Regulator of calcineurin 1 (Rcan1) has a protective role in brain ischemia/reperfusion injury.

BACKGROUND: An increase in intracellular calcium concentration [Ca2+]i is one of the first events to take place after brain ischemia. A key [Ca2+]i-regulated signaling molecule is the phosphatase calcineurin (CN), which plays important roles in the modulation of inflammatory cascades. Here, we have analyzed the role of endogenous regulator of CN 1 (Rcan1) in response to experimental ischemic stroke induced by middle cerebral artery occlusion. METHODS: Animals were subjected to focal cerebral ischemia with reperfusion. To assess the role of Rcan1 after stroke, we measured infarct volume after 48 h of reperfusion in Rcan1 knockout (KO) and wild-type (WT) mice. In vitro studies were performed in astrocyte-enriched cortical primary cultures subjected to 3% oxygen (hypoxia) and glucose deprivation (HGD). Adenoviral vectors were used to analyze the effect of overexpression of Rcan1-4 protein. Protein expression was examined by immunohistochemistry and immunoblotting and expression of mRNA by quantitative real-time Reverse-Transcription Polymerase Chain Reaction (real time qRT-PCR). RESULTS: Brain ischemia/reperfusion (I/R) injury in vivo increased mRNA and protein expression of the calcium-inducible Rcan1 isoform (Rcan1-4). I/R-inducible expression of Rcan1 protein occurred mainly in astroglial cells, and in an in vitro model of ischemia, HGD treatment of primary murine astrocyte cultures induced Rcan1-4 mRNA and protein expression. Exogenous Rcan1-4 overexpression inhibited production of the inflammatory marker cyclo-oxygenase 2. Mice lacking Rcan1 had higher expression of inflammation associated genes, resulting in larger infarct volumes. CONCLUSIONS: Our results support a protective role for Rcan1 during the inflammatory response to stroke, and underline the importance of the glial compartment in the inflammatory reaction that takes place after ischemia. Improved understanding of non-neuronal mechanisms in ischemic injury promises novel approaches to the treatment of acute ischemic stroke.

A novel G21R mutation of the GJB2 gene causes autosomal dominant non-syndromic congenital deafness in a Cuban family

Deafness is a complex disorder affecting 1/1000 infants. In developed countries, more than 50 percent of deafness cases are thought to have a genetic cause. At least 40 loci for dominant non-syndromic deafness and another 30 for recessive non-syndromic deafness have been described. Mutations in the GJB2 gene are the cause of an important number of cases of non-syndromic recessive deafness but are not as common in non-syndromic dominant deafness cases. We describe here a new dominant mutation (G21R) in the GJB2 gene which causes deafness and has been identified in a three generation Cuban family with dominant non-syndromic congenital sensorineural profound deafness.

Slc7a9-deficient mice develop cystinuria non-I and cystine urolithiasis.

Cystinuria is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in urolithiasis of cystine. Cystinuria is caused by defects in the amino acid transport system b0,+ (i.e. the rBAT/b0,+AT heteromeric complex). Mutations in SLC3A1, encoding rBAT, cause cystinuria type A, characterized by a silent phenotype in heterozygotes (phenotype I). Mutations in SLC7A9, encoding b0,+AT, cause cystinuria type B, in which heterozygotes in most cases hyperexcrete cystine and dibasic amino acids (phenotype non-I). To facilitate in vivo investigation of b0,+AT in cystinuria, Slc7a9 knockout mice have been generated. Expression of b0,+AT protein is completely abolished in the kidney of Slc7a9-/- mice ('Stones'). In contrast, Stones expressed significant amounts of rBAT protein, which is covalently linked to unidentified light subunit(s). Stones mice present a dramatic hyperexcretion of cystine and dibasic amino acids, while Slc7a9+/- mice show moderate but significant hyperexcretion of these amino acids (phenotype non-I). Forty-two per cent of Stones mice develop cystine calculi in the urinary system. Calculi develop during the first month of life and grow throughout the life span of the animals. Histopathology in kidney reveals typical changes for urolithiasis (tubular and pelvic dilatation, tubular necrosis, tubular hyaline droplets and chronic interstitial nephritis). The fact that some Stones mice, generated in a mixed genetic background, develop cystine calculi from an early age, while others do not develop them in their first year of life, suggests the involvement of modifier genes in the lithiasis phenotype. Thus, Stones provide a valid model of cystinuria which can be used in the study of genetic, pharmacological and environmental factors involved in cystine urolithiasis.

Human connexin26 (GJB2) deafness mutations affect the function of gap junction channels at different levels of protein expression.

Mutations in the connexin26 (GJB2) gene account for about half of inherited non-syndromic deafness cases in Western countries. The connexin26 protein is a subunit of gap junctions that form a network of intercellular communication among supporting cells and fibrocytes in the mammalian inner ear. Here we describe functional implications of mutations in the coding region of connexin26 genes (M1V, M34T, L90P, R127H, F161S, P173R, and R184P), identified in patients and stably transfected in human HeLa cells. While all mutated connexin26 cDNAs were transcribed, only M34T, L90P, R127H, F161S, and R184P were translated in HeLa cells. Analysis of indirect immunofluorescence showed membranous localization, strong for M34T, L90P, R127H, and very weak for F161S, but no signal corresponding to M1V, P173R and R184P. Tracer coupling experiments revealed diffusion of microinjected neurobiotin into neighbouring cells in the case of M34T and R127H, whereas M1V, L90P, F161S, P173R and R184P mutants did not show intercellular coupling. The results of oligomerisation studies suggested a partly disturbed assembly of hemichannels in M34T and L90P mutants but complete absence of hemichannel formation in the R184P mutant. The R127H mutation did not affect channel formation and is likely to represent a polymorphism. Our results show that mutations in the connexin26 gene can affect gap junctional intercellular communication at the level of protein translation, trafficking or assembly of hemichannels.

Connexin mutations in hearing loss, dermatological and neurological disorders.

Gap junctions are important structures in cell-to-cell communication. Connexins, the protein units of gap junctions, are involved in several human disorders. Mutations in beta-connexin genes cause hearing, dermatological and peripheral nerve disorders. Recessive mutations in the gene encoding connexin 26 (GJB2) are the most common cause of childhood-onset deafness. The combination of mutations in the GJB2 and GJB6 (Cx30) genes also cause childhood hearing impairment. Although both recessive and dominant connexin mutants are functionally impaired, dominant mutations might have in addition a dominant-negative effect on wild-type connexins. Some dominant mutations in beta-connexin genes have a pleiotropic effect at the level of the skin, the auditory system and the peripheral nerves. Understanding the genotype-phenotype correlations in diseases caused by mutations in connexin genes might provide important insight into the mechanisms that lead to these disorders.

A common frameshift mutation and other variants in GJB4 (connexin 30.3): Analysis of hearing impairment families.

Mutations in GJB1, GJB2, GJB3 and GJB6 are involved in hearing impairment. GJB2, GJB3 and GJB6 are also mutated in patients with hyperproliferative skin disorders. The human GJB4 gene has been deduced in silico and a mutation in a family with erythrokeratodermia variabilis has been reported. We describe here the analysis of the GJB4 gene in hearing impairment patients and control subjects. We have identified a common (4%) frameshift mutation (154del4) in GJB4 in both affected and hearing subjects, one patient being homozygous for the mutation. We have also detected five amino acid variants (R103C, R124Q, R160C, C169W and E204A) in individuals that have not skin disorders. While mutation 154del4 is not associated with hearing impairment the involvement of some of the amino acid variants detected here is uncertain. These GJB4 variants should help to define the putative role of connexin 30.3 in both skin disorders and hearing impairment.

Mutations in the TMPRSS3 gene are a rare cause of childhood nonsyndromic deafness in Caucasian patients.

Two loci for nonsyndromic recessive deafness located on chromosome 21q22.3 have previously been reported, DFNB8 and DFNB10. Recently a gene which encodes a transmembrane serine protease, TMPRSS3 or ECHOS1, was found to be responsible for both the DFNB8 and DFNB10 phenotypes. To determine the contribution of TMPRSS3 mutations in the general congenital/childhood nonsyndromic deaf population we performed mutation analysis of the TMPRSS3 gene in 448 unrelated deaf patients from Spain, Italy, Greece, and Australia who did not have the common 35delG GJB2 mutation. From the 896 chromosomes studied we identified two novel pathogenic mutations accounting for four mutant alleles and at least 16 nonpathogenic sequence variants. The pathogenic mutations were a 1-bp deletion resulting in a frameshift and an amino acid substitution in the LDLRA domain of TMPRSS3. From this and another study we estimate the frequency of TMPRSS3 mutations in our sample as 0.45%, and approximately 0.38% in the general Caucasian childhood deaf population. However, TMPRSS3 is still an important contributor to genetic deafness in populations with large consanguineous families.