Accumulation of sulfatide in neuronal and glial cells of arylsulfatase A deficient mice

Arylsulfatase A (ASA) degrades sulfatide, seminolipid and lactosylceramide sulfate, glycolipids recognized by the Sulph I antibody although sulfatide is considered the main antigen. Sulfatide is myelin associated but studies have shown a minor distribution also in non-myelin forming cells. The aim of this work was to further study sulfatide in neurons and astrocytes by immunohistochemistry, facilitated by investigation of tissue from adult ASA deficient (ASA ?/?) mice. Cells with a low presence of sulfatide might be detected due to lack of ASA activity and accumulation of Sulph I antigens. Sulfatide positive astrocytes and neurons were more numerous and intensely stained in ASA ?/? mice, demonstrating a sulfatide accumulation compared to controls. Sulph I staining was especially increased in the molecular layer of cerebellum, in which Purkinje cell dendrites displayed an altered morphology, and in layer IV–VI of cerebral cortex. In hippocampus, immunostaining was found in neuronal cytoplasm in ASA ?/? but in nuclear membranes of control mice. We observed a gray matter astrogliosis, which appeared to be associated to sulfatide accumulation. In addition, the developmental change (<20 months) of Sulph I antigens, galactosylceramide, phospholipids and cholesterol were followed by lipid analyses which verified sulfatide and seminolipid accumulation in adult ASA ?/? mice, although no lactosylceramide sulfate could be detected. In addition to demonstrating sulfatide in neurons and astrocytes, this study supports the value of ASA ?/? mice as a model for metachromatic leukodystrophy and suggests that accumulation of sulfatide beyond myelin might contribute to the pathology of this disease.     

Neonatal endocrine abnormalities in myelin deficient Jimpy-tabby mice.

The Jimpy mouse is a sex-linked recessive mutant characterized by a paucity of myelin in the Central Nervous System. These mice are bred with another sex-linked gene bearing a fur mutation known as Tabby. The endocrine system of these animals was examined for histologic abnormalites because growth hormone and thyroxine play important roles in controlling myelinogenesis. The results of the present study show that the pituitary and thyroid are abnormal in appearance by two days after birth. The number of somatotrophs in the Jimpy-Tabby pituitary is reduced about 30% but the number of granules in these cells is increased 25%. The endoplasmic reticulum of the thyroid follicular cells in the mutants is dilated and occupies most of the cytoplasm. The results of this study demonstrate for the first time that Jimpy-Tabby mice exhibit abnormalities outside the Central Nervous System. The neonatal changes observed in the endocrine system are the basis for continued studies to determine if they are responsible for the myelin deficit.     

Thymus,kidney and craniofacial abnormalities in Six 1 deficient mice

Six genes are widely expressed during vertebrate embryogenesis, suggesting that they are implicated in diverse differentiation processes. To determine the functions of the Six1 gene, we constructed Six1-deficient mice by replacing its first exon by the beta-galactosidase gene. We have previously shown that mice lacking Six1 die at birth due to thoracic skeletal defects and severe muscle hypoplasia affecting most of the body muscles. Here, we report that Six1(-/-) neonates also lack a kidney and thymus, as well as displaying a strong disorganisation of craniofacial structures, namely the inner ear, the nasal cavity, the craniofacial skeleton, and the lacrimal and parotid glands. These organ defects can be correlated with Six1 expression in the embryonic primordium structures as revealed by X-Gal staining at different stages of embryogenesis. Thus, the fetal abnormalities of Six1(-/-) mice appear to result from the absence of the Six 1 homeoprotein during early stages of organogenesis. Interestingly, these Six1 defects are very similar to phenotypes caused by mutations of Eya 1, which are responsible for the BOR syndrome in humans. Close comparison of Six1 and Eya 1 deficient mice strongly suggests a functional link between these two factors. Pax gene mutations also lead to comparable phenotypes, suggesting that a regulatory network including the Pax, Six and Eya genes is required for several types of organogenesis in mammals.     

Hemorheological abnormalities in lipoprotein lipase deficient mice with severe hypertriglyceridemia

Severe hypertriglyceridemia (HTG) is a metabolic disturbance often seen in clinical practice. It is known to induce life-threatening acute pancreatitis, but its role in atherogenesis remains elusive. Hemorheological abnormality was thought to play an important role in pathogenesis of both pancreatitis and atherosclerosis. However, hemorheology in severe HTG was not well investigated. Recently, we established a severe HTG mouse model deficient in lipoprotein lipase (LPL) in which severe HTG was observed to cause a significant increase in plasma viscosity. Disturbances of erythrocytes were also documented, including decreased deformability, electrophoresis rate, and membrane fluidity, and increased osmotic fragility. Scanning electron microscopy demonstrated that most erythrocytes of LPL deficient mice deformed with protrusions, irregular appearances or indistinct concaves. Analysis of erythrocyte membrane lipids showed decreased cholesterol (Ch) and phospholipid (PL) contents but unaltered Ch/PL ratio. The changes of membrane lipids may be partially responsible for the hemorheological and morphologic abnormalities of erythrocytes. This study indicated that severe HTG could lead to significant impairment of hemorheology and this model may be useful in delineating the role of severe HTG in the pathogenesis of hyperlipidemic pancreatitis and atherosclerosis.     

Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin

Mice homozygous for the mutation myelin deficient (mld), an allele of shiverer, exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mld mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.     

Altered synaptic plasticity and behavioral abnormalities in CNGA3‐deficient mice

The role of the cyclic nucleotide‐gated (CNG) channel CNGA3 is well established in cone photoreceptors and guanylyl cyclase‐D‐expressing olfactory neurons. To assess a potential function of CNGA3 in the mouse amygdala and hippocampus, we examined synaptic plasticity and performed a comparative analysis of spatial learning, fear conditioning and step‐down avoidance in wild‐type mice and CNGA3 null mutants (CNGA3^?/?). CNGA3^?/? mice showed normal basal synaptic transmission in the amygdala and the hippocampus. However, cornu Ammonis (CA1) hippocampal long‐term potentiation (LTP) induced by a strong tetanus was significantly enhanced in CNGA3^?/? mice as compared with their wild‐type littermates. Unlike in the hippocampus, LTP was not significantly altered in the amygdala of CNGA3^?/? mice. Enhanced hippocampal LTP did not coincide with changes in hippocampus‐dependent learning, as both wild‐type and mutant mice showed a similar performance in water maze tasks and contextual fear conditioning, except for a trend toward higher step‐down latencies in a passive avoidance task. In contrast, CNGA3^?/? mice showed markedly reduced freezing to the conditioned tone in the amygdala‐dependent cued fear conditioning task. In conclusion, our study adds a new entry on the list of physiological functions of the CNGA3 channel. Despite the dissociation between physiological and behavioral parameters, our data describe a so far unrecognized role of CNGA3 in modulation of hippocampal plasticity and amydgala‐dependent fear memory.     

Characterization of cell- and region-specific abnormalities in the epididymis of cathepsin A deficient mice

Cathepsin A (PPCA), a lysosomal carboxypeptidase that functions as a protective protein for alpha-neuraminidase and beta-galactosidase in a multi-enzyme complex, has been shown to be expressed in the epithelial cells of the epididymis. In the present study, the epididymis of PPCA-/- mice from 2 to 10 months of age was compared with those of their wild-type counterparts. Major accumulations of pale vacuoles, corresponding to lysosomes, were noted in principal and narrow/apical cells in PPCA-/- mice, and clear cells also appearing highly vacuolated, were grossly enlarged in size. This was especially evident in the caput and corpus regions, where quantitative analyses confirmed that the epithelium of the tubules in these regions was expanding in profile area. In addition, the base of the epithelium in these regions was often greatly vacuolated, corresponding to cells that presented no identifiable features and appeared to be degenerating. Halo cells dispersed at various levels in the epithelium also appeared to be abnormal, accumulating pale lysosomes. Furthermore, numerous macrophages were observed in the intertubular space of the entire duct, presenting a large size and plethora of pale lysosomes. Taken together, the present data indicate major lysosomal abnormalities in the epididymis of PPCA-/- mice in a cell type and region specific manner. In addition, it is suggested that the compromised halo cells, due to PPCA deficiency within their lysosomes, cannot function properly and as a result there is a recruitment of macrophages in the intertubular space.     

Structural abnormalities in the primary somatosensory cortex and a normal behavioral profile in Contactin-5 deficient mice

Contactin-5 (Cntn5) is an immunoglobulin cell adhesion molecule that is exclusively expressed in the central nervous system. In view of its association with neurodevelopmental disorders, particularly autism spectrum disorder (ASD), this study focused on Cntn5-positive areas in the forebrain and aimed to explore the morphological and behavioral phenotypes of the Cntn5 null mutant (Cntn5^?/?) mouse in relation to these areas and ASD symptomatology. A newly generated antibody enabled us to elaborately describe the spatial expression pattern of Cntn5 in P7 wild type (Cntn5^+/+) mice. The Cntn5 expression pattern included strong expression in the cerebral cortex, hippocampus and mammillary bodies in addition to described previously brain nuclei of the auditory pathway and the dorsal thalamus. Thinning of the primary somatosensory (S1) cortex was found in Cntn5^?/? mice and ascribed to a misplacement of Cntn5-ablated cells. This phenotype was accompanied by a reduction in the barrel/septa ratio of the S1 barrel field. The structure and morphology of the hippocampus was intact in Cntn5^?/? mice. A set of behavioral experiments including social, exploratory and repetitive behaviors showed that these were unaffected in Cntn5^?/? mice. Taken together, these data demonstrate a selective role of Cntn5 in development of the cerebral cortex without overt behavioral phenotypes.     

NMDA receptor‐deficient mice display sexual dimorphism in the onset and severity of behavioural abnormalities

N‐methyl‐d ‐aspartate (NMDA) receptor‐deficient mice can be used to understand the role that NMDA receptors (NMDARs) play in the pathophysiology of neurodevelopmental disorders such as schizophrenia. Genetically modified mice with low levels of NR1 subunit (NR1 knockdown mice) have reduced receptor levels throughout development, and have robust abnormalities in behaviours that are relevant to schizophrenia. We traced the onset and severity of these behaviours at three developmental stages to understand when in development the underlying circuits depend on intact NMDAR function. We examined social behaviour, working memory, executive function, locomotor activity and stereotypy at 3, 6 and 12 weeks of age in NR1 knockdown mice and their wild‐type littermates. We discovered that each of these behaviours had a unique developmental trajectory in mutant mice, and males showed an earlier onset and severity than females in several behaviours. Hyperlocomotion was most substantial in juvenile mice and plateaued in adult mice, whereas stereotypy progressively worsened with age. Impairments in working memory and sociability were sexually dimorphic, with deficits first detected in peri‐adolescent males but only detected in adult females. Interestingly, executive function was most impaired in peri‐adolescent mice of either sex. Furthermore, while juvenile mutant mice had some ability to problem solve in the puzzle box test, the same mice lost this ability when tested 4 weeks later. Our studies highlight key developmental periods for males and females in the expression of behaviours that are relevant to psychiatric disorders.     

Poster Sessions BP03: Myelin,Demyelination and Diseases of Myelin. Myelin and myelination in PLP‐null mice

The myelin proteolipid protein (PLP) is the major structural protein of CNS myelin, accounting for approximately half of total myelin protein. We studied synthesis and accumulation of myelin components for two months postnatally in PLP‐null mice and age‐matched controls. Accumulation of myelin, as assayed by levels of whole brain cerebroside and myelin basic protein, was normal in the knockout mice. The rate of cerebroside synthesis in the knockout mice was also normal. Myelin was isolated at several ages during development, using a standard subcellular fractionation protocol. The yield of ‘purified myelin’ isolated from a large particle (crude mitochondrial) fraction was reduced in PLP‐null mice, but increased amounts of ‘myelin’ were obtained in the small particle (crude microsomal) fraction. This ‘myelin’ in the crude microsomal fraction was identified as such by flotation on 0.85 m sucrose and the myelin‐characteristic 2 : 1 molar ratio of cholesterol to cerebroside. This suggests myelin from PLP‐null mice is physically more fragile than normal myelin, and that during tissue dispersion, much more PLP‐null myelin is fragmented into small vesicles than is the case for normal myelin. Three hours after intracranial injection of tritiated acetate into PLP‐null mice, cerebroside in myelin isolated from the large particle fraction was at a similar specific radioactivity to that isolated from the small particle (crude microsomal) fraction, suggesting that the most recently deposited PLP‐null myelin is not preferentially unstable. The increased fragility evident during tissue dispersion is indicative of an underlying structural abnormality in PLP‐null myelin. Whether this inherent structural instability affects myelin metabolism is under investigation. Acknowledgements: Supported by USPHS & NMSS grants.     

Myelin instability and oligodendrocyte metabolism in myelin-deficient mutant mice

During the active phase of myelination in myelin-deficient mutant mice (mld), myelin basic protein (MBP) synthesis is defective and the myelin lamellae are uncompacted. In these mutants, we found a fast metabolism of the myelin-associated glycoprotein (MAG) and of sulfatides, and the presence of cholesterol esters and a degradation product of MAG, dMAG, indicating that mld myelin was unstable. The increased synthesis of MAG and Wolfgram protein, two proteins present in uncompacted myelin sheath and paranodal loops, was demonstrated by high levels of messengers. Simultaneously, we found an accumulation of inclusion bodies, vacuoles, and rough endoplasmic reticulum in mld oligodendrocytes. This material was heavily immunostained for MAG. Furthermore, the developmental change between the two molecular forms of MAG (p72MAG/p67MAG) was delayed in mld mice. In 85-d-old mld mice, the MBP content increased and myelin lamellae became better compacted. In these mutants, dMAG was absent and MAG mRNAs were found in normal amounts. Furthermore, the fine structure of mld oligodendrocytes was normal and the MAG immunostaining was similar to age-matched controls. These results support a functional role for MBP in maintaining the metabolic stability and the compact structure of myelin. Furthermore, in the absence of MBP and myelin compaction, the regulation of the synthesis of at least two membrane proteins related to myelin cannot proceed.     

Elevated sulfatide levels in neurons cause lethal audiogenic seizures in mice

Galactosylceramide (GalCer) and 3- O -sulfo-GalCer (sulfatide) are abundant sphingolipids in myelinating glial cells. However, low levels of GalCer and sulfatide have also been found in neurons, though their physiological role in these cells is unknown. Transgenic mice over-expressing UDP-galactose : ceramide galactosyltransferase (CGT) under control of the Thy1.2 promoter synthesize C18 : 0 fatty acid containing GalCer and sulfatide in neurons. Depending on the genetic background, these transgenic mice have a significantly reduced life span. Transgenic mice were extremely sensitive to sound stimuli and displayed lethal audiogenic seizures after relatively mild acoustic stimulation, i.e., key jangling. CGT-transgenic mice additionally over-expressing the adenosine 3'-phospho 5'-phosphosulfate : cerebroside sulfotransferase were more sensitive to audiogenic seizure induction than mice expressing only the CGT-transgene. This correlated with the higher sulfatide content in neuronal plasma membranes of the double-transgenic mice compared with CGT-transgenic mice, and strongly suggests that lethal audiogenic seizures are caused by elevated sulfatide levels in transgenic neurons. CGT-transgenic mice will be a useful model to further investigate how sulfatide affects functional properties of neurons.     

Circadian behaviour in neuroglobin deficient mice

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night.     

Vascular abnormalities in mice deficient for the G protein-coupled receptor GPR4 that functions as a pH sensor

GPR4 is a G protein-coupled receptor expressed in the vasculature, lung, kidney, and other tissues. In vitro ectopic overexpression studies implicated GPR4 in sensing extracellular pH changes leading to cyclic AMP (cAMP) production. To investigate its biological roles in vivo, we generated GPR4-deficient mice by homologous recombination. Whereas GPR4-null adult mice appeared phenotypically normal, neonates showed a higher frequency of perinatal mortality. The average litter size from GPR4(-/-) intercrosses was approximately 30% smaller than that from GPR4(+/+) intercrosses on N3 and N5 C57BL/6 genetic backgrounds. A fraction of knockout embryos and neonates had spontaneous hemorrhages, dilated and tortuous subcutaneous blood vessels, and defective vascular smooth muscle cell coverage. Mesangial cells in kidney glomeruli were also significantly reduced in GPR4-null neonates. Some neonates exhibited respiratory distress with airway lining cell metaplasia. To examine whether GPR4 is functionally involved in vascular pH sensing, an ex vivo aortic ring assay was used under defined pH conditions. Compared to wild-type aortas, microvessel outgrowth from GPR4-null aortas was less inhibited by acidic extracellular pH. Treatment with an analog of cAMP, a downstream effector of GPR4, abolished microvessel outgrowth bypassing the GPR4-knockout phenotype. These results suggest that GPR4 deficiency leads to partially penetrant vascular abnormalities during development and that this receptor functions in blood vessel pH sensing.     

Kif3a deficiency reverses the skeletal abnormalities in Pkd1 deficient mice by restoring the balance between osteogenesis and adipogenesis

Pkd1 localizes to primary cilia in osteoblasts and osteocytes. Targeted deletion of Pkd1 in osteoblasts results in osteopenia and abnormalities in Runx2-mediated osteoblast development. Kif3a, an intraflagellar transport protein required for cilia function, is also expressed in osteoblasts. To assess the relationship between Pkd1 and primary cilia function on bone development, we crossed heterozygous Pkd1- and Kif3a-deficient mice to create compound Pkd1 and Kif3a-deficient mice. Pkd1 haploinsufficiency (Pkd1(+/Δ)) resulted in osteopenia, characterized by decreased bone mineral density, trabecular bone volume, and cortical thickness. In addition, deficiency of Pkd1 resulted in impaired osteoblastic differentiation and enhanced adipogenesis in both primary osteoblasts and/or bone marrow stromal cell cultures. These changes were associated with decreased Runx2 expression, increased PPARγ expression, and impaired hedgehog signaling as evidenced by decreased Gli2 expression in bone and osteoblast cultures. In contrast, heterozygous Kif3a(+/Δ) mice display no abnormalities in skeletal development or osteoblast function, but exhibited decreased adipogenic markers in bone and impaired adipogenesis in vitro in association with decreased PPARγ expression and upregulation of Gli2. Superimposed Kif3a deficiency onto Pkd1(+/Δ) mice paradoxically corrected the effects of Pkd1 deficiency on bone mass, osteoblastic differentiation, and adipogenesis. In addition, Runx2, PPARγ and Gli2 expression in bone and osteoblasts were normalized in compound double Pkd1(+/Δ) and Kif3a(+/Δ) heterozygous mice. The administration of sonic hedgehog, overexpression of Gli2, and the PC1 C-tail construct all increased Gli2 and Runx2-II expression, but decreased PPARγ2 gene expression in C3H10T1/2 cells. Our findings suggest a role for Pkd1 and Kif3a to counterbalance the regulation of osteogenesis and adipogenesis through differential regulation of Runx2 and PPARγ by Gli2.