Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model

BACKGROUND: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4(+/+)) or lack a complete Slc26a4 gene (Slc26a4(-/-)). METHODS: Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K(+ )concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry. RESULTS: Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4(-/- )mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4(-/- )mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K(+ )channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K(+ )concentrations were normal and membrane proteins necessary for K(+ )secretion were present, including the K(+ )channel KCNQ1 and KCNE1, Na(+)/2Cl(-)/K(+ )cotransporter SLC12A2 and the gap junction GJB2. CONCLUSIONS: These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome

Macrophage invasion contributes to degeneration of stria vascularis in Pendred syndrome mouse model

Background: Pendred syndrome, an autosomal-recessive disorder characterized by deafness and goiter, is caused by a mutation of SLC26A4, which codes for the anion exchanger pendrin. We investigated the relationship between pendrin expression and deafness using mice that have (Slc26a4+/+ or Slc26a4+/-) or lack (Slc26a4-/-) a complete Slc26a4 gene. Previously, we reported that stria vascularis of adult Slc26a4-/- mice is hyperpigmented and that marginal cells appear disorganized. Here we determine the time course of hyperpigmentation and marginal cell disorganization, and test the hypothesis that inflammation contributes to this tissue degeneration. Methods: Slc26a4-/- and age-matched control (Slc26a4+/+ or Slc26a4+/-) mice were studied at four postnatal (P) developmental stages: before and after the age that marks the onset of hearing (P10 and P15, respectively), after weaning (P28-41) and adult (P74-170). Degeneration and hyperpigmentation stria vascularis was evaluated by confocal microscopy. Gene expression in stria vascularis was analyzed by microarray and quantitative RT-PCR. In addition, the expression of a select group of genes was quantified in spiral ligament, spleen and liver to evaluate whether expression changes seen in stria vascularis are specific for stria vascularis or systemic in nature. Results: Degeneration of stria vascularis defined as hyperpigmentation and marginal cells disorganization was not seen at P10 or P15, but occurred after weaning and was associated with staining for CD68, a marker for macrophages. Marginal cells in Slc26a4-/-, however, had a larger apical surface area at P10 and P15. No difference in the expression of Lyzs, C3 and Cd45 was found in stria vascularis of P15 Slc26a4+/- and Slc26a4-/- mice. However, differences in expression were found after weaning and in adult mice. No difference in the expression of markers for acute inflammation, including Il1a, Il6, Il12a, Nos2 and Nos3 were found at P15, after weaning or in adults. The expression of macrophage markers including Ptprc (= Cd45), Cd68, Cd83, Lyzs, Lgals3 (= Mac2 antigen), Msr2, Cathepsins B, S, and K (Ctsb, Ctss, Ctsk) and complement components C1r, C3 and C4 was significantly increased in stria vascularis of adult Slc26a4-/- mice compared to Slc26a4+/+ mice. Expression of macrophage markers Cd45 and Cd84 and complement components C1r and C3 was increased in stria vascularis but not in spiral ligament, liver or spleen of Slc26a4-/- compared to Slc26a4+/- mice. The expression of Lyzs was increased in stria vascularis and spiral ligament but not in liver or spleen. Conclusion: The data demonstrate that hyperpigmentation of stria vascularis and marginal cell reorganization in Slc26a4-/- mice occur after weaning, coinciding with an invasion of macrophages. The data suggest that macrophage invasion contributes to tissue degeneration in stria vascularis, and that macrophage invasion is restricted to stria vascularis and is not systemic in nature. The delayed onset of degeneration of stria vascularis suggests that a window of opportunity exists to restore/preserve hearing in mice and therefore possibly in humans suffering from Pendred syndrome

Generation of a mouse model of Primary Hyperoxaluria Type 1 via CRISPR/Cas9 mediated gene editing

Background: Primary Hyperoxaluria Type 1 (PH1) is an inborn error of metabolism caused by mutations in the AGXT gene, which encodes for the hepatocyte-specific enzyme alanine: glyoxylate aminotransferase (AGT). AGT catalyzes the conversion of glyoxylate to glycine in the peroxisome and prevents the build-up of oxalate which occurs in PH1. This causes nephrocalcinosis, systemic oxalosis, and end-stage renal disease. Liver transplant is currently the only curative treatment available. Although a mouse model has previously been generated, the severity of the reported disease phenotype varies, and a better understanding of the genotype-phenotype relationship in both the mouse model and human disease is needed. Methods: We developed an Agxt-/- mouse model using CRISPR/Cas9-mediated gene editing. We performed a natural history study and ethylene glycol (EG) challenge to evaluate the phenotype of this mouse. Results: Agxt-/- mice had elevated plasma glycolate, urine glycolate, and urine oxalate levels compared to Agxt+/+ mice. A small subset of Agxt-/- mice developed minimal nephrocalcinosis (1/8 at 12 weeks, 1/8 at 26 weeks, 0/8 at 39 weeks, and 3/7 at 52 weeks of age). When challenged with 0.7% or 1.2% EG in drinking water for 3 weeks, 2/10 Agxt-/- mice developed nephrocalcinosis. Agxt2mRNA and protein expression were unchanged between Agxt-/- and Agxt+/+ mice. Hydroxy acid oxidase 1(Hao1) messenger ribonucleic acid (mRNA) levels were unchanged, but the corresponding glycolate oxidase protein was increased in Agxt-/- mice. Conclusion: We have created an Agxt-/- mouse model which resembles much of the clinical phenotype of PH1 patients and will be a useful tool in developing novel therapies for this devastating disease. [JBCGenetics 2019; 2(1.000): 28-39