Fabian Runkel

Morphologic and molecular characterization of two novel Krt71 (Krt2-6g) mutations: Krt71rco12 and Krt71rco13

We have analyzed two novel mouse mutant strains, Rco12 and Rco13, displaying a wavy pelage and curly vibrissae that have been identified in an ENU screen for dominant mutations affecting the pelage. The mutations were mapped to mouse Chromosome 15 and identified as missense point mutations in the first exon of the Krt71 (formerly called Krt2-6g) gene causing alterations of amino acid residue 143 from alanine to glycine (Rco12) and residue 146 from isoleucine to phenylalanine. The morphologic analyses demonstrated that both mutations cause identical phenotypes leading to the formation of filamentous aggregates in Henle’s and Huxley’s layers of the inner root sheath (IRS) of the hair follicle that leads to the bending of the hair shaft. Both novel mutations are located in the immediate vicinity of previously identified mutations in murine Krt71 that cause similar phenotypes and alter the helix initiation motif of the keratin. The characterization of these mutants demonstrates the importance of this Krt71 domain for the formation of linear IRS intermediate filaments.

Promoter-like sequences regulating transcriptional activity in neurexin and neuroligin genes.

Synapse function requires the cell‐adhesion molecules neurexins (Nrxn) and neuroligins (Nlgn). Although these molecules are essential for neurotransmission and prefer distinct isoform combinations for interaction, little is known about their transcriptional regulation. Here, we started to explore this important aspect because expression of Nrxn1‐3 and Nlgn1‐3 genes is altered in mice lacking the transcriptional regulator methyl‐CpG‐binding protein2 (MeCP2). Since MeCP2 can bind to methylated CpG‐dinucleotides and Nrxn/Nlgn contain CpG‐islands, we tested genomic sequences for transcriptional activity in reporter gene assays. We found that their influence on transcription are differentially activating or inhibiting. As we observed an activity difference between heterologous and neuronal cell lines for distinct Nrxn1 and Nlgn2 sequences, we dissected their putative promoter regions. In both genes, we identify regions in exon1 that can induce transcription, in addition to the alternative transcriptional start points in exon2. While the 5′‐regions of Nrxn1 and Nlgn2 contain two CpG‐rich elements that show distinct methylation frequency and binding to MeCP2, other regions may act independently of this transcriptional regulator. These data provide first insights into regulatory sequences of Nrxn and Nlgn genes that may represent an important aspect of their function at synapses in health and disease.

Grey, a novel mutation in the murine Lyst gene, causes the beige phenotype by skipping of exon 25.

The murine beige mutant phenotype and the human Chediak-Higashi syndrome are caused by mutations in the murine Lyst (lysosomal trafficking regulator) gene and the human CHS gene, respectively. In this report we have analyzed a novel murine mutant Lyst allele, called Lystbg-grey, that had been found in an ENU mutation screen and named grey because of the grey coat color of affected mice. The phenotype caused by the Lystbg-grey mutation was inherited in a recessive fashion. Melanosomes of melanocytes associated with hair follicles and the choroid layer of the eye, as well as melanosomes in the neural tube-derived pigment epithelium of the retina, were larger and irregularly shaped in homozygous mutants compared with those of wild-type controls. Secretory vesicles in dermal mast cells of the mutant skin were enlarged as well. Test crosses with beige homozygous mutant mice (Lystbg) showed that double heterozygotes (Lystbg/Lystbg-grey) were phenotypically indistinguishable from either homozygous parent, demonstrating that the ENU mutation was an allele of the murine Lyst gene. RT-PCR analyses revealed the skipping of exon 25 in Lystbg-grey mutants, which is predicted to cause a missense D2399E mutation and the loss of the following 77 amino acids encoded by exon 25 but leave the C-terminal end of the protein intact. Analysis of the genomic Lyst locus around exon 25 showed that the splice donor at the end of exon 25 showed a T-to-C transition point mutation. Western blot analysis suggests that the Lystbg-grey mutation causes instability of the LYST protein. Because the phenotype of Lystbg and Lystbg-grey mutants is indistinguishable, at least with respect to melanosomes and secretory granules in mast cells, the Lystbg-grey mutation defines a critical region for the stability of the murine LYST protein.

Functional significance of a highly conserved glutamate residue of the human noradrenaline transporter

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+‐ and Cl–‐dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site‐directed mutagenesis and the mutants were expressed in transfected COS‐7 or HEK‐293 cells. Cell surface expression of hE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in Vmax values for noradrenaline and dopamine uptake of 2–3‐fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82–260‐fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.

Alopecia and male infertility in oligotriche mutant mice are caused by a deletion on distal chromosome 9

The recessive mutation oligotriche (olt) affects the coat and male fertility in the mouse. In homozygous (olt/olt) mutants, the coat is sparse, most notably in the inguinal and medial femoral region. In these regions, almost all hair shafts are bent and distorted in their course through the dermis and rarely penetrate the epidermis because the hair cortex is not fully keratinized. During hair follicle morphogenesis, mutant hair follicles exit from anagen one day before those of normal littermates and show a prolongation of the catagen stage. The oligotriche (olt) locus was mapped to distal chromosome 9 within a 5-Mbp interval distal to D9Mit279. Analysis of candidate gene expression revealed that olt/olt mutant mice do not express functional phospholipase C delta 1 (Plcd1) mRNA. This deficiency is the consequence of a 234-kbp deletion involving not only the Plcd1 locus but also the chromosomal segment harboring the genes Vill (villin-like), Dlec1 (deleted in lung and esophageal cancer 1), Acaa1b (acetyl-Coenzyme A acyltransferase 1B, synonym thiolase B), and parts of the genes Ctdspl (carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase-like) and Slc22a14 (solute carrier family 22 member 14). Offspring of olt/olt females, mated with Plcd1−/− knockout males, exhibit coat defects similar to those observed in homozygous olt/olt mutant mice but the spermiogenesis in male offspring is normal. We conclude that the 234-kbp deletion from chromosome 9 harbors a gene involved in spermiogenesis and we propose that the oligotriche mutant be used as a model for the study of the putative tumor suppressor genes Dlec1, Ctdspl, and Vill. We also suggest that the oligotriche locus be named Del(9Ctdspl-Slc22a14)1Pas.

Fxyd3 and Lgi4 expression in the adult mouse: a case of endogenous antisense expression.

We have investigated the expression of Fxyd3 and Lgi4 in the adult mouse by Northern blot analyses and in situ hybridization. Murine Fxyd3 and Lgi4 have been mapped to the same locus on mouse Chromosome (Chr) 7, where the last exon of Fxyd3 completely overlaps with the 3’UTR in the last exon of Lgi4, which is transcribed in the opposite orientation. The Fxyd3 gene (formerly called Mat-8) encodes an 8-kDa transmembrane protein that is upregulated in mammary tumors and can induce a chloride conductance upon RNA injection into Xenopus oocytes. Fxyd3 is a member of the Fxyd family of which several members are tissue-specific regulators of ion channels. Murine Lgi4 is a recently described member of the leucine-rich-repeat gene family Lgi. Northern blot analyses demonstrated a 0.6-kb Fxyd3 transcript with abundant expression in the murine skin, colon, and mammary gland, but low level expression in the brain. In contrast, a 3.2-kb Lgi4 transcript was abundant in brain, with lower level expression in colon. Lgi4 transcription in the skin was detectable only by RT-PCR. A Fxyd3-specific sense cRNA probe hybridized to a transcript in Northern blots of brain and colon RNA that co-migrated with the Lgi4 mRNA. In situ hybridization experiments revealed that both Fxyd3 and Lgi4 were expressed in the same tissue compartments in skin, uterus, intestine, mammary gland, and brain. These results demonstrate that Fxyd3 and Lgi4 transcripts potentially form double-stranded RNA molecules in many cell types in vivo, which may impact on their respective expression.

Alopecia in a Viable Phospholipase C Delta 1 and Phospholipase C Delta 3 Double Mutant

Background Inositol 1,4,5trisphosphate (IP3) and diacylglycerol (DAG) are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD) forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia), whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. Methodology/Principal Findings We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3mNab) that resulted from the insertion of an intracisternal A particle (IAP) into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3mNab alleles are phenotypically normal. However, the presence of one Plcd3mNab allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9)olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9)olt1Pas and the Plcd3mNab mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. Conclusions/Significance The Plcd3mNab mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface.

Mutants and Variants of the Human Antidepressant-Sensitive Norepinephrine Transporter

The norepinephrine transporter (NET) is responsible for the rapid re-uptake of synaptically released NE. Substrate transport by neurotransmitter transporters (NTTs) is principially posible in both directions. In Na+- dependent transporters the normal transport direction is given by the Na+ gradient mainted by Na+/K+-ATPase. The ions Na+ and Cl- are cosubstrates of the NET and both ions are also needed for substrate (e.g., NE) transport and binding of NET inhibitors such as nisoxetine or the tricyclic antidepressant desipramine (DMI). In transfected cells overexpressing the NET, a channel-like mode of the NET has also been described.1 The NET transports the catecholamines NE, DA and epinephrine and amines like amphetamine, and the NET is a primary target of cocaine and of clinically important antidepressants (such as DMI, doxepine and reboxetine). The NET is not only expressed in noradrenergic neurons of the central and peripheral nervous system and in adrenal medullary cells but also in the placenta and in endothelial cells of small blood vessels of the lung where the NET is involved in the inactivation of circulating catecholamines.2