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1.  A novel nonsense mutation in the NDP gene in a Chinese family with Norrie disease 
Molecular Vision  2010;16:2653-2658.
Purpose
Norrie disease (ND), a rare X-linked recessive disorder, is characterized by congenital blindness and, occasionally, mental retardation and hearing loss. ND is caused by the Norrie Disease Protein gene (NDP), which codes for norrin, a cysteine-rich protein involved in ocular vascular development. Here, we report a novel mutation of NDP that was identified in a Chinese family in which three members displayed typical ND symptoms and other complex phenotypes, such as cerebellar atrophy, motor disorders, and mental disorders.
Methods
We conducted an extensive clinical examination of the proband and performed a computed tomography (CT) scan of his brain. Additionally, we performed ophthalmic examinations, haplotype analyses, and NDP DNA sequencing for 26 individuals from the proband’s extended family.
Results
The proband’s computed tomography scan, in which the fifth ventricle could be observed, indicated cerebellar atrophy. Genome scans and haplotype analyses traced the disease to chromosome Xp21.1-p11.22. Mutation screening of the NDP gene identified a novel nonsense mutation, c.343C>T, in this region.
Conclusions
Although recent research has shown that multiple different mutations can be responsible for the ND phenotype, additional research is needed to understand the mechanism responsible for the diverse phenotypes caused by mutations in the NDP gene.
PMCID: PMC3002970  PMID: 21179243
2.  CLRN1 Is Nonessential in the Mouse Retina but Is Required for Cochlear Hair Cell Development 
PLoS Genetics  2009;5(8):e1000607.
Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5–6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT–PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT–PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration.
Author Summary
Usher syndrome (USH) is a progressive disease affecting two primary senses: vision and hearing. Often by the third decade of life, affected persons have lost the majority of their rod photoreceptors, which leads to night blindness and peripheral vision loss. Similarly, hearing loss often progresses into the third or fourth decade. By the fourth decade, patients typically approach legal blindness and hearing impairment continues to decline. The proteins that when mutated cause USH are frequently found in primary sensory cells, photoreceptor and hair cells, that directly respond to light and sound, respectively. Similar to other forms of USH, the mRNA coding for the protein responsible for USH type 3 (CLRN1) is expressed in cochlear hair cells of the inner ear. However, as demonstrated in the current study, and unlike other USH disease proteins in the retina, we show that the Clrn1 is expressed in glial cells in the retina (Müller cells) and is not expressed in the photoreceptors themselves. For reasons that remain unclear, the Clrn1 knockout mouse does not have a retinal degeneration phenotype but does become deaf soon after birth. In the current paper, we characterize the expression pattern in the retina and analyze the effects of removing the Clrn1 gene on vision and hearing.
doi:10.1371/journal.pgen.1000607
PMCID: PMC2719914  PMID: 19680541
3.  Maternal xNorrin, a Canonical Wnt Signaling Agonist and TGF-β Antagonist, Controls Early Neuroectoderm Specification in Xenopus 
PLoS Biology  2012;10(3):e1001286.
Xenopus maternal Norrin, which activates Wnt signaling but inhibits TGF-β family molecules, is essential for neuroectoderm formation. Loss of TGF-β inhibition in Norrin may contribute to the development of Norrie disease.
Dorsal–ventral specification in the amphibian embryo is controlled by β-catenin, whose activation in all dorsal cells is dependent on maternal Wnt11. However, it remains unknown whether other maternally secreted factors contribute to β-catenin activation in the dorsal ectoderm. Here, we show that maternal Xenopus Norrin (xNorrin) promotes anterior neural tissue formation in ventralized embryos. Conversely, when xNorrin function is inhibited, early canonical Wnt signaling in the dorsal ectoderm and the early expression of the zygotic neural inducers Chordin, Noggin, and Xnr3 are severely suppressed, causing the loss of anterior structures. In addition, xNorrin potently inhibits BMP- and Nodal/Activin-related functions through direct binding to the ligands. Moreover, a subset of Norrin mutants identified in humans with Norrie disease retain Wnt activation but show defective inhibition of Nodal/Activin-related signaling in mesoderm induction, suggesting that this disinhibition causes Norrie disease. Thus, xNorrin is an unusual molecule that acts on two major signaling pathways, Wnt and TGF-β, in opposite ways and is essential for early neuroectoderm specification.
Author Summary
A key step during early embryogenesis is the generation of neural precursors, which later form the central nervous system. In vertebrates, this process requires proper dorsal–ventral axis specification, and we know that the canonical Wnt and BMP signaling pathways help pattern the dorsal ectoderm. In this study, we examine other factors that are involved in neuroectoderm development in the frog species Xenopus laevis. We find that maternal Xenopus Norrin (xNorrin) is required for canonical Wnt signaling in the dorsal ectoderm, functions upstream of neural inducers, and is required for neural formation. We also find that xNorrin not only activates Wnt signaling, but also inhibits BMP/Nodal-related signaling. In humans, mutations in Norrin cause Norrie disease. Using Norrin mutants identified in patients with Norrie disease, we find that some Norrin mutants fail to inhibit BMP/Nodal-related signaling (specifically, TGF-β) but retain the ability to activate the Wnt pathway, suggesting that loss of TGF-β inhibition may contribute to Norrie disease development.
doi:10.1371/journal.pbio.1001286
PMCID: PMC3308935  PMID: 22448144
4.  Cellular Localization, Expression, and Structure of the Nuclear Dot Protein 52 
The Journal of Cell Biology  1997;138(2):435-448.
Nuclear dots containing PML and Sp100 proteins (NDs) play a role in the development of acute promyelocytic leukemia, are modified after infection with various viruses, and are autoimmunogenic in patients with primary biliary cirrhosis (PBC). PML and Sp100 gene expression is strongly enhanced by interferons (IFN). Based on immunostaining with a monoclonal antibody (mAb C8A2), a third protein, nuclear dot protein 52 (NDP52), was recently localized in NDs. Here we analyzed the cellular localization, expression, and structure of NDP52 in more detail. Our NDP52-specific sera revealed mainly cytoplasmic staining but no ND pattern, neither in untreated nor in IFN-treated cells. Cells transfected with NDP52 expression vectors showed exclusively cytoplasmic staining. In subcellular fractionation experiments, NDP52 was found in cytoplasmic and nuclear fractions. Unlike as described for Sp100 and PML, NDP52 mRNA and protein levels were only marginally enhanced by IFN γ and not enhanced at all by IFN β. NDP52 homodimerization but no heterodimerization with Sp100 or PML could be demonstrated. None of the 93 PBC sera tested contained autoantibodies against NDP52. Finally, mAb C8A2 reacted not only with NDP52 but also with a conformation-dependent epitope on the Sp100 protein. These data imply that NDP52 forms homodimers but no heterodimers with Sp100 and PML, lacks autoantigenicity in PBC, localizes mainly in the cytoplasm, and is associated with the nucleus, but not with NDs. Finally, unlike Sp100 and PML, NDP52 expression is neither markedly enhanced nor localization detectably altered by type I and II IFNs.
PMCID: PMC2138200  PMID: 9230084
5.  Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity 
Cell  2012;151(6):1332-1344.
Norrin/Frizzled4 (Fz4) signaling activates the canonical Wnt pathway to control retinal vascular development. Using genetically engineered mice, we show that precocious Norrin production leads to premature retinal vascular invasion and delayed Norrin production leads to characteristic defects in intra-retinal vascular architecture. In genetic mosaics, wild type endothelial cells (ECs) instruct neighboring Fz4−/− ECs to produce an architecturally normal mosaic vasculature, a cell non-autonomous effect. However, over the ensuing weeks, Fz4−/− ECs are selectively eliminated from the mosaic vasculature, implying the existence of a quality control program that targets defective ECs. In the adult retina and cerebellum, gain or loss of Norrin/Fz4 signaling results in a cell-autonomous gain or loss, respectively, of blood retina barrier (BRB) and blood brain barrier (BBB) function, indicating an ongoing requirement for Frizzled signaling in barrier maintenance and substantial plasticity in mature CNS vascular structure.
doi:10.1016/j.cell.2012.10.042
PMCID: PMC3535266  PMID: 23217714
6.  α-MSH Analogue Attenuates Blood Pressure Elevation in DOCA-Salt Hypertensive Mice 
PLoS ONE  2013;8(8):e72857.
Melanocyte-stimulating hormones, α-, β- and γ-MSH, regulate important physiological functions including energy homeostasis, inflammation and sodium metabolism. Previous studies have shown that α-MSH increases sodium excretion and promotes vascular function in rodents, but it is unexplored whether these characteristics of α-MSH could translate into therapeutic benefits in the treatment of hypertension. Therefore, we first assessed the diuretic and natriuretic properties of the stable α-MSH analogue [Nle4, D-Phe7]-α-MSH (NDP-α-MSH) and investigated whether it has protective effects in deoxycorticosterone acetate (DOCA)-salt hypertensive mice. Adult male C57Bl/6N mice were subjected to DOCA-salt treatment and randomized to receive intraperitoneal injections of either saline as vehicle or NDP-α-MSH (0.3 mg/kg/day for 14 days) starting 7 days after the DOCA-salt treatment. Systemic hemodynamics, serum and urine electrolytes, and oxidative stress markers were assessed in control sham-operated and DOCA-salt mice. NDP-α-MSH elicited marked diuretic and natriuretic responses that were reversible with the MC3/4 receptor antagonist SHU9119. Chronic NDP-α-MSH treatment attenuated blood pressure elevation in DOCA-salt mice without affecting the blood pressure of normotensive control animals. Owing to the enhanced sodium excretion, NDP-α-MSH-treated mice were protected from DOCA-salt-induced hypernatremia. DOCA-salt treatment mildly increased oxidative stress at the tissue level, but NDP-α-MSH had no significant effects on the oxidative stress markers. In conclusion, treatment with NDP-α-MSH increases urinary sodium excretion and protects against DOCA-salt-induced hypertension. These findings point to the potential future use of α-MSH analogues in the treatment of hypertension.
doi:10.1371/journal.pone.0072857
PMCID: PMC3745458  PMID: 23977363
7.  The Role of Adenosine Kinase in Cochlear Development and Response to Noise 
Journal of neuroscience research  2010;88(12):2598-2609.
Adenosine signalling has an important role in cochlear protection from oxidative stress. In most tissues, intracellular adenosine kinase (ADK) is the primary route of adenosine metabolism and the key regulator of intracellular and extracellular adenosine levels. The present study provides the first evidence for ADK distribution in the adult and developing rat cochlea. In the adult cochlea ADK was localised to the nuclear or perinuclear region of spiral ganglion neurones, lateral wall tissues and epithelial cells lining scala media. In the developing cochlea, ADK was strongly expressed in multiple cell types at birth, and reached its peak level of expression at postnatal day 21 (P21). Ontogenetic changes in ADK expression were evident in the spiral ganglion, organ of Corti and stria vascularis. In the spiral ganglion, ADK showed a shift from predominantly satellite cell immunolabelling at P1 to neuronal expression from P14 onwards. In contrast to the role of ADK in various aspects of cochlear development, ADK contribution to the cochlear response to noise stress was less obvious. Transcript and protein levels of ADK were unaltered in the cochlea exposed to broadband noise (90–110dBSPL, 24 hours) and the selective inhibition of ADK in the cochlea with ABT-702 failed to restore hearing thresholds after exposure to traumatic noise. This study indicates that ADK is involved in purine salvage pathways for nucleotide synthesis in the adult cochlea, but its role in the regulation of adenosine signalling under physiological and pathological conditions is yet to be established.
doi:10.1002/jnr.22421
PMCID: PMC3041170  PMID: 20648650
adenosine kinase; cochlea; development; noise; ABT-702
8.  Vacuolization and alterations of lysosomal membrane proteins in cochlear marginal cells contribute to hearing loss in neuraminidase 1 – deficient mice 
Biochimica et biophysica acta  2009;1802(2):259.
The neuraminidase-1 (Neu1) knockout mouse model is a phenocopy of the lysosomal storage disease (LSD) sialidosis, characterized by multisystemic and neuropathic symptoms, including hearing loss. We have characterized the auditory defects in Neu1−/− mice and found that hearing loss involves both conductive and sensorineural components. Auditory brainstem response (ABR) thresholds were significantly elevated in Neu1−/− mice at P21 (48~55 dB), and hearing loss appeared progressive (ABR threshold elevation 53~66 dB at P60). At these ages Neu1−/− mice accumulated cerumen in the external ear canal and had a thickened mucosa and inflammation in the middle ear. In cochleae of adult wild-type mice, Neu1 was expressed in several cell types in the stria vascularis, the organ of Corti, and spiral ganglion. Progressive morphological abnormalities such as extensive vacuolization were detected in the Neu1−/− cochleae as early as P9. These early morphologic changes in Neu1−/− cochleae were associated with oversialylation of several lysosomal associated membrane proteins (Lamps) in the stria vascularis. A marked increase in the expression and apical localization of Lamp-1 in marginal cells of the stria vascularis predicts exacerbation of lysosomal exocytosis into the endolymph. Consequently, the endolymphatic potential in Neu1−/− mice was reduced by approximately 20 mV at ages P31–P44, which would cause dysfunction of transduction in sensory hair cells. This study suggests a molecular mechanism that contributes to hearing loss in sialidosis and identifies potential therapeutic targets.
doi:10.1016/j.bbadis.2009.10.008
PMCID: PMC2818351  PMID: 19857571
9.  Delta/Notch-Like EGF-Related Receptor (DNER) is Expressed in Hair Cells and Neurons in the Developing and Adult Mouse Inner Ear 
The Notch signaling pathway is known to play important roles in inner ear development. Previous studies have shown that the Notch1 receptor and ligands in the Delta and Jagged families are important for cellular differentiation and patterning of the organ of Corti. Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) is a novel Notch ligand expressed in developing and adult CNS neurons known to promote maturation of glia through activation of Notch. Here we use in situ hybridization and an antibody against DNER to carry out expression studies of the mouse cochlea and vestibule. We find that DNER is expressed in spiral ganglion neuron cell bodies and peripheral processes during embryonic development of the cochlea and expression in these cells is maintained in adults. DNER becomes strongly expressed in auditory hair cells during postnatal maturation in the mouse cochlea and immunoreactivity for this protein is strong in hair cells and afferent and efferent peripheral nerve endings in the adult organ of Corti. In the vestibular system, we find that DNER is expressed in hair cells and vestibular ganglion neurons during development and in adults. To investigate whether DNER plays a functional role in the inner ear, perhaps similar to its described role in glial maturation, we examined cochleae of DNER−/− mice using immunohistochemical markers of mature glia and supporting cells as well as neurons and hair cells. We found no defects in expression of markers of supporting cells and glia or myelin, and no abnormalities in hair cells or neurons, suggesting that DNER plays a redundant role with other Notch ligands in cochlear development.
doi:10.1007/s10162-009-0203-x
PMCID: PMC2862923  PMID: 20058045
cochlea; vestibular; development; maturation; Notch
10.  Norrin, Frizzled4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization 
Cell  2009;139(2):285-298.
SUMMARY
Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled4 (Fz4) receptor, Lrp5 co-receptor, or Norrin ligand cause retinal hypovascularization, but the role of Norrin/Fz4/Lrp signaling in vascular development has not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, while excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is up-regulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.
doi:10.1016/j.cell.2009.07.047
PMCID: PMC2779707  PMID: 19837032
11.  Differential Distribution of Stem Cells in the Auditory and Vestibular Organs of the Inner Ear 
The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.
doi:10.1007/s10162-006-0058-3
PMCID: PMC2538418  PMID: 17171473
cochlea; utricle; hair cell; regeneration; spiral ganglion; neurosphere
12.  Unilateral sporadic retinal dysplasia: Results of histopathologic, immunohistochemical, chromosomal, genetic, and VEGF-A analyses 
PURPOSE
To describe new findings in a case of unilateral retinal dysplasia.
METHODS
Histopathologic evaluation of an enucleated globe and analysis with immunohistochemical probes, karyotyping, and genetic analysis for the Norrie gene, and aqueous assay for vascular endothelial growth factor A (VEGF-A).
RESULTS
Histopathological examination of the globe revealed retinal dysplasia with pseudorosette formation, abnormal or absent retinal nuclear lamination, a paucity of disorganized retinal microvasculature, retinal infoldings, advanced gliosis, persistent hyperplastic vitreous, exuberant neovascularization of the vitreous, and iris neovascularization (identical to the findings observed in bilateral Norrie disease). Immunohistochemistry disclosed GFAP-positive and GLUT-1-positive gliosis and retinal and persistent hyperplastic vitreous microvessels that were CD34-positive and GLUT-1-negative. Ki-67-positive retinal cells were polarized toward the subretinal space and absent in the retinal invaginations and pseudorosettes. A normal karyotype was found, and DNA sequencing revealed no known mutation in the region of the Norrie gene (NDP) in sputum or retinal DNA. Aqueous obtained immediately after enucleation contained an exceptionally high concentration of VEGF-A (4.5 ng/mL).
CONCLUSIONS
Despite the failure to find an abnormal NDP allele, other unexplored NDP regions, an undetected defect restricted to retinal tissues, or an autosomal mutation coupled with disrupted signaling pathways may be responsible for the condition. High aqueous VEGF-A suggests that this cytokine may play a role in pathogenesis in conjunction with other pathways.
doi:10.1016/j.jaapos.2011.08.009
PMCID: PMC4242509  PMID: 22153404
13.  Spinster Homolog 2 (Spns2) Deficiency Causes Early Onset Progressive Hearing Loss 
PLoS Genetics  2014;10(10):e1004688.
Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.
Author Summary
Progressive hearing loss is common in the human population but we know very little about the molecular mechanisms involved. Mutant mice are useful for investigating these mechanisms and have revealed a wide range of different abnormalities that can all lead to the same outcome: deafness. We report here our findings of a new mouse line with a mutation in the Spns2 gene, affecting the release of a lipid called sphingosine-1-phosphate, which has an important role in several processes in the body. For the first time, we report that this molecular pathway is required for normal hearing through a role in generating a voltage difference that acts like a battery, allowing the sensory hair cells of the cochlea to detect sounds at extremely low levels. Without the normal function of the Spns2 gene and release of sphingosine-1-phosphate locally in the inner ear, the voltage in the cochlea declines, leading to rapid loss of sensitivity to sound and ultimately to complete deafness. The human version of this gene, SPNS2, may be involved in human deafness, and understanding the underlying mechanism presents an opportunity to develop potential treatments for this form of hearing loss.
doi:10.1371/journal.pgen.1004688
PMCID: PMC4214598  PMID: 25356849
14.  ROLE OF LEUKOTRIENES IN N-(3,5-DICHLOROPHENYL)SUCCINIMIDE (NDPS) AND NDPS METABOLITE NEPHROTOXICITY IN MALE FISCHER 344 RATS 
Toxicology  2012;300(1-2):92-99.
The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) can induce marked nephrotoxicity in rats following a single intraperitoneal (ip) administration of 0.4 mmol/kg or greater. Although NDPS induces direct renal proximal tubular toxicity, a role for renal vascular effects may also be present. The purpose of this study was to examine the possible role of vasoconstrictor leukotrienes in NDPS and NDPS metabolite nephrotoxicity. Male Fischer 344 rats (4 rats/group) were administered diethylcarbamazine (DEC; 250 or 500 mg/kg, ip), an inhibitor of LTA4 synthesis, 1h before NDPS (0.4 mmol/kg, ip), N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS, 0.1, 0.2, or 0.4 mmol/kg, ip), or N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA, 0.1 mmol/kg, ip) or vehicle. In a separate set of experiments, the LTD4 receptor antagonist LY171883 (100 mg/kg, po) was administered 0.5 h before and again 6 h after NDHS (0.1 mmol/kg, ip) or 2-NDHSA (0.1 mmol/kg, ip) or vehicle. Renal function was monitored for 48 h post-NDPS or NDPS metabolite. DEC markedly reduced the nephrotoxicity induced by NDPS and its metabolites, while LY171883 treatments provided only partial attenuation of NDHS and 2-NDHSA nephrotoxicity. These results suggest that leukotrienes contribute to the mechanisms of NDPS nephrotoxicity.
doi:10.1016/j.tox.2012.06.003
PMCID: PMC3412395  PMID: 22706168
Leukotrienes; Kidney; Rats; Nephrotoxicity; N-(3, 5-Dichlorophenyl)succinimide
15.  Expression and cellular localization of the Mas receptor in the adult and developing mouse retina 
Molecular Vision  2014;20:1443-1455.
Purpose
Recent studies have provided evidence that a local renin-angiotensin system (RAS) exists in the retina and plays an important role in retinal neurovascular function. We have recently shown that increased expression of ACE2 and angiotensin (1-7) [Ang (1-7)], two components of the protective axis of the RAS, in the retina via adeno-associated virus (AAV)-mediated gene delivery, conferred protection against diabetes-induced retinopathy. We hypothesized that the protective molecular and cellular mechanisms of Ang (1-7) are mediated by its receptor, Mas, and the expression level and cellular localization dictate the response to Ang (1-7) and activation of subsequent protective signaling pathways. We tested this hypothesis by examining the expression and cellular localization of the Mas receptor in adult and developing mouse retinas.
Methods
The cellular localization of the Mas receptor protein was determined with immunofluorescence of the eyes of adult and postnatal day 1 (P1), P5, P7, P15, and P21 mice using the Mas receptor-specific antibody, and mRNA was detected with in situ hybridization of paraffin-embedded sections. Western blotting and real-time reverse-transcription (RT)–PCR analysis were performed to determine the relative levels of the Mas protein and mRNA in adult and developing retinas, as well as in cultured retinal Müller glial and RPE cells.
Results
In the adult eye, the Mas receptor protein was abundantly present in retinal ganglion cells (RGCs) and photoreceptor cells; a lower level of expression was observed in endothelial cells, Müller glial cells, and other neurons in the inner nuclear layer of the retina. In the developing retina, Mas receptor mRNA and protein expression was detected in the inner retina at P1, and the expression levels increased with age to reach the adult level and pattern by P15. In the adult mouse retina, Mas receptor mRNA was expressed at a much higher level when compared to angiotensin II (Ang II) type I (AT1R) and type II (AT2R) receptor mRNA.
Conclusions
The Mas receptor is expressed in developing and adult mouse retinas, and is more abundant in retinal neurons than in endothelial and Müller glial cells. These observations suggest that Mas receptor-mediated signaling may play important roles that extend beyond mediating the vascular effects of Ang (1-7) in developing and adult retinas. In addition, the relatively high expression of the Mas receptor when compared to AT1R suggests that they may play a more important role in maintaining normal retinal physiology than previously considered.
PMCID: PMC4203581  PMID: 25352750
16.  Epithelial Cell Stretching and Luminal Acidification Lead to a Retarded Development of Stria Vascularis and Deafness in Mice Lacking Pendrin 
PLoS ONE  2011;6(3):e17949.
Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4−/−, are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO3− transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4−/− mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4+/− and Slc26a4−/− mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4−/− mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4−/− mice, and possibly in humans, lacking functional pendrin expression.
doi:10.1371/journal.pone.0017949
PMCID: PMC3056798  PMID: 21423764
17.  Canonical WNT signaling components in vascular development and barrier formation 
The Journal of Clinical Investigation  2014;124(9):3825-3846.
Canonical WNT signaling is required for proper vascularization of the CNS during embryonic development. Here, we used mice with targeted mutations in genes encoding canonical WNT pathway members to evaluate the exact contribution of these components in CNS vascular development and in specification of the blood-brain barrier (BBB) and blood-retina barrier (BRB). We determined that vasculature in various CNS regions is differentially sensitive to perturbations in canonical WNT signaling. The closely related WNT signaling coreceptors LDL receptor–related protein 5 (LRP5) and LRP6 had redundant functions in brain vascular development and barrier maintenance; however, loss of LRP5 alone dramatically altered development of the retinal vasculature. The BBB in the cerebellum and pons/interpeduncular nuclei was highly sensitive to decrements in canonical WNT signaling, and WNT signaling was required to maintain plasticity of barrier properties in mature CNS vasculature. Brain and retinal vascular defects resulting from ablation of Norrin/Frizzled4 signaling were ameliorated by stabilizing β-catenin, while inhibition of β-catenin–dependent transcription recapitulated the vascular development and barrier defects associated with loss of receptor, coreceptor, or ligand, indicating that Norrin/Frizzled4 signaling acts predominantly through β-catenin–dependent transcriptional regulation. Together, these data strongly support a model in which identical or nearly identical canonical WNT signaling mechanisms mediate neural tube and retinal vascularization and maintain the BBB and BRB.
doi:10.1172/JCI76431
PMCID: PMC4151216  PMID: 25083995
18.  Macrophage invasion contributes to degeneration of stria vascularis in Pendred syndrome mouse model 
BMC Medicine  2006;4:37.
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.
doi:10.1186/1741-7015-4-37
PMCID: PMC1796619  PMID: 17187680
19.  A novel signaling pathway regulates colon cancer angiogenesis through Norrin 
Scientific Reports  2014;4:5630.
Norrin binds to the frizzled-4 receptor, stimulating canonical Wnt signaling. We investigate here the role of colorectal cancer (CRC) produced Norrin in endothelial cell growth, motility, and blood vessel formation, as well as the expression of the Norrin signaling pathway components in the CRC tumor microenvironment. Norrin conditioned medium produced by CRC cell line CaCO2 transfected with Norrin expression construct increased endothelial cell motility. Blocking Norrin signaling reduced endothelial cell motility, branch point number (1/mm2), and the network length (mm/mm2) during in vitro angiogenesis. Colorectal tumors express Norrin protein. Endothelial cells in the colorectal tumor microenvironment contain all of the components of the Norrin signaling pathway needed to respond to Norrin protein. This study presents data that Norrin may play a role in the regulation of angiogenesis in the colorectal cancer tumor microenvironment.
doi:10.1038/srep05630
PMCID: PMC4088094  PMID: 25005225
20.  Comparative Study of Pax2 Expression in Glial Cells in the Retina and Optic Nerve of Birds and Mammals 
The Journal of comparative neurology  2010;518(12):10.1002/cne.22335.
Little is known about the expression of Pax2 in mature retina or optic nerve. Here we probed for the expression of Pax2 in late stages of embryonic development and in mature chick retina. We find two distinct Pax2 isoforms expressed by cells within the retina and optic nerve. Surprisingly, Müller glia in central regions of the retina express Pax2, and levels of expression are decreased with increasing distance from the nerve head. In Müller glia, the expression levels of Pax2 are increased by acute retinal damage or treatment with growth factors. At the optic nerve, Pax2 is expressed by peripapillary glia, at the junction of the neural retina and optic nerve head and by glia within the optic nerve. In addition, we assayed for Pax2 expression in glial cells in mammalian retinas. In mammalian retinas, unlike the case in chick retina, the Müller glia do not express Pax2. Pax2-expressing cells are found in the optic nerve and astrocytes within the mouse retina. By comparison, Pax2-positive cells are not found within the guinea pig retina; Pax2-expressing glia are confined to the optic nerve. In dog and monkey (Macaca fascicularis), Pax2 is expressed by astrocytes that are scattered across inner retinal layers and by numerous glia within the optic nerve. Interestingly, Pax2-positive glial cells are found at the peripheral edge of the dog retina, but only in older animals. We conclude that the expression of Pax2 in the vertebrate eye is restricted to retinal astrocytes, peripapillary glia, and glia within the optic nerve.
doi:10.1002/cne.22335
PMCID: PMC3840394  PMID: 20437530
retina; Pax2; Müller glia; astrocytes; optic nerve
21.  TAK1 Expression in the Cochlea: A Specific Marker for Adult Supporting Cells 
Transforming growth factor-β-activated kinase-1 (TAK1) is a mitogen activated protein kinase kinase kinase that is involved in diverse biological roles across species. Functioning downstream of TGF-β and BMP signaling, TAK1 mediates the activation of the c-Jun N-terminal kinase signaling pathway, serves as the target of pro-inflammatory cytokines, such as TNF-α, mediates NF-κβ activation, and plays a role in Wnt/Fz signaling in mesenchymal stem cells. Expression of TAK1 in the cochlea has not been defined. Data mining of previously published murine cochlear gene expression databases indicated that TAK1, along with TAK1 interacting proteins 1 (TAB1), and 2 (TAB2), is expressed in the developing and adult cochlea. The expression of TAK1 in the developing cochlea was confirmed using RT-PCR and immunohistochemistry. Immunolabeling of TAK1 in embryonic, neonatal, and mature cochleas via DAB chromogenic and fluorescent immunohistochemistry indicated that TAK1 is broadly expressed in both the developing otocyst and periotic mesenchyme at E12.5 but becomes more restricted to specific types of supporting cells as the organ of Corti matures. By P1, TAK1 immunolabeling is found in cells of the stria vascularis, hair cells, supporting cells, and Kölliker’s organ. By P16, TAK1 labeling is limited to cochlear supporting cells. In the adult cochlea, TAK1 immunostaining is only present in the cytoplasm of Deiters’ cells, pillar cells, inner phalangeal cells, and inner border cells, with no expression in any other cochlear cell types. While the role of TAK1 in the inner ear is unclear, TAK1 expression may be used as a novel marker for specific sub-populations of supporting cells.
doi:10.1007/s10162-011-0265-4
PMCID: PMC3123448  PMID: 21472480
cochlea; Deiters’ cell; phalangeal cell; pillar cell; development; gene expression
22.  Neonatal Maternal Deprivation Response and Developmental Changes in Gene Expression Revealed by Hypothalamic Gene Expression Profiling in Mice 
PLoS ONE  2010;5(2):e9402.
Neonatal feeding problems are observed in several genetic diseases including Prader-Willi syndrome (PWS). Later in life, individuals with PWS develop hyperphagia and obesity due to lack of appetite control. We hypothesized that failure to thrive in infancy and later-onset hyperphagia are related and could be due to a defect in the hypothalamus. In this study, we performed gene expression microarray analysis of the hypothalamic response to maternal deprivation in neonatal wild-type and Snord116del mice, a mouse model for PWS in which a cluster of imprinted C/D box snoRNAs is deleted. The neonatal starvation response in both strains was dramatically different from that reported in adult rodents. Genes that are affected by adult starvation showed no expression change in the hypothalamus of 5 day-old pups after 6 hours of maternal deprivation. Unlike in adult rodents, expression levels of Nanos2 and Pdk4 were increased, and those of Pgpep1, Ndp, Brms1l, Mett10d, and Snx1 were decreased after neonatal deprivation. In addition, we compared hypothalamic gene expression profiles at postnatal days 5 and 13 and observed significant developmental changes. Notably, the gene expression profiles of Snord116del deletion mice and wild-type littermates were very similar at all time points and conditions, arguing against a role of Snord116 in feeding regulation in the neonatal period.
doi:10.1371/journal.pone.0009402
PMCID: PMC2827556  PMID: 20195375
23.  Identification of two novel LRP5 mutations in families with familial exudative vitreoretinopathy 
Molecular Vision  2014;20:395-409.
Purpose
To investigate the clinical features and disease-causing mutations in two Chinese families with familial exudative vitreoretinopathy (FEVR).
Methods
Clinical data and genomic DNA were collected for patients with FEVR. The coding exons and adjacent intronic regions of FZD4, LRP5, TSPAN12, and NDP were amplified with PCR, and the resulting amplicons were analyzed with Sanger sequencing. Wild-type and mutant LRP5 proteins were assayed for the Norrin/β-catenin pathway by luciferase reporter assays.
Results
Two novel heterozygous mutations in the LRP5 gene were identified in two relatives—p.A422T and p.L540P. Typical FEVR fundus change and mild reduced bone mineral density (BMD) was found in the two patients and the affected parent. In the luciferase studies, both p.A422T and p.L540P mutants displayed a significant reduction of the luciferase activity in SuperTopFlash (STF) cells in response to Norrin (87% reduction for p.A422T and 97% reduction for p.L540P). Both patients had an additional LRP5 sequence change (p.Q816P in Patient 1 from the unaffected mother and p.T852M in Patient 2 verified as a new mutation). Luciferase assay showed no reduction for p.Q816P and 94.9% reduction for the new mutation p.T852M, suggesting that p.Q816P may be not pathogenic and p.T852M may be pathogenic.
Conclusions
Our findings demonstrated two new novel LRP5 mutations in Chinese patients with FEVR and mild reduced BMD. They emphasize the complexity of FEVR mutations and phenotypes.
PMCID: PMC3976684  PMID: 24715757
24.  Sox10 Expressing Cells in the Lateral Wall of the Aged Mouse and Human Cochlea 
PLoS ONE  2014;9(6):e97389.
Age-related hearing loss (presbycusis) is a common human disorder, affecting one in three Americans aged 60 and over. Previous studies have shown that presbyacusis is associated with a loss of non-sensory cells in the cochlear lateral wall. Sox10 is a transcription factor crucial to the development and maintenance of neural crest-derived cells including some non-sensory cell types in the cochlea. Mutations of the Sox10 gene are known to cause various combinations of hearing loss and pigmentation defects in humans. This study investigated the potential relationship between Sox10 gene expression and pathological changes in the cochlear lateral wall of aged CBA/CaJ mice and human temporal bones from older donors. Cochlear tissues prepared from young adult (1–3 month-old) and aged (2–2.5 year-old) mice, and human temporal bone donors were examined using quantitative immunohistochemical analysis and transmission electron microscopy. Cells expressing Sox10 were present in the stria vascularis, outer sulcus and spiral prominence in mouse and human cochleas. The Sox10+ cell types included marginal and intermediate cells and outer sulcus cells, including those that border the scala media and those extending into root processes (root cells) in the spiral ligament. Quantitative analysis of immunostaining revealed a significant decrease in the number of Sox10+ marginal cells and outer sulcus cells in aged mice. Electron microscopic evaluation revealed degenerative alterations in the surviving Sox10+ cells in aged mice. Strial marginal cells in human cochleas from donors aged 87 and older showed only weak immunostaining for Sox10. Decreases in Sox10 expression levels and a loss of Sox10+ cells in both mouse and human aged ears suggests an important role of Sox10 in the maintenance of structural and functional integrity of the lateral wall. A loss of Sox10+ cells may also be associated with a decline in the repair capabilities of non-sensory cells in the aged ear.
doi:10.1371/journal.pone.0097389
PMCID: PMC4041576  PMID: 24887110
25.  Identification and characterization of Pannexin expression in the mammalian cochlea 
The gap junction in vertebrates is encoded by the connexin gene family. Recently, a new gene family termed pannexin (Panx) has been identified in vertebrates and found to encode gap junctional proteins as well. To date, three pannexin isoforms (Panx1, 2 and 3) have been cloned from mouse and human genomes. In this study, expression of pannexins in the mouse and rat cochlea was investigated. PCR and Western blot analysis showed that all three pannexin isoforms were expressed in the cochlea. Immunofluorescent staining showed that Panx1 expression was extensive. In the organ of Corti, Panx1 labeling was found in supporting cells, including pillar cells, Hensen cells, Claudius cells and Boettcher cells. Both surface plaque-like punctate labeling and diffuse-cytoplasmic labeling were visible. However, the labeling was weak and rare in Deiters cells. No labeling was found in the hair cells. Intense labeling for Panx1 was also observed in the interdental cells in the spiral limbus, the inner and outer sulcus cells, and the type II fibrocytes in the spiral prominence and central region in the cochlear lateral wall. However, no overlapping labeling was observed. In addition, Panx1 labeling was detectable in the Reissner's membrane and strial blood vessel cells. Panx2 labeling was restricted to the basal cells in the stria vascularis and was also detectable in the spiral ganglion neurons. However, no overlapping labeling for Panx1 and Panx2 was observed. Finally, Panx3 labeling was exclusively observed in the cochlear bone. Thus, Panx1, 2 and 3 are abundantly expressed in the mammalian cochlea and demonstrate distinct cellular distributions. Like connexins, they may play an important role in hearing.
doi:10.1002/cne.21898
PMCID: PMC2630187  PMID: 19009624
Gap junction; hemichannel; cochlea; connexin; intercellular communication; deafness

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