The PGRMC1 Antagonist AG-205 Inhibits Synthesis of Galactosylceramide and Sulfatide.

Sulfatide synthesis in the human renal cancer cell line SMKT-R3 was strongly inhibited in the presence of low µM concentrations of AG-205, a progesterone receptor membrane component 1 (PGRMC1) antagonist. This was also the case in Chinese hamster ovary (CHO) cells stably transfected with UDP-galactose: ceramide galactosyltransferase and cerebroside sulfotransferase, the two enzymes required for sulfatide synthesis. In CHO cells synthesizing galactosylceramide but not sulfatide, galactosylceramide was also strongly reduced, suggesting an effect at the level of galactolipid synthesis. Notably, AG-205 inhibited galactosylceramide synthesis to a similar extent in wild type CHO cells and cells that lack PGRMC1 and/or PGRMC2. In vitro enzyme activity assays showed that AG-205 is an inhibitor of UDP-galactose: ceramide galactosyltransferase, but not cerebroside sulfotransferase. This study shows that PGRMC1 is only one of several targets of AG-205 and should be used with caution, especially in studies using cells synthesizing galactosylceramide and sulfatide.

Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor.

Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition.

Endogenous ligands of natural killer T cells are alpha-linked glycosylceramides.

The nature of the endogenous ligands for natural killer T (NKT) cells has been debated for more than a decade. Because the mammalian glycosylceramide synthases are invertases, it is believed that in mammals all glycosylceramides are ß anomers. However, the possibility that an alternative enzymatic pathway, an unfaithful enzyme, or unique physico-chemical environments could allow the production of small quantities of α anomers should be entertained. Classic biochemical and chemical analysis approaches are not well suited for this challenge as they lack sensitivity. Using a combination of biological assays and new technological approaches, we have unequivocally demonstrated that α glycosylceramides were constitutively produced by mammalian immune cells, loaded onto CD1d and presented to NKT cells both in the thymus and in the periphery. Their amount is controlled tightly by catabolic enzymes, and can be altered in vitro and in vivo to modify NKT cell behavior.

Expression of sulfatide and sulfated lactosylceramide among histological types of human ovarian carcinomas.

Among negatively charged lipids, sulfoglycolipids are known to be expressed by specific cell populations and to be involved in their functions, including in adhesion with functional proteins, modification of ion channels and induction of cellular differentiation. Accordingly, we determined their amounts in several histologically defined types of ovarian carcinoma tissues. Sulfoglycolipids were determined by TLC-immunostaining with monoclonal anti-sulfatide antibodies and the gene expression of their synthetic enzymes was by RT-PCR. All types of ovarian carcinomas were revealed to exhibit potential to synthesize sulfoglycolipids, either sulfatide (I(3)SO3-GalCer) or sulfated lactosylceramides (II(3)SO3-LacCer), which were expressed at the following frequencies, 6 out of 6 mucinous cystadenocarcinomas, 4 out of 7 serous cystadenocarcinomas, 2 out of 3 endometrioid carcinomas, and 2 out of 3 clear cell adenocarcinomas. All mucinous cystadenocarcinoma tissues preferentially contained sulfatide in amounts of 0.61-1.13 µg per mg dry weight, the molecular species being similar with those of GalCer. Whereas the other carcinomas contained either sulfatide or sulfated LacCer, the latter being detected in 4 out of 6 specimens with sulfoglycolipids. The expression of sulfatide and sulfated LacCer was found to be positively correlated with the amounts of GalCer and LacCer as substrates for sulfotransferase and expression of the genes for GalCer sulfotransferase and ceramide galactosyltransferase. Sulfoglycolipids in ovarian carcinoma tissues were revealed to be expressed in morphologically defined type-characteristic manners, in contrast to the ubiquitous distribution of GM3.

Ceramide galactosyltransferase expression is regulated positively by Nkx2.2 and negatively by OLIG2.

Myelin, a multilamellar structure extended from oligodendrocytes or Schwann cells, plays a critical role in maintenance of neuronal function, and damage or loss of myelin causes demyelinating diseases such as multiple sclerosis. For precise alignment of the myelin sheath, there is a requirement for expression of galactosylceramide (GalCer), a major glycosphingolipid in myelin. Synthesis of GalCer is strictly limited in oligodendrocytes in a developmental stage-specific manner. Ceramide galactosyltransferase (CGT), a key enzyme for biosynthesis of GalCer, exhibits restricted expression in oligodendrocytes but the mechanism is poorly understood. Based on our assumption that particular oligodendrocyte-lineage-specific transcription factors regulate CGT expression, we co-expressed a series of candidate transcription factors with the human CGT promoter-driving luciferase expression in oligodendroglioma cells to measure the promoter activity. We found that Nkx2.2 strongly activated the CGT promoter. In addition, we identified a novel repressive DNA element in the first intron of CGT and OLIG2, an oligodendrocyte-specific transcription factor, as a binding protein of this element. Moreover, overexpression of OLIG2 completely canceled the activating effect of Nkx2.2 on CGT promoter activity. Expression of CGT mRNA was also upregulated by Nkx2.2, but this upregulation was cancelled by co-expression of OLIG2 with Nkx2.2. Our study suggests that CGT expression is controlled by balanced expression of the negative modulator OLIG2 and positive regulator Nkx2.2, providing new insights into how expression of GalCer is tightly regulated in cell-type- and stage-specific manners.

Altered phospholipid molecular species and glycolipid composition in brain, liver and fibroblasts of Zellweger syndrome.

We studied the altered molecular species of lipids in brain and liver tissues, and fibroblasts from patients with Zellweger syndrome (ZS). ZS cerebellum samples contained a higher amount of sphingomyelin with shorter chain fatty acids compared to that in normal controls. The amount of phosphatidylethanolamine (PE) was less than half of that in controls, with the absence of the PE-type of plasmalogen. Gangliosides were accumulated in the brains and fibroblasts of ZS patients. To investigate whether or not impaired beta-oxidation of very long chain fatty acids and/or plasmalogen synthesis affects glycolipids metabolism, RNAi of peroxisomal acylCo-A oxidase (ACOX1) and glyceronephosphate O-acyltransferase (GNPAT) was performed using cultured neural cells. In neuronal F3-Ngn1 cells, ACOX1 and GNPAT silencing up-regulated ceramide galactosyltransferase (UGT8) mRNA expression, and down-regulated UDP-glucose ceramide glucosyltransferase (UGCG). These results suggest that both impaired beta-oxidation of very long chain fatty acids and plasmalogen synthesis affect glycolipid metabolism in neuronal cells.

Ceramide galactosyltransferase (UGT8) as a molecular marker of canine mammary tumor malignancy.

Thirty-two canine mammary tubulopapillary carcinomas and 14 simple adenomas were studied by immunohistochemistry for the expression of UDP-galactose:ceramide galactosyltransferase (UGT8). The majority of tissue specimens (57%) representing adenomas had no or weak reaction with anti-UGT8 antibodies (0-2 pts according to IRS scale) in comparison to the majority of carcinomas (90%) which stained with high intensities (3-9 pts according to IRS scale). When the average values of the reaction intensities (IRS) for malignant and benign tumors were compared, using the Mann-Whitney U-test, significant differences in UGT8 expression between them were found (P < 0.001). Mammary tubulopapillary carcinomas were further analyzed by IHC and the same rabbit polyclonal antibody directed against UGT8 according to their malignancy grade. It was found that the level of UGT8 increased in tumor specimens together with their grading. A comparison of the average values of the reaction intensity (IRS scale) revealed a significant difference (Mann-Whitney U-test, P < 0.05) in UGT8 expression between tumors representing malignancy grades G3 and G1. Based on the obtained results, it is proposed that UGT8 is associated with malignancy of canine mammary gland cells and may have a potential value as a diagnostic marker.

The lifetime of UDP-galactose:ceramide galactosyltransferase is controlled by a distinct endoplasmic reticulum-associated degradation (ERAD) regulated by sigma-1 receptor chaperones.

The glycosphingolipid biosynthesis is initiated by monoglycosylation of ceramides, the action of which is catalyzed either by UDP-glucose:ceramide glucosyltransferase or by UDP-galactose:ceramide galactosyltransferase (CGalT). CGalT is expressed predominantly at the endoplasmic reticulum (ER) of oligodendrocytes and is responsible for synthesizing galactosylceramides (GalCer) that play an important role in regulation of axon conductance. However, despite the importance of ceramide monoglycosylation enzymes in a spectrum of cellular functions, the mechanism that fine tunes activities of those enzymes is largely unknown. In the present study, we demonstrated that the sigma-1 receptor (Sig-1R) chaperone, the mammalian homologue of a yeast C8-C7 sterol isomerase, controls the protein level and activity of the CGalT enzyme via a distinct ER-associated degradation system involving Insig. The Sig-1R forms a complex with Insig via its transmembrane domain partly in a sterol-dependent manner and associates with CGalT at the ER. The knockdown of Sig-1Rs dramatically prolonged the lifetime of CGalT without affecting the trimming of N-linked oligosaccharides at CGalT. The increased lifetime leads to the up-regulation of CGalT protein as well as elevated enzymatic activity in CHO cells stably expressing CGalT. Knockdown of Sig-1Rs also decreased CGalT degradation endogenously expressed in D6P2T-schwannoma cells. Our data suggest that Sig-1Rs negatively regulate the activity of GalCer synthesis under physiological conditions by enhancing the degradation of CGalT through regulation of the dynamics of Insig in the lipid-activated ER-associated degradation system. The GalCer synthesis may thus be influenced by sterols at the ER.

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

BACKGROUND: 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 (Cgt-/-) 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 Cgt-/- mice with an additional deletion of the fatty acid 2-hydroxylase (Fa2h) gene. RESULTS: Fa2h-/-/Cgt-/- double-deficient mice lack sulfatide, GalCer, and in addition HFA-GlcCer and sphingomyelin. Interestingly, compared to Cgt-/- mice the amount of GlcCer in CNS myelin was strongly reduced in Fa2h-/-/Cgt-/- mice by more than 80%. This was accompanied by a significant increase in sphingomyelin, which was the predominant sphingolipid in Fa2h-/-/Cgt-/- mice. Despite these significant changes in myelin sphingolipids, compact myelin was formed in Fa2h-/-/Cgt-/- mice, and g-ratios of myelinated axons in the spinal cord of 4-week-old Fa2h-/-/Cgt-/- mice did not differ significantly from that of Cgt-/- mice, and there was no obvious phenotypic difference between Fa2h-/-/Cgt-/- and Cgt-/- mice CONCLUSIONS: 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 Cgt-/- mice does not functionally compensate the loss of HFA-GalCer.

Quantification of seminolipid by LC-ESI-MS/MS-multiple reaction monitoring: compensatory levels in Cgt(+/⁻) mice.

Seminolipid, also known as sulfogalactosylglycerolipid (SGG), plays important roles in male reproduction. Therefore, an accurate and sensitive method for SGG quantification in testes and sperm is needed. Here we compare SGG quantitation by the traditional colorimetric Azure A assay with LC-ESI-MS/MS using multiple reaction monitoring (MRM). Inclusion of deuterated SGG as the internal standard endowed accuracy to the MRM method. The results showed reasonable agreement between the two procedures for purified samples, but for crude lipid extracts, the colorimetric assay significantly overestimated the SGG content. Using ESI-MS/MS MRM, C16:0-alkyl/C16:0-acyl SGG of Cgt(+/⁻) mice was quantified to be 406.06 ± 23.63 µg/g testis and 0.13 ± 0.02 µg/million sperm, corresponding to 78% and 87% of the wild-type values, respectively. CGT (ceramide galactosyltransferase) is a critical enzyme in the SGG biosynthesis pathway. Cgt⁻/⁻ males depleted of SGG are infertile due to spermatogenesis arrest. However, Cgt(+/⁻) males sire offspring. The higher than 50% expression level of SGG in Cgt(+/⁻) animals, compared with the wild-type expression, might be partly due to compensatory translation of the active CGT enzyme. The results also indicated that 78% of SGG levels in Cgt(+/⁻) mice were sufficient for normal spermatogenesis.

Modification of sphingoglycolipids and sulfolipids in kidney cell lines under heat stress: activation of monohexosylceramide synthesis as a ceramide scavenger.

Heat stress on Madin-Darby canine kidney cells increased ceramide content to 187% at 40 degrees C for 24 h, and the de novo synthesis from serine increased to 146%. Glucosylceramide (GlcCer) and galactosylceramide (GalCer) synthesis from ceramide, the first glycosylation step of sphingolipid metabolism in kidney cells, increased to 290% (GalCer) and 143% (GlcCer) after metabolic labeling with (14)C-glucose at 42 degrees C for 20 h. The more complex glycolipid lactosylceramide also increased to 151%, whereas sulfatide and ganglioside GM3 decreased to 21% and 43%, respectively. Sulfatide (SM4s) showed optimal sulfation at 37 degrees C, whereas cholesterol sulfate was optimally sulfated at 40 degrees C. The gene expression of ceramide glucosyltransferase (GluT), ceramide galactosyltransferase, and GalCer sulfotransferase (GST) after 24 h culture at 42 degrees C significantly increased to 714%, 221%, and 174%, respectively. Another kidney cell line, COS7, showed less activation of these transferases by heat stress. Although GST gene expression was higher under heat stress, SM4s synthesis decreased, which may have been due to increased GST sensitivity to a temperature higher than 37 degrees C. When we introduced the HSP70 gene into the expression vector and transfected the plasmid (pCDM-dHSP70) into kidney cells, GlcCer synthesis increased significantly. From these results, we speculated that HSP70 may play a role in GluT gene expression to increase GlcCer and decrease intracellular ceramide level.

Myelin protein composition is altered in mice lacking either sulfated or both sulfated and non-sulfated galactolipids.

Myelin is highly enriched in galactocerebroside (GalCer) and its sulfated form sulfatide. Mice, unable to synthesize GalCer and sulfatide (CGT(null)) or sulfatide alone (CST(null)), exhibit disorganized paranodal structures and progressive dysmyelination. To obtain insights into the molecular mechanisms underlying these defects, we examined myelin composition of these mutants by two-dimensional differential fluorescence intensity gel electrophoresis proteomic approach and immunoblotting. We identified several proteins whose expressions were significantly altered in these mutants. These proteins are known to regulate cytoskeletal dynamics, energy metabolism, vesicular trafficking or adhesion, suggesting a disruption in these physiological processes in the absence of myelin galactolipids. Further analysis of one of these proteins, nucleotide diphosphate kinase (NDK)/Nm23, showed that it was reduced in myelin of CGT(null) and increased in CST(null), but not in whole brain homogenate. Immunostaining showed an increase in its expression in the cell bodies of CGT(null)- and a decrease in CST(null)-oligodenrocytes, together leading to the hypothesis that transport of NDK/Nm23 from oligodenrocyte cell bodies into myelin may be differentially dysregulated in the absence of these galactolipids. This study provides new insights into the changes that occur in the composition/distribution of myelin proteins in mice lacking either unsulfated and/or sulfated galactolipids and reinforces the role of these lipids in intracellular trafficking.

Nur7 is a nonsense mutation in the mouse aspartoacylase gene that causes spongy degeneration of the CNS.

Aspartoacylase (ASPA) is an oligodendrocyte-restricted enzyme that catalyzes the hydrolysis of neuronally derived N-acetylaspartate (NAA) to acetate and aspartic acid. ASPA deficiency leads to the fatal childhood autosomal recessive leukodystrophy Canavan disease (CD). Here we demonstrate that the previously described ENU-induced nur7 mouse mutant is caused by a nonsense mutation, Q193X, in the Aspa gene (Aspa(nur7)). Homozygous Aspa(nur7nur7) mice do not express detectable Aspa protein and display an early-onset spongy degeneration of CNS myelin with increased NAA levels similar to that observed in CD patients. In addition, CNS regions rich in neuronal cell bodies also display vacuolization. Interestingly, distinct myelin rich areas, such as the corpus callosum, optic nerve, and spinal cord white matter appear normal in Aspa(nur7/nur7) mice. Reduced cerebroside synthesis has been demonstrated in CD patients and animal models. To determine the potential relevance of this observation in disease pathogenesis, we generated Aspa(nur7/nur7) mice that were heterozygous for a null allele of the gene that encodes the enzyme UDP-galactose:ceramide galactosyltransferase (Cgt), which is responsible for catalyzing the synthesis of the abundant myelin galactolipids. Despite reduced amounts of cerebrosides, the Aspa(nur7/nur7);Cgt(+/-) mice were not more severely affected than the Aspa(nur7) mutants, suggesting that diminished cerebroside synthesis is not a major contributing factor in disease pathogenesis. Furthermore, we found that myelin degeneration leads to significant axonal loss in the cerebellum of older Aspa(nur7) mutants. This finding suggests that axonal pathology caused by CNS myelin defects may underlie the neurological disabilities that CD patients develop at late stages of the disease.

Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set.

Since the first use of streptomycin as an effective antibiotic drug in the treatment of tuberculosis, aminoglycoside antibiotics have been widely used against a variety of bacterial infections for over six decades. However, the pathways for aminoglycoside biosynthesis still remain unclear, mainly because of difficulty in genetic manipulation of actinomycetes producing this class of antibiotics. Gentamicin belongs to the group of 4,6-disubstituted aminoglycosides containing a characteristic core aminocyclitol moiety, 2-deoxystreptamine (2-DOS), and the recent discovery of its biosynthetic gene cluster in Micromonospora echinospora has enabled us to decipher its biosynthetic pathway. To determine the minimal set of genes and their functions for the generation of gentamicin A(2), the first pseudotrisaccharide intermediate in the biosynthetic pathway for the gentamicin complex, various sets of candidate genes from M. echinospora and other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain of Streptomyces venezuelae. Heterologous expression of different combinations of putative 2-DOS biosynthetic genes revealed that a subset, gtmB-gtmA-gacH, is responsible for the biosynthesis of this core aminocyclitol moiety of gentamicin. Expression of gtmG together with gtmB-gtmA-gacH led to production of 2'-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that adds N-acetyl-d-glucosamine (GLcNA) to 2-DOS. Expression of gtmM in a 2'-N-acetylparomamine-producing recombinant S. venezuelae strain generated paromamine. Expression of gtmE in an engineered paromamine-producing strain of S. venezuelae successfully generated gentamicin A(2), indicating that GtmE is another glycosyltransferase that attaches d-xylose to paromamine. These results represent in vivo evidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside.

Metabolic responses of sulfatide and related glycolipids in Madin-Darby canine kidney (MDCK) cells under osmotic stresses.

Incorporation of (35)S-sulfate into the polar molecular species of sulfoglycolipids (SM4s) in Madin-Darby canine kidney cells increased in a hypertonic medium (500 mOsm/L) supplemented with sodium chloride. The unknown sulfoglycolipid (SX) was identified as GlcCer sulfate based on the results of TLC, GLC, and mass spectra. The synthesis of SX increased in the hypotonic medium unlike that of SM4s and SM3. TLC showed that hypertonic stress induced the accumulation of GalCer as a precursor of SM4s, whereas hypotonic stress increased GlcCer as a precursor of GlcCer sulfate. The level of ceramide as a precursor of both GalCer and GlcCer increased under hypertonic stress and decreased under hypotonic stress. Cerebroside sulfotransferase mRNA was shown to be elevated in the hyperosmotic condition but not in the hypotonic condition. The increase in SM4s under hypertonic stress was induced by the activation of both the ceramide galactosyltransferase and the cerebroside sulfotransferase genes, whereas the increase in GlcCer sulfate under hypotonic stress was caused by the accumulation of GlcCer as the result of activation of ceramide glucosyltransferase.

Sulfatide storage in neurons causes hyperexcitability and axonal degeneration in a mouse model of metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by deficiency in the sulfolipid degrading enzyme arylsulfatase A (ASA). In the absence of a functional ASA gene, 3-O-sulfogalactosylceramide (sulfatide; SGalCer) and other sulfolipids accumulate. The storage is associated with progressive demyelination and various finally lethal neurological symptoms. Lipid storage, however, is not restricted to myelin-producing cells but also occurs in neurons. It is unclear whether neuronal storage contributes to symptoms of the patients. Therefore, we have generated transgenic ASA-deficient [ASA(-/-)] mice overexpressing the sulfatide synthesizing enzymes UDP-galactose:ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST) in neurons to provoke neuronal lipid storage. CGT-transgenic ASA(-/-) [CGT/ASA(-/-)] mice showed an accumulation of C18:0 fatty acid-containing SGalCer in the brain. Histochemically, an increase in sulfolipid storage could be detected in central and peripheral neurons of both CGT/ASA(-/-) and CST/ASA(-/-) mice compared with ASA(-/-) mice. CGT/ASA(-/-) mice developed severe neuromotor coordination deficits and weakness of hindlimbs and forelimbs. Light and electron microscopic analyses demonstrated nerve fiber degeneration in the spinal cord of CGT/ASA(-/-) mice. CGT/ASA(-/-) and, to a lesser extent, young ASA(-/-) mice exhibited cortical hyperexcitability, with recurrent spontaneous cortical EEG discharges lasting 5-15 s. These observations suggest that SGalCer accumulation in neurons contributes to disease phenotype.

Modest phenotypic improvements in ASA-deficient mice with only one UDP-galactose:ceramide-galactosyltransferase gene.

BACKGROUND: Arylsulfatase A (ASA)-deficient mice are a model for the lysosomal storage disorder metachromatic leukodystrophy. This lipidosis is characterised by the lysosomal accumulation of the sphingolipid sulfatide. Storage of this lipid is associated with progressive demyelination. We have mated ASA-deficient mice with mice heterozygous for a non-functional allele of UDP-galactose:ceramide-galactosyltransferase (CGT). This deficiency is known to lead to a decreased synthesis of galactosylceramide and sulfatide, which should reduce sulfatide storage and improve pathology in ASA-deficient mice. RESULTS: ASA-/- CGT+/- mice, however, showed no detectable decrease in sulfatide storage. Neuronal degeneration of cells in the spiral ganglion of the inner ear, however, was decreased. Behavioural tests showed small but clear improvements of the phenotype in ASA-/- CGT+/- mice. CONCLUSION: Thus the reduction of galactosylceramide and sulfatide biosynthesis by genetic means overall causes modest improvements of pathology.

Disruption of axo-glial junctions causes cytoskeletal disorganization and degeneration of Purkinje neuron axons.

Axo-glial junctions (AGJs) play a critical role in the organization and maintenance of molecular domains in myelinated axons. Neurexin IV/Caspr1/paranodin (NCP1) is an important player in the formation of AGJs because it recruits a paranodal complex implicated in the tethering of glial proteins to the axonal membrane and cytoskeleton. Mice deficient in either the axonal protein NCP1 or the glial ceramide galactosyltransferase (CGT) display disruptions in AGJs and severe ataxia. In this article, we correlate these two phenotypes and show that both NCP1 and CGT mutants develop large swellings accompanied by cytoskeletal disorganization and degeneration in the axons of cerebellar Purkinje neurons. We also show that alphaII spectrin is part of the paranodal complex and that, although not properly targeted, this complex is still formed in CGT mutants. Together, these findings establish a physiologically relevant link between AGJs and axonal cytoskeleton and raise the possibility that some neurodegenerative disorders arise from disruption of the AGJs.

The molecular relationship between deficient UDP-galactose uridyl transferase (GALT) and ceramide galactosyltransferase (CGT) enzyme function: a possible cause for poor long-term prognosis in classic galactosemia.

Classic galactosemia is an autosomal recessive disorder that is caused by activity deficiency of the UDP-galactose uridyl transferase (GALT). The clinical spectrum of classic galactosemia differs according to the type and number of mutations in the GALT gene. Short-term clinical symptoms such as jaundice, hepatomegaly, splenomegaly and E. coli sepsis are typically associated with classic galactosemia. These symptoms are often severe but quickly ameliorate with dietary restriction of galactose. However, long-term symptoms such as mental retardation and primary ovarian failure do not resolve irrespective of dietary intervention or the period of initial dietary intervention. There seem to be an association between deficient galactosylation of cerebrosides and classic galactosemia. Galactocerebrosides and glucocerebrosides are the primary products of the enzyme UDP-galactose:cerebroside galactosyl transferase (CGT). There has been an observation of deficient galactosylation coupled with over glucosylation in the brain tissue specimens sampled from deceased classic galactosemia patients. The plausible mechanism with which the association between GALT and CGT had not been explained before. Yet, UDP-galactose serves as the product of GALT as well as a substrate for CGT. In classic galactosemia, there is a consistent deficiency in cerebroside galactosylation. We postulate that the molecular link between defective GALT enzyme, which result in classic galactosemia; and the cerebroside galactosyl transferase, which is responsible for galactosylation of cerebrosides is dependent on the cellular concentrations of UDP-galactose. We further hypothesize that a threshold concentration of UDP-galactose exist below which the integrity of cerebroside galactosylation suffers.