Redundant contribution of a Transient Receptor Potential cation channel Member 1 exon 11 single nucleotide polymorphism to equine congenital stationary night blindness.

BACKGROUND: Congenital stationary night-blindness (CSNB) is a recessive autosomal defect in low-light vision in Appaloosa and other horse breeds. This condition has been mapped by linkage analysis to a gene coding for the Transient Receptor Potential cation channel Member 1 (TRPM1). TRPM1 is normally expressed in the ON-bipolar cells of the inner nuclear layer of the retina. Down-regulation of TRPM1 expression in CSNB results from a transposon-like insertion in intron 1 of the TRPM1 gene. Stop transcription signals in this transposon significantly reduce TRPM1 primary transcript levels in CSNB horses. This study describes additional contributions by a second mutation of the TRPM1 gene, the ECA1 108,249,293 C > T SNP, to down-regulation of transcription of the TRPM1 gene in night-blind horses. This TRPM1 SNP introduces a consensus binding site for neuro-oncological ventral antigen 1 (Nova-1) protein in the primary transcript. Nova-1 binding disrupts normal splicing signals, producing unstable, non-functional mRNA transcripts. RESULTS: Retinal bipolar cells express both TRPM1 and Nova-1 proteins. In vitro addition of Nova-1 protein retards electrophoretic migration of TRPM1 RNA containing the ECA1 108,249,293 C > T SNP. Up-regulating Nova-1 expression in primary cultures of choroidal melanocytes carrying the intron 11 SNP caused an average log 2-fold reduction of ~6 (64-fold) of TRPM1 mRNA expression. CONCLUSIONS: These finding suggest that the equine TRPM1 SNP can act independently to reduce survival of TRPM1 mRNA escaping the intron 1 transcriptional stop signals in CSNB horses. Coexistence and co-inheritance of two independent TRPM1 mutations across 1000 equine generations suggests a selective advantage for the apparently deleterious CSNB trait.

Relative quantification of white blood cell mitochondrial DNA and assessment of mitochondria by use of transmission electron microscopy in English Springer Spaniels with and without retinal dysplasia.

OBJECTIVE: To compare relative amounts of WBC mitochondrial DNA (mtDNA; assessed via real-time PCR assay) and morphology of lymphocyte mitochondria (assessed via transmission electron microscopy [TEM]) in blood samples collected from English Springer Spaniels with and without retinal dysplasia. ANIMALS: 7 and 5 client-owned English Springer Spaniels (1 to 11 years old) with and without retinal dysplasia, respectively. PROCEDURES: Blood samples were obtained from affected and unaffected dogs via venipuncture. Genomic DNA was extracted from WBCs of the 7 affected and 5 unaffected dogs, and relative quantification of the cytochrome c oxidase subunit 1 gene (COX1) was determined via analysis of real-time PCR results. White blood cells from 3 affected and 4 unaffected dogs were embedded in epoxide resin for TEM; cross sections were examined for lymphocytes, which were measured. The mitochondria within lymphocytes were quantified, and the mitochondrial surface area per lymphocyte cross section was calculated. A masked technique was used to compare mitochondrial morphology between the 2 groups. RESULTS: Compared with the smallest measured quantity of mtDNA among unaffected dogs, mtDNA amounts varied among unaffected (1.08- to 4.76-fold differences) and affected dogs (1- to 2.68-fold differences). Analysis of lymphocyte measurements and mitochondrial surface area, morphology, and quantity revealed no significant differences between affected and unaffected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: No significant differences were detected in relative amounts of WBC mtDNA or the size, number, or morphology of lymphocyte mitochondria in English Springer Spaniels affected with retinal dysplasia, compared with results for unaffected control dogs.

Differential mitochondrial DNA and gene expression in inherited retinal dysplasia in miniature Schnauzer dogs.

PURPOSE: To investigate the molecular basis of inherited retinal dysplasia in miniature Schnauzers. METHODS: Retina and retinal pigment epithelial tissues were collected from canine subjects at the age of 3 weeks. Total RNA isolated from these tissues was reverse transcribed to make representative cDNA pools that were compared for differences in gene expression by using a subtractive hybridization technique referred to as representational difference analysis (RDA). Expression differences identified by RDA were confirmed and quantified by real-time reverse-transcription PCR. Mitochondrial morphology from leukocytes and skeletal muscle of normal and affected miniature Schnauzers was examined by transmission electron microscopy. RESULTS: RDA screening of retinal pigment epithelial cDNA identified differences in mRNA transcript coding for two mitochondrial (mt) proteins--cytochrome oxidase subunit 1 and NADH dehydrogenase subunit 6--in affected dogs. Contrary to expectations, these identified sequences did not contain mutations. Based on the implication of mt-DNA-encoded proteins by the RDA experiments we used real-time PCR to compare the relative amounts of mt-DNA template in white blood cells from normal and affected dogs. White blood cells of affected dogs contained less than 30% of the normal amount of two specific mtDNA sequences, compared with the content of the nuclear-encoded glyceraldehyde-3-phosphate dehydrogenase (GA-3-PDH) reference gene. Retina and RPE tissue from affected dogs had reduced mRNA transcript levels for the two mitochondrial genes detected in the RDA experiment. Transcript levels for another mtDNA-encoded gene as well as the nuclear-encoded mitochondrial Tfam transcription factor were reduced in these tissues in affected dogs. Mitochondria from affected dogs were reduced in number and size and were unusually electron dense. CONCLUSIONS: Reduced levels of nuclear and mitochondrial transcripts in the retina and RPE of miniature Schnauzers affected with retinal dysplasia suggest that the pathogenesis of the disorder may arise from a lowered energy supply to the retina and RPE.

Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery.

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.

L'objectif de la présente étude était de développer un système murin de culture d'explant de rétine/choroïde/sclérotique afin de faciliter la livraison intra-vitréenne de vecteurs. Des explants de segments postérieurs provenant de souris adultes de deux groupes d'âge différents (4 sem et 15 sem) furent cultivés dans un milieu sans sérum pour des périodes de temps variables. La viabilité tissulaire fut évaluée par morphologie macroscopique, quantification de la survie cellulaire, expression de la caspase-3 activée, et immunohistochimie. Afin d'imiter la thérapie génique oculaire, les explants furent exposés pendant 48 h à des unités de transduction variables d'un vecteur lentiviral exprimant le gène pour la protéine fluorescente verte. Les explants de cellules de la rétine sont demeurés viables pour environ 1 sem, bien que dans la couche de cellules ganglionnaires on nota le développement de l'apoptose entre 4 à 7 j. Suite à l'infusion de vecteur dans le segment postérieur, la transduction virale fut notée dans plusieurs couches rétiniennes des animaux des deux groupes d'âge. Une influence de l'âge de la souris donneuse fut notée et chez les souris plus âgées la transduction ne se faisait pas aussi bien que chez les jeunes souris. Ce modèle d'explant permet la gestion facile de culture de segment oculaire postérieur avec un minimum de dérangement du tissu, et pourrait être utile pour étudier des méthodes visant à augmenter la thérapie génique sous conditions contrôlées.(Traduit par Docteur Serge Messier).

Differences in expression of retinal pigment epithelium mRNA between normal canines.

A reference database of differences in mRNA expression in normal healthy canine retinal pigment epithelium (RPE) has been established. This database identifies non-informative differences in mRNA expression that can be used in screening canine RPE for mutations associated with clinical effects on vision. Complementary DNA (cDNA) pools were prepared from mRNA harvested from RPE, amplified by PCR, and used in a subtractive hybridization protocol (representational differential analysis) to identify differences in RPE mRNA expression between canines. The effect of relatedness of the test canines on the frequency of occurrence of differences was evaluated by using 2 unrelated canines for comparison with 2 female sibling canines of blue heeler/bull terrier lineage. Differentially expressed cDNA species were cloned, sequenced, and identified by comparison to public database entries. The most frequently observed differentially expressed sequence from the unrelated canine comparison was cDNA with 21 base pairs (bp) identical to the human epithelial membrane protein 1 gene (present in 8 of 20 clones). Different clones from the same-sex sibling RPE contained repetitions of several short sequence motifs including the human epithelial membrane protein 1 (4 of 25 clones). Other prevalent differences between sibling RPE included sequences similar to a chicken genetic marker sequence motif (5 of 25), and 6 clones with homology to porcine major histocompatibility loci. In addition to identifying several repetitively occurring, noninformative, differentially expressed RPE mRNA species, the findings confirm that fewer differences occurred between siblings, highlighting the importance of using closely related subjects in representational difference analysis studies.

Mitochondrial transcription factor A (Tfam) gene sequencing and mitochondrial evaluation in inherited retinal dysplasia in miniature schnauzer dogs.

Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (µm²) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

Structure and function of CLCA proteins.

CLCA proteins were discovered in bovine trachea and named for a calcium-dependent chloride conductance found in trachea and in other secretory epithelial tissues. At least four closely located gene loci in the mouse and the human code for independent isoforms of CLCA proteins. Full-length CLCA proteins have an unprocessed mass ratio of approximately 100 kDa. Three of the four human loci code for the synthesis of membrane-associated proteins. CLCA proteins affect chloride conductance, epithelial secretion, cell-cell adhesion, apoptosis, cell cycle control, mucus production in asthma, and blood pressure. There is a structural and probable functional divergence between CLCA isoforms containing or not containing beta4-integrin binding domains. Cell cycle control and tumor metastasis are affected by isoforms with the binding domains. These isoforms are expressed prominently in smooth muscle, in some endothelial cells, in the central nervous system, and also in secretory epithelial cells. The isoform with disrupted beta4-integrin binding (hCLCA1, pCLCA1, mCLCA3) alters epithelial mucus secretion and ion transport processes. It is preferentially expressed in secretory epithelial tissues including trachea and small intestine. Chloride conductance is affected by the expression of several CLCA proteins. However, the dependence of the resulting electrical signature on the expression system rather than the CLCA protein suggests that these proteins are not independent Ca2+-dependent chloride channels, but may contribute to the activity of chloride channels formed by, or in conjunction with, other proteins.

Complex expression and localization of inactivating Kv channels in cultured hippocampal astrocytes.

Voltage-gated potassium channels are well established as critical for setting action potential frequency, membrane potential, and neurotransmitter release in neurons. However, their role in the "nonexcitable" glial cell type is yet to be fully understood. We used whole cell current kinetics, pharmacology, immunocytochemistry, and RT-PCR to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that approximately 70, 10, and <5% of total A current is associated with Kv4, Kv3, and Kv1 channels, respectively. In addition, pharmacology and kinetics provide evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Given this complex A-type channel expression pattern, we assessed the role of A currents in membrane voltage oscillations in response to current injections. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. As in neurons, this effect may enable astrocytes to respond rapidly to high-frequency synaptic events. Our results indicate that hippocampal astrocytes in vitro express multiple A-type Kv channel alpha-subunits with accessory, possibly Ca(2+)-sensitive, cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Function of these channels remains to be determined in a physiological setting. However, this study suggests that they enable astrocytes to respond rapidly with membrane voltage oscillations to high-frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity.

The calcium-dependent chloride conductance mediator pCLCA1.

The regulatory behavior, inhibitor sensitivity, and properties of the whole cell chloride conductance observed in cells expressing the cDNA coding for a chloride conductance mediator isoform of the CLCA gene family, pCLCA1, have been studied. Common C-kinase consensus phosphorylation sites between pCLCA1 and the closely related human isoform hCLCA1 are consistent with a role for calcium in channel activation. Both channels are activated rapidly on exposure to the calcium ionophore ionomycin. Direct involvement of calcium in the activation of pCLCA1 was supported by the finding that treatment with the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM reduced the rate of chloride efflux from NIH/3T3 cells expressing the pCLCA1 channel. No combination of A-kinase activators used was effective in activating chloride efflux via this channel despite the presence of a unique strong A-kinase consensus site in pCLCA1. Notable differences of pCLCA1 from the reported properties of CLCA family members include the failure of phorbol 12-myristate 13-acetate to activate chloride efflux in cells expressing pCLCA1 and a lack of inhibition of chloride efflux from these cells after treatment with DIDS or dithiothreitol. However, selected inhibitors of anionic conductance inhibited pCLCA1-dependent anion efflux. The electrogenic nature of the ionomycin-dependent efflux of chloride from cells expressing pCLCA1 was confirmed by detection of outwardly rectifying chloride current and inhibition of this current by chloride conductance inhibitors in a whole cell patch-clamp study.

pCLCA1 lacks inherent chloride channel activity in an epithelial colon carcinoma cell line.

The effects of CLCA protein expression on the regulation of Cl(-) conductance by intracellular Ca(2+) and cAMP have been studied previously in nonepithelial cell lines chosen for low backgrounds of endogenous Cl(-) conductance. However, CLCA proteins have been cloned from, and normally function in, differentiated epithelial cells. In this study, we examine the effects of differentiation of the Caco-2 epithelial colon carcinoma cell line on modulation of Cl(-) conductance by pCLCA1 protein expression. Cl(-) transport was measured as (36)Cl(-) efflux, as transepithelial short-circuit currents, and as whole cell patch-clamp current-voltage relations. The rate of (36)Cl(-) efflux and amplitude of currents in patch-clamp studies after the addition of the Ca(2+) ionophore A-23187 were increased significantly by pCLCA1 expression in freshly passaged Caco-2 cells. However, neither endogenous nor pCLCA1-dependent Ca(2+)-sensitive Cl(-) conductance could be detected in 14-day-postpassage cells. In contrast to Ca(2+)-sensitive Cl(-) conductance, endogenous cAMP-dependent Cl(-) conductance does not disappear on Caco-2 differentiation. cAMP-dependent Cl(-) conductance was modulated by pCLCA1 expression in Caco-2 cells, and this modulation was observed in freshly passaged and in mature 14-day-postpassage Caco-2 cultures. pCLCA1 mRNA expression, antigenic pCLCA1 protein epitope expression, and pCLCA1 function as a modulator of cAMP-dependent Cl(-) conductance were retained through differentiation in Caco-2 cells, whereas Ca(2+)-dependent Cl(-) conductance disappeared. We conclude that pCLCA1 expression may increase the sensitivity of preexisting endogenous Cl(-) channels to Ca(2+) and cAMP agonists but apparently lacks inherent Cl(-) channel activity under growth conditions where endogenous channels are not expressed.

pCLCA1 becomes a cAMP-dependent chloride conductance mediator in Caco-2 cells.

Members of the CLCA protein family are expressed in airway and intestinal epithelium, where they may participate in secretory activity as mediators of chloride conductance. A calcium-dependent chloride conductance has been observed upon expression of CLCA proteins in non-epithelial cell lines. The pCLCA1 gene, cloned in our laboratory, codes for a product containing a unique A-kinase consensus acceptor site not found in other CLCA proteins. Calcium-dependent, but not cAMP-dependent, chloride conductance increased when pCLCA1 was expressed in NIH/3T3 fibroblasts. We transfected the Caco-2 human colon carcinoma cell line with pCLCA1 to investigate the regulation of CLCA-associated chloride conductance in this differentiated epithelial cell line. Expression of pCLCA1 in the Caco-2 cell line enhanced cAMP-responsive 36Cl efflux, short circuit current, and whole cell chloride current in these cells. This cAMP-dependent chloride conductance was localized to the apical membrane of polarized Caco-2 cells.

CLCA protein and chloride transport in canine retinal pigment epithelium.

Problems in ion and fluid transfer across the retinal pigment epithelium (RPE) are a probable cause of inappropriate accumulations of fluid between the photoreceptors of the retina and the RPE. The activities of Cl- transporters involved in basal fluid transfer across the RPE have been compared to determine whether Ca2+- or cAMP-dependent channels may be responsible for basal housekeeping levels of secretory activity in this tissue. The role of a candidate Ca2+-dependent CLCA protein in the basal RPE transport of Cl- has been investigated. Low concentrations of the Cl- conductance inhibitors glibenclamide and 5-nitro-2-(3-phenylpropylamino)benzoate reduced the short-circuit current in dog RPE preparations mounted in Ussing chambers and decreased the Ca2+-dependent Cl- efflux from fibroblasts expressing the pCLCA1 Cl- conductance regulator. However, these same agents did not inhibit the rate of Cl- release from cultured fibroblasts expressing the cystic fibrosis transmembrane regulator (CFTR) conductive Cl- channel. Addition of ionomycin to primary cultures of canine RPE cells or to fibroblasts expressing the pCLCA1 channel regulator increased the rate of release of Cl- from both types of cultured cells. However, the presence of pCLCA1 also increased cAMP-dependent Cl- release from fibroblasts expressing CFTR. We conclude that Ca2+-dependent Cl- transport may be more important than cAMP-dependent Cl- transport for normal fluid secretion across the RPE. Furthermore, CLCA proteins expressed in the RPE appear to regulate the activity of other Cl- transporters, rather than functioning as primary ion transport proteins.