Usher syndrome IIIA: a review of the disorder and preclinical research advances in therapeutic approaches.

Usher syndrome (USH) is an autosomal recessive disorder characterized by sensorineural hearing loss, progressive pigmentary retinopathy, and vestibular dysfunction. The degree and onset of hearing loss vary among subtypes I, II, and III, while blindness often occurs in the second to fourth decades of life. Usher type III (USH3), characterized by postlingual progressive sensorineural hearing loss, varying levels of vestibular dysfunction, and varying degrees of visual impairment, typically manifests in the first to second decades of life. While USH3 is rare, it is highly prevalent in certain populations. RP61, USH3, and USH3A symbolize the same disorder, with the latter symbol used more frequently in recent literature. This review focuses on the clinical features, epidemiology, molecular genetics, treatment, and research advances for sensory deficits in USH3A.

Unconventional secretory pathway activation restores hair cell mechanotransduction in an USH3A model.

The pathogenic variant c.144T>G (p.N48K) in the clarin1 gene (CLRN1) results in progressive loss of vision and hearing in Usher syndrome IIIA (USH3A) patients. CLRN1 is predicted to be an essential protein in hair bundles, the mechanosensory structure of hair cells critical for hearing and balance. When expressed in animal models, CLRN1 localizes to the hair bundle, whereas glycosylation-deficient CLRN1N48K aggregates in the endoplasmic reticulum, with only a fraction reaching the bundle. We hypothesized that the small amount of CLRN1N48K that reaches the hair bundle does so via an unconventional secretory pathway and that activation of this pathway could be therapeutic. Using genetic and pharmacological approaches, we find that clarin1 knockout (clrn1KO/KO ) zebrafish that express the CLRN1c.144T>G pathogenic variant display progressive hair cell dysfunction, and that CLRN1N48K is trafficked to the hair bundle via the GRASP55 cargo-dependent unconventional secretory pathway (GCUSP). On expression of GRASP55 mRNA, or on exposure to the drug artemisinin (which activates GCUSP), the localization of CLRN1N48K to the hair bundles was enhanced. Artemisinin treatment also effectively restored hair cell mechanotransduction and attenuated progressive hair cell dysfunction in clrn1KO/KO larvae that express CLRN1c.144T>G , highlighting the potential of artemisinin to prevent sensory loss in CLRN1c.144T>G patients.

A biallelic variant in CLRN2 causes non-syndromic hearing loss in humans.

Deafness, the most frequent sensory deficit in humans, is extremely heterogeneous with hundreds of genes involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein, as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 14.96 Mb locus on chromosome 4p15.32p15.1 containing a likely pathogenic missense variant in CLRN2 (c.494C > A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C > A variant leads to two events: (1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and (2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

A small molecule mitigates hearing loss in a mouse model of Usher syndrome III.

Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844). To test the efficacy of BF844, we developed a mouse model that mimicked the progressive hearing loss associated with USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the CLRN1(N48K) mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in patients with USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.

Zebrafish Models for the Mechanosensory Hair Cell Dysfunction in Usher Syndrome 3 Reveal That Clarin-1 Is an Essential Hair Bundle Protein.

Usher syndrome type III (USH3) is characterized by progressive loss of hearing and vision, and varying degrees of vestibular dysfunction. It is caused by mutations that affect the human clarin-1 protein (hCLRN1), a member of the tetraspanin protein family. The missense mutation CLRN1(N48K), which affects a conserved N-glycosylation site in hCLRN1, is a common causative USH3 mutation among Ashkenazi Jews. The affected individuals hear at birth but lose that function over time. Here, we developed an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. Immunolabeling demonstrated that Clrn1 localized to the hair cell bundles (hair bundles). The clrn1 mutants generated by zinc finger nucleases displayed aberrant hair bundle morphology with diminished function. Two transgenic zebrafish that express either hCLRN1 or hCLRN1(N48K) in hair cells were produced to examine the subcellular localization patterns of wild-type and mutant human proteins. hCLRN1 localized to the hair bundles similarly to zebrafish Clrn1; in contrast, hCLRN1(N48K) largely mislocalized to the cell body with a small amount reaching the hair bundle. We propose that this small amount of hCLRN1(N48K) in the hair bundle provides clarin-1-mediated function during the early stages of life; however, the presence of hCLRN1(N48K) in the hair bundle diminishes over time because of intracellular degradation of the mutant protein, leading to progressive loss of hair bundle integrity and hair cell function. These findings and genetic tools provide an understanding and path forward to identify therapies to mitigate hearing loss linked to the CLRN1 mutation. SIGNIFICANCE STATEMENT: Mutations in the clarin-1 gene affect eye and ear function in humans. Individuals with the CLRN1(N48K) mutation are born able to hear but lose that function over time. Here, we develop an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. This approach illuminates the role of clarin-1 and the molecular mechanism linked to the CLRN1(N48K) mutation in sensory hair cells of the inner ear. Additionally, the investigation provided an in vivo model to guide future drug discovery to rescue the hCLRN1(N48K) in hair cells.

ACF7 is a hair-bundle antecedent, positioned to integrate cuticular plate actin and somatic tubulin.

The precise morphology of the mechanosensitive hair bundle requires seamless integration of actin and microtubule networks. Here, we identify Acf7a (actin crosslinking family protein 7a) as a protein positioned to bridge these distinct cytoskeletal networks in hair cells. By imaging Acf7a-Citrine fusion protein in zebrafish and immunolabeling of vestibular and cochlear mouse hair cells, we show that Acf7a and ACF7 circumscribe, underlie, and are interwoven into the cuticular plate (CP), and they also encircle the basal body of the kinocilium. In cochlear hair cells, ACF7 localization is graded, with the highest concentration near each fonticulus--an area free of F-actin in the region of the CP that contains the basal body. During hair-cell development and regeneration, Acf7a precedes formation of the hair bundle and CP. Finally, electron tomography demonstrates that the ends of microtubules insert into the CP and are decorated with filamentous linkers connecting microtubules to the CP. These observations are consistent with ACF7 being a linker protein, which may shape the cytoskeleton of the hair cell early during hair-bundle genesis.

Noddy, a mouse harboring a missense mutation in protocadherin-15, reveals the impact of disrupting a critical interaction site between tip-link cadherins in inner ear hair cells.

In hair cells of the inner ear, sound or head movement increases tension in fine filaments termed tip links, which in turn convey force to mechanosensitive ion channels to open them. Tip links are formed by a tetramer of two cadherin proteins: protocadherin 15 (PCDH15) and cadherin 23 (CDH23), which have 11 and 27 extracellular cadherin (EC) repeats, respectively. Mutations in either protein cause inner ear disorders in mice and humans. We showed recently that these two cadherins bind tip-to-tip in a "handshake" mode that involves the EC1 and EC2 repeats of both proteins. However, a paucity of appropriate animal models has slowed our understanding both of the interaction and of how mutations of residues within the predicted interface compromise tip link integrity. Here, we present noddy, a new mouse model for hereditary deafness. Identified in a forward genetic screen, noddy homozygotes lack inner ear function. Mapping and sequencing showed that noddy mutant mice harbor an isoleucine-to-asparagine (I108N) mutation in the EC1 repeat of PCDH15. Residue I108 interacts with CDH23 EC2 in the handshake and its mutation impairs the interaction in vitro. The noddy mutation allowed us to determine the consequences of blocking the handshake in vivo: tip link formation and bundle morphology are disrupted, and mechanotransduction channels fail to remain open at rest. These results offer new insights into the interaction between PCDH15 and CDH23 and help explain the etiology of human deafness linked to mutations in the tip-link interface.

Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling.

Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway.

The mechanosensory structure of the hair cell requires clarin-1, a protein encoded by Usher syndrome III causative gene.

Mutation in the clarin-1 gene (Clrn1) results in loss of hearing and vision in humans (Usher syndrome III), but the role of clarin-1 in the sensory hair cells is unknown. Clarin-1 is predicted to be a four transmembrane domain protein similar to members of the tetraspanin family. Mice carrying null mutation in the clarin-1 gene (Clrn1(-/-)) show loss of hair cell function and a possible defect in ribbon synapse. We investigated the role of clarin-1 using various in vitro and in vivo approaches. We show by immunohistochemistry and patch-clamp recordings of Ca(2+) currents and membrane capacitance from inner hair cells that clarin-1 is not essential for formation or function of ribbon synapse. However, reduced cochlear microphonic potentials, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] loading, and transduction currents pointed to diminished cochlear hair bundle function in Clrn1(-/-) mice. Electron microscopy of cochlear hair cells revealed loss of some tall stereocilia and gaps in the v-shaped bundle, although tip links and staircase arrangement of stereocilia were not primarily affected by Clrn1(-/-) mutation. Human clarin-1 protein expressed in transfected mouse cochlear hair cells localized to the bundle; however, the pathogenic variant p.N48K failed to localize to the bundle. The mouse model generated to study the in vivo consequence of p.N48K in clarin-1 (Clrn1(N48K)) supports our in vitro and Clrn1(-/-) mouse data and the conclusion that CLRN1 is an essential hair bundle protein. Furthermore, the ear phenotype in the Clrn1(N48K) mouse suggests that it is a valuable model for ear disease in CLRN1(N48K), the most prevalent Usher syndrome III mutation in North America.

Usher syndrome IIIA gene clarin-1 is essential for hair cell function and associated neural activation.

Usher syndrome 3A (USH3A) is an autosomal recessive disorder characterized by progressive loss of hearing and vision due to mutation in the clarin-1 (CLRN1) gene. Lack of an animal model has hindered our ability to understand the function of CLRN1 and the pathophysiology associated with USH3A. Here we report for the first time a mouse model for ear disease in USH3A. Detailed evaluation of inner ear phenotype in the Clrn1 knockout mouse (Clrn1(-/-)) coupled with expression pattern of Clrn1 in the inner ear are presented here. Clrn1 was expressed as early as embryonic day 16.5 in the auditory and vestibular hair cells and associated ganglionic neurons, with its expression being higher in outer hair cells (OHCs) than inner hair cells. Clrn1(-/-) mice showed early onset hearing loss that rapidly progressed to severe levels. Two to three weeks after birth (P14-P21), Clrn1(-/-) mice showed elevated auditory-evoked brainstem response (ABR) thresholds and prolonged peak and interpeak latencies. By P21, approximately 70% of Clrn1(-/-) mice had no detectable ABR and by P30 these mice were deaf. Distortion product otoacoustic emissions were not recordable from Clrn1(-/-) mice. Vestibular function in Clrn1(-/-) mice mirrored the cochlear phenotype, although it deteriorated more gradually than cochlear function. Disorganization of OHC stereocilia was seen as early as P2 and by P21 OHC loss was observed. In sum, hair cell dysfunction and prolonged peak latencies in vestibular and cochlear evoked potentials in Clrn1(-/-) mice strongly indicate that Clrn1 is necessary for hair cell function and associated neural activation.

Actin Crosslinking Family Protein 7 Deficiency Does Not Impair Hearing in Young Mice.

To enable hearing, the sensory hair cell contains specialized subcellular structures at its apical region, including the actin-rich cuticular plate and circumferential band. ACF7 (actin crosslinking family protein 7), encoded by the gene Macf1 (microtubule and actin crosslinking factor 1), is a large cytoskeletal crosslinking protein that interacts with microtubules and filamentous actin to shape cells. ACF7 localizes to the cuticular plate and the circumferential band in the hair cells of vertebrates. The compelling expression pattern of ACF7 in hair cells, combined with conserved roles of this protein in the cytoskeleton of various cell types in invertebrates and vertebrates, led to the hypothesis that ACF7 performs a key function in the subcellular architecture of hair cells. To test the hypothesis, we conditionally target Macf1 in the inner ears of mice. Surprisingly, our data show that in young, but mature, conditional knockout mice cochlear hair cell survival, planar cell polarity, organization of the hair cells within the organ of Corti, and capacity to hear are not significantly impacted. Overall, these results fail to support the hypothesis that ACF7 is an essential hair cell protein in young mice, and the purpose of ACF7 expression in the hair cell remains to be understood.

Mitochondrial calcium uniporter is essential for hearing and hair cell preservation in congenic FVB/NJ mice.

Mitochondrial Ca2+ regulates a wide range of cell processes, including morphogenesis, metabolism, excitotoxicity, and survival. In cochlear hair cells, the activation of mechano-electrical transduction and voltage-gated Ca2+ channels result in a large influx of Ca2+. The intracellular rise in Ca2+ is partly balanced by the mitochondria which rapidly uptakes Ca2+ via a highly selective channel comprised of the main pore-forming subunit, the mitochondrial Ca2+ uniporter (MCU), and associated regulatory proteins. MCU thus contributes to Ca2+ buffering, ensuring cytosolic homeostasis, and is posited to have a critical role in hair cell function and hearing. To test this hypothesis, Ca2+ homeostasis in hair cells and cochlear function were investigated in FVB/NJ mice carrying the knockout allele of Mcu (Mcu+/- or Mcu-/-). The Mcu knockout allele, which originated in C57BL/6 strain cosegregated along with Cdh23ahl allele to the FVB/NJ strain, due to the close proximity of these genes. Neither Mcu+/- nor Mcu-/- genotypes affected cochlear development, morphology, or Ca2+ homeostasis of auditory hair cells in the first two postnatal weeks. However, Mcu-/- mice displayed high-frequency hearing impairment as early as 3 weeks postnatal, which then progressed to profound hearing loss at all frequencies in about 6 months. In Mcu+/- mice, significantly elevated ABR thresholds were observed at 6 months and 9 months of age only at 32 kHz frequency. In three-month-old Mcu-/- mice, up to 18% of the outer hair cells and occasionally some inner hair cells were missing in the mid-cochlear region. In conclusion, mitochondrial Ca2+ uniporter is not required for the development of cochlea in mice, but is essential for hearing and hair cell preservation in congenic FVB/NJ mice.

A nonsynonymous SNP within PCDH15 is associated with lipid traits in familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCHL) is a common lipid disorder characterized by the presence of multiple lipoprotein phenotypes that increase the risk of premature coronary heart disease. In a previous study, we identified an intragenic microsatellite marker within the protocadherin 15 (PCDH15) gene to be associated with high triglycerides (TGs) in Finnish dyslipidemic families. In this study we analyzed all four known nonsynonymous SNPs within PCDH15 in 1,268 individuals from Finnish and Dutch multigenerational families with FCHL. Association analyses of quantitative traits for SNPs were performed using the QTDT test. The nonsynonymous SNP rs10825269 resulted in a P = 0.0006 for the quantitative TG trait. Additional evidence for association was observed with the same SNP for apolipoprotein B levels (apo-B) (P = 0.0001) and total cholesterol (TC) levels (P = 0.001). None of the other three SNPs tested showed a significant association with any lipid-related trait. We investigated the expression of PCDH15 in different human tissues and observed that PCDH15 is expressed in several tissues including liver and pancreas. In addition, we measured the plasma lipid levels in mice with loss-of-function mutations in Pcdh15 (Pcdh15(av-Tg) and Pcdh15(av-3J)) to investigate possible abnormalities in their lipid profile. We observed a significant difference in plasma TG and TC concentrations for the Pcdh15(av-3J) carriers when compared with the wild type (P = 0.013 and P = 0.044, respectively). Our study suggests that PCDH15 is associated with lipid abnormalities.

Clarin-2 is essential for hearing by maintaining stereocilia integrity and function.

Hearing relies on mechanically gated ion channels present in the actin-rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound-receptive structure is limited. Utilizing a large-scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2clarinet/clarinet mice (p.Trp4* mutation) exhibit a progressive, early-onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non-syndromic progressive hearing loss. Our in-depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin-2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin-2 leads to loss of mechano-electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance.

A mouse model with postnatal endolymphatic hydrops and hearing loss.

Endolymphatic hydrops (ELH), hearing loss and neuronal degeneration occur together in a variety of clinically significant disorders, including Meniere's disease (MD). However, the sequence of these pathological changes and their relationship to each other are not well understood. In this regard, an animal model that spontaneously develops these features postnatally would be useful for research purposes. A search for such a model led us to the Phex Hyp-Duk mouse, a mutant allele of the Phex gene causing X-linked hypophosphatemic rickets. The hemizygous male (Phex Hyp-Duk/Y) was previously reported to exhibit various abnormalities during adulthood, including thickening of bone, ELH and hearing loss. The reported inner-ear phenotype was suggestive of progressive pathology and spontaneous development of ELH postnatally, but not conclusive. The main focuses of this report are to further characterize the inner ear phenotype in Phex Hyp-Duk/Y mice and to test the hypotheses that (a) the Phex Hyp-Duk/Y mouse develops ELH and hearing loss postnatally, and (b) the development of ELH in the Phex Hyp-Duk/Y mouse is associated with obstruction of the endolymphatic duct (ED) due to thickening of the surrounding bone. Auditory brainstem response (ABR) recordings at various times points and histological analysis of representative temporal bones reveal that Phex Hyp-Duk/Y mice typically develop adult onset, asymmetric, progressive hearing loss closely followed by the onset of ELH. ABR and histological data show that functional degeneration precedes structural degeneration. The major degenerative correlate of hearing loss and ELH in the mutants is the primary loss of spiral ganglion cells. Further, Phex Hyp-Duk/Y mice develop ELH without evidence of ED obstruction, supporting the idea that ELH can be induced by a mechanism other than the blockade of longitudinal flow of endolymphatic fluid, and occlusion of ED is not a prerequisite for the development of ELH in patients.

Tympanometry assessment of 61 inbred strains of mice.

Otitis media (OM) accounts for more than 20 million clinic visits in the United States every year. Resistance to antibiotics has hampered current management of the disease. Identification of genetic factors underlying susceptibility to OM is greatly needed in order to develop alternative treatment strategies. Genetically defined inbred mouse strains offer a powerful tool for dissecting genetic and environmental factors that may lead to OM in mice. Here, we report a study of middle ear function of 61 genetically diverse inbred strains of mice using tympanometry. Of the 61 inbred strains tested, the 129P1/ReJ, 129P3/J, 129S1/SvImJ, 129X1/SvJ, A/HeJ, BALB/cJ, BUB/BnJ, C57L/J, EL/SuzSeyFrkJ, FVB/NJ, I/LnJ, LP/J, NZB/BlNJ, PL/J and YBR/Ei strains exhibited tympanograms that were statistically different from other healthy strains according to parameters including middle ear pressure, volume and compliance. These differences are most likely the result of genetic factors that, when understood, will facilitate prevention and treatment of otitis media in humans. In addition, a negative correlation between age and compliance of the tympanic membrane was discovered. This is the first report to successfully use tympanometry to measure mouse middle ear function, which has been a challenge for the hearing research field because of the mouse's tiny ear size.

N-Acetyl L-cysteine does not protect against premature age-related hearing loss in C57BL/6J mice: a pilot study.

A compound capable of preventing age-related hearing loss would be very useful in an aging population. N-acetyl-L-cysteine (L-NAC) has been shown to be protective against noise exposure, a condition that leads to increased oxidative stress. Not withstanding environmental factors, there is evidence that age-related hearing loss (AHL) in the mouse is linked to more than one genetic loci and, by extension, in humans. Our hypothesis is that AHL defect results in increased sensitivity to oxidative stress and L-NAC would be able to protect the hearing of a mouse model of pre-mature AHL, the C57BL/6J (B6) mouse strain. L-NAC was added to the regular water bottle of B6 mice (experimental group) and available ad lib. The other group received normal tap water. Hearing was tested monthly by the ability to generate the auditory brainstem response (ABR). After the final ABR test, mice were sacrificed by an overdose of Avertin, ears were harvested and hair cell loss was quantified. There was no difference in ABR thresholds or in histopathology between the control group and the group receiving L-NAC in their drinking water. In contrast to the protective effects of L-NAC against noise-induced hearing loss, the lack of protective effect in this study may be due to (i) the dosage level; (ii) the duration of treatment; (iii) the biochemical mechanisms underlying age-induced hearing loss; or (iv) how the mouse metabolizes L-NAC.

Optimization of ribonucleic acid detection from archival Guinea pig temporal bone specimens.

HYPOTHESIS: The choice of ribonucleic acid (RNA) isolation protocol coupled with modifications to RNA extraction and detection procedures may result in a more reliable method to detect gene expression in archived temporal bones. BACKGROUND: A large number of archival temporal bones exist. Retrospective analysis of these specimens using techniques of RNA extraction will greatly enrich our understanding of the pathophysiology of specific otologic diseases. However, archival human temporal bones are aged and embedded in paraffin or celloidin, rendering isolation and manipulation of nucleic acid in preserved specimens difficult, especially as it pertains to RNA degradation. Despite some reports of moderate success in the recent past, RNA isolation and gene expression using polymerase chain reaction (PCR) analysis continues to be challenging and unreliable. Archival guinea pig temporal bone specimens were used to develop and optimize a protocol for RNA extraction and gene expression analysis using PCR and quantitative PCR methods. The genes amplified comprise housekeeping genes and genes associated with the glutamate pathway. METHODS: Archival celloidin-embedded guinea pig temporal bones were collected from the senior author's collection of experimental hydropic inner ear specimens. RNA from this tissue was extracted using the protocol described previously in 16animals and using a modified trizol extraction technique in 10 animals. Gene expression analysis was performed on the extracted RNA. Analysis included two housekeeping genes, GAPDH and 18S, as well as three mediators of the glutamate pathway, glutamate aspartate transporter, glutamate synthetase, and inducible nitric oxide synthase. RESULTS: Compared with the standard extraction protocol, the trizol-based extraction technique showed greater reliability and reproducibility of RNA detection. The housekeeping gene GAPDH or 18S was detected in 7 of 36 attempts with the standard protocol versus 9 of 9 using the modified extraction method (P < 0.001). The gene of interest, glutamate aspartate transporter, was detected in 3 of 26 attempts with the standard protocol versus 12 of 13 attempts using the modified extraction method (P < 0.001). Quantification of messenger RNA levels was then achieved using quantitative PCR methods. CONCLUSION: Improved reliability for detection of gene expression and demonstration of reproducibility were accomplished by modification of RNA extraction technique and standard reverse transcriptase PCR protocol. In addition, we also showed that gene expression from archival material can be quantified by real-time PCR.

Molecular changes associated with the endolymphatic hydrops model.

HYPOTHESIS: Hearing loss and cochlear degeneration in the guinea pig model of endolymphatic hydrops (ELH) results, in part, from toxic levels of excitatory amino acids (EAAs) such as glutamate, which in turn leads to changes in the expression of genes linked to intracellular glutamate homeostasis and apoptosis, leading to neuronal cell death. BACKGROUND: EAAs have been shown to play a role in normal auditory signal transmission in mammalian cochlea, but have also been implicated in neurotoxicity when levels are elevated. Changes in the expression of specific genes involved in the glutamatergic and apoptotic pathway would serve as evidence for excitotoxicity linked to elevated levels of glutamate. METHODS: Guinea pigs underwent surgical obliteration of the endolymphatic duct, and then a timed harvest of the treated (right) and control (left) cochlea and subsequent quantification of gene expression via real-time quantitative polymerase chain reaction. RESULTS: Quantitative polymerase chain reaction data show significant upregulation of glutamate aspartate transporter and neuronal nitric oxide synthase mRNA levels 3 weeks postsurgery and Caspase 3 mRNA levels 1 week postsurgery. No significant changes were detected in glutamine synthetase expression levels. CONCLUSION: Upregulation of genes involved in glutamate homeostasis and the apoptotic pathway in animals treated with endolymphatic duct obstruction (usually associated with secondary ELH) support the hypothesis that EAAs may play a role in the pathophysiology of ELH-related cochlear injury. Inhibitors to these pathways can be useful for the study of new avenues to delay or prevent ELH-related hearing loss.

Spatiotemporal changes in the distribution of LHFPL5 in mice cochlear hair bundles during development and in the absence of PCDH15.

Mechanosensory transduction by vertebrate hair cells depends on a protein complex at the tips of shorter stereocilia associated with mechanoelectrical transduction channels activated by tip links in the hair bundle. In mammalian hair cells, this complex includes transmembrane channel-like protein subunit 1 (TMC1), lipoma HMGIC fusion partner-like 5 protein (LHFPL5) and protocadherin 15 (PCDH15), a lower-end component of the tip link. TMC1 interacts with LHFPL5 and PCDH15 but how the complex develops to maturity, and the relationships between these proteins, remains uncertain. Here we evaluate the spatiotemporal development of LHFPL5 distributions in mouse cochlear hair bundles by immunofluorescence and immunogold transmission electron microscopy, from postnatal day 0 (P0) through P21 in wild type and PCDH15-deficient mice. At P0, hair bundles contain many short microvilli-like processes which we term unranked stereocilia, and a subset of lengthening rows, adjacent to a kinocilium. LHFPL5 is distributed throughout the bundle, including on stereocilia tips and the kinocilium. At P3, 4-to-6 rows of ranked stereocilia are evident, total LHFPL5 expression peaks, and LHFPL5 is localised to ranked stereocilia tips of all rows and to lower shaft/ankle links. By P12, the bundle has a mature pattern with 3 ranked rows but virtually no unranked stereocilia or kinocilium; LHFPL5 expression has declined and become restricted to the tips of shorter stereocilia. Throughout development from P0, expression of LHFPL5 is greater overall on apical than basal bundles, but there is, on average, an equal amount of labelling per labelled tip. In P3 mice lacking PCDH15, LHFPL5 labelling is not at the tips but is primarily on unranked stereocilia and lower lateral links. These data show that LHFPL5 is already present in the MET apparatus at P0 but requires PCDH15 at P3 to remain there. Shaft/ankle link localisation suggests it interacts with link proteins other than PCDH15.