Transgenic Tg(Kcnj10-ZsGreen) fluorescent reporter mice allow visualization of intermediate cells in the stria vascularis.

The stria vascularis (SV) is a stratified epithelium in the lateral wall of the mammalian cochlea, responsible for both endolymphatic ion homeostasis and generation of the endocochlear potential (EP) critical for normal hearing. The SV has three layers consisting predominantly of basal, intermediate, and marginal cells. Intermediate and marginal cells form an intricate interdigitated network of cell projections making discrimination of the cells challenging. To enable intermediate cell visualization, we engineered by BAC transgenesis, reporter mouse lines expressing ZsGreen fluorescent protein under the control of Kcnj10 promoter and regulatory sequences. Kcnj10 encodes KCNJ10 protein (also known as Kir4.1 or Kir1.2), an ATP-sensitive inwardly-rectifying potassium channel critical to EP generation, highly expressed in SV intermediate cells. In these transgenic mice, ZsGreen fluorescence mimics Kcnj10 endogenous expression in the cochlea and was detected in the intermediate cells of the SV, in the inner phalangeal cells, Hensen's, Deiters' and pillar cells, in a subset of spiral ganglion neurons, and in glial cells. We show that expression of the transgene in hemizygous mice does not alter auditory function, nor EP. These transgenic Tg(Kcnj10-ZsGreen) mice allow live and fixed tissue visualization of ZsGreen-expressing intermediate cells and will facilitate future studies of stria vascularis cell function.

Emerging Mechanisms in the Pathogenesis of Menière's Disease: Evidence for the Involvement of Ion Homeostatic or Blood-Labyrinthine Barrier Dysfunction in Human Temporal Bones.

HYPOTHESIS: Analysis of human temporal bone specimens of patients with Menière's disease (MD) may demonstrate altered expression of gene products related to barrier formation and ionic homeostasis within cochlear structures compared with control specimens. BACKGROUND: MD represents a challenging otologic disorder for investigation. Despite attempts to define the pathogenesis of MD, there remain many gaps in our understanding, including differences in protein expression within the inner ear. Understanding these changes may facilitate the identification of more targeted therapies for MD. METHODS: Human temporal bones from patients with MD (n = 8) and age-matched control patients (n = 8) were processed with immunohistochemistry stains to detect known protein expression related to ionic homeostasis and barrier function in the cochlea, including CLDN11, CLU, KCNJ10, and SLC12A2. Immunofluorescence intensity analysis was performed to quantify protein expression in the stria vascularis, organ of Corti, and spiral ganglion neuron (SGN). RESULTS: Expression of KCNJ10 was significantly reduced in all cochlear regions, including the stria vascularis (9.23 vs 17.52, p = 0.011), OC (14.93 vs 29.16, p = 0.014), and SGN (7.69 vs 18.85, p = 0.0048) in human temporal bone specimens from patients with MD compared with control, respectively. CLDN11 (7.40 vs 10.88, p = 0.049) and CLU (7.80 vs 17.51, p = 0.0051) expression was significantly reduced in the SGN. CONCLUSION: The results of this study support that there may be differences in the expression of proteins related to ionic homeostasis and barrier function within the cochlea, potentially supporting the role of targeted therapies to treat MD.

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining.

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

Transgenic Tg(Kcnj10-ZsGreen) Fluorescent Reporter Mice Allow Visualization of Intermediate Cells in the Stria Vascularis.

The stria vascularis (SV) is a stratified epithelium in the lateral wall of the mammalian cochlea, responsible for both endolymphatic ion homeostasis and generation of the endocochlear potential (EP) critical for normal hearing. The SV has three layers consisting predominantly of basal, intermediate, and marginal cells. Intermediate and marginal cells form an intricate interdigitated network of cell projections making discrimination of the cells challenging. To enable intermediate cell visualization, we engineered by BAC transgenesis, reporter mouse lines expressing ZsGreen fluorescent protein under the control of Kcnj10 promoter and regulatory sequences. Kcnj10 encodes KCNJ10 protein (also known as Kir4.1 or Kir1.2), an ATP-sensitive inwardly-rectifying potassium channel critical to EP generation, highly expressed in SV intermediate cells. In these transgenic mice, ZsGreen fluorescence mimics Kcnj10 endogenous expression in the cochlea and was detected in the intermediate cells of the SV, in the inner phalangeal cells, Hensen's, Deiters' and pillar cells, in a subset of spiral ganglion neurons, and in glial cells. We show that expression of the transgene in hemizygous mice does not alter auditory function, nor EP These transgenic Tg(Kcnj10-ZsGreen) mice allow live and fixed tissue visualization of ZsGreen-expressing intermediate cells and will facilitate future studies of stria vascularis cell function.

Identification of harmine and ß-carboline analogs from a high-throughput screen of an approved drug collection; profiling as differential inhibitors of DYRK1A and monoamine oxidase A and for in vitro and in vivo anti-cancer studies.

DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is highly expressed in glioma, an aggressive brain tumor, and has been proposed as a therapeutic target for cancer. In the current study, we have used an optimized and validated time-resolved fluorescence energy transfer (TR-FRET)-based DYRK1A assay for high-throughput screening (HTS) in 384-well format. A small-scale screen of the FDA-approved Prestwick drug collection identified the ß-carboline, harmine, and four related analogs as DYRK1A inhibitors. Hits were confirmed by dose response and in an orthogonal DYRK1A assay. Harmine's potential therapeutic use has been hampered by its off-target activity for monoamine oxidase A (MAO-A) which impacts multiple nervous system targets. Selectivity profiling of harmine and a broader collection of analogs allowed us to map some divergent SAR (structure-activity relationships) for the DYRK1A and MAO-A activities. The panel of harmine analogs had varying activities in vitro in glioblastoma (GBM) cell lines when tested for anti-proliferative effects using a high content imaging assay. In particular, of the identified analogs, harmol was found to have the best selectivity for DYRK1A over MAO-A and, when tested in a glioma tumor xenograft model, harmol demonstrated a better therapeutic window compared to harmine.

Pharmacological targeting of GLI1 inhibits proliferation, tumor emboli formation and in vivo tumor growth of inflammatory breast cancer cells.

Activation of the Hedgehog (Hh) pathway effector GLI1 is linked to tumorigenesis and invasiveness in a number of cancers, with targeting of GLI1 by small molecule antagonists shown to be effective. We profiled a collection of GLI antagonists possessing distinct mechanisms of action for efficacy in phenotypic models of inflammatory and non-inflammatory breast cancer (IBC and non-IBC) that we showed expressed varying levels of Hh pathway mediators. Compounds GANT61, HPI-1, and JK184 decreased cell proliferation, inhibited GLI1 mRNA expression and decreased the number of colonies formed in TN-IBC (SUM149) and TNBC (MDA-MB-231 and SUM159) cell lines. In addition, GANT61 and JK184 significantly down-regulated GLI1 targets that regulate cell cycle (cyclin D and E) and apoptosis (Bcl2). GANT61 reduced SUM149 spheroid growth and emboli formation, and in orthotopic SUM149 tumor models significantly decreased tumor growth. We successfully utilized phenotypic profiling to identify a subset of GLI1 antagonists that were prioritized for testing in in vivo models. Our results indicated that GLI1 activation in TN-IBC as in TNBC, plays a vital role in promoting cell proliferation, motility, tumor growth, and formation of tumor emboli.