Ana Carolina Breier

Amyloid-β induced toxicity involves ganglioside expression and is sensitive to GM1 neuroprotective action.

The effect of Aβ25-35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C(14)] galactose and results showed that Aβ25-35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aβ25-35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10 μM was able to prevent the toxicity triggered by the fibrillar Aβ25-35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24h) upon the Aβ-induced alterations on GSK3β dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aβ-induced dephosphorylation (activation) of GSK3β, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimers disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimers disease experimental models and suggest a protective role for GM1 in Aβ-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimers treatment.

Reduction of gangliosides, phospholipids and cholesterol content in cerebral cortex of rats caused by chronic hypermethioninemia

Neurological dysfunction is observed in patients with severe hypermethioninemia, whose physiopathology is still poorly understood. In the current study we investigated the effect of chronic administration of methionine on the content and species of gangliosides and phospholipids, as well as on the concentration of cholesterol in rat cerebral cortex. Wistar rats received subcutaneous injections of methionine (1.34–2.68 μmol/g of body weight), twice a day, from the 6th to the 28th day of age and controls received saline. Animals were killed 12 h after the last injection. Results showed that methionine administration significantly decreased the total content of lipids in cerebral cortex of rats. We also observed that this amino acid significantly reduced the absolute quantity of the major brain gangliosides (GM1, GD1a, GD1b and GT1b) and phospholipids (sphingomyelin, phosphatidylcholine and phosphatidylethanolamine). We also showed that Na+,K+‐ATPase activity and TBARS were changed in cerebral cortex of rats subjected to hypermethioninemia. If confirmed in human beings, these data could suggest that the alteration in lipid composition, Na+,K+‐ATPase activity and TBARS caused by methionine might contribute to the neurophysiopathology observed in hypermethioninemic patients.

Resveratrol prevents global cerebral ischemia-induced decrease in lipid content

Abstract Objective: The present study was undertaken to evaluate whether resveratrol (RSV) modulates membrane lipid composition, as well as on ganglioside profile in ischemia/reperfusion injury. Methods: Global cerebral ischemia was induced by four-vessel occlusion for 10 minutes. RSV (30 mg/kg) or vehicle was intraperitoneally administered to rats 7 days prior to ischemia. Brain structures were homogenized with chloroform/methanol for ganglioside, phospholipids, and cholesterol levels. Results: RSV significantly prevented the reduction in the total content of gangliosides, phospholipids, and cholesterol in hippocampi and cerebral cortex induced by global cerebral ischemia. Although ischemia/reperfusion decreased ganglioside content, the ganglioside profiles were apparently not modified. Conclusions: Our experiments suggest that lipid metabolism is important for development of ischemic damage and indicate that RSV treatment 7 days prior to ischemia may prevent membrane lipid loss.

Determination of the lysosomal hydrolase activity in blood collected on filter paper, an alternative to screen high risk populations.

This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease.

Screening of high-risk Gaucher disease patients in Brazil using miniaturized dried blood spots and leukocyte techniques.

This study investigates the miniaturization of the screening technique using dried blood spots on filter paper (DBS) to measure GBA and CT activities, and GBA and β-galactosidase activities in leukocytes. 274 DBS from individuals with suspected GD were screened for 1.5 years. Of these, we confirmed the diagnosis in 13.5%. The miniaturization of the DBS and leukocyte techniques afforded to reduce costs and sample size appropriate for a reliable diagnosis.

Effects of chronic proline administration on lipid contents of rat brain.

In the present work we investigated the effects of chronic proline administration on ganglioside, cholesterol and phospholipid total contents, as well as on ganglioside profile in cerebral cortex, hippocampus, hypothalamus and cerebellum of rats. We also evaluated the ganglioside content and profile in detergent‐soluble and resistant microdomains isolated from synaptic membranes obtained from cerebral cortex. Proline solution (hyperprolinemic) or saline (control) were subcutaneously administered to rats from 6th to 28th post‐natal day, according to body weight. Twelve hours after the last injection, the animals were sacrificed by decapitation without anaesthesia. Brain structures were homogenized with chloroform:methanol for lipid extraction. Synaptic membranes were obtained by differential centrifugation and detergent‐soluble and resistant microdomains were isolated by cold Triton X‐100 treatment. Results showed that rats subjected to chronic proline treatment presented a significant increase of ganglioside content in cortex and hippocampus, while this membrane lipid content was not altered in hypothalamus and cerebellum. Besides, phospholipid and cholesterol contents were not modified in all structures studied. On the other hand, ganglioside content decreased in detergent‐soluble and resistant microdomains isolated from synaptic membrane obtained from hyperprolinemic cortex. Although ganglioside profiles were apparently not modified, the individual absolute quantities were altered in cortex and hippocampus total lipid extract and membrane microdomains. Our findings suggest that chronic proline treatment affects in a distinct manner different cerebral regions concerning the lipid composition of the cell membranes, reflecting on its distribution in the cortex membrane microdomains. Among these phenomena consequences, distinct modulations in synaptic transmission may be suggested which might contribute to the impairment in cognition and/or other neurological dysfunctions found in hyperprolinemia type II patients.

Quantification of glucosylceramide in plasma of Gaucher disease patients

Gaucher disease is a sphingolipidosis that leads to an accumulation of glucosylceramide. The objective of this study was to develop a methodology, based on the extraction, purification and quantification of glucosylceramide from blood plasma, for use in clinical research laboratories. Comparison of the glucosylceramide content in plasma from Gaucher disease patients, submitted to enzyme replacement therapy or otherwise, against that from normal individuals was also carried out. The glucosylceramide, separated from other glycosphingolipids by high performance thin layer chromatography (HPTLC) was chemically developed (CuSO4 / H3PO4) and the respective band confirmed by immunostaining (human anti-glucosylceramide antibody / peroxidase-conjugated secondary antibody). Chromatogram quantification by densitometry demonstrated that the glucosylceramide content in Gaucher disease patients was seventeen times higher than that in normal individuals, and seven times higher than that in patients on enzyme replacement therapy. The results obtained indicate that the methodology established can be used in complementary diagnosis and for treatment monitoring of Gaucher disease patients.

Comparing the alpha-galactosidase A biochemical properties from healthy individuals and Fabry disease patients

BACKGROUND Due to the importance and the difficulty still present in determining the biochemical diagnosis of Fabry disease (FD), the aim of this study was to establish and compare the biochemical and kinetic properties of alpha-galactosidase A (GLA) in dried blood spots (DBS), plasma and leukocyte samples of FD patients and healthy subjects to evaluate the possible use of these parameters as an auxiliary tool in the diagnosis of this disease. METHODS GLA activity in DBS, plasma and leukocyte samples from Fabry disease patients and healthy subjects was compared and characterized in terms of optimal pH, Km and Vmax and heat stability. RESULTS A difference was observed between the Km and Vmax of FD patients and healthy controls using DBS, plasma and leukocyte samples. In leukocytes, pre-incubation at 50°C for 60 min was effective to differentiate FD patients from healthy controls. CONCLUSION These results can be used as an auxiliary method to the FD diagnosis, especially in cases of patients whose GLA activity is within normal range.

Alpha-l-iduronidase and arylsulfatase B in dried blood spots on filter paper: Biochemical parameters and time stability

BACKGROUND The goal of this study was to assess the biochemical parameters of the enzymes α-l-iduronidase (IDUA) and arylsulfatase B (ASB), which are deficient in mucopolysaccharidosis (MPS) I and VI, respectively, in dried blood spot (DBS) samples impregnated on filter paper. METHODS AND RESULTS The optimal pH, Km, and Vmax of IDUA and ASB in DBS are hereby presented. After these analyses, the reference values for the activities of these enzymes in DBS with cutoff of 3.65nmol/h/mL for IDUA and 6.80nmol/h/mL for ASB were established. The research also showed that the stability (21days) of the IDUA activity is lower than ASB, which maintained its enzymatic activity stable up until 60days of analysis, after impregnating the filter paper with blood. CONCLUSION Currently, DBS ensures important advantages in handling storage and transportation of samples with respect to neonatal screening programs. This study contributes to characterizing and differentiating the biochemistry of deficient enzymes in MPSs I and VI of DBS samples.