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.

A dominant vimentin mutant upregulates Hsp70 and the activity of the ubiquitin-proteasome system, and causes posterior cataracts in transgenic mice.

Vimentin is the main intermediate filament (IF) protein of mesenchymal cells and tissues. Unlike other IF-/- mice, vimentin-/- mice provided no evidence of an involvement of vimentin in the development of a specific disease. Therefore, we generated two transgenic mouse lines, one with a (R113C) point mutation in the IF-consensus motif in coil1A and one with the complete deletion of coil 2B of the rod domain. In epidermal keratins and desmin, point mutations in these parts of the alpha-helical rod domain cause keratinopathies and desminopathies, respectively. Here, we demonstrate that substoichiometric amounts of vimentin carrying the R113C point mutation disrupted the endogenous vimentin network in all tissues examined but caused a disease phenotype only in the eye lens, leading to a posterior cataract that was paralleled by the formation of extensive protein aggregates in lens fibre cells. Unexpectedly, central, postmitotic fibres became depleted of aggregates, indicating that they were actively removed. In line with an increase in misfolded proteins, the amounts of Hsp70 and ubiquitylated vimentin were increased, and proteasome activity was raised. We demonstrate here for the first time that the expression of mutated vimentin induces a protein-stress response that contributes to disease pathology in mice, and hypothesise that vimentin mutations cause cataracts in humans.

Absence of 2-hydroxylated sphingolipids is compatible with normal neural development but causes late-onset axon and myelin sheath degeneration.

Sphingolipids containing 2-hydroxylated fatty acids are among the most abundant lipid components of the myelin sheath and therefore are thought to play an important role in formation and function of myelin. To prove this hypothesis, we generated mice lacking a functional fatty acid 2-hydroxylase (FA2H) gene. FA2H-deficient (FA2H(-/-)) mice lacked 2-hydroxylated sphingolipids in the brain and in peripheral nerves. In contrast, nonhydroxylated galactosylceramide was increased in FA2H(-/-) mice. However, oligodendrocyte differentiation examined by in situ hybridization with cRNA probes for proteolipid protein and PDGFalpha receptor and the time course of myelin formation were not altered in FA2H(-/-) mice compared with wild-type littermates. Nerve conduction velocity measurements of sciatic nerves revealed no significant differences between FA2H(-/-) and wild-type mice. Moreover, myelin of FA2H(-/-) mice up to 5 months of age appeared normal at the ultrastructural level, in the CNS and peripheral nervous system. Myelin thickness and g-ratios were normal in FA2H(-/-) mice. Aged (18-month-old) FA2H(-/-) mice, however, exhibited scattered axonal and myelin sheath degeneration in the spinal cord and an even more pronounced loss of stainability of myelin sheaths in sciatic nerves. These results show that structurally and functionally normal myelin can be formed in the absence of 2-hydroxylated sphingolipids but that its long-term maintenance is strikingly impaired. Because axon degeneration appear to start rather early with respect to myelin degenerations, these lipids might be required for glial support of axon function.

Down-regulation of polysialic acid is required for efficient myelin formation.

Oligodendrocyte precursor cells modify the neural cell adhesion molecule (NCAM) by the attachment of polysialic acid (PSA). Upon further differentiation into mature myelinating oligodendrocytes, however, oligodendrocyte precursor cells down-regulate PSA synthesis. In order to address the question of whether this down-regulation is a necessary prerequisite for the myelination process, transgenic mice expressing the polysialyltransferase ST8SiaIV under the control of the proteolipid protein promoter were generated. In these mice, postnatal down-regulation of PSA in oligodendrocytes was abolished. Most NCAM-120, the characteristic NCAM isoform in oligodendrocytes, carried PSA in the transgenic mice at all stages of postnatal development. Polysialylated NCAM-120 partially co-localized with myelin basic protein and was present in purified myelin. The permanent expression of PSA-NCAM in oligodendrocytes led to a reduced myelin content in the forebrains of transgenic mice during the period of active myelination and in the adult animal. In situ hybridizations indicated a significant decrease in the number of mature oligodendrocytes in the forebrain. Thus, down-regulation of PSA during oligodendrocyte differentiation is a prerequisite for efficient myelination by mature oligodendrocytes. Furthermore, myelin of transgenic mice exhibited structural abnormalities like redundant myelin and axonal degeneration, indicating that the down-regulation of PSA is also necessary for myelin maintenance.

A spontaneously immortalized Schwann cell line to study the molecular aspects of metachromatic leukodystrophy.

The arylsulfatase A (ASA)-deficient mouse is a murine model of human metachromatic leukodystrophy (MLD) caused by a genetic defect in the ASA gene. Deficiency of ASA causes accumulation of cerebroside-3-sulfate (sulfatide) in visceral organs and in the central and peripheral nervous system, which subsequently causes demyelination in these areas. To investigate further the cellular pathomechanism of MLD, we established spontaneously immortalized Schwann cell lines from ASA-deficient mice. Cells showed marked sulfatide storage in the late endosomal/lysosomal compartment. This sulfatide accumulation can be further increased by external treatment with sulfatide using a lipid based transfection reagent as a cargo. The accumulated sulfatide was degraded in response to ASA treatment and first examination revealed that alteration on the molecular level found in ASA-deficient mice can also be observed in the presented cell culture model. Hence, these cells could be a suitable model to study MLD at a molecular level.

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.

Keratin 5 knockout mice reveal plasticity of keratin expression in the corneal epithelium.

We have recently demonstrated that the keratin K3 gene, which is active in the suprabasal human corneal epithelium, is missing in the genome of the mouse. We show that a normal K3 gene exists in a wide variety of mammals while in rodents the gene is converted to a pseudogene with a very strong sequence drift. The availability of K5-/- mice provides a unique opportunity to investigate type-specific keratin function during corneal differentiation in the absence of both K5 and K3. Here, we report that the deletion of K5, which in wild-type mice forms a cytoskeleton with K12, does neither cause keratin aggregation nor cytolysis in the cornea. This is due to the induction of K4 in corneal epithelial cells, normally restricted to corneal stem stem cells residing in the limbus. Using a combination of antibodies and RT-PCR, we identified additional keratins expressed in the mouse cornea including K23 which was previously thought to be specific for pancreatic carcinomas. This reflects an unexpected complexity of keratin expression in the cornea. Our data suggest that in the absence of mechanical stress, corneal differentiation does not depend on distinct keratin pairs, supporting a concept of functional redundancy, at least for certain keratins.

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 Lyst(bg-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 Lyst(bg-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 (Lyst(bg)) showed that double heterozygotes (Lyst(bg)/Lyst(bg-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 Lyst(bg-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 Lyst(bg-grey) mutation causes instability of the LYST protein. Because the phenotype of Lyst(bg) and Lyst(bg-grey) mutants is indistinguishable, at least with respect to melanosomes and secretory granules in mast cells, the Lyst(bg-grey) mutation defines a critical region for the stability of the murine LYST protein.

Loss-of-function mutations in the keratin 5 gene lead to Dowling-Degos disease.

Dowling-Degos disease (DDD) is an autosomal dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation of the flexures. We performed a genomewide linkage analysis of two German families and mapped DDD to chromosome 12q, with a total LOD score of 4.42 ( theta =0.0) for marker D12S368. This region includes the keratin gene cluster, which we screened for mutations. We identified loss-of-function mutations in the keratin 5 gene (KRT5) in all affected family members and in six unrelated patients with DDD. These represent the first identified mutations that lead to haploinsufficiency in a keratin gene. The identification of loss-of-function mutations, along with the results from additional functional studies, suggest a crucial role for keratins in the organization of cell adhesion, melanosome uptake, organelle transport, and nuclear anchorage.

Expression pattern and functional characterization of connexin29 in transgenic mice.

Using newly generated transgenic mice in which the coding region of the connexin29 (Cx29) gene was replaced by the lacZ reporter gene, we confirmed previous immunochemical results that Cx29 is expressed in Schwann cells, oligodendrocytes and Bergmann glia cells. In addition, we detected lacZ/Cx29 in Schwann cells of the sciatic nerve and in particular of the spiral ganglion in the inner ear, as well as at low abundance in the stria vascularis. Furthermore, we found lacZ/Cx29 expression in nonmyelinating Schwann cells of the adrenal gland, in chondrocytes of intervertebral discs and the epiphysis of developing bones. Electron microscopic analyses of myelin sheaths in the central and peripheral nervous system of Cx29-deficient mice detected no abnormalities. The nerve conduction in the sciatic nerve of adult Cx29-deficient mice and the auditory brain stem response as well as visually evoked potentials in 4- to 10-week-old Cx29-deficient mice were not different from wild-type littermate controls. Thus, in contrast to connexin32 and connexin47, which are also expressed in myelinating cells, Cx29 does not contribute to the function of myelin in adult mice.

Reversal of non-hydroxy:alpha-hydroxy galactosylceramide ratio and unstable myelin in transgenic mice overexpressing UDP-galactose:ceramide galactosyltransferase.

The sphingolipids galactosylceramide and sulfatide are important for the formation and maintenance of myelin. Transgenic mice overexpressing the galactosylceramide synthesizing enzyme UDP-galactose:ceramide galactosyltransferase in oligodendrocytes display an up to four-fold increase in UDP-galactose:ceramide galactosyltransferase activity, which correlates with an increase in its products monogalactosyl diglyceride and non-hydroxy fatty acid-containing galactosylceramide. Surprisingly, however, we observed a concomitant decrease in alpha-hydroxylated galactosylceramide such that total galactosylceramide in transgenic mice was almost unaltered. These data suggest that UDP-galactose:ceramide galactosyltransferase activity does not limit total galactosylceramide level. Furthermore, the predominance of alpha-hydroxylated galactosylceramide appeared to be determined by the extent to which non-hydroxylated ceramide was galactosylated rather than by the higher affinity of UDP-galactose:ceramide galactosyltransferase for alpha-hydroxy fatty acid ceramide. The protein composition of myelin was unchanged with the exception of significant up-regulation of the myelin and lymphocyte protein. Transgenic mice were able to form myelin, which, however, was apparently unstable and uncompacted. These mice developed a progressive hindlimb paralysis and demyelination in the CNS, demonstrating that tight control of UDP-galactose:ceramide galactosyltransferase expression is essential for myelin maintenance.

Oligodendrocyte-specific ceramide galactosyltransferase (CGT) expression phenotypically rescues CGT-deficient mice and demonstrates that CGT activity does not limit brain galactosylceramide level.

Galactosylceramide (GalC) is the major sphingolipid of the myelin membrane. Mice lacking GalC due to ceramide galactosyltransferase (CGT) deficiency form unstable and functionally affected myelin and exhibit a progressive demyelination, accompanied by severe motor coordination deficits. In addition to oligodendrocytes, CGT is also expressed in other cells, e.g., neurons and astrocytes. We examined the possibility that lack of CGT in these cells contributes to the phenotype of CGT-deficient mice. Toward this aim, we generated transgenic mice expressing CGT under the control of oligodendrocyte-specific proteolipid protein (PLP) promoter and examined the possibility of a transgenic rescue of CGT-deficient mice. CGT-deficient mice expressing the PLP-CGT transgene did not show any behavioral abnormalities, normal myelin structure, and MBP levels. CGT activity as well as GalC and sulfatide levels of rescued mice were not significantly different from wild-type controls. Thus, transgenic rescue with the PLP-CGT transgene was apparently complete. In contrast to wild-type and rescued mice, PLP-CGT transgenic mice on a wild-type background exhibited significantly elevated CGT activity which directly correlated with an increase in non-hydroxy fatty acid (NFA)-GalC, but not alpha-hydroxy fatty acid (HFA)-GalC. HFA-GalC decreased in adult transgenic mice, indicating that NFA-GalC, but not HFA-GalC levels are limited by CGT activity. As a consequence, the total amount of GalC is unchanged over a rather wide range of CGT expression levels in the mouse brain. Our results indicate that loss of CGT in oligodendrocytes is exclusively responsible for the myelin structural deficits, demyelination, and behavioral abnormalities in CGT-deficient mice.

Missense mutations as a cause of metachromatic leukodystrophy. Degradation of arylsulfatase A in the endoplasmic reticulum.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). Biosynthesis studies of ASA with various structure-sensitive monoclonal antibodies reveal that some epitopes of the enzyme form within the first minutes of biosynthesis whereas other epitopes form later, between 10 and 25 min. When we investigated 12 various ASAs, with amino acid substitutions according to the missense mutations found in metachromatic leukodystrophy patients, immunoprecipitation with monoclonal antibodies revealed folding deficits in all 12 mutant ASA enzymes. Eleven of the 12 mutants show partial expression of the early epitopes, but only six of these show, in addition, incomplete expression of late epitopes. In none of the mutant enzymes were the late forming epitopes found in the absence of early epitopes. Thus, data from the wild-type and mutant enzymes indicate that the enzyme folds in a sequential manner and that the folding of early forming epitopes is a prerequisite for maturation of the late epitopes. All mutant enzymes in which the amino acid substitution prevents the expression of the late forming epitopes are retained in the endoplasmic reticulum (ER). In contrast, all mutants in which a single late epitope is at least partially expressed can leave the ER. Thus, irrespective of the missense mutation, the expression of epitopes forming late in biosynthesis correlates with the ability of the enzyme to leave the ER. The degradation of ER-retained enzymes can be reduced by inhibitors of the proteasome and ER alpha1,2-mannosidase I, indicating that all enzymes are degraded via the proteasome. Inhibition of degradation did not lead to an enhanced delivery from the ER for any of the mutant enzymes.

Exocytosis of storage material in a lysosomal disorder.

Lysosomal exocytosis is a ubiquitously occurring process, which has a physiological role in repair of wounds of the plasma membrane. Lysosomal storage disorders are a group of more than 40 different diseases, which are characterized by intralysosomal storage of various substances. Metachromatic leukodystrophy is a lysosomal disease caused by the deficiency of arylsulfatase A, which results in the storage of the sphingolipid 3-O-sulfogalactosylceramide (sulfatide) in, e.g., oligodendrocytes and distal tubule kidney cells. Here we show that sulfatide storing cultured primary kidney cells of arylsulfatase A deficient mice can undergo calcium induced lysosomal exocytosis and that this results in the delivery of storage material to the culture medium. In metachromatic leukodystrophy extracellular sulfatide has been found in urine and cerebrospinal fluid. Lysosomal exocytosis may explain the presence of sulfatide in these body fluids.

Functional integration of embryonic stem cell-derived neurons in vivo.

Pluripotency and the potential for continuous self-renewal make embryonic stem (ES) cells an attractive donor source for neuronal cell replacement. Despite recent encouraging results in this field, little is known about the functional integration of transplanted ES cell-derived neurons on the single-cell level. To address this issue, ES cell-derived neural precursors exhibiting neuron-specific enhanced green fluorescent protein (EGFP) expression were introduced into the developing brain. Donor cells implanted into the cerebral ventricles of embryonic rats migrated as single cells into a variety of brain regions, where they acquired complex morphologies and adopted excitatory and inhibitory neurotransmitter phenotypes. Synaptic integration was suggested by the expression of PSD-95 (postsynaptic density-95) on donor cell dendrites, which in turn were approached by multiple synaptophysin-positive host axon terminals. Ultrastructural and electrophysiological data confirmed the formation of synapses between host and donor cells. Ten to 21 d after birth, all EGFP-positive donor cells examined displayed active membrane properties and received glutamatergic and GABAergic synaptic input from host neurons. These data demonstrate that, at the single-cell level, grafted ES cell-derived neurons undergo morphological and functional integration into the host brain circuitry. Antibodies to the region-specific transcription factors Bf1, Dlx, En1, and Pax6 were used to explore whether functional donor cell integration depends on the acquisition of a regional phenotype. Our data show that incorporated neurons frequently exhibit a lacking or ectopic expression of these transcription factors. Thus, the lack of an appropriate regional "code" does not preclude morphological and synaptic integration of ES cell-derived neurons.

Functional expression of connexin57 in horizontal cells of the mouse retina.

Horizontal cells are interneurons of the vertebrate retina that exhibit strong electrical and tracer coupling but the identity of the channel-forming connexins has remained elusive. Here we show that horizontal cells of the mouse retina express connexin57 (Cx57). We have generated Cx57-deficient mice by replacing the Cx57 coding region with a lacZ reporter gene, expressed under control of the endogenous Cx57 promoter. These mice were fertile and showed no obvious anatomical or behavioural abnormalities. Cx57 mRNA was expressed in the retina of wild-type littermates but was absent from the retina of Cx57-deficient mice. Previously reported results that the Cx57 gene was very weakly expressed in several other mouse tissues turned out to be unspecific. Cx57 mRNA is abundantly expressed in the retina and weakly in the thymus of adult mice but absent in all other adult tissues tested, including brain. Furthermore, Cx57 is expressed in embryonic kidney at E16.5 to E18.5 days post-conception, as indicated by the pattern of lacZ expression. Within the retina, lacZ signals were assigned exclusively to horizontal cells based on co-localization with cell-type-specific marker proteins. Microinjection of Neurobiotin into horizontal cells of isolated retinae revealed less than 1% of tracer coupling in Cx57-deficient retinae compared with wild-type controls. Cx57 is the first connexin identified in mammalian horizontal cells and the first connexin whose expression is apparently restricted to only one type of neuron.

Connexin 47 (Cx47)-deficient mice with enhanced green fluorescent protein reporter gene reveal predominant oligodendrocytic expression of Cx47 and display vacuolized myelin in the CNS.

To further characterize the recently described gap junction gene connexin 47 (Cx47), we generated Cx47-null mice by replacing the Cx47 coding DNA with an enhanced green fluorescent protein (EGFP) reporter gene, which was thus placed under control of the endogenous Cx47 promoter. Homozygous mutant mice were fertile and showed no obvious morphological or behavioral abnormalities. Colocalization of EGFP fluorescence and immunofluorescence of cell marker proteins revealed that Cx47 was mainly expressed in oligodendrocytes in highly myelinated CNS tissues and in few calcium-binding protein S100beta subunit-positive cells but not in neurons or peripheral sciatic nerve. This corrects our previous conclusion that Cx47 mRNA is expressed in brain and spinal cord neurons (Teubner et al., 2001). Cx47 protein was detected by Western blot analysis after immunoprecipitation in CNS tissues of wild-type mice but not in heart or Cx47-deficient tissues. Electron microscopic analysis of CNS white matter in Cx47-deficient mice revealed a conspicuous vacuolation of nerve fibers, particularly at the site of the optic nerve where axons are first contacted by oligodendrocytes and myelination starts. Initial analyses of Cx32/Cx47-double-deficient mice showed that these mice developed an action tremor and died on average at 51 d after birth. The central white matter of these double-deficient mice exhibited much more abundant vacuolation in nerve fibers than mice deficient only in Cx47.

Oligodendrocytes expressing exclusively the DM20 isoform of the proteolipid protein gene: myelination and development.

Oligodendroglia and Schwann cells synthesize myelin-specific proteins and lipids for the assembly of the highly organized myelin membrane of the motor-sensory axons in the central (CNS) and peripheral nervous system (PNS), respectively, allowing rapid saltatory conduction. The isoforms of the main myelin proteins, the peripheral myelin basic isoproteins (MBP) and the integral proteolipid proteins, PLP and DM20, arise from alternative splicing. Activation of a cryptic splice site in exon III of plp leads to the deletion of 105 bp encoding the PLP-specific 35 amino acid residues within the cytosolic loop 3 of the four-transmembrane domain (TMD) integral membrane protein. To study the different proposed functions of DM20 during the development of oligodendrocytes and in myelination, we targeted the plp locus in embryonic stem cells by homologous recombination by a construct, which allows solely the expression of the DM20 specific exon III sequence. The resulting dm20(only) mouse line expresses exclusively DM20 isoprotein, which is functionally assembled into the membrane, forming a highly ordered and tightly compacted myelin sheath. The truncated cytosolic loop devoid of the PLP-specific 35 amino acid residues, including two thioester groups, had no impact on the periodicity of CNS myelin. In contrast to the PLP/DM20-deficient mouse, mutant CNS of dm20(only) mice showed no axonal swellings and neurodegeneration but a slow punctuated disintegration of the compact layers of the myelin sheath and a rare oligodendrocyte death developing with aging.