential for normal prestin activity.

Altered traveling wave propagation and reduced endocochlear potential associated with cochlear dysplasia in the BETA2/NeuroD1 null mouse.

The BETA2/NeuroD1 null mouse has cochlear dysplasia. Its cochlear duct is shorter than normal, there is a lack of spiral ganglion neurons, and there is hair cell disorganization. We measured vertical movements of the tectorial membrane at acoustic frequencies in excised cochleae in response to mechanical stimulation of the stapes using laser doppler vibrometry. While tuning curve sharpness was similar between wild-type, heterozygotes, and null mice in the base, null mutants had broader tuning in the apex. At both the base and the apex, null mice had less phase lag accumulation with increasing stimulus frequency than wild-type or heterozygote mice. In vivo studies demonstrated that the null mouse lacked distortion product otoacoustic emissions, and the cochlear microphonic and endocochlear potential were found to be severely reduced. Electrically evoked otoacoustic emissions could be elicited, although the amplitudes were lower than those of wild-type mice. Cochlear cross-sections revealed an incomplete partition malformation, with fenestrations within the modiolus that connected the cochlear turns. Outer hair cells from null mice demonstrated the normal pattern of prestin expression within their lateral walls and normal FM 1-43 dye entry. Overall, these data demonstrate that while tonotopicity can exist with cochlear dysplasia, traveling wave propagation is abnormally fast. Additionally, the presence of electrically evoked otoacoustic emissions suggests that outer hair cell reverse transduction is present, although the acoustic response is shaped by the alterations in cochlear mechanics.

The nuclear receptor Nor-1 is essential for proliferation of the semicircular canals of the mouse inner ear.

Nor-1 belongs to the nur subfamily of nuclear receptor transcription factors. The precise role of Nor-1 in mammalian development has not been established. However, recent studies indicate a function for this transcription factor in oncogenesis and apoptosis. To examine the spatiotemporal expression pattern of Nor-1 and the developmental and physiological consequences of Nor-1 ablation, Nor-1-null mice were generated by insertion of the lacZ gene into the Nor-1 genomic locus. Disruption of the Nor-1 gene results in inner ear defects and partial bidirectional circling behavior. During early otic development, Nor-1 is expressed exclusively in the semicircular canal forming fusion plates. After formation of the membranous labyrinth, Nor-1 expression in the vestibule is limited to nonsensory epithelial cells localized at the inner edge of the semicircular canals and to the ampullary and utricular walls. In the absence of Nor-1, the vestibular walls fuse together as normal; however, the endolymphatic fluid space in the semicircular canals is diminished and the roof of the ampulla appears flattened due to defective continual proliferative growth of the semicircular canals.

Functional expression and microdomain localization of prestin in cultured cells.

INTRODUCTION: Prestin is an essential component of the molecular motor of cochlear outer hair cells that contribute to frequency selectivity and sensitivity of mammalian hearing. A model system to study prestin employs its transfection into cultured HEK 293 cells. Our goal was to characterize prestin's trafficking pathway and localization in the plasma membrane. METHODS: We used immuno-colocalization of prestin with intracellular and plasma membrane markers and sucrose density fractionation to analyze prestin in membrane compartments. Voltage clamping was used to measure nonlinear capacitance (NLC), prestin's electrical signature. RESULTS & DISCUSSION: Prestin targets to the membrane by 24 hours post-transfection when NLC is measurable. Prestin then concentrates into membrane foci that colocalize and fractionate with membrane microdomains. Depleting membrane cholesterol content altered prestin localization and NLC. CONCLUSION: Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.

Helper-dependent adenovirus-mediated gene transfer into the adult mouse cochlea.

BACKGROUND: Gene therapy may provide a way to restore cochlear function to deaf patients. The most successful techniques for cochlear gene therapy have been injection of early-generation adenoviral vectors into scala media in guinea pigs. However, it is important to be able to perform gene therapy research in mice because there is wide availability of transgenic strains with hereditary hearing loss. PURPOSE: We demonstrate our technique for delivery of a third-generation adenoviral vector, helper-dependent adenovirus (HDAd), to the adult mouse cochlea. METHODS: Mice were injected with an HDAd that contained a reporter gene for either beta-galactosidase or green fluorescent protein into scala media. After 4 days, the cochleae were harvested for analyses. Auditory brainstem response monitoring of cochlear function was performed before making a cochleostomy, after making a cochleostomy, and before killing the animal. RESULTS: Beta-galactosidase was identified in the spiral ligament, the organ of Corti, and spiral ganglion cells by light microscopy. Green fluorescent protein epifluorescence was assessed in whole-mount organ of Corti preparations using confocal microscopy. This demonstrated transduction of inner hair cells, outer hair cells, and supporting cells. Paraffin-embedded cross sections similarly revealed gene transduction within the organ of Corti. Threshold shifts of 39.8 +/- 5.4 and 37.7 +/- 5.5 dB were observed in mice injected with HDAd or control buffer, respectively. CONCLUSION: The technique of scala media HDAd injection reliably infects the adult mouse cochlea, including cells within the organ of Corti, although the procedure itself adversely affects hearing.