Barbera Veldhuisen

PKD2, a Gene for Polycystic Kidney Disease That Encodes an Integral Membrane Protein

A second gene for autosomal dominant polycystic kidney disease was identified by positional cloning. Nonsense mutations in this gene (PKD2) segregated with the disease in three PKD2 families. The predicted 968-amino acid sequence of the PKD2 gene product has six transmembrane spans with intracellular amino- and carboxyl-termini. The PKD2 protein has amino acid similarity with PKD1, the Caenorhabditis elegans homolog of PKD1, and the family of voltage-activated calcium (and sodium) channels, and it contains a potential calcium-binding domain.

A spectrum of mutations in the second gene for autosomal dominant polycystic kidney disease (PKD2).

Recently the second gene for autosomal dominant polycystic kidney disease (ADPKD), located on chromosome 4q21-q22, has been cloned and characterized. The gene encodes an integral membrane protein, polycystin-2, that shows amino acid similarity to the PKD1 gene product and to the family of voltage-activated calcium (and sodium) channels. We have systematically screened the gene for mutations by single-strand conformation-polymorphism analysis in 35 families with the second type of ADPKD and have identified 20 mutations. So far, most mutations found seem to be unique and occur throughout the gene, without any evidence of clustering. In addition to small deletions, insertions, and substitutions leading to premature translation stops, one amino acid substitution and five possible splice-site mutations have been found. These findings suggest that the first step toward cyst formation in PKD2 patients is the loss of one functional copy of polycystin-2.

Alpha-1 antitrypsin Null mutations and severity of emphysema

BACKGROUND Alpha-1 antitrypsin (AAT) deficiency is an autosomal-codominant disorder, caused by mutations in the SERPINA1 gene on chromosome 14. Individuals affected by the most common mutations, SZ and ZZ, have serum AAT concentrations of 25% and 15% of normal levels, and present a higher risk of emphysema. Mutations causing total absence of serum AAT (Null mutations) were suggested to be associated with very early onset emphysema but their clinical phenotype is poorly known. HYPOTHESIS Absence of AAT in Null mutations results in more severe emphysema as compared to ZZ and SZ. METHODS We genotyped all known Dutch subjects (n=12) with absent serum AAT, and compared their lung function values (FEV1 and KCO) with those of individuals with ZZ and SZ genotype, matched for age and smoking history. RESULTS All subjects with absent serum AAT presented homozygous Null mutations. In three subjects, a new mutation in exon 2 of the SERPINA1 gene was found. Subjects with Null mutations showed significantly lower lung function values than SZ and ZZ individuals (p=0.000 and 0.001 for FEV1 and KCO, respectively). In all groups, there was a positive correlation between serum AAT and lung function values (p=0.025 and 0.014 for FEV1 and KCO, respectively). CONCLUSIONS Serum levels of AAT are correlated with the severity of pulmonary phenotype. Subjects with Null mutations should be considered a subgroup at particularly high risk of emphysema within AAT deficiency (AATD). Early detection of carriers of this genotype would be important for preventive and therapeutic interventions.

Multiple transcripts generated by the DCAMKL gene are expressed in the rat hippocampus

We have recently cloned a novel Doublecortin CaMK-like kinase (rDCAMKL) cDNA, and a related cDNA called CaMK-related peptide (CARP) from the rat hippocampus. These genes are structurally highly similar to the human DCAMKL-1 gene and doublecortin, a gene associated with X-linked lissencephaly and subcortical band heterotopia. Here we report on the genomic organization of the murine DCAMKL gene and its products. Our results show that DCAMKL and CARP are alternative splice products of the same gene. The DCAMKL gene also generates three alternatively-spliced rDCAMKL transcripts of which we have cloned the corresponding cDNAs and which potentially generate different DCAMKL proteins. In situ hybridization experiments show that the different rDCAMKL transcripts are all expressed in the adult rat hippocampus. We conclude that alternative splicing of the DCAMKL gene may generate different but similar proteins in the adult rat hippocampus thereby regulating different but overlapping aspects of DCAMKL controlled neuronal plasticity.

Hippocampal CARP over-expression solidifies consolidation of contextual fear memories

The Doublecortin-Like Kinase (DCLK) gene is involved in neuronal migration during development. Through alternative splicing the DCLK gene also produces a transcript called Ca(2+)/calmodulin dependent protein kinase (CaMK)-related peptide (CARP) that is expressed exclusively during adulthood in response to neuronal activity. The function of CARP, however, is poorly understood. To study CARP function, we have generated transgenic mice with over-expression of the CARP transcript in, amongst other brain areas, the hippocampus. We aimed to characterize possible behavioral adaptations of these mice by using a Pavlovian fear conditioning approach. This type of fear conditioning, in which both the hippocampus and amygdala are critically involved, allows studying the formation and extinction of fear related memories. We here report on the behavioral adaptations of two distinct transgenic lines: one with high levels of CARP in the hippocampus and amygdala, whilst the other has high levels of CARP in the hippocampal formation, but not in the amygdala. We tested both mouse lines separately by comparing them to their wild-type littermate controls. We provide evidence suggesting consolidation of contextual fear memories is strengthened in mice of both transgenic lines.

Over-expression of δC-DCLK-short in mouse brain results in a more anxious behavioral phenotype

Products of the Doublecortin-Like Kinase (DCLK) gene are associated with cortical migration and hippocampal maturation during embryogenesis. However, the functions of those DCLK gene transcripts that encode kinases and are expressed during adulthood are incompletely understood. To elucidate potential functions of these DCLK gene splice variants we have generated and analyzed transgenic mice with neuronal over-expression of a truncated, constitutively active form of DCLK-short, designated δC-DCLK-short. Previously, we have performed an extensive molecular characterization of these transgenic δC-DCLK-short mice and established that a specific subunit of the GABA(A) receptor, which is involved in anxiety-related GABAergic neurotransmission, is down-regulated in the hippocampus. Here we show that δC-DCLK-short mRNA is highly expressed in the hippocampus, cortex and amygdala of transgenic mice. We provide evidence that the δC-DCLK-short protein is expressed and functional. In addition, we examined anxiety-related behavior in δC-DCLK-short mice in the elevated plus maze. Interestingly, δC-DCLK-short mice spend less time, move less in the open arms of the maze and show a reduction in the number of rim dips. These behaviors indicate that δC-DCLK-short mice display a more anxious behavioral phenotype.

Over-expression of the DCLK gene transcript CARP decreases CA3/CA1 network excitability

Products of the Doublecortin Like Kinase (DCLK) gene are implicated in cortical migration and hippocampal maturation during embryogenesis. However, one of its splice variants, called CaMK Related Peptide (CARP), is expressed during adulthood in response to neurological stimuli, such as kainic acid-induced seizures and BDNF-LTP. The function of this transcript of the DCLK gene is poorly understood. To elucidate its function during adulthood we have created transgenic mice with over-expression of CARP in the brain. To study potential functions of CARP in the hippocampus we performed an electrophysiological characterization of the CA3/CA1 network of transgenic and wild-type mice and showed that field excitatory post synaptic potentials (fEPSPs) are highly increased in transgenic mice, while population spike amplitudes (PSAs) remained equal between genotypes. Consequently, hippocampal CA3/CA1 network excitability was decreased in transgenic mice. In addition we show a 2-fold up-regulation of the Ca(2+)-binding protein calretinin and a down-regulation of Rapgef4, a guanine exchange factor for Rap1, in the hippocampus. Given previously established conditions during which CARP is induced and our current data, we propose that this DCLK gene product affects glutamatergic neuronal transmission in response to neurological stimuli.