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In the metagenetic life-cycle of the scyphozoan Cassiopea xamachana metamorphosis of planula-larvae or larva-like buds to polyps is triggered by specific external cues which are transmitted inside the larva or bud where internal signals finally coordinate the initiation of metamorphosis. This study deals with an endogenous metamorphosis inducer present in planulae and buds of Cassiopea. The inductive cue is localized in the basal part of the buds and can be characterized as a peptide with an apparent molecular weight of about 7,000 Da. Further purification was performed via reversed phase HPLC on a C18 column. Additional inhibitor assays revealed that protein kinase C and PI3 kinase, two known elements of the metamorphosis-inducing signal transduction cascade in Cassiopea, may act downstream of the endogenous inducing peptide.

Methods for isolation of neutral lysoglycosphingolipids (n-lyso-GSLs) such as glucosylsphingosine and galactosylsphingosine normally involve mild alkaline or acid hydrolysis followed by multiple chromatography steps, yielding relatively low recoveries of n-lyso-GSLs and neutral glycosphingolipids (n-GSLs). We now describe a new technique for isolating these compounds using one chromatography step, resulting in quantitative recovery of n-GSLs and n-lyso-GSLs. Lipids are extracted using a modified Folch procedure in which recovery is optimized by reextracting the Folch upper phase with water-saturated butanol. The extract is applied to an aminopropyl solid phase column from which both n-GSLs and n-lyso-GSLs elute in the same fraction. Separation is achieved using a new two-dimensional thin-layer chromatography procedure. The usefulness of this technique for biological samples was tested by examining Glc[4,5-(3)H]ceramide and Glc[4,5-(3)H]sphingosine accumulation in metabolically-labeled neurons treated with an inhibitor of lysosomal glucocerebrosidase. Accurate quantification of both lipids was obtained with Glc[4,5-(3)H]ceramide and Glc[4,5-(3)H]sphingosine accumulating at levels of 20 nmol/mg DNA and 40 pmol/mg DNA, respectively. This simple and rapid technique can therefore be used for the analysis of lyso-GSLs and GSLs in the same tissue, which may permit the determination of their metabolic pathways in normal and in pathological tissues, such as those taken from Gaucher and Krabbe's disease patients.

Krabbe disease (globoid cell leukodystrophy) is an inherited recessive autosomal leukodystrophy caused by deficiency of the enzyme galactocerebrosidase. The lack of this enzyme leads to the build-up of galactolipids that will promote the death of oligodendrocytes and the demyelination of the central and peripheral nervous systems. There are two clinical forms: early onset and late onset. This article reports a case of late onset Krabbe disease and discusses the importance of early diagnosis for its prognosis. Doença de Krabbe (leucodistrofia de células globoides) é uma leucodistrofia de herança autossômica recessiva causada pela deficiência da enzima galactocerebrosidase. A falta dessa enzima leva ao acúmulo de galactolipídeos que irão promover a morte dos oligodendrócitos e a desmielinização do sistema nervoso central e periférico. Possui duas formas clínicas: de início precoce e de aparecimento tardio. O presente artigo relata um caso da apresentação tardia da doença de Krabbe e discute a importância do diagnóstico precoce para o seu prognóstico.

Globoid cell leukodystrophy (GLD) is an autosomal recessive disorder of infants, caused by deficient activity of cerebroside-β-galactosidase resulting in loss of myelin accompanied by loss of oligodendrocytes. The loss of oligodendrocyte population is accompanied by accumulation of psychosine, which is considered as the molecule responsible for the observed pathophysiology of GLD. We were able to detect apoptotic cells by terminal dUTP nick-end labeling assay and nuclear localization of p53 in postmortem brain tissue of Krabbe's disease patients, which were not detected in the control brain. To study the role of psychosine in cell death, we investigated the effect of psychosine on C6 glial cell survival by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Similar to ceramide (43.8% loss) the galactopsychosine and glucopsychosine treatment killed up to 46.3 and 48.75% of cells, respectively. On the other hand, sphingosine had no effect. DNA laddering assay confirmed these results. Moreover, psychosine-induced detection of annexin-V positive cells supports a role for psychosine in C6 glial cell death via the apoptotic pathway. These results indicate that psychosine may play a role in apoptotic cell loss observed in GLD brain.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of clinical manifestations including neuronopathic and non-neuronopathic forms. While the lipid glucosylceramide is stored in both patients with Gaucher disease and in a null allele mouse model of Gaucher disease, elevated levels of a second potentially toxic substrate, glucosylsphingosine, are also found. Using high performance liquid chromatography, glucosylsphingosine levels were measured in tissues from patients with type 1, 2, and 3 Gaucher disease. Glucosylsphingosine was measured in 16 spleen samples (8 type 1; 4 type 2; and 4, type 3) and levels ranged from 54 to 728 ng/mg protein in the patients with type 1 disease, 133 to 1200 ng/mg protein in the patients with type 2, and 109 to 1298 ng/mg protein in the type 3 samples. The levels of splenic glucosylsphingosine bore no relation to the type of Gaucher disease, the age of the patient, the genotype, nor the clinical course. In the same patients, hepatic glucosylsphingosine levels were lower than in spleen. Glucosylsphingosine was also measured in brains from 13 patients (1 type 1; 8 type 2; and 4 type 3). While the glucosylsphingosine level in the brain from the type 1 patient, 1.0 ng/mg protein, was in the normal range, the levels in the type 3 samples ranged from 14 to 32 ng/mg protein, and in the type 2 samples from 24 to 437 ng/mg protein, with the highest values detected in two fetuses with hydrops fetalis. The elevated levels found in brains from patients with neuronopathic Gaucher disease support the hypothesis that glucosylsphingosine may contribute to the nervous system involvement in these patients.

Modifier genes may account for the phenotypic variability observed in the late-onset forms of globoid cell leukodystrophy (GCL) in humans. In order to begin a search for modifier genes, the effect of genetic background on the clinical and pathological manifestations of GCL was investigated in twitcher mice. Twitcher mice on a C57BL/6 × CAST/Ei background had an increased life span (61.4 ± 2.5 vs 37.0 ± 0.6 days), a delayed onset of tremor (24 vs 21 days), and a delayed decline in walking ability compared to C57BL/6 twitcher mice. Pathologically, C57BL/6 × CAST/Ei twitcher mice had fewer lectin-positive globoid cells, less gliosis, and a greater preservation of myelin compared to C57BL/6 twitcher mice under moribund conditions. Similar concentrations of psychosine, the toxic species that accumulates in GCL, were measured by tandem mass spectrometry between moribund C57BL/6 twitcher mice (286.5 pmol/mg protein), 40-day C57BL/6 × CAST/Ei twitcher mice (276.5 pmol/mg), and moribund C57BL/6 × CAST/Ei twitcher mice (247.0 pmol/mg), suggesting that the milder phenotype in CAST/Ei × C57BL/6 twitcher mice did not correlate with less psychosine. In summary, the introduction of modifier genes from the wild, inbred CAST/Ei strain had a phenotypic effect resulting in a significantly slower disease course.

The twitcher mouse is a murine model of human globoid cell leukodystrophy (GLD; Krabbe disease) caused by a genetic defect in the activity of galactosylceramidase (GALC). An accumulation of cytotoxic metabolite, galactosylsphingosine (psychosine), in myelin forming cells (oligodendrocytes and Schwann cells) of the twitcher mouse as well as patients with GLD has been suggested to cause dysfunction of these cells and subsequent demyelination in the central and peripheral nervous system. To investigate further the cellular pathomechanism of GLD, we established spontaneously immortalized Schwann cell lines from the twitcher mouse. Long-term cultures of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of 3-week-old twitcher mice were maintained for 6 months, and spontaneously developed colonies were expanded further and characterized. One of the cell lines, designated TwS1, showed distinct Schwann cell phenotypes, was passaged twice a week and maintained for over 10 months without phenotypic alterations. The TwS1 cells had a nonsense mutation in the GALC genome, and showed markedly reduced GALC activity and elevated psychosine levels. Ultrastructurally, varieties of cytoplasmic inclusions were demonstrated in TwS1 cells. When TwS1 cells were infected with a retrovirus vector encoding GALC, GALC activity was markedly increased and psychosine levels were significantly decreased. These immortalized Schwann cells can be useful in studies on the nervous system lesions in GLD.

Globoid cell leukodystrophy (Krabbe disease) is characterized by the accumulation of a toxic metabolite, psychosine (galactosylsphingosine), which is a substrate for the deficient enzyme (galactocerebroside beta-galactosidase). This study underscores the possible role of psychosine in the effect of inducible nitric oxide synthase (iNOS) -derived NO in the pathophysiology of this demyelinating disease. For the first time, we provide evidence of the expression of iNOS in CNS of Krabbe patient and show that the iNOS-expressing cells in the CNS were astrocytes. Psychosine potentiated the LPS-induced production of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) in primary rat astrocytes and regulated the cytokine-mediated production of NO in C6 glioma and primary rat astrocyte. Psychosine induced cytokine-mediated nuclear translocation of AP-1 and C/EBP by potentiating the expression of Fra-1 and C/EBP-delta proteins. This suggests that psychosine maintained or sustained the cytokine-primed expression of iNOS by further potentiating the nuclear translocation of AP-1 and C/EBP without modulating the cytokine-mediated transcription activity of NF-kappaB. This study hypothesizes that accumulated psychosine leads to production of cytokines and iNOS expression. The ensuing excessive production of NO and ONOO- may play a role in pathogenesis of Krabbe disease.

During the course of studies on natural occurrence of sphingosine base in brain, cationic glycosphingolipids bound to carboxymethyl-Sephadex and eluted with triethylamine in organic solvents were isolated and characterized. Four classes of compounds were identified: (i) plasmalopsychosine-A and -B; (ii) glyceroplasmalopsychosine; (iii) glycosphingolipids having de-N-acetyl-hexosamine, e.g., de-N-acetyl-Lc3Cer; (iv) glycosylsphingosine, i.e., lysoglycosphingolipid. Only two kinds, galactosylsphingosine (psychosine) and lactosylsphingosine, were found to occur naturally in brain. All these compounds were isolated from extract of brain white matter. Their occurrence, quantity, and distribution pattern differ from one species to another. Their quantity is much lower than that of regular acidic and neutral glycosphingolipids. They may interact with regular glycosphingolipids in glycosphingolipid-enriched microdomains to elicit signal transduction, to modify cellular phenotype, although studies along this line are highly limited at this time.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a rare and often fatal demyelinating disease caused by mutations in the galactocerebrosidase (galc) gene that result in accumulation of galactosylsphingosine (psychosine). We recently reported that the extracellular matrix (ECM) protease, matrix metalloproteinase-3, is elevated in GLD and that it regulates psychosine-induced microglial activation. Here, we examined central nervous system ECM component expression in human GLD patients and in the twitcher mouse model of GLD using immunohistochemistry. The influence of ECM proteins on primary murine microglial responses to psychosine was evaluated using ECM proteins as substrates and analyzed by quantitative real-time polymerase chain reaction, immunocytochemistry, and ELISA. Functional analysis of microglial cytotoxicity was performed on oligodendrocytes in coculture, and cell death was measured by lactose dehydrogenase assay. Tenascin-C (TnC) was expressed at higher levels in human GLD and in twitcher mice versus controls. Microglial responses to psychosine were enhanced by TnC, as determined by an increase in globoid-like cell formation, matrix metalloproteinase-3 mRNA expression, and higher toxicity toward oligodendrocytes in culture. These findings were consistent with a shift toward the M1 microglial phenotype in TnC-grown microglia. Thus, elevated TnC expression in GLD modified microglial responses to psychosine. These data offer a novel perspective and enhance understanding of the microglial contribution to GLD pathogenesis.

Deficiency of glucocerebrosidase (GBA) leads to Gaucher disease (GD), an inherited disorder characterised by storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. Recently, we reported marked increases of deacylated GlcCer, named glucosylsphingosine (GlcSph), in plasma of GD patients. To improve quantification, [5-9] (13)C5-GlcSph was synthesised for use as internal standard with quantitative LC-ESI-MS/MS. The method was validated using plasma of 55 GD patients and 20 controls. Intra-assay variation was 1.8% and inter-assay variation was 4.9% for GlcSph (m/z 462.3). Plasma GlcSph levels with the old and new methods closely correlate (r=0.968, slope=1.038). Next, we analysed GlcSph in 24h urine samples of 30 GD patients prior to therapy. GlcSph was detected in the patient samples (median 1.20nM, range 0.11-8.92nM), but was below the limit of quantification in normal urine. Enzyme replacement therapy led to a decrease of urinary GlcSph of GD patients, coinciding with reductions in plasma GlcSph and markers of Gaucher cells (chitotriosidase and CCL18). In analogy to globotriaosylsphingsone in urine of Fabry disease patients, additional isoforms of GlcSph differing in structure of the sphingosine moiety were identified in GD urine samples. In conclusion, GlcSph can be sensitively detected by LC-ESI-MS/MS with an internal isotope standard. Abnormalities in urinary GlcSph are a hallmark of Gaucher disease allowing biochemical confirmation of diagnosis.

Niemann-Pick disease type C (NP-C) is a devastating, neurovisceral lysosomal storage disorder which is characterised by variable manifestation of visceral signs, progressive neuropsychiatric deterioration and premature death, caused by mutations in the NPC1 and NPC2 genes. Due to the complexity of diagnosis and the availability of an approved therapy in the EU, improved detection of NP-C may have a huge impact on future disease management. At the cellular level dysfunction or deficiency of either the NPC1 or NPC2 protein leads to a complex intracellular endosomal/lysosomal trafficking defect, and organ specific patterns of sphingolipid accumulation. Lysosphingolipids have been shown to be excellent biomarkers of sphingolipidosis in several enzyme deficient lysosomal storage disorders. Additionally, in a recent study the lysosphingolipids, lysosphingomyelin (SPC) and glucosylsphingosine (GlcSph), appeared to be elevated in the plasma of three adult NP-C patients. In order to investigate the clinical utility of SPC and GlcSph as diagnostic markers, an in-depth fit for purpose biomarker assay validation for measurement of these biomarkers in plasma by liquid chromatography-tandem mass spectrometry was performed. Plasma SPC and GlcSph are stable and can be measured accurately, precisely and reproducibly. In a retrospective analysis of 57 NP-C patients and 70 control subjects, median plasma SPC and GlcSph were significantly elevated in NP-C by 2.8-fold and 1.4-fold respectively. For miglustat-naive NP-C patients, aged 2–50 years, the area under the ROC curve was 0.999 for SPC and 0.776 for GlcSph. Plasma GlcSph did not correlate with SPC levels in NP-C patients. The data indicate excellent potential for the use of lysosphingomyelin in NP-C diagnosis, where it could be used to identify NP-C patients for confirmatory genetic testing.

Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes, particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore, we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition, we created induced pluripotent stem cell (iPSC)–derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages, reduced glycolipid storage, and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development.

Demyelination is a major contributor to the general decay of neural functions in children with Krabbe disease. However, recent reports have indicated a significant involvement of neurons and axons in the neuropathology of the disease. In this study, we have investigated the nature of cellular inclusions in the Krabbe brain. Brain samples from the twitcher mouse model for Krabbe disease and from patients affected with the infantile and late-onset forms of the disease were examined for the presence of neuronal inclusions. Our experiments demonstrated the presence of cytoplasmic aggregates of thioflavin-S-reactive material in both human and murine mutant brains. Most of these inclusions were associated with neurons. A few inclusions were detected to be associated with microglia and none were associated with astrocytes or oligodendrocytes. Thioflavin-S-reactive inclusions increased in abundance, paralleling the development of neurological symptoms, and distributed throughout the twitcher brain in areas of major involvement in cognition and motor functions. Electron microscopy confirmed the presence of aggregates of stereotypic β-sheet folded proteinaceous material. Immunochemical analyses identified the presence of aggregated forms of α-synuclein and ubiquitin, proteins involved in the formation of Lewy bodies in Parkinson's disease and other neurodegenerative conditions. In vitro assays demonstrated that psychosine, the neurotoxic sphingolipid accumulated in Krabbe disease, accelerated the fibrillization of α-synuclein. This study demonstrates the occurrence of neuronal deposits of fibrillized proteins including α-synuclein, identifying Krabbe disease as a new α-synucleinopathy. Copyright © 2014 Pathological Society of Great Britain and Ireland. Fil: Smith, Benjamin R.. University Of Ilinois Chicago; Estados Unidos Fil: Santos, Marta B.. University Of Ilinois Chicago; Estados Unidos Fil: Marshall, Michael S.. University Of Ilinois Chicago; Estados Unidos Fil: Cantuti-Castelvetri, Ludovico. University Of Ilinois Chicago; Estados Unidos Fil: Lopez-Rosas, Aurora. University Of Ilinois Chicago; Estados Unidos Fil: Li, Guannan. University Of Ilinois Chicago; Estados Unidos Fil: Van Breemen, Richard B.. University Of Ilinois Chicago; Estados Unidos Fil: Claycomb, Kumiko I.. University of Connecticut; Estados Unidos Fil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Crocker, Stephen J.. University of Connecticut; Estados Unidos Fil: Givogri, Maria I.. University of Illinois; Estados Unidos Fil: Bongarzone, Ernesto R.. University of Illinois; Estados Unidos

To identify novel anti-cancer agents, we created and screened a unique nutraceutical library for activity against acute myeloid leukemia (AML) cells. From this screen, we determined that glucopsychosine was selectively toxic toward AML cell lines and primary AML patient samples with no effect toward normal hematopoietic cells. It delayed tumor growth and reduced tumor weights in mouse xenograft models without imparting toxicity. Glucopsychosine increased cytosolic calcium and induced apoptosis through calpain enzymes. Extracellular calcium was functionally important for glucopsychosine-induced AML cell death and surface calcium channel expression is altered in AML cells highlighting a unique mechanism of glucopsychosine’s selectivity.

Demyelination is a major contributor to the general decay of neural functions in children with Krabbe disease. However, recent reports have indicated a significant involvement of neurons and axons in the neuropathology of the disease. In this study, we have investigated the nature of cellular inclusions in the Krabbe brain. Brain samples from the twitcher mouse model for Krabbe disease and from patients affected with the infantile and late-onset forms of the disease were examined for the presence of neuronal inclusions. Our experiments demonstrated the presence of cytoplasmic aggregates of thioflavin-S-reactive material in both human and murine mutant brains. Most of these inclusions were associated with neurons. A few inclusions were detected to be associated with microglia and none were associated with astrocytes or oligodendrocytes. Thioflavin-S-reactive inclusions increased in abundance, paralleling the development of neurological symptoms, and distributed throughout the twitcher brain in areas of major involvement in cognition and motor functions. Electron microscopy confirmed the presence of aggregates of stereotypic β-sheet folded proteinaceous material. Immunochemical analyses identified the presence of aggregated forms of α-synuclein and ubiquitin, proteins involved in the formation of Lewy bodies in Parkinson's disease and other neurodegenerative conditions. In vitro assays demonstrated that psychosine, the neurotoxic sphingolipid accumulated in Krabbe disease, accelerated the fibrillization of α-synuclein. This study demonstrates the occurrence of neuronal deposits of fibrillized proteins including α-synuclein, identifying Krabbe disease as a new α-synucleinopathy. Fil: Smith, Benjamin R.. University of Illinois; Estados Unidos Fil: Santos, Marta B.. University of Illinois; Estados Unidos Fil: Marshal, Michael S.. University of Illinois; Estados Unidos Fil: Cantuti Castelvetri, Ludovico. University of Illinois; Estados Unidos Fil: Lopez Rosas, Aurora. University of Illinois; Estados Unidos Fil: Li, Guannan. University of Illinois; Estados Unidos Fil: Van Breemen, Richard B.. University of Illinois; Estados Unidos Fil: Claycomb, Kumiko I.. University Of Connecticut; Estados Unidos Fil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Crocker, Stephen. University Of Connecticut; Estados Unidos Fil: Givogri, Maria I.. University of Illinois; Estados Unidos Fil: Bongarzone, Ernesto R.. University of Illinois; Estados Unidos

Globoid cell leukodystrophy (GLD) is a metabolic disease caused by mutations in the galactocerebrosidase (GALC) gene. GALC is a lysosomal enzyme whose function is to degrade galacto-lipids, including galactosyl-ceramide and galactosyl-sphingosine (psychosine, PSY). GALC loss of function causes progressive intracellular accumulation of PSY. It is widely held that PSY is the main trigger for the degeneration of myelinating cells and progressive white-matter loss. However, still little is known about the molecular mechanisms by which PSY imparts toxicity. Here, we address the role of calcium dynamics during PSY-induced cell death. Using the human oligodendrocyte cell line MO3.13, we report that cell death by PSY is accompanied by robust cytosolic and mitochondrial calcium (Ca2+) elevations, and by mitochondrial reactive oxygen species (ROS) production. Importantly, we demonstrate that the reduction of extracellular calcium content by the chelating agent ethylenediaminetetraacetic acid can decrease intra-mitochondrial ROS production and enhance cell viability. Antioxidant administration also reduces mitochondrial ROS production and cell loss, but this treatment does not synergize with Ca2+ chelation. Our results disclose novel intracellular pathways involved in PSY-induced death that may be exploited for therapeutic purposes to delay GLD onset and/or slow down its progression.

Objective Previous studies have shown that ulcerative colitis (UC) is associated with the presence of lamina propria non-invariant (Type II) NKT cells producing IL-13 and mediating epithelial cell cytotoxicity. Here we sought to define the antigen(s) stimulating the NKT cells and to quantitate these cells in the UC lamina propria. Design Detection of Type II NKT cells in UC lamina propria mononuclear cells (LPMC) with lyso-sulfatide loaded tetramer and quantum dot-based flow cytometry and staining. Culture of UC LPMCs with lyso-sulfatide glycolipid to determine sulfatide induction of epithelial cell cytotoxicity, IL-13 production and IL-13Rα2 expression. Blinded quantum dot-based phenotypic analysis to assess UC LPMC expression of IL-13Rα2, CD161 and IL-13. Results Approximately 36% of UC LPMC were lyso-sulfatide tetramer positive, whereas few, if any, control LPMCs were positive. When tested, the positive cells were also CD3 and IL-13Rα2 positive. Culture of UC LPMC with lyso-sulfatide glycolipid showed that sulfatide stimulates UC LPMC production of IL-13 and induces UC CD161 LPMC-mediated cytotoxicity of activated epithelial cells; additionally, lyso-sulfatide induces enhanced expression of IL-13Rα2. Finally, blinded phenotypic analysis of UC LP MC using multicolour quantum dot-staining technology showed that approximately 60% of the LPMC bear both IL-13Rα2 and CD161 and most of these cells also produce IL-13. Conclusions These studies show that UC lamina propria is replete with Type II NKT cells responsive to lyso-sulfatide glycolipid and bearing IL-13Rα2. Since lyso-sulfatide is a self-antigen, these data suggest that an autoimmune response is involved in UC pathogenesis.

Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive inherited neurodegenerative disorder caused by the deficiency of the lysosomal enzyme β-galactosylceramidase. The pathogenesis of the disorder has been proposed to arise from the accumulation of the cytotoxic metabolite galactosylsphingosine (psychosine). The twitcher mouse is a naturally occurring murine model of globoid cell leukodystrophy. We have developed a rapid, sensitive, and specific mass spectrometric method for determining the galactosylsphingosine concentration in the tissues of twitcher mice. Galactosylsphingosine is extracted from the tissues in methanol, isolated using strong cation-exchange and C18 solid-phase extraction chromatography, and then directly analyzed using electrospray ionization-tandem mass spectrometry. A lactosylsphingosine internal standard has been employed for quantification. The assay demonstrated significant accumulation of galactosylsphingosine in the brain, spinal cord, and kidney of twitcher mice. It is anticipated that this method may be of use in the monitoring of experimental therapies for globoid cell leukodystrophy.—Whitfield, P. D., P. C. Sharp, R. Taylor, and P. Meikle. Quantification of galactosylsphingosine in the twitcher mouse using electrospray ionization-tandem mass spectrometry. J. Lipid Res. 2001. 42: 2092–2095.

Twitcher mouse is a murine model of human globoid cell leukodystrophy (Krabbe disease), which is characterized by a genetic deficiency in galactocerebrosidase (GALC) activity. The nervous system is affected early and severely by demyelination in the white matter. So far, there is no effective treatment for Krabbe disease except bone marrow transplantation (BMT). However, BMT has inherent limitations such as unavailability of donors and graft-versus-host disease. In this study, we injected recombinant adenovirus encoding GALC into the lateral ventricle of twitcher mice at postnatal day 0 (PND 0) and the therapeutic effects were evaluated. Our results showed slight, but significant improvements in motor functions, body weight and twitching and a prolonged life span. In brain, GALC activity was increased to 15% that of normal littermates and psychosine concentration was decreased to 55% that of untreated twitcher mice at PND 15. The number of PAS-positive globoid cells in brain stem was also reduced significantly at PND 35. In contrast, when adenoviruses were injected to the twitcher mice at PND 15, almost no improvements were observed. These results demonstrate that the timing of treatment may be of great importance in Krabbe disease.

Globoid cell leukodystrophy (Krabbe disease) is a severe leukodystrophy caused by mutations in the galactocerebrosidase (GALC) gene leading to extremely low (less than 5% of normal activity) GALC activity. Human patients include primarily severely affected infants as well as patients with a later onset of symptoms. The infants usually die before 2 years of age, but it is difficult to predict the clinical course in older patients. In addition to these patients, additional individuals identified in this laboratory have 10--20% of normal GALC activity measured in accessible tissues. These individuals have a wide range of clinical presentations involving neurological degeneration. On molecular analysis of the GALC gene they all have three or more mutations considered to be normal polymorphisms resulting in amino acid changes in the two copies of the GALC gene. In order to investigate the role these amino acid changes may play on clinical, biochemical, and pathological findings, a new transgenic mouse was generated by homologous recombination. After preliminary studies determined what effect each amino acid change had on mouse GALC activity in transient transfection experiments, mice containing a cysteine residue at codon 168 instead of histidine (H168C) were produced. These mice developed symptoms, but they were delayed by 10--15 days from the well-characterized twitcher (twi) mouse. They accumulated psychosine slightly slower than twi mice, showed pathological changes less severe than twi mice in the central and peripheral nervous systems, and live about 15 days longer than twi mice. They have large litters and will play a role in therapy trials using new procedures currently under development.

Sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins derived from a common precursor protein (prosaposin) encoded by a single gene. They are required for in vivo degradation of sphingolipids with short carbohydrate chains. Six cysteines and one glycosylation site are strictly conserved in all four saposins. Total deficiency of all saposins and specific deficiency of saposin B or C are known among human patients. A mouse model of total saposin deficiency closely mimics the human disease. However, no specific saposin A or D deficiency is known. We introduced an amino acid substitution (C106F) into the saposin A domain by the Cre/loxP system which eliminated one of the three conserved disulfide bonds. Saposin A(-/-) mice developed slowly progressive hind leg paralysis with clinical onset at approximately 2.5 months and survival up to 5 months. Tremors and shaking, prominent in other myelin mutants, were not obvious until the terminal stage. Pathology and analytical biochemistry were qualitatively identical to, but generally much milder than, that seen in the typical infantile globoid cell leukodystrophy (GLD) in man (Krabbe disease) and in several other mammalian species, due to genetic deficiency of lysosomal galactosylceramidase (GALC) (EC 3.2.1.46). Thus, saposin A is indispensable for in vivo degradation of galactosylceramide by GALC. It should now be recognized that, in addition to GALC deficiency, genetic saposin A deficiency could also cause chronic GLD. Genetic saposin A deficiency might be anticipated among human patients with undiagnosed late-onset chronic leukodystrophy without GALC deficiency.

One of the most exciting aspects of modern cell biology is the potential to make connections between disparate areas of research, and thus open up new lines of enquiry. Two recent papers have done just this ([Kanazawa et al. 2000][1]; [Im et al. 2001][2], this issue). Building on the biochemical

Globoid cell leukodystrophy (GLD) is characterized histopathologically by apoptosis of oligodendrocytes, progressive demyelination, and the existence of large, multinuclear (globoid) cells derived from perivascular microglia. The glycosphingolipid, psychosine (d-galactosyl-β-1,1′ sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase. Here we document that an orphan G protein–coupled receptor, T cell death–associated gene 8, is a specific psychosine receptor. Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells. Our discovery of a molecular target for psychosine suggests a mechanism for the globoid cell histology characteristic of GLD, provides a tool with which to explore the disjunction of mitosis and cytokinesis in cell cultures, and provides a platform for developing a medicinal chemistry for psychosine.

Our earlier studies demonstrated that high-density lipoproteins (HDLs) stimulate multiple signaling pathways, including activation of phosphatidylcholine-specific phospholipases C and D (PC-PLs) and phosphatidylinositol-specific phospholipase C (PI-PLC). However, only activation of PC-PLs was linked to the HDL-induced cholesterol efflux. In the study presented here, the role of HDL-induced PI-PLC activation was studied. In human skin fibroblasts, HDL potently induced PI-PLC as inferred from enhanced phosphatidylinositol bisphosphate (PtdInsP(2)) turnover and Ca(2+) mobilization. The major protein component of HDL, apo A-I, did not induce PtdInsP(2) turnover or Ca(2+) mobilization in these cells. Both HDL and apo A-I promoted cellular cholesterol efflux, whereas only HDL induced fibroblast proliferation. Inhibition of PI-PLC with U73122 or blocking intracellular Ca(2+) elevation with Ni(2+) or EGTA markedly reduced the extent of HDL-induced cell proliferation but had no effect on cholesterol efflux. In fibroblasts from patients with Tangier disease which are characterized by defective cholesterol efflux, neither HDL-induced PtdInsP(2) breakdown and Ca(2+) mobilization nor cell proliferation was impaired. HDL-induced fibroblast proliferation, PtdInsP(2) turnover, and Ca(2+) mobilization were fully mimicked by the lipid fraction isolated from HDL. Analysis of this fraction with high-performance liquid chromatography (HPLC) and time-of-flight secondary ion mass spectroscopy (TOF-SIMS) revealed that the PI-PLC-inducing activity is identical with two bioactive lysosphingolipids, namely, lysosulfatide (LSF) and sphingosylphosphorylcholine (SPC). Like native HDL, LSF and SPC induced PtdInsP(2) turnover, Ca(2+) mobilization, and fibroblast proliferation. However, both compounds did not promote cholesterol efflux. In conclusion, two agonist activities are carried by HDL. Apo A-I stimulates phosphatidylcholine breakdown and thereby facilitates cholesterol efflux, whereas LSF and SPC trigger PI-PLC activation and thereby stimulate cell proliferation.

Gaucher disease, the most common of the sphingolipidoses, results from the inherited deficiency of the enzyme glucocerebrosidase (EC 3.2.1.45). Although type 2 (acute neuronopathic) Gaucher disease is associated with rapidly progressive and fatal neurologic deterioration, the pathophysiologic mechanisms leading to the neurologic symptoms and early demise remain uncharacterized. While the pathology encountered in Gaucher disease has been attributed to glucocerebroside storage, glucosylsphingosine (Glc-sph), a cytotoxic compound, also accumulates in the tissues. Elevations of brain Glc-sph have been reported in patients with types 2 and 3 Gaucher disease. In this study, Glc-sph levels were measured using HPLC in tissues from mice with type 2 Gaucher disease created with a null glucocerebrosidase allele. Compared with unaffected littermates, homozygous mice with type 2 Gaucher disease had approximately a 100-fold elevation of Glc-sph in brain, as well as elevated levels in other tissues. This accumulation was detected in utero by E 13 and increased progressively throughout gestation. Similarly, elevated Glc-sph levels were seen in human fetuses with type 2 Gaucher disease, indicating that therapy initiated after birth may be too late to prevent the sequaelae of progressive neurologic damage that begins early in gestation. These findings suggest that the accumulation of Glc-sph may be responsible for the rapid demise of mice with type 2 Gaucher disease and the devastating clinical course seen in patients with type 2 Gaucher disease.

In a search for possible endogenous ligands of nuclear receptors that are activated by peroxisome proliferators (PPARs), a solid phase binding assay was developed employing recombinant mouse PPAR-alpha, containing a myc-epitope, a histidine repeat and a kinase A domain. After in vitro labelling with 32P-gamma-ATP, the binding of purified 32P-PPAR-alpha to a panel of different natural and synthetic lipids, immobilized on silica layers, was evaluated. Autoradiographs of the silica layers revealed binding to two main classes of lipophilic compounds. A first class comprised (poly)unsaturated fatty acids. Compounds belonging to a second class were characterized by the presence of an overall positive charge such as long chain amines, sphingoid bases (sphingenine), and lysoglycosphingolipids (psychosine). PPAR-alpha did not bind to N-acylated sphingoid bases (ceramides) or to sphingenine phosphorylated at the primary hydroxy group (sphingenine-1-phosphate). The binding of PPAR-alpha to sphingoid bases might be of interest given the role of PPAR-alpha and sphingolipids in various cellular processes.

Cross-breeding of mouse mutants, each defective in either synthesis (CGT knockout) or degradation (twitcher) of galactosylceramide, generates hybrids with a genotype of galc-/-, cgt+/-, in addition to doubly deficient mice. They are ideally suited to test the potential usefulness of limiting synthesis of the substrate as a treatment of genetic disorders due to degradative enzyme defects. The rate of accretion of galactosylceramide in the brain of CGT knockout carrier mice (cgt+/-) is approximately two-thirds of the normal, suggesting a gene-level compensation for the reduced gene dosage. Phenotype of twitcher mice with a single dose of normal cgt gene was indeed milder with statistical significance, albeit only slightly. Compared among 10 paired littermates, the difference in the life span was 7 ± 3.9 days (S.D.) and the difference in the maximum attained body weight was 1.9 ± 1.2 g (S.D.). Neuropathologists were able to distinguish blindly galc-/-, cgt+/- mice from galc-/-, cgt+/+ mice. The brain psychosine level in galc-/-, cgt+/- mice was also approximately two-thirds of the galc-/-, cgt+/+ mice. These observations indicate that reduction of galactosylceramide synthesis to two-thirds of the normal level results in minor but clearly detectable phenotypic improvements. Because of the detrimental consequences of drastic reduction in galactosylceramide synthesis that may be required for pragmatically meaningful improvements, this approach by itself is unlikely to be useful as the sole treatment but may be helpful as a supplement to other therapies. J. Neurosci. Res. 59:179–187, 2000 © 2000 Wiley-Liss, Inc.

As part of our program directed toward the design and synthesis of high-affinity ligands for the GalCer-binding site on the HIV cell surface glycoprotein, gp120, we required a reliable method for qualitatively assessing relative binding affinities for related analogues. Due to the hydrophilic nature of these synthetic conjugates, difficulties were encountered with typical ELISA methods, which rely upon hydrophobic interactions to anchor the ligand to a microtiter plate. Other types of assays were also problematic due to nonspecific binding of gp120. Therefore, we developed a general method for plating water-soluble ligands on microtiter plates using biotin/NeutrAvidin recognition for adhesion. A water-soluble GalCer analogue was prepared by conjugating psychosine to biotin using a novel tetraethylene glycol linker. In a similar manner, LacCer and GlcCer analogues were prepared and these conjugates were plated into microtiter wells containing NeutrAvidin. Unoccupied sites were blocked using biotin functionalized as a primary amide. Gp120 binding to galactosyl sphingosine, GalSph (19), GlcSph (22), and LacSph (23) conjugates was assessed through incubation with recombinant HRP-gp120. It was determined that LacSph has the strongest interaction with gp120. The binding affinities of GalSph and GlcSph were similar to each other and less strong than LacSph. These data contradict earlier studies where HPTLC showed that LacCer and GlcCer do not significantly bind gp120. They also contradict liposome-based assays that reported psychosine is not recognized by gp120. The extent of plating for each biotinylated molecule was quantified using HRP-biotin, allowing direct comparison of ligand plating efficiencies for the first time. Several other synthetic biotin conjugates were prepared and tested, demonstrating the feasibility of performing ELISA on water-soluble ligands.

The o-phthaldialdehyde precolumn derivatives of psychosine, sphinganine and sphingosine extracted from brain and spinal cord tissues were determined by high-performance liquid chromatography-fluorescence detection. This method was developed with the purpose of detecting an endogenous amount of psychosine, sphingosine and sphinganine using small aliquots of brain tissues and spinal cord in rats. These sphingolipid bases were extracted in various ratios of chloroform-methanol and several pH values. Recovery of the method is about 81% in 12 ng/tube (final volume, 320 microl), 90-95% in 45 ng/tube of sphingosine and sphinganine within 2-12% relative standard deviation. Detection limits of these sphingoid bases were about 0.05 pmol/mg brain tissue. In the forebrain, brainstem and spinal cord of rats at three different ages of postnatal days (PND) 1, PND 13 and 6 months old, the endogenous concentrations of psychosine, sphingosine and sphinganine were determined. From these results, this method is suitable for the determination of sphingoid bases in small aliquot of brain and spinal cord tissues.

To define the thermotropic behavior of galactosylceramides (GalCer) containing cis monounsaturated acyl chains, N-X:1Delta(X-9) cis galactosylsphingosines (GalSph) were synthesized (where X=24, 22, 20, or 18) and investigated by differential scanning calorimetry (DSC). After hydration of dried glycolipid, aqueous dispersions were prepared by repetitive heating and freeze-thaw cycles. The DSC data clearly showed that introducing a single cis double bond into the acyl chain of GalCer lowers the transition temperature of the main endothermic peak and affects the kinetics of formation of various metastable and stable gel phases. More importantly, the data emphasize the role that double bond location in concert with acyl chain length play in modulating the thermotropic behavior of GalCers. In contrast to the 18:1 GalCer and 20:1 GalCer endotherms which remain unchanged after identical repetitive heating scans and low temperature incubations, the thermotropic responses of 22:1 GalCer and 24:1 GalCer depended directly upon incubation time at lower temperatures following a heating scan. Only after extended incubation (4-5 days) did the endotherms revert to behavior observed during the initial heating scan that followed sample preparation by cyclic heating and freeze-thaw methods. The extended incubation times required for 22:1 GalCer and 24:1 GalCer to assume their more stable packing motifs appear to be consistent with nucleation events that promote transbilayer interdigitation. Yet, due to the slow kinetics of the process, the presence of cis monounsaturation in very long acyl chains that are common to GalCer may effectively inhibit transbilayer lipid interdigitation under physiological conditions.

An increasing number of sphingolipids, glycosphingolipids and some of their degradation products have been recognized in recent years as second messengers involved in signal transduction and as modulators of numerous cellular functions. These can be converted into inert, caged compounds, introduced into cells and tissues and subsequently photolysed to active compounds thus enabling the study of fast biological processes. The novel, potentially caged compounds synthesized here are substituted 2-nitrobenzyl urethans and 2-nitrobenzyl amines derived from sphingosine, dihydrosphingosine, N-methylsphingosine, N-methyldihydrosphingosine, psychosine and glucosylsphingosine. Upon irradiation of the afore mentioned compounds they release, or are expected to release, the free biologically active amines.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a devastating, degenerative neurological disorder caused by mutations in the galactosylceramidase (GALC) gene that severely affect enzyme activity. Currently, treatment options for this disorder are very limited. Enzyme replacement therapy (ERT) has been shown to be effective in lysosomal storage disorders with predominantly peripheral manifestations such as type I Gaucher's and Fabry's disease. Little however is known about the possible benefit of ERT in GLD, which has a substantial central nervous system component. In this study, we examined the effect of peripheral GALC injections in the twitcher mouse model of the disease. Although we were unable to block the precipitous decline that normally occurs just before death, we did observe significant early improvements in motor performance, a substantial attenuation in the initial failure to thrive, and an increase in life span. Immunohistochemical and activity analyses demonstrated GALC uptake in multiple tissues, including the brain. This was associated with a decrease in the abnormal accumulation of the GALC substrate psychosine, which is thought to play a pivotal role in disease pathology. These results indicate that peripheral ERT is likely to be beneficial in GLD.

Background Gaucher disease (GD) is the most common lysosomal storage disorder (LSD). Based on a deficient β-glucocerebrosidase it leads to an accumulation of glucosylceramide. Standard diagnostic procedures include measurement of enzyme activity, genetic testing as well as analysis of chitotriosidase and CCL18/PARC as biomarkers. Even though chitotriosidase is the most well-established biomarker in GD, it is not specific for GD. Furthermore, it may be false negative in a significant percentage of GD patients due to mutation. Additionally, chitotriosidase reflects the changes in the course of the disease belatedly. This further enhances the need for a reliable biomarker, especially for the monitoring of the disease and the impact of potential treatments. Methodology Here, we evaluated the sensitivity and specificity of the previously reported biomarker Glucosylsphingosine with regard to different control groups (healthy control vs. GD carriers vs. other LSDs). Findings Only GD patients displayed elevated levels of Glucosylsphingosine higher than 12 ng/ml whereas the comparison controls groups revealed concentrations below the pathological cut-off, verifying the specificity of Glucosylsphingosine as a biomarker for GD. In addition, we evaluated the biomarker before and during enzyme replacement therapy (ERT) in 19 patients, demonstrating a decrease in Glucosylsphingosine over time with the most pronounced reduction within the first 6 months of ERT. Furthermore, our data reveals a correlation between the medical consequence of specific mutations and Glucosylsphingosine. Interpretation In summary, Glucosylsphingosine is a very promising, reliable and specific biomarker for GD.

Krabbe disease is a devastating pediatric leukodystrophy caused by mutations in the galactocerebrosidase (GALC) gene. A significant subset of the infantile form of the disease is due to missense mutations that result in aberrant protein production. The currently used mouse model, twitcher, has a nonsense mutation not found in Krabbe patients, although it is similar to the human 30 kb deletion in abrogating GALC expression. Here, we identify a spontaneous mutation in GALC, GALCtwi-5J, that precisely matches the E130K missense mutation in patients with infantile Krabbe disease. GALCtwi-5J homozygotes show loss of enzymatic activity despite normal levels of precursor protein, and manifest a more severe phenotype than twitcher, with half the life span. Although neuropathological hallmarks such as gliosis, globoid cells and psychosine accumulation are present throughout the nervous system, the CNS does not manifest significant demyelination. In contrast, the PNS is severely hypomyelinated and lacks large diameter axons, suggesting primary dysmyelination, rather than a demyelinating process. Our data indicate that early demise is due to mechanisms other than myelin loss and support an important role for neuroinflammation in Krabbe disease progression. Furthermore, our results argue against a causative relationship between psychosine accumulation, white matter loss and gliosis.

Krabbe disease is a lethal, demyelinating condition caused by genetic deficiency of galactocerebrosidase (GALC) and resultant accumulation of its cytotoxic substrate, psychosine (galactosylsphingosine), primarily in oligodendrocytes (OLs). Psychosine is generated by galactosylation of sphingosine by UDP-galactose:ceramide galactosyltransferase (CGT), a galactosylceramide synthesizing enzyme which is primarily expressed in OLs. The expression of CGT and the synthesis of galactosyl-sphingolipids are associated with the terminal differentiation of OL, but little is known about the participation of endogenous psychosine accumulation in OL differentiation under GALC deficient conditions. In this study, we report that accumulation of endogenous psychosine under GALC deficient Krabbe conditions impedes OL differentiation process both by decreasing the expression of myelin lipids and protein and by inducing the cell death of maturating OLs. The psychosine pathology under GALC deficient conditions involves participation of secretory phospholipase A2 (sPLA2) activation and increase in its metabolites, as evidenced by attenuation of psychosine-induced pathology by treatment with pharmacological inhibitor of sPLA2 7,7-dimethyleicosadienoic acid (DEDA). These observations suggest for potential therapeutic efficacy of sPLA2 inhibitor in Krabbe disease.

Globoid cell leukodystrophy (GLD) or Krabbe disease, is a fatal demyelinating disease attributed to mutations in the galactocerebrosidase (GALC) gene. Loss of function mutations in GALC result in accumulation of the glycolipid intermediate, galactosylsphingosine (psychosine). Due to the cytotoxicity of psychosine, it has been hypothesized that accumulated psychosine underlie the pathophysiology of GLD. However, the cellular mechanisms of GLD pathophysiology remain unclear. Globoid cells, multinucleated microglia/macrophages in the central nervous system (CNS), are a defining characteristic of GLD. Here we report that exposure of primary glial cultures to psychosine induces the expression and the production of matrix metalloproteinase (MMP)-3 that mediated a morphological transformation of microglia into a multinucleated globoid cell type. Additionally, psychosine-induced globoid cell formation from microglia was prevented by either genetic ablation or chemical inhibition of MMP-3. These effects are microglia-specific as peripheral macrophages exposed to psychosine did not become activated or express increased levels of MMP-3. In the brain from twitcher mice, a murine model of human GLD, elevated MMP-3 expression relative to wild-type littermates was contemporaneous with disease onset and further increased with disease progression. Further, bone marrow transplantation (BMT), currently the only therapeutically beneficial treatment for GLD, did not mitigate the elevated expression of MMP-3 in twitcher mice. Hence, elevated expression of MMP-3 in GLD may promote microglial responses to psychosine that may represent an important pathophysiological process in this disease and its treatment.

Mutations of GBA1 , the gene encoding glucocerebrosidase, represent a common genetic risk factor for developing the synucleinopathies Parkinson disease (PD) and dementia with Lewy bodies. PD patients with or without GBA1 mutations also exhibit lower enzymatic levels of glucocerebrosidase in the central nervous system (CNS), suggesting a possible link between the enzyme and the development of the disease. Previously, we have shown that early treatment with glucocerebrosidase can modulate α-synuclein aggregation in a presymptomatic mouse model of Gaucher-related synucleinopathy ( Gba1 D409V/D409V ) and ameliorate the associated cognitive deficit. To probe this link further, we have now evaluated the efficacy of augmenting glucocerebrosidase activity in the CNS of symptomatic Gba1 D409V/D409V mice and in a transgenic mouse model overexpressing A53T α-synuclein. Adeno-associated virus-mediated expression of glucocerebrosidase in the CNS of symptomatic Gba1 D409V/D409V mice completely corrected the aberrant accumulation of the toxic lipid glucosylsphingosine and reduced the levels of ubiquitin, tau, and proteinase K-resistant α-synuclein aggregates. Importantly, hippocampal expression of glucocerebrosidase in Gba1 D409V/D409V mice (starting at 4 or 12 mo of age) also reversed their cognitive impairment when examined using a novel object recognition test. Correspondingly, overexpression of glucocerebrosidase in the CNS of A53T α-synuclein mice reduced the levels of soluble α-synuclein, suggesting that increasing the glycosidase activity can modulate α-synuclein processing and may modulate the progression of α-synucleinopathies. Hence, increasing glucocerebrosidase activity in the CNS represents a potential therapeutic strategy for GBA1 -related and non- GBA1 –associated synucleinopathies, including PD.

Globoid cell leukodystrophy (Krabbe disease) is a neurological disorder of infants caused by genetic deficiency of the lysosomal enzyme β-galactosylceramidase leading to accumulation of the neurotoxic metabolite 1-β-d-galactosylsphingosine (psychosine) in the central nervous system. Angiogenesis plays a pivotal role in the physiology and pathology of the brain. Here, we demonstrate that psychosine has anti-angiogenic properties by causing the disassembling of endothelial cell actin structures at micromolar concentrations as found in the brain of patients with globoid cell leukodystrophy. Accordingly, significant alterations of microvascular endothelium were observed in the post-natal brain of twitcher mice, an authentic model of globoid cell leukodystrophy. Also, twitcher endothelium showed a progressively reduced capacity to respond to pro-angiogenic factors, defect that was corrected after transduction with a lentiviral vector harbouring the murine β-galactosylceramidase complementary DNA. Finally, RNA interference-mediated β-galactosylceramidase gene silencing causes psychosine accumulation in human endothelial cells and hampers their mitogenic and motogenic response to vascular endothelial growth factor. Accordingly, significant alterations were observed in human microvasculature from brain biopsy of a globoid cell leukodystrophy case. Together these data demonstrate that β-galactosylceramidase deficiency induces significant alterations in endothelial neovascular responses that may contribute to central nervous system and systemic damages that occur in globoid cell leukodystrophy.

Sphingosine, sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC) each elicited transient elevations in intracellular free Ca2+ ([Ca2+]i) as a result of Ca2+ release from an intracellular store in MC3T3-E1 pre-osteoblasts. No elevation in [Ca2+]i was detected in response to the application of N-acetyl sphingosine. Psychosine (1-galactosyl sphingosine) also caused Ca2+ release from intracellular stores, which suggests that phosphorylation of the 1-carbon of sphingosine is not required for its action as a Ca2+ release agonist in MC3T3-E1 cells. Sequential additions of combinations of sphingosine, SPP, SPC and psychosine revealed Ca2+ transients only after the application of the first sphingolipid, despite the fact that the intracellular Ca2+ stores refilled. This indicates that each agent desensitized the MC3T3-E1 cells to the action of the others and suggests that all of the sphingolipids tested employ the same receptor or a common intracellular messenger.

To study the neuronal deficits in neuronopathic Gaucher Disease (nGD), the chronological behavioral profiles and the age of onset of brain abnormalities were characterized in a chronic nGD mouse model (9V/null). Progressive accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) in the brain of 9V/null mice were observed at as early as 6 and 3 months of age for GC and GS, respectively. Abnormal accumulation of α-synuclein was present in the 9V/null brain as detected by immunofluorescence and Western blot analysis. In a repeated open-field test, the 9V/null mice (9 months and older) displayed significantly less environmental habituation and spent more time exploring the open-field than age-matched WT group, indicating the onset of short-term spatial memory deficits. In the marble burying test, the 9V/null group had a shorter latency to initiate burying activity at 3 months of age, whereas the latency increased significantly at ≥12 months of age; 9V/null females buried significantly more marbles to completion than the WT group, suggesting an abnormal response to the instinctive behavior and an abnormal activity in non-associative anxiety-like behavior. In the conditional fear test, only the 9V/null males exhibited a significant decrease in response to contextual fear, but both genders showed less response to auditory-cued fear compared to age- and gender-matched WT at 12 months of age. These results indicate hippocampus-related emotional memory defects. Abnormal gait emerged in 9V/null mice with wider front-paw and hind-paw widths, as well as longer stride in a gender-dependent manner with different ages of onset. Significantly higher liver- and spleen-to-body weight ratios were detected in 9V/null mice with different ages of onsets. These data provide temporal evaluation of neurobehavioral dysfunctions and brain pathology in 9V/null mice that can be used for experimental designs to evaluate novel therapies for nGD.

Background New York State has screened over 1.2 million newborns for Krabbe disease, and we identified 4 newborns with infantile Krabbe disease. In addition, 6 other newborns were identified with very low galactosylcerebrosidase (GALC) activity. Because these patients remain asymptomatic, we investigated whether psychosine levels could be a useful marker for disease. Methods HPLC–MS/MS methodology was used to determine the psychosine concentrations in dried blood spots (DBS) collected from the following cohorts: known Krabbe patients, screened babies that were determined to have infantile Krabbe disease, asymptomatic infants with low GALC activity, and normal controls. Results The psychosine concentrations from the known Krabbe patients ranged from 7 to 50 ng/ml. Newborns identified by screening who were confirmed with infantile Krabbe disease ranged from 23 to 73 ng/ml. Asymptomatic individuals with low GALC activity had concentrations ranging from 1.7 to 5.7 ng/ml. Concentrations in newborns with normal GALC activity were all Conclusions The psychosine concentrations in DBS from confirmed infantile patients are at least four times higher than the asymptomatic newborns and nearly an order of magnitude greater than normal newborns. Further studies are needed to determine if psychosine can be used as a predictor of disease status/progression in screen positive newborns.