NAAG synthetase deficiency has only low influence on pathogenesis in a Canavan disease mouse model.

Canavan disease (CD) is a leukodystrophy caused by mutations in the N-acetylaspartate (NAA) hydrolase aspartoacylase (ASPA). Inability to degrade NAA and its accumulation in the brain results in spongiform myelin degeneration. NAA is mainly synthesized by neurons, where it is also a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). Hydrolysis of this peptide by glutamate carboxypeptidases is an additional source of extracellular NAA besides the instant neuronal release of NAA. This study examines to what extent NAA released from NAAG contributes to NAA accumulation and pathogenesis in the brain of Aspanur7/nur7 mutant mice, an established model of CD. Towards this aim, Aspanur7/nur7 mice with additional deficiencies in NAAG synthetase genes Rimklb and/or Rimkla were generated. Loss of myelin in Aspanur7/nur7 mice was not significantly affected by Rimkla and Rimklb deficiency and there was also no obvious change in the extent of brain vacuolation. Astrogliosis was slightly reduced in the forebrain of Rimkla and Rimklb double deficient Aspanur7/nur7 mice. However, only minor improvements at the behavioral level were found. The brain NAA accumulation in CD mice was, however, not significantly reduced in the absence of NAAG synthesis. In summary, there was only a weak tendency towards reduced pathogenic symptoms in Aspanur7/nur7 mice deficient in NAAG synthesis. Therefore, we conclude that NAAG metabolism has little influence on NAA accumulation in Aspanur7/nur7 mice and development of pathological symptoms in CD.

Decreased turnover of the CNS myelin protein Opalin in a mouse model of hereditary spastic paraplegia 35.

Spastic paraplegia 35 (SPG35) (OMIM: 612319) or fatty acid hydroxylase-associated neurodegeneration (FAHN) is caused by deficiency of fatty acid 2-hydroxylase (FA2H). This enzyme synthesizes sphingolipids containing 2-hydroxylated fatty acids, which are particularly abundant in myelin. Fa2h-deficient (Fa2h-/-) mice develop symptoms reminiscent of the human disease and therefore serve as animal model of SPG35. In order to understand further the pathogenesis of SPG35, we compared the proteome of purified CNS myelin isolated from wild type and Fa2h-/- mice at different time points of disease progression using tandem mass tag labeling. Data analysis with a focus on myelin membrane proteins revealed a significant increase of the oligodendrocytic myelin paranodal and inner loop protein (Opalin) in Fa2h-/- mice, whereas the concentration of other major myelin proteins was not significantly changed. Western blot analysis revealed an almost 6-fold increase of Opalin in myelin of Fa2h-/- mice aged 21-23 months. A concurrent unaltered Opalin gene expression suggested a decreased turnover of the Opalin protein in Fa2h-/- mice. Supporting this hypothesis, Opalin protein half-life was reduced significantly when expressed in CHO cells synthesizing 2-hydroxylated sulfatide, compared to cells synthesizing only non-hydroxylated sulfatide. Degradation of Opalin was inhibited by inhibitors of lysosomal degradation but unaffected by proteasome inhibitors. Taken together, these results reveal a new function of 2-hydroxylated sphingolipids namely affecting the turnover of a myelin membrane protein. This may play a role in the pathogenesis of SPG35.

An enzymatic fluorimetric assay for determination of N-acetylaspartate.

N-acetylaspartate (NAA) is an abundant metabolite in the mammalian brain and a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). The physiological role of NAA is not fully understood and requires further studies. We here describe the development of a coupled enzymatic fluorimetric assay for the determination of NAA in biological samples. Deproteinized tissue extracts are first passed through a strong cation exchange column to remove aspartate. NAA in the sample is hydrolysed by aspartoacylase and released aspartate oxidized using l-aspartate oxidase. Generated H2O2 is measured with peroxidase in a fluorimetric assay using Ampliflu Red. The limit of detection and the lower limit of quantification are 1.0 µM (10 pmol/well) and 3.3 µM (33 pmol/well), respectively, with a linear range to 100 µM. Specificity of the assay was confirmed using samples from mice deficient in NAA synthase Nat8l that were spiked with NAA. Analysis of samples from aspartoacylase-deficient mice showed a 2 to 3-fold increase in brain NAA concentration, in line with previous reports. Mice lacking NAAG synthetases had a slightly reduced (-10%) brain NAA level. Thus, the new fluorimetric enzymatic assay is useful to perform sensitive and large scale quantification of NAA in biological samples without the need for expensive equipment.

Citrylglutamate synthase deficient male mice are subfertile with impaired histone and transition protein 2 removal in late spermatids.

Chromatin remodelling in spermatids is an essential step in spermiogenesis and involves the exchange of most histones by protamines, which drives chromatin condensation in late spermatids. The gene Rimklb encodes a citrylglutamate synthase highly expressed in testes of vertebrates and the increase of its reaction product, ß-citrylglutamate, correlates in time with the appearance of spermatids. Here we show that deficiency in a functional Rimklb gene leads to male subfertility, which could be partially rescued by in vitro fertilization. Rimklb-deficient mice are impaired in a late step of spermiogenesis and produce spermatozoa with abnormally shaped heads and nuclei. Sperm chromatin in Rimklb-deficient mice was less condensed and showed impaired histone to protamine exchange and retained transition protein 2. These observations suggest that citrylglutamate synthase, probably via its reaction product ß-citrylglutamate, is essential for efficient chromatin remodelling during spermiogenesis and may be a possible candidate gene for male subfertility or infertility in humans.

Fatty Acid 2-Hydroxylase and 2-Hydroxylated Sphingolipids: Metabolism and Function in Health and Diseases.

Sphingolipids containing acyl residues that are hydroxylated at C-2 are found in most, if not all, eukaryotes and certain bacteria. 2-hydroxylated sphingolipids are present in many organs and cell types, though they are especially abundant in myelin and skin. The enzyme fatty acid 2-hydroxylase (FA2H) is involved in the synthesis of many but not all 2-hydroxylated sphingolipids. Deficiency in FA2H causes a neurodegenerative disease known as hereditary spastic paraplegia 35 (HSP35/SPG35) or fatty acid hydroxylase-associated neurodegeneration (FAHN). FA2H likely also plays a role in other diseases. A low expression level of FA2H correlates with a poor prognosis in many cancers. This review presents an updated overview of the metabolism and function of 2-hydroxylated sphingolipids and the FA2H enzyme under physiological conditions and in diseases.

Optical genome mapping reveals additional prognostic information compared to conventional cytogenetics in AML/MDS patients.

Cytogenetic diagnostics play a crucial role in risk stratification and classification of myeloid malignancies such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), thus influencing treatment decisions. Optical genome mapping (OGM) is a novel whole genome method for the detection of cytogenetic abnormalities. Our study assessed the applicability and practicality of OGM as diagnostic tool in AML and MDS patients. In total, 27 patients with AML or MDS underwent routine diagnostics including classical karyotyping and fluorescence in situ hybridization (FISH) or real-time PCR analysis wherever indicated as well as OGM following a recently established workflow. Methods were compared regarding concordance and content of information. In 93%, OGM was concordant to classical karyotyping and a total of 61 additional variants in a predefined myeloid gene-set could be detected. In 67% of samples the karyotype could be redefined by OGM. OGM offers a whole genome approach to cytogenetic diagnostics in AML and MDS with a high concordance to classical cytogenetics. The method has the potential to enter routine diagnostics as a gold standard for cytogenetic diagnostics widely superseding FISH. Furthermore, OGM can serve as a tool to identify genetic regions of interest and future research regarding tumor biology.

Absence of endogenous carnosine synthesis does not increase protein carbonylation and advanced lipoxidation end products in brain, kidney or muscle.

Carnosine and other histidine-containing dipeptides are expected to be important anti-oxidants in vertebrates based on various in vitro and in vivo studies with exogenously administered carnosine or its precursor ß-alanine. To examine a possible anti-oxidant role of endogenous carnosine, mice lacking carnosine synthase (Carns1-/-) had been generated and were examined further in the present study. Protein carbonylation increased significantly between old (18 months) and aged (24 months) mice in brain and kidney but this was independent of the Carns1 genotype. Lipoxidation end products were not increased in 18-month-old Carns1-/- mice compared to controls. We also found no evidence for compensatory increase of anti-oxidant enzymes in Carns1-/- mice. To explore the effect of carnosine deficiency in a mouse model known to suffer from increased oxidative stress, Carns1 also was deleted in the type II diabetes model Leprdb/db mouse. In line with previous studies, malondialdehyde adducts were elevated in Leprdb/db mouse kidney, but there was no further increase by additional deficiency in Carns1. Furthermore, Leprdb/db mice lacking Carns1 were indistinguishable from conventional Leprdb/db mice with respect to fasting blood glucose and insulin levels. Taken together, Carns1 deficiency appears not to reinforce oxidative stress in old mice and there was no evidence for a compensatory upregulation of anti-oxidant enzymes. We conclude that the significance of the anti-oxidant activity of endogenously synthesized HCDs is limited in mice, suggesting that other functions of HCDs play a more important role.

Carnosine synthase deficiency is compatible with normal skeletal muscle and olfactory function but causes reduced olfactory sensitivity in aging mice.

Carnosine (ß-alanyl-l-histidine) and anserine (ß-alanyl-3-methyl-l-histidine) are abundant peptides in the nervous system and skeletal muscle of many vertebrates. Many in vitro and in vivo studies demonstrated that exogenously added carnosine can improve muscle contraction, has antioxidant activity, and can quench various reactive aldehydes. Some of these functions likely contribute to the proposed anti-aging activity of carnosine. However, the physiological role of carnosine and related histidine-containing dipeptides (HCDs) is not clear. In this study, we generated a mouse line deficient in carnosine synthase (Carns1). HCDs were undetectable in the primary olfactory system and skeletal muscle of Carns1-deficient mice. Skeletal muscle contraction in these mice, however, was unaltered, and there was no evidence for reduced pH-buffering capacity in the skeletal muscle. Olfactory tests did not reveal any deterioration in 8-month-old mice lacking carnosine. In contrast, aging (18-24-month-old) Carns1-deficient mice exhibited olfactory sensitivity impairments that correlated with an age-dependent reduction in the number of olfactory receptor neurons. Whereas we found no evidence for elevated levels of lipoxidation and glycation end products in the primary olfactory system, protein carbonylation was increased in the olfactory bulb of aged Carns1-deficient mice. Taken together, these results suggest that carnosine in the olfactory system is not essential for information processing in the olfactory signaling pathway but does have a role in the long-term protection of olfactory receptor neurons, possibly through its antioxidant activity.

Mice deficient in the NAAG synthetase II gene Rimkla are impaired in a novel object recognition task.

N-acetylaspartylglutamate (NAAG) is an abundant neuropeptide in the mammalian nervous system, synthesized by two related NAAG synthetases I and II (NAAGS-I and -II) encoded by the genes Rimklb and Rimkla, respectively. NAAG plays a role in cognition and memory, according to studies using inhibitors of the NAAG hydrolase glutamate carboxypeptidase II that increase NAAG concentration. To examine consequences of reduced NAAG concentration, Rimkla-deficient (Rimkla-/- ) mice were generated. These mice exhibit normal NAAG level at birth, likely because of the intact Rimklb gene, but have significantly reduced NAAG levels in all brain regions in adulthood. In wild type mice NAAGS-II was most abundant in brainstem and spinal cord, as demonstrated using a new NAAGS-II antiserum. In the hippocampus, NAAGS-II was only detectable in neurons expressing parvalbumin, a marker of GABAergic interneurons. Apart from reduced open field activity, general behavior of adult (6 months old) Rimkla-/- mice examined in different tests (dark-light transition, optokinetic behavior, rotarod, and alternating T-maze) was not significantly altered. However, Rimkla-/- mice were impaired in a short-term novel object recognition test. This was also the case for mice lacking NAA synthase Nat8l, which are devoid of NAAG. Together with results from previous studies showing that inhibition of the NAAG degrading enzyme glutamate carboxypeptidase II is associated with a significant improvement in object recognition, these results suggest a direct involvement of NAAG synthesized by NAAGS-II in the memory consolidation underlying the novel object recognition task.

CNS myelin protein 36K regulates oligodendrocyte differentiation through Notch.

In contrast to humans and other mammals, zebrafish can successfully regenerate and remyelinate central nervous system (CNS) axons following injury. In addition to common myelin proteins found in mammalian myelin, 36K protein is a major component of teleost fish CNS myelin. Although 36K is one of the most abundant proteins in zebrafish brain, its function remains unknown. Here we investigate the function of 36K using translation-blocking Morpholinos. Morphant larvae showed fewer dorsally migrated oligodendrocyte precursor cells as well as upregulation of Notch ligand. A gamma secretase inhibitor, which prevents activation of Notch, could rescue oligodendrocyte precursor cell numbers in 36K morphants, suggesting that 36K regulates initial myelination through inhibition of Notch signaling. Since 36K like other short chain dehydrogenases might act on lipids, we performed thin layer chromatography and mass spectrometry of lipids and found changes in lipid composition in 36K morphant larvae. Altogether, we suggest that during early development 36K regulates membrane lipid composition, thereby altering the amount of transmembrane Notch ligands and the efficiency of intramembrane gamma secretase processing of Notch and thereby influencing oligodendrocyte precursor cell differentiation and further myelination. Further studies on the role of 36K short chain dehydrogenase in oligodendrocyte precursor cell differentiation during remyelination might open up new strategies for remyelination therapies in human patients.

A progesterone receptor membrane component 1 antagonist induces large vesicles independent of progesterone receptor membrane component 1 expression.

Treatment of different cell lines with progesterone receptor membrane component 1 (PGRMC1) antagonist AG-205 rapidly induces the formation of large vesicular structures that likely represent endosomes. Crispr/Cas9 was used to target the PGRMC1 and progesterone receptor membrane component 2 (PGRMC2) genes in CHO-K1 and HeLa. Unexpectedly, deficiency in one of these or both genes did not inhibit the formation of enlarged vesicles by AG-205, demonstrating additional molecular target(s) of this compound besides PGRMC1. Thus, AG-205 cannot be regarded as a PGRMC1-specific antagonist. However, provided that its currently unknown target(s) will be identified, AG-205 may serve as a new reagent to study endosomal trafficking.

N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

N-acetylaspartate (NAA) is synthesized by aspartate N-acetyltransferase (gene: Nat8l) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) Nat8l-ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of Nat8l-ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as Nat8l-ko and Nat8l-ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, Nat8l-deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased de novo lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

BDNF and NGF signals originating from sensory ganglia promote cranial motor axon growth.

After exiting the hindbrain, branchial motor axons reach their targets in association with sensory ganglia. The trigeminal ganglion has been shown to promote motor axon growth from rhombomeres 2/3 and 4/5, but it is unknown whether this effect is ganglion specific and through which signals it is mediated. Here, we addressed these questions by co-cultures of ventral rhombomere 8 explants with cranial and spinal sensory ganglia in a collagen gel matrix. Our results show that all cranial sensory ganglia and even a trunk dorsal root ganglion can promote motor axon growth and that ganglia isolated from older embryos had a stronger effect on the axonal growth than younger ones. We found that brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are necessary and sufficient for this effect. Altogether, our results demonstrate that the promoting effect of sensory ganglia on cranial motor axon growth is stage dependent, but not ganglion specific and is mediated by BDNF and NGF signals.

Transgenic overexpression of polysialyltransferase ST8SiaIV under the control of a neuron-specific promoter does not affect brain development but impairs exploratory behavior.

A large body of the literature has demonstrated that the polysialic acid (polySia) modification of the neural cell adhesion molecule (NCAM) is a key regulator of cellular interactions during brain development, maintenance and plasticity. To properly fulfill these functions, polySia concentration has to be carefully controlled. This is done by the regulation of the expression of the two polySia-synthesizing enzymes ST8SiaII and ST8SiaIV. From this point of view we and others have demonstrated that downregulation of ST8SiaIV during oligodendrocyte differentiation is a prerequisite for efficient myelin formation and maintenance. Here, we addressed the question whether the prevention of polySia downregulation in neurons affects brain and particularly myelin development and functioning. For this purpose, we developed transgenic (tg) mouse lines overexpressing the polysialyltransferase ST8SiaIV in neurons. tg expression of ST8SiaIV prevented the postnatal downregulation of polySia, and most of the polySias in the forebrain and brain stem of adult tg mice were associated with NCAM-140 and NCAM-180 isoforms. Structural examination of the brain revealed no overt abnormalities of axons and myelin. In addition, ultrastructural and western blot analyses indicated normal myelin development. However, behavioral studies revealed reduced rearing activity, a measure for exploratory behavior, while parameters of motor activity were not affected in tg mice. Taken together, these results suggest that a persisting presence of polySia in neurons has no major effect on brain structure, myelination and myelin maintenance, but causes mild behavioral changes.

Identification of progesterone receptor membrane component-1 as an interaction partner and possible regulator of fatty acid 2-hydroxylase.

The fatty acid 2-hydroxylase (FA2H) is essential for synthesis of 2-hydroxylated fatty acids in myelinating and other cells, and deficiency of this enzyme causes a complicated form of hereditary spastic paraplegia also known as fatty acid hydroxylase-associated neurodegeneration. Despite its important role in sphingolipid metabolism, regulation of FA2H and its interaction with other proteins involved in the same or other metabolic pathways is poorly understood. To identify potential interaction partners of the enzyme, quantitative mass spectrometry using stable isotope labeling of cells was combined with formaldehyde cross-linking and proximity biotinylation, respectively. Besides other enzymes involved in sphingolipid synthesis and intermembrane transfer of ceramide, and putative redox partners of FA2H, progesterone receptor membrane component-1 (PGRMC1) and PGRMC2 were identified as putative interaction partners. These two related heme-binding proteins are known to regulate several cytochrome P450 enzymes. Bimolecular fluorescence complementation experiments confirmed the interaction of FA2H with PGRMC1. Moreover, the PGRMC1 inhibitor AG-205 significantly reduced synthesis of hydroxylated ceramide and glucosylceramide in FA2H-expressing cells. This suggests that PGRMC1 may regulate FA2H activity, possibly through its heme chaperone activity.

Diastereomer-specific quantification of bioactive hexosylceramides from bacteria and mammals.

Mammals synthesize, cell-type specifically, the diastereomeric hexosylceramides, ß-galactosylceramide (GalCer) and ß-glucosylceramide (GlcCer), which are involved in several diseases, such as sphingolipidosis, diabetes, chronic kidney diseases, or cancer. In contrast, Bacteroides fragilis, a member of the human gut microbiome, and the marine sponge, Agelas mauritianus, produce α-GalCer, one of the most potent stimulators for invariant natural killer T cells. To dissect the contribution of these individual stereoisomers to pathologies, we established a novel hydrophilic interaction chromatography-based LC-MS2 method and separated (R > 1.5) corresponding diastereomers from each other, independent of their lipid anchors. Testing various bacterial and mammalian samples, we could separate, identify (including the lipid anchor composition), and quantify endogenous ß-GlcCer, ß-GalCer, and α-GalCer isomers without additional derivatization steps. Thereby, we show a selective decrease of ß-GlcCers versus ß-GalCers in cell-specific models of GlcCer synthase-deficiency and an increase of specific ß-GlcCers due to loss of ß-glucoceramidase 2 activity. Vice versa, ß-GalCer increased specifically when cerebroside sulfotransferase (Gal3st1) was deleted. We further confirm ß-GalCer as substrate of globotriaosylceramide synthase for galabiaosylceramide synthesis and identify additional members of the human gut microbiome to contain immunogenic α-GalCers. Finally, this method is shown to separate corresponding hexosylsphingosine standards, promoting its applicability in further investigations.

N-Acetylaspartate Synthase Deficiency Corrects the Myelin Phenotype in a Canavan Disease Mouse Model But Does Not Affect Survival Time.

Canavan disease (CD) is a severe, lethal leukodystrophy caused by deficiency in aspartoacylase (ASPA), which hydrolyzes N-acetylaspartate (NAA). In the brains of CD patients, NAA accumulates to high millimolar concentrations. The pathology of the disease is characterized by loss of oligodendrocytes and spongy myelin degeneration in the CNS. Whether accumulating NAA, absence of NAA-derived acetate, or absence of any unknown functions of the ASPA enzyme is responsible for the pathology of the disease is not fully understood. We generated ASPA-deficient (Aspa(nur7/nur7)) mice that are also deficient for NAA synthase Nat8L (Nat8L(-/-)/Aspa(nur7/nur7)). These mice have no detectable NAA. Nevertheless, they exhibited normal myelin content, myelin sphingolipid composition, and full reversal of spongy myelin and axonal degeneration. Surprisingly, although pathology was fully reversed, the survival time of the mice was not prolonged. In contrast, Aspa(nur7/nur7) mice with only one intact Nat8L allele accumulated less NAA, developed a less severe pathology, phenotypic improvements, and, importantly, an almost normal survival time. Therefore, inhibition of NAA synthase is a promising therapeutic option for CD. The reduced survival rate of Nat8L(-/-)/Aspa(nur7/nur7) mice, however, indicates that complete inhibition of NAA synthase may bear unforeseeable risks for the patient. Furthermore, we demonstrate that acetate derived from NAA is not essential for myelin lipid synthesis and that loss of NAA-derived acetate does not cause the myelin phenotype of Aspa(nur7/nur7) mice. Our data clearly support the hypothesis that NAA accumulation is the major factor in the development of CD.

Sequential C-H Arylation and Enantioselective Hydrogenation Enables Ideal Asymmetric Entry to the Indenopiperidine Core of an 11ß-HSD-1 Inhibitor.

A concise asymmetric synthesis of an 11ß-HSD-1 inhibitor has been achieved using inexpensive starting materials with excellent step-economy at low catalyst loadings. The catalytic enantioselective total synthesis of 1 was accomplished in 7 steps and 38% overall yield aided by the development of an innovative, sequential strategy involving Pd-catalyzed pyridinium C-H arylation and Ir-catalyzed asymmetric hydrogenation of the resulting fused tricyclic indenopyridinium salt highlighted by the use of a unique P,N-ligand (MeO-BoQPhos) with 1000 ppm of [Ir(COD)Cl]2.

Arylsulfatase A Overexpressing Human iPSC-derived Neural Cells Reduce CNS Sulfatide Storage in a Mouse Model of Metachromatic Leukodystrophy.

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disorder resulting from a functional deficiency of arylsulfatase A (ARSA), an enzyme that catalyzes desulfation of 3-O-sulfogalactosylceramide (sulfatide). Lack of active ARSA leads to the accumulation of sulfatide in oligodendrocytes, Schwann cells and some neurons and triggers progressive demyelination, the neuropathological hallmark of MLD. Several therapeutic approaches have been explored, including enzyme replacement, autologous hematopoietic stem cell-based gene therapy, intracerebral gene therapy or cell-based gene delivery into the central nervous system (CNS). However, long-term treatment of the blood-brain-barrier protected CNS remains challenging. Here we used MLD patient-derived induced pluripotent stem cells (iPSCs) to generate long-term self-renewing neuroepithelial stem cells and astroglial progenitors for cell-based ARSA replacement. Following transplantation of ARSA-overexpressing precursors into ARSA-deficient mice we observed a significant reduction of sulfatide storage up to a distance of 300 µm from grafted cells. Our data indicate that neural precursors generated via reprogramming from MLD patients can be engineered to ameliorate sulfatide accumulation and may thus serve as autologous cell-based vehicle for continuous ARSA supply in MLD-affected brain tissue.

Loss of electrostatic cell-surface repulsion mediates myelin membrane adhesion and compaction in the central nervous system.

During the development of the central nervous system (CNS), oligodendrocytes wrap their plasma membrane around axons to form a multilayered stack of tightly attached membranes. Although intracellular myelin compaction and the role of myelin basic protein has been investigated, the forces that mediate the close interaction of myelin membranes at their external surfaces are poorly understood. Such extensive bilayer-bilayer interactions are usually prevented by repulsive forces generated by the glycocalyx, a dense and confluent layer of large and negatively charged oligosaccharides. Here we investigate the molecular mechanisms underlying myelin adhesion and compaction in the CNS. We revisit the role of the proteolipid protein and analyze the contribution of oligosaccharides using cellular assays, biophysical tools, and transgenic mice. We observe that differentiation of oligodendrocytes is accompanied by a striking down-regulation of components of their glycocalyx. Both in vitro and in vivo experiments indicate that the adhesive properties of the proteolipid protein, along with the reduction of sialic acid residues from the cell surface, orchestrate myelin membrane adhesion and compaction in the CNS. We suggest that loss of electrostatic cell-surface repulsion uncovers weak and unspecific attractive forces in the bilayer that bring the extracellular surfaces of a membrane into close contact over long distances.