Biomedical subjects

Christian A Hübner

Publications and source records attributed to Christian A Hübner.

At least 19 recordsLinked to original sources

PubMed2006

Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery.

We report a signaling mechanism in rats between mother and fetus aimed at preparing fetal neurons for delivery. In immature neurons, gamma-aminobutyric acid (GABA) is the primary excitatory neurotransmitter. We found that, shortly before delivery, there is a transient reduction in the intracellular chloride concentration and an excitatory-to-inhibitory switch of GABA actions. These events were triggered by oxytocin, an essential maternal hormone for labor. In vivo administration of an oxytocin receptor antagonist before delivery prevented the switch of GABA actions in fetal neurons and aggravated the severity of anoxic episodes. Thus, maternal oxytocin inhibits fetal neurons and increases their resistance to insults during delivery.

Action Potentials↗

PubMed2006

Targeted disruption of the murine retinal dehydrogenase gene Rdh12 does not limit visual cycle function.

RDH12 codes for a member of the family of short-chain alcohol dehydrogenases/reductases proposed to function in the visual cycle that supplies the chromophore 11-cis retinal to photoreceptor cells. Mutations in RDH12 cause severe and progressive childhood onset autosomal-recessive retinal dystrophy, including Leber congenital amaurosis. We generated Rdh12 knockout mice, which exhibited grossly normal retinal histology at 10 months of age. Levels of all-trans and 11-cis retinoids in dark- and light-adapted animals and scotopic and photopic electroretinogram (ERG) responses were similar to those for the wild type, as was recovery of the ERG response following bleaching, for animals matched for an Rpe65 polymorphism (p.L450M). Lipid peroxidation products and other measures of oxidative stress did not appear to be elevated in Rdh12(-/-) animals. RDH12 was localized to photoreceptor inner segments and the outer nuclear layer in both mouse and human retinas by immunohistochemistry. The present findings, together with those of earlier studies showing only minor functional deficits in mice deficient for Rdh5, Rdh8, or Rdh11, suggest that the activity of any one isoform is not rate limiting in the visual response.

Alcohol Oxidoreductases↗

PubMed2006

Germ cell nuclear factor is a repressor of CRIPTO-1 and CRIPTO-3.

The pluripotency of embryonic stem and embryonic carcinoma cells is maintained by the expression of a set of "stemness" genes. Whereas these genes are down-regulated upon induction of differentiation, the germ cell nuclear factor (GCNF) is transiently up-regulated and represses several pluripotency genes. CRIPTO-1, a co-receptor for the morphogen nodal, is strongly expressed in undifferentiated cells and is rapidly down-regulated during retinoic acid-induced differentiation. Although CRIPTO-1 is expressed at very low levels in adult tissues under normal conditions, it is found highly expressed in a broad range of tumors, where it acts as a potent oncogene. We show that expression of CRIPTO-1 is directly repressed by GCNF during differentiation of the human teratocarcinoma cell line, NT2. GCNF bound to a DR0 element of the CRIPTO-1 promoter in vitro, as shown by electrophoretic mobility shift assays, and in vivo, as demonstrated by chromatin immunoprecipitation. Reporter gene assays demonstrated that GCNF-mediated repression of the CRIPTO-1 promoter is dependent upon the DR0 site. Overexpression of GCNF in NT2 cells resulted in repression of CRIPTO-1 transcription, whereas expression of the transcription-activating fusion construct GCNF-VP16 led to an induction of the CRIPTO-1 gene and prevented its retinoic acid-induced down-regulation. Furthermore, we demonstrated that CRIPTO-3, a processed pseudogene of CRIPTO-1 on the X chromosome, is expressed in undifferentiated NT2 cells and is regulated by GCNF in parallel to CRIPTO-1. Thus, our study supports the hypothesis of GCNF playing a central role during differentiation of stem cells by repression of stem cell-specific genes.

Base Sequence↗

PubMed2006

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6.

Mammalian CLC proteins function as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies. The disruption of ClC-6 in mice reduced their pain sensitivity and caused moderate behavioral abnormalities. Neuronal tissues showed autofluorescence at initial axon segments. At these sites, electron microscopy revealed electron-dense storage material that caused a pathological enlargement of proximal axons. These deposits were positive for several lysosomal proteins and other marker proteins typical for neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. However, the lysosomal pH of Clcn6(-/-) neurons appeared normal. CLCN6 is a candidate gene for mild forms of human NCL. Analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients but failed to prove a causative role of CLCN6 in that disease.

Animals↗

PubMed2006

Multiplex ligation-dependent probe amplification for rapid detection of proteolipid protein 1 gene duplications and deletions in affected males and carrier females with Pelizaeus-Merzbacher disease.

BACKGROUND: Pelizaeus-Merzbacher disease is a rare X-linked neurodegenerative disorder caused by sequence variations in the proteolipid protein 1 gene (PLP1). PLP1 gene duplications account for approximately 50%-75% of cases and point variations for approximately 15%-20% of cases; deletions and insertions occur infrequently. We used multiplex ligation-dependent probe amplification (MLPA) to detect PLP1 gene alterations, especially gene duplications and deletions. METHODS: We performed MLPA on 102 samples from individuals with diverse PLP1 gene abnormalities and from controls, including 50 samples previously characterized for the PLP1 gene by quantitative PCR but which were anonymized for prior results and sex. RESULTS: All males with PLP1 gene duplications (n = 13), 1 male with a triplication, 2 males with whole gene deletions, and all controls (n = 72) were unambiguously assigned to their correct genotype. Of 4 female carriers tested by MLPA and quantitative PCR, 3 were duplication carriers by both methods, and 1 was a triplication carrier by MLPA and a duplication carrier by quantitative PCR. For 1 sample with a partial deletion, MLPA showed exon 3 deleted but PCR showed exons 3 and 4 deleted. Sequence analysis of 2 samples with reduced dosage for exons 3 and 5 revealed point variations overlapping the annealing site for the corresponding MLPA probe. The precision of MLPA analysis was excellent and comparable to or better than quantitative PCR, with CVs of 4.3%-9.8%. CONCLUSIONS: MLPA is a rapid and reliable method to determine PLP1 gene copies. Samples with partial PLP1 gene dosage alterations require confirmation with a non-MLPA method.

Carrier State↗

PubMed2006

Neurogenic mechanisms contribute to hypertension in mice with disruption of the K-Cl cotransporter KCC3.

The neurodegenerative disorder Andermann syndrome is caused by mutations of the K-Cl cotransporter KCC3. Mice with a targeted disruption of the corresponding gene, Slc12a6, reproduce neurodegeneration of the peripheral and central nervous system (CNS) and display arterial hypertension. Kcc3 is expressed in numerous tissues, including the CNS and vascular smooth muscle cells. As the intracellular chloride concentration may influence myogenic tone and hence blood pressure, we measured the chloride concentration in vascular smooth muscle cells. It was indeed increased in superficial brain arteries and saphenous arteries of Kcc3(-/-) mice. Isolated saphenous arteries and their third-order branches, however, reacted indistinguishably to changes in intravascular pressure, stimulation of alpha1-adrenoreceptors, exogenous nitric oxide, or blockade of calcium-activated chloride channels. Likewise, the responses to alpha1-adrenergic stimulation or exogenous nitric oxide in vivo were identical in both genotypes. These results argue against a major vascular-intrinsic component of arterial hypertension in Kcc3(-/-) mice. In contrast, either alpha1-adrenergic blockade or inhibition of ganglionic transmission abolished the difference in arterial blood pressure between both genotypes. This demonstrates a neurogenic component in the maintenance of this phenotype, which is further supported by an increase of urinary norepinephrine and epinephrine excretion in Kcc3(-/-) mice. Our data indicate that local control of myogenic tone does not require KCC3 and that hypertension in Kcc3(-/-) mice depends on an elevated sympathetic tone.

Aldosterone↗

PubMed2006

The forkhead transcription factor Foxi1 directly activates the AE4 promoter.

Intercalated cells are highly specialized cells within the renal collecting duct epithelium and play an important role in systemic acid-base homoeostasis. Whereas type A intercalated cells secrete protons via an apically localized H+-ATPase, type B intercalated cells secrete HCO3-. Type B intercalated cells specifically express the HCO3-/Cl- exchanger AE4 (anion exchanger 4), encoded by Slc4a9. Mice with a targeted disruption of the gene for the forkhead transcription factor Foxi1 display renal tubular acidosis due to an intercalated cell-differentiation defect. Collecting duct cells in these mice are characterized by the absence of inter-calated cell markers including AE4. To test whether Slc4a9 is a direct target gene of Foxi1, an AE4 promoter construct was generated for a cell-based reporter gene assay. Co-transfection with the Foxi1 cDNA resulted in an approx. 100-fold activation of the AE4 promoter construct. By truncating the AE4 promoter at the 5'-end, we demonstrate that a fragment of approx. 462 bp upstream of the transcription start point is sufficient to mediate activation by Foxi1. Sequence analysis of this region revealed at least eight potential binding sites for Foxi1 in both sense and antisense orientation. Only one element was bound by recombinant Foxi1 protein in bandshift assays. Mutation of this site abolished both binding in bandshift assays and transcriptional activation by co-transfection of Foxi1 in the reporter gene assay. We thus identify the AE4 promoter as a direct target of Foxi1.

Animals↗

PubMed2006

Mice with a targeted disruption of the Cl-/HCO3- exchanger AE3 display a reduced seizure threshold.

Neuronal activity results in significant pH shifts in neurons, glia, and interstitial space. Several transport mechanisms are involved in the fine-tuning and regulation of extra- and intracellular pH. The sodium-independent electroneutral anion exchangers (AEs) exchange intracellular bicarbonate for extracellular chloride and thereby lower the intracellular pH. Recently, a significant association was found with the variant Ala867Asp of the anion exchanger AE3, which is predominantly expressed in brain and heart, in a large cohort of patients with idiopathic generalized epilepsy. To analyze a possible involvement of AE3 dysfunction in the pathogenesis of seizures, we generated an AE3-knockout mouse model by targeted disruption of Slc4a3. AE3-knockout mice were apparently healthy, and neither displayed gross histological and behavioral abnormalities nor spontaneous seizures or spike wave complexes in electrocorticograms. However, the seizure threshold of AE3-knockout mice exposed to bicuculline, pentylenetetrazole, or pilocarpine was reduced, and seizure-induced mortality was significantly increased compared to wild-type littermates. In the pyramidal cell layer of the hippocampal CA3 region, where AE3 is strongly expressed, disruption of AE3 abolished sodium-independent chloride-bicarbonate exchange. These findings strongly support the hypothesis that AE3 modulates seizure susceptibility and, therefore, are of significance for understanding the role of intracellular pH in epilepsy.

Animals↗

PubMed2005

Retinal degeneration associated with RDH12 mutations results from decreased 11-cis retinal synthesis due to disruption of the visual cycle.

Retinoid dehydrogenases/reductases catalyze key oxidation-reduction reactions in the visual cycle that converts vitamin A to 11-cis retinal, the chromophore of the rod and cone photoreceptors. It has recently been shown that mutations in RDH12, encoding a retinol dehydrogenase, result in severe and early-onset autosomal recessive retinal dystrophy (arRD). In a cohort of 1011 individuals diagnosed with arRD, we have now identified 20 different disease-associated RDH12 mutations, of which 16 are novel, in a total of 22 individuals (2.2%). Haplotype analysis suggested a founder mutation for each of the three common mutations: p.L99I, p.T155I and c.806_810delCCCTG. Patients typically presented with early disease that affected the function of both rods and cones and progressed to legal blindness in early adulthood. Eleven of the missense variants identified in our study exhibited profound loss of catalytic activity when expressed in transiently transfected COS-7 cells and assayed for ability to convert all-trans retinal to all-trans retinol. Loss-of-function appeared to result from decreased protein stability, as expression levels were significantly reduced. For the p.T49M variant, differing activity profiles were associated with each of the alleles of the common p.R161Q RDH12 polymorphism, suggesting that genetic background may act as a modifier of mutation effect. A locus (LCA3) for Leber congenital amaurosis, a severe, early-onset form of arRD, maps close to RDH12 on chromosome 14q24. Haplotype analysis in the family in which LCA3 was mapped excluded RDH12 as the LCA3 gene and thus suggests the presence of a novel arRD gene in this region.

Alcohol Oxidoreductases↗

PubMed2005

Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses.

The development of GABAergic synapses is associated with an excitatory to inhibitory shift of the actions of GABA because of a reduction of [Cl-]i. This is due to a delayed postnatal expression of the K+ -Cl- cotransporter KCC2, which has low levels at birth and peaks during the first few postnatal weeks. Whether the expression of the cotransporter and the excitatory to inhibitory shift have other consequences on the operation of GABA(A) receptors and synapses is not yet known. We have now expressed KCC2 in immature neurones at an early developmental stage and determined the consequences on the formation of GABA and glutamate synapses. We report that early expression of the cotransporter selectively enhances GABAergic synapses: there is a significant increase of the density of GABA(A) receptors and synapses and an increase of the frequency of GABAergic miniature postsynaptic currents. The density of glutamate synapses and frequency of AMPA miniature postsynaptic currents are not affected. We conclude that the expression of KCC2 and the reduction of [Cl-]i play a critical role in the construction of GABAergic networks that extends beyond the excitatory to inhibitory shift of the actions of GABA.

Action Potentials↗

PubMed2005

Seventeen novel PLP1 mutations in patients with Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a rare X-chromosomal neurodegenerative disorder that affects primarily the white matter of the central nervous system and is caused by mutations of the PLP1 (proteolipid protein 1) gene. We performed mutation analysis of 133 male patients with suspected PMD. Following SSCP analysis of all coding exons of PLP1, we found most likely pathogenic mutations (single base substitutions and small rearrangements) including 17 novel sequence variants in 21 (15.8%) patients. Most patients with missense mutations had a severe phenotype. Twelve patients (9.0%) carried a duplication of the entire gene, as demonstrated by quantitative real-time PCR, and presented with a variable clinical phenotype including mild, classical, and severe courses of disease. Two patients had large deletions, spanning approximately 115 kb, that included the PLP1 gene. In total, we identified pathogenic mutations involving PLP1 in 35 (26.3%) of the 133 patients analyzed.

Amino Acid Substitution↗

PubMed2004

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte.

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

Animals↗

PubMed2004

Mutations in RDH12 encoding a photoreceptor cell retinol dehydrogenase cause childhood-onset severe retinal dystrophy.

We identified three consanguineous Austrian kindreds with 15 members affected by autosomal recessive childhood-onset severe retinal dystrophy, a genetically heterogeneous group of disorders characterized by degeneration of the photoreceptor cells. A whole-genome scan by microarray analysis of single-nucleotide polymorphisms (ref. 2) identified a founder haplotype and defined a critical interval of 1.53 cM on chromosome 14q23.3-q24.1 that contains the gene associated with this form of retinal dystrophy. RDH12 maps in this region and encodes a retinol dehydrogenase proposed to function in the visual cycle. A homozygous 677A-->G transition (resulting in Y226C) in RDH12 was present in all affected family members studied, as well as in two Austrian individuals with sporadic retinal dystrophy. We identified additional mutations in RDH12 in 3 of 89 non-Austrian individuals with retinal dystrophy: a 5-nucleotide deletion (806delCCCTG) and the transition 565C-->T (resulting in Q189X), each in the homozygous state, and 146C-->T (resulting in T49M) and 184C-->T (resulting in R62X) in compound heterozygosity. When expressed in COS-7 cells, Cys226 and Met49 variants had diminished and aberrant activity, respectively, in interconverting isomers of retinol and retinal. The severe visual impairment of individuals with mutations in RDH12 is in marked contrast to the mild visual deficiency in individuals with fundus albipunctatus caused by mutations in RDH5, encoding another retinal dehydrogenase. Our studies show that RDH12 is associated with retinal dystrophy and encodes an enzyme with a unique, nonredundant role in the photoreceptor cells.

Adult↗

PubMed2004

Expression of the KCl cotransporter KCC2 parallels neuronal maturation and the emergence of low intracellular chloride.

Fast synaptic inhibition in the adult central nervous system (CNS) is mediated by GABA and glycine. During early development GABA acts as an excitatory neurotransmitter, which is deemed to be important for the maturation of the CNS. During development GABAergic responses undergo a switch from excitatory to inhibitory. This switch is correlated with upregulation of KCC2, the neuronal isoform of the potassium-chloride cotransporter family. KCC2 lowers the intraneuronal chloride concentration below its electrochemical equilibrium. KCC2 activity is thought to depend on phosphorylation by endogenous tyrosine kinases. Here, we analyzed the expression pattern of KCC2 during murine embryonic and postnatal development by in situ hybridization and Western blot analysis. KCC2 expression paralleled neuronal differentiation and preceded the decline of the GABA reversal potential (EGABA) in spinal cord motoneurons and hippocampal pyramidal cells. The adult inhibitory response to GABA was established earlier in the spinal cord than in the hippocampus. Phosphorylated KCC2 protein was already present early in development when the functional GABA switch had not yet occurred. Thus, tyrosine-phosphorylation seems to be less important than the transcriptional upregulation of KCC2.

Animals↗

PubMed2004

Intronic mutations in the L1CAM gene may cause X-linked hydrocephalus by aberrant splicing.

L1 disease is a clinically heterogeneous X-chromosomal neurodevelopmental disorder that is frequently associated with mental retardation and congenital hydrocephalus in males. It is caused by mutations in L1CAM that encodes a multifunctional transmembrane neuronal cell adhesion molecule. We report our findings on 6 novel intronic L1CAM sequence variants (c.523+5G>A, c.1123+1G>A, c.1547-13delC, c.3323-17dupG, c.3457+3A>T, and c.3457+18C>T), and a recurrent one (c.523+12C>T). While the pathogenic potential of nucleotide changes within the evolutionarily well-conserved splice consensus sequence (c.523+5G>A, c.1123+1G>A, and c.3457+3A>T) is widely accepted, it is not always straight forward to assess the disease relevance of intronic mutations, if they lie outside the consensus. The c.523+12C>T variant co-segregated with X-linked hydrocephalus in two unrelated families. In the mutated allele, a preferentially used novel splice donor site is generated that results in a frame shift due to insertion of the first 10 bp of intron 5 in the mature mRNA, a largely truncated protein, and most likely a functional null allele. The c.1547-13delC mutation creates a new acceptor site resulting in the insertion of 4 additional amino acids at the end of the immunoglobulin like domain 5. In contrast, c.3323-17dupG and c.3457+18C>T seem to be non-pathogenic L1CAM variants.

Base Sequence↗

PubMed2004

Ion channels: function unravelled by dysfunction.

Ion channels allow the passage of specific ions and electrical charge. Plasma membrane channels are, for example, important for electrical excitability and transepithelial transport, whereas intracellular channels have roles in acidifying endosomes or in releasing Ca(2+) from stores. The function of several channels emerged from mutations in humans or mice. The resulting phenotypes include kidney stones resulting from impaired endocytosis, hypertension, defective insulin secretion, cardiac arrhythmias, neurological diseases like epilepsy or deafness and even 'developmental' defects such as osteopetrosis.

Animals↗

PubMed2004

The three sorCS genes are differentially expressed and regulated by synaptic activity.

We have isolated the murine sorCS3 gene, a new member of the family of receptors containing a Vps10p-domain. Receptors of this family facilitate rapid endocytosis and are thought to be involved in intracellular sorting. SorCS3 and the highly homologous sorCS1 and sorCS2 genes were expressed in a combinatorial, mostly non-overlapping pattern in both the developing and mature central nervous system. During development, distribution and abundancy of their transcripts was regulated. Moreover, their expression was differentially influenced by neuronal activity in the hippocampus of adult mice. Although kainic acid-induced seizures had no effect on sorCS2 mRNA levels, they dramatically increased the expression of sorCS1 and sorCS3. The activity-dependent induction of sorCS1 expression required de novo protein synthesis, whereas that of sorCS3 did not. Our results imply that the three sorCS genes have diverse, but partly overlapping functions in the developing and mature central nervous system.

Animals↗

PubMed2003

Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold.

K-Cl co-transporters are encoded by four homologous genes and may have roles in transepithelial transport and in the regulation of cell volume and cytoplasmic chloride. KCC3, an isoform mutated in the human Anderman syndrome, is expressed in brain, epithelia and other tissues. To investigate the physiological functions of KCC3, we disrupted its gene in mice. This severely impaired cell volume regulation as assessed in renal tubules and neurons, and moderately raised intraneuronal Cl(-) concentration. Kcc3(-/-) mice showed severe motor abnormalities correlating with a progressive neurodegeneration in the peripheral and CNS. Although no spontaneous seizures were observed, Kcc3(-/-) mice displayed reduced seizure threshold and spike-wave complexes on electrocorticograms. These resembled EEG abnormalities in patients with Anderman syndrome. Kcc3(-/-) mice also displayed arterial hypertension and a slowly progressive deafness. KCC3 was expressed in many, but not all cells of the inner ear K(+) recycling pathway. These cells slowly degenerated, as did sensory hair cells. The present mouse model has revealed important cellular and systemic functions of KCC3 and is highly relevant for Anderman syndrome.

Animals↗