Systemic deficiency of GM1 ganglioside in Parkinson’s disease tissues and its relation to the disease etiology

Following our initial reports on subnormal levels of GM1 in the substantia nigra and occipital cortex of Parkinson’s disease (PD) patients, we have examined additional tissues from such patients and found these are also deficient in the ganglioside. These include innervated tissues intimately involved in PD pathology such as colon, heart and others, somewhat less intimately involved, such as skin and fibroblasts. Finally, we have analyzed GM1 in peripheral blood mononuclear cells, a type of tissue apparently with no direct innervation, and found those too to be deficient in GM1. Those patients were all afflicted with the sporadic form of PD (sPD), and we therefore conclude that systemic deficiency of GM1 is a characteristic of this major type of PD. Age is one factor in GM1 decline but is not sufficient; additional GM1 suppressive factors are involved in producing sPD. We discuss these and why GM1 replacement offers promise as a disease-altering therapy.

Metabolic cardiomyopathy in GM1 gangliosidosis: Worse prognosis factor?

GM1 gangliosidosis is an autosomal recessive lysosomal storage disorder due to deficiency of the β-galactosidase enzyme which hydrolyzes the terminal β-galactosyl residues from GM1 ganglioside, glycoproteins, and glycosaminoglycans. Patients with infantile GM1 gangliosidosis present at birth or shortly thereafter with visceral changes and severe neurological deterioration leading to early death. In this report, we presented a case of infantile GM1 gangliosidosis associated with multiple organomegaly.

Turning the spotlight on the oligosaccharide chain of GM1 ganglioside

It is well over a century that glycosphingolipids are matter of interest in different fields of research. The hydrophilic oligosaccharide and the lipid moiety, the ceramide, both or separately have been considered in different moments as the crucial portion of the molecule, responsible for the role played by the glycosphingolipids associated to the plasma-membranes or to any other subcellular fraction. Glycosphingolipids are a family of compounds characterized by thousands of structures differing in both the oligosaccharide and the ceramide moieties, but among them, the nervous system monosialylated glycosphingolipid GM1, belonging to the group of gangliosides, has gained particular attention by a multitude of Scientists. In recent years, a series of studies have been conducted on the functional roles played by the hydrophilic part of GM1, its oligosaccharide, that we have named “OligoGM1”. These studies allowed to shed new light on the mechanisms underlying the properties of GM1 defining the role of the OligoGM1 in determining precise interactions with membrane proteins instrumental for the neuronal functions, leaving to the ceramide the role of correctly positioning the GM1 in the membrane crucial for the oligosaccharide-protein interactions. In this review we aim to report the recent studies on the cascade of events modulated by OligoGM1, as the bioactive portion of GM1, to support neuronal differentiation and trophism together with preclinical studies on its potential to modify the progression of Parkinson’s disease.

The characterization of exosomes from fibrosarcoma cell and the useful usage of Dynamic Light Scattering (DLS) for their evaluation

Exosomes are a type of extracellular vesicles containing mRNA, miRNA, and proteins of origin cells, which can control the characteristics of other cells or surroundings. Despite increasing evidence on oncogenic properties of tumor-derived exosomes, fibrosarcoma-derived exosomes remain largely unrevealed. While the proper extraction and characterization of exosomes is critical in exosomes research, there are various limitations in techniques to measure the size and homogeneity of exosomes. Here, we analyzed exosomes from a fibrosarcoma cell line WEHI-164 compared with a breast cancer cell line MDA-MD-231 as a control. Results from dot blot and western blot analysis demonstrated that GM1 ganglioside, and TSG101, HSC70 and GAPDH proteins were contained in exosomes from the WEHI-164 fibrosarcoma cell line. The existence of tetraspanins such as CD81, CD63 and CD9 was confirmed in the exosomes by ExoView analysis. The results obtained from TEM showed their sphere-like shapes of around 50 to 70 nm in radius. Through DLS, we found out that the mean radius of the exosomes derived from WEHI-164 and MDA-MB-231 cell lines was 94.4 nm and 107.8 nm, respectively, with high homogeneity. When comparing the radius measured by TEM with the radius measured by DLS, it was revealed that the difference between the two methods was about 40 nm. This study has significance in characterizing the molecular properties of exosomes from a fibrosarcoma, which has not been researched much before, and in providing clear evidence that DLS can be used as an efficient, convenient and noninvasive technique to simply check the homogeneity and size of exosomes.

Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation

Background: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. Objective: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. Methods: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. Results: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. Conclusion: The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.

Histological and Clinical Findings in Rabbits Sensitized with GM1 Ganglioside

BACKGROUND: Acute motor axonal neuropathy (AMAN) is a peripheral nerve disorder that attacks motor axons and occurs acutely. AMAN is one type of Guillain–Barre syndrome (GBS) which often attacks men of productive age. Until now, although patients have undergone intravenous immunoglobulin (IVIG) therapy and/or plasmapheresis, long-standing disability remains a problem. In Indonesia, the availability and cost of these therapies are constraints. AIM: Our study aimed to find a proper animal model suitable for AMAN and can be executed in our institution, Naval Health Institute with a hope to find new therapeutic modalities in healing with AMAN. METHODS: GM1 ganglioside immunized in New Zealand male white rabbits with complete Freund’s adjuvant, every 3 weeks until 20 weeks. We evaluated the effects GM1 ganglioside on body weight, functional score, and axon degeneration’s scale. Functional score was examined based on Tarlov’s. Hematoxylin-eosin was used to stain this slide. RESULTS: Rabbits that being immunized with GM1 ganglioside experience a number of neurological signs and symptoms that resemble AMAN, that is, sluggish righting reflex, muscular weakness, flaccid hyper paralysis, and body weight loss. Pathological examination shows extensive degeneration of peripheral nerves, infiltration of macrophages, and perineuritis. CONCLUSION: This histological and clinical findings support that this neuropathy is induced by an autoimmune response delivered by cells that respond to gangliosides.

Cholera Toxin Subunit B for Sensitive and Rapid Determination of Exosomes by Gel Filtration

We developed a sensitive fluorescence-based assay for determination of exosome concentration. In our assay, Cholera toxin subunit B (CTB) conjugated to a fluorescence probe and a gel filtration technique (size-exclusion chromatography) are used. Exosomal membranes are particularly enriched in raft-forming lipids (cholesterol, sphingolipids, and saturated phospholipids) and in GM1 ganglioside. CTB binds specifically and with high affinity to exosomal GM1 ganglioside residing in rafts only, and it has long been the probe of choice for membrane rafts. The CTB-gel filtration assay allows for detection of as little as 3 × 108 isolated exosomes/mL in a standard fluorometer, which has a sensitivity comparable to other methods using advanced instrumentation. The linear quantitation range for CTB-gel filtration assay extends over one order of magnitude in exosome concentration. Using 80 nM fluorescence-labeled CTB, we quantitated 3 × 108 to 6 × 109 exosomes/mL. The assay ranges exhibited linear fluorescence increases versus exosome concentration (r2 = 0.987). The assay was verified for exosomal liposomes. The assay is easy to use, rapid, and does not require any expensive or sophisticated instrumentation.