Accumulation of lysosulfatide in the brain of arylsulfatase A-deficient mice

Lysosomal storage diseases are a group of disorders where accumulation of catabolites is manifested in the lysosomes of different cell types. In metachromatic leukodystrophy (Arylsulfatase A [EC.3.1.6.8] deficiency) storage of the glycosphingolipid sulfatide in the brain leads to demyelination, resulting in neuromotor co-ordination deficits and regression. In a mouse model for metachromatic leukodystrophy, the ASA null mutant mouse, the accumulation of sulfatide in correlation to phenotype has been thoroughly investigated. Another lipid species reported to accumulate in patients with metachromatic leukodystrophy is the sulfatide related lipid lysosulfatide. Lysosulfatide was shown to be a cytotoxic compound in cell culture experiments and thus suggested to be involved in the pathology of metachromatic leukodystrophy. In this study, we further investigated the developmental profile of lysosulfatide in the brain of ASA null mutant mice by using high performance liquid chromatography. Lysosulfatide could be detected in the brain of normal mice (ASA +/+) from 1.8 months up to 23.1 months of age. From the age of 8.8 months the lysosulfatide levels remained constant at 1 pmol/mg wet tissue. The developmental change (< 20 months) of brain lysosulfatide showed an accumulation in ASA null mutant mice at ages above one month compared to its normal counterpart (ASA +/+). Thus, the ASA null mutant mouse might be a suitable model to further investigate the role of lysosulfatide in the pathogenesis of metachromatic leukodystrophy

Myelination in the absence of UDP-galactose:ceramide galactosyl-transferase and fatty acid 2 -hydroxylase

<p>Abstract</p> <p>Background</p> <p>The sphingolipids galactosylceramide (GalCer) and sulfatide are major myelin components and are thought to play important roles in myelin function. The importance of GalCer and sulfatide has been validated using UDP-galactose:ceramide galactosyltransferase-deficient (<it>Cgt</it>^-/-) mice, which are impaired in myelin maintenance. These mice, however, are still able to form compact myelin. Loss of GalCer and sulfatide in these mice is accompanied by up-regulation of 2-hydroxylated fatty acid containing (HFA)-glucosylceramide in myelin. This was interpreted as a partial compensation of the loss of HFA-GalCer, which may prevent a more severe myelin phenotype. In order to test this hypothesis, we have generated <it>Cgt</it>^-/-mice with an additional deletion of the fatty acid 2-hydroxylase (<it>Fa2h</it>) gene.</p> <p>Results</p> <p><it>Fa2h</it>^-/-/Cgt^-/-double-deficient mice lack sulfatide, GalCer, and in addition HFA-GlcCer and sphingomyelin. Interestingly, compared to <it>Cgt</it>^-/-mice the amount of GlcCer in CNS myelin was strongly reduced in <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice by more than 80%. This was accompanied by a significant increase in sphingomyelin, which was the predominant sphingolipid in <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice. Despite these significant changes in myelin sphingolipids, compact myelin was formed in <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice, and g-ratios of myelinated axons in the spinal cord of 4-week-old <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-mice did not differ significantly from that of <it>Cgt</it>^-/-mice, and there was no obvious phenotypic difference between <it>Fa2h</it>^-/-/<it>Cgt</it>^-/-and <it>Cgt</it>^-/-mice</p> <p>Conclusions</p> <p>These data show that compact myelin can be formed with non-hydroxylated sphingomyelin as the predominant sphingolipid and suggest that the presence of HFA-GlcCer and HFA-sphingomyelin in <it>Cgt</it>^-/-mice does not functionally compensate the loss of HFA-GalCer.</p

Synthesis and structure-activity relationships of cerebroside analogues as substrates of cerebroside sulphotransferase and discovery of a competitive inhibitor

Metachromatic leukodystrophy (MLD) is a rare genetic disease characterised by a dysfunction of the enzyme arylsulphatase A leading to the lysosomal accumulation of cerebroside sulphate (sulphatide) causing subsequent demyelination in patients. The enzyme galactosylceramide (cerebroside) sulphotransferase (CST) catalyses the transfer of a sulphate group from 3 '-phosphoadenosine-5'-phosphosulphate (PAPS) to cerebrosides producing sulphatides. Substrate reduction therapy for arylsulphatase A by inhibition of CST was proposed as a promising therapeutic approach. To identify competitive CST inhibitors, we synthesised and investigated analogues of the substrate galactosylceramide with variations at the anomeric position, the acyl substituent and the carbohydrate moiety, and investigated their structure-activity relationships. While most of the compounds behaved as substrates, alpha-galactosylceramide16was identified as the first competitive CST inhibitor. Compound16can serve as a new lead structure for the development of drugs for the treatment of this devastating disease, MLD, for which small molecule therapeutics are currently not available

Expression of Hepatoma-derived growth factor family members in the adult central nervous system

BACKGROUND: Hepatoma-derived growth factor (HDGF) belongs to a polypeptide family containing five additional members called HDGF related proteins 1–4 (HRP-1 to -4) and Lens epithelial derived growth factor. Whereas some family members such as HDGF and HRP-2 are expressed in a wide range of tissues, the expression of others is very restricted. HRP-1 and -4 are only expressed in testis, HRP-3 only in the nervous system. Here we investigated the expression of HDGF, HRP-2 and HRP-3 in the central nervous system of adult mice on the cellular level by immunohistochemistry. In addition we performed Western blot analysis of various brain regions as well as neuronal and glial cell cultures. RESULTS: HDGF was rather evenly expressed throughout all brain regions tested with the lowest expression in the substantia nigra. HRP-2 was strongly expressed in the thalamus, prefrontal and parietal cortex, neurohypophysis, and the cerebellum, HRP-3 in the bulbus olfactorius, piriform cortex and amygdala complex. HDGF and HRP-2 were found to be expressed by neurons, astrocytes and oligodendrocytes. In contrast, strong expression of HRP-3 in the adult nervous system is restricted to neurons, except for very weak expression in oligodendrocytes in the brain stem. Although the majority of neurons are HRP-3 positive, some like cerebellar granule cells are negative. CONCLUSION: The coexpression of HDGF and HRP-2 in glia and neurons as well as the coexpression of all three proteins in many neurons suggests different functions of members of the HDGF protein family in cells of the central nervous system that might include proliferation as well as cell survival. In addition the restricted expression of HRP-3 point to a special function of this family member for neuronal cells

Lysosomal di-N-acetylchitobiase-deficient mouse tissues accumulate Man2GlcNAc2 and Man3GlcNAc2

AbstractMost lysosomal storage diseases are caused by defects in genes encoding for acidic hydrolases. Deficiency of an enzyme involved in the catabolic pathway of N-linked glycans leads to the accumulation of the respective substrate and consequently to the onset of a specific storage disorder. Di-N-acetylchitobiase and core specific α1–6mannosidase represent the only exception. In fact, to date no lysosomal disease has been correlated to the deficiency of these enzymes. We generated di-N-acetylchitobiase-deficient mice by gene targeting of the Ctbs gene in murine embryonic stem cells. Accumulation of Man2GlcNAc2 and Man3GlcNAc2 was evaluated in all analyzed tissues and the tetrasaccharide was detected in urines. Multilamellar inclusion bodies reminiscent of polar lipids were present in epithelia of a scattered subset of proximal tubules in the kidney. Less constantly, enlarged Kupffer cells were observed in liver, filled with phagocytic material resembling partly digested red blood cells. These findings confirm an important role for lysosomal di-N-acetylchitobiase in glycans degradation and suggest that its deficiency could be the cause of a not yet described lysosomal storage disease

Overexpression of hepatoma-derived growth factor in melanocytes does not lead to oncogenic transformation

<p>Abstract</p> <p>Background</p> <p>HDGF is a growth factor which is overexpressed in a wide range of tumors. Importantly, expression levels were identified as a prognostic marker in some types of cancer such as melanoma.</p> <p>Methods</p> <p>To investigate the presumed oncogenic/transforming capacity of HDGF, we generated transgenic mice overexpressing HDGF in melanocytes. These mice were bred with mice heterozygous for a defective copy of the Ink4a tumor suppressor gene and were exposed to UV light to increase the risk for tumor development both genetically and physiochemically. Mice were analyzed by immunohistochemistry and Western blotting. Furthermore, primary melanocytes were isolated from different strains created.</p> <p>Results</p> <p>Transgenic animals overexpressed HDGF in hair follicle melanocytes. Interestingly, primary melanocytes isolated from transgenic animals were not able to differentiate <it>in vitro </it>whereas cells isolated from wild type and HDGF-deficient animals were. Although, HDGF^-/-/Ink4a^+/-mice displayed an increased number of epidermoid cysts after exposure to UV light, no melanomas or premelanocytic alterations could be detected in this mouse model.</p> <p>Conclusions</p> <p>The results therefore provide no evidence that HDGF has a transforming capacity in tumor development. Our results in combination with previous findings point to a possible role in cell differentiation and suggest that HDGF promotes tumor progression after secondary upregulation and may represent another protein fitting into the concept of non-oncogene addiction of tumor tissue.</p

N-glycosylation is required for full enzymic activity of the murine galactosylceramide sulphotransferase.

3- O -Sulphogalactosylceramide (sulphatide) is a major lipid component of myelin membranes, and is required for proper myelin formation. Sulphatide is synthesized in the Golgi apparatus by galactosylceramide sulphotransferase (CST; EC 2.8.2.11). Murine and human CSTs contain two putative N-glycosylation sites (Asn-66 and Asn-312). The second site is conserved among all galactose 3-O-sulphotransferases cloned to date. In order to study the functional relevance of N-glycosylation, we generated epitope-tagged CST and soluble Protein A-CST fusion proteins lacking both N-glycosylation sites, separately or in combination. Our results show that both sites are glycosylated when CST is expressed in Chinese hamster ovary (CHO) or COS cells. Moreover, transfecting CST mutants lacking both N-glycosylation sites, or only Asn-312, reduced significantly the amount of sulphatide synthesized, whereas substituting Asn-66 with a glutamine residue did not. In contrast, activity in vitro was reduced by approx. 50% in the Asn-66-->Gln (N66Q) mutant, and was almost undetectable in N312Q and N66/312Q transfectants. Furthermore, soluble Protein A-CST expressed in the presence of tunicamycin was almost inactive, and accumulated in transfected cells. Expression of fully active CST in a CHO-glycosylation mutant lacking N-acetylglucosaminyltransferase I demonstrated that condensation of the N-linked pentamannosyl-core structure is sufficient to form a fully active enzyme