Search Results (147)

Neuroinflammation driven by microglial activation and α-synuclein (αSyn) aggregation is one of the central features driving Parkinson’s disease (PD) pathogenesis. GM1 ganglioside’s oligosaccharide moiety (OligoGM1) has shown neuroprotective potential in PD neuronal models, but its direct effects on inflammation remain poorly defined. This study investigated the ability of OligoGM1 to modulate microglial activation and αSyn handling in a human in vitro model. Human embryonic microglial (HMC3) cells were exposed to αSyn pre-formed fibrils (PFFs) in the presence or absence of OligoGM1. Microglial activation markers, intracellular αSyn accumulation, and cytokine release were assessed by immunofluorescence and ELISA. OligoGM1 had no effect on microglial morphology or cytokine release under basal conditions. Upon αSyn challenge, cells exhibited increased amounts of ionized calcium-binding adaptor molecule 1 (Iba1), triggered receptor expressed on myeloid cells 2 (TREM2), elevated αSyn accumulation, and secreted pro-inflammatory cytokines. OligoGM1 pre-treatment significantly reduced the number and area of Iba1(+) cells, the intracellular αSyn burden in TREM2(+) microglia, and the release of interleukin 6 (IL-6). OligoGM1 selectively attenuated αSyn-induced microglial activation and enhanced αSyn clearance without compromising basal immune function. These findings confirm and support the potential of OligoGM1 as a multitarget therapeutic candidate for PD that is capable of modulating glial reactivity and neuroinflammatory responses. Full article

Tay–Sachs disease (TSD) is a rare and severe neurodegenerative disorder inherited in an autosomal recessive manner. It is caused by a deficiency of the enzyme hexosaminidase A, which is responsible for the degradation of GM2 gangliosides—lipids that accumulate in the nerve cells of the central nervous system. The inability to break down these lipids leads to their progressive accumulation, resulting in irreversible brain damage. Mechanistically, TSD is caused by mutations in the HEXA gene, which encodes the alpha subunit of hexosaminidase A. These mutations disrupt enzyme activity and alter cellular pathways involved in lysosomal lipid degradation. Although Tay–Sachs specifically involves the alpha subunit, similar clinical features can be seen in Sandhoff disease, a related disorder caused by mutations in the HEXB gene, which encodes the beta subunit shared by hexosaminidase A and B. Tay–Sachs is classified into three clinical forms according to age of onset and symptom severity: the classic infantile form, which is the most common and severe; a juvenile (subacute) form; and an adult-onset form, which progresses more slowly and tends to present with milder symptoms. Diagnosis is based on enzymatic testing showing reduced or absent hexosaminidase A activity, confirmed by genetic testing. Prenatal diagnosis and genetic counseling play a key role in prevention and reproductive decision-making, especially in high-risk populations. Although no curative treatment currently exists, ongoing research is exploring gene therapy, enzyme replacement, and pharmacological approaches. Certain compounds, such as gemfibrozil, have shown potential to slow symptom progression. Early diagnosis and multidisciplinary care are essential to improving quality of life, although therapeutic options remain limited due to the progressive nature of the disease. Full article

Background: Tay–Sachs disease (TSD) is an autosomal recessive lysosomal storage disorder characterized by the accumulation of GM2 ganglioside due to mutations in the HEXA gene, which encodes the α-subunit of β-Hexosaminidase A. This accumulation leads to significant neuropathological effects and premature death in affected individuals. No effective treatments exist, but enzyme replacement therapies are under investigation. In our previous work, we demonstrated the internalization and efficacy of human recombinant lysosomal β-hexosaminidase A (rhHex-A), produced in the methylotrophic yeast Pichia pastoris, in reducing lipids and lysosomal mass levels in fibroblasts and neural stem cells derived from patient-induced pluripotent stem cells (iPSCs). In this study, we further evaluated the potential of rhHex-A to prevent GM2 accumulation using fibroblast and neuroglia cells from a TSD patient alongside a relevant mouse model. Methods: Fibroblasts and neuroglial cell lines derived from a murine model and TSD patients were treated with 100 nM rhHexA for 72 h. After treatment, cells were stained by anti-GM2 (targeting GM2 ganglioside; KM966) and anti-LAMP1 (lysosomal-associated membrane protein 1) colocalization staining and incubated with 50 nM LysoTracker Red DND-99 to label lysosomes. In addition, GM2AP and HEXB expression were analyzed to assess whether rhHex-A treatment affected the levels of enzymes involved in GM2 ganglioside degradation. Results: Immunofluorescence staining for LysoTracker and colocalization studies of GM2 and Lamp1 indicated reduced lysosomal mass and GM2 levels. Notably, rhHex-A treatment also affected the expression of the HEXB gene, which is involved in GM2 ganglioside metabolism, highlighting a potential regulatory interaction within the metabolic pathway. Conclusions: Here, we report that rhHex-A produced in yeast can efficiently degrade GM2 ganglioside and rescue lysosomal accumulation in TSD cells. Full article

Background/Objectives: Tay–Sachs disease (TSD) is a neurodegenerative disorder caused by a deficiency in β-hexosaminidase A (HexA), which accumulates GM2 gangliosides, primarily in neurons. Currently, therapeutic options are limited, highlighting the need for new strategies such as gene therapy. Despite their effectiveness, viral vectors can elicit adverse immune responses; consequently, non-viral vectors are being explored as an alternative. We have previously investigated the use of CRISPR/Cas9 nickase (nCas9) as a potential tool for treating TSD. Here, we expanded our study by evaluating the PP6D5 polymer as a novel non-viral vector for delivering the CRISPR/nCas9 system to restore HexA activity. Methods: First, we evaluated the PP6D5-mediated CRISPR/nCas9 system’s transfection efficiency in NIH-3T3 fibroblasts, U87MG astrocytoma, SHSY5Y neuroblastoma, and TSD fibroblasts. We then evaluated the potential of PP6D5 to correct the gene defect in TSD fibroblasts. Results: The results showed that PP6D5 exhibited significantly higher transfection efficiency compared to lipofectamine 3000 in all tested cell models. In TSD fibroblasts, transfection with both HEXA and HEXB cDNAs increased the HexA activity levels by up to 7.4-fold, compared to a 3.2-fold increase in cells transfected only with HEXA cDNA after 15 days post-transfection. These levels were up to 4.5-fold higher than those observed in lipofectamine-mediated transfection. Additionally, PP6D5-mediated CRISPR/nCas9-based genome editing led to a significant reduction in the lysosomal mass of TSD fibroblasts. Conclusions: This study provides promising evidence for the use of the PP6D5 polymer as a non-viral vector for delivering CRISPR/nCas9-based gene therapy in TSD. The use of the PP6D5 polymer may offer some advantages that viral vectors cannot, such as a reduction in cytotoxicity and higher TE in difficult-to-transfect cell lines. Furthermore, this type of polymeric vector has not been extensively explored for gene therapy, making this study an important contribution to the development of non-viral delivery systems for the treatment of neurodegenerative diseases. Full article

Background: Racotumomab-alum is an anti-idiotype vaccine targeting the NeuGcGM3 tumor-associated ganglioside. Clinical trials in advanced cancer patients have demonstrated low toxicity, high immunogenicity and clinical benefit. The goal of this study was to identify circulating biomarkers of clinical outcome. Methods: Eighteen patients with stage IIIb/IV non-small-cell lung cancer (NSCLC) were injected with racotumomab-alum as switch maintenance therapy after first-line chemotherapy. Treatment was administered until severe performance status worsening or toxicity. The frequencies of innate and adaptive lymphocytes were assessed by flow cytometry. Circulating factors were measured using multi-analyte flow assay kits. Results: The median overall survival was 16.5 months. Twenty-seven percent of patients were classified as long-term survivors. Patients with lower baseline frequencies of CD4+Tregs and central memory (CM) CD8+T cells displayed longer survival rates. Furthermore, higher baseline frequencies of NKT cells and a high CD8+T/CD4+Treg ratio were associated with longer survival. Interestingly, patients with significantly lower levels of effector memory (EM) CD8+T cells survived longer. The levels of NKT cells and terminal effector memory (EMRA) CD8+T cells were higher in long-term survivors in comparison with short-term survivors in post-immune samples. As expected, the ratio of CD8+T/CD4+Tregs showed significantly higher values during treatment in patients with clinical benefits. Regarding serum factors, pro-tumorigenic cytokines significantly increased during treatment in poor survivors. Conclusions: In advanced NSCLC patients receiving racotumomab-alum vaccine, longer survival could be associated with a unique profile of circulating lymphocyte subsets at baseline and during treatment. Additionally, certain pro-tumor-related cytokines increased in short-term survivors. These results should be confirmed in larger randomized clinical trials. This clinical trial was registered in the Cuban Clinical Trials Register (RPCE00000279). Full article

Background: GM3 Synthase Deficiency (GM3SD) is a rare autosomal recessive neurodevelopmental disease characterized by recurrent seizures and neurological deficits. The disorder stems from mutations in the ST3GAL5 gene, encoding GM3 synthase (GM3S), a key enzyme in ganglioside biosynthesis. While enzyme deficiencies affecting ganglioside catabolism are well-documented, the consequences of impaired ganglioside biosynthesis remain less explored. Methods: To investigate GM3SD, we used a Human Embryonic Kidney 293-T (HEK293-T) knockout (KO) cell model generated via CRISPR/Cas9 technology. Lipid composition was assessed via high-performance thin-layer chromatography (HPTLC); glycohydrolase activity in lysosomal and plasma membrane (PM) fractions was enzymatically analyzed. Lysosomal homeostasis was evaluated through protein content analysis and immunofluorescence, and cellular bioenergetics was measured using a luminescence-based assay. Results: Lipidome profiling revealed a significant accumulation of lactosylceramide (LacCer), the substrate of GM3S, along with increased levels of monosialyl-globoside Gb5 (MSGb5), indicating a metabolic shift in glycosphingolipid biosynthesis. Lipid raft analysis revealed elevated cholesterol levels, which may impair microdomain fluidity and signal transduction. Furthermore, altered activity of lysosomal and plasma membrane (PM)-associated glycohydrolases suggests secondary deregulation of glycosphingolipid metabolism, potentially contributing to abnormal lipid patterns. In addition, we observed increased lysosomal mass, indicating potential lysosomal homeostasis dysregulation. Finally, decreased adenosine triphosphate (ATP) levels point to impaired cellular bioenergetics, emphasizing the metabolic consequences of GM3SD. Conclusions: Together, these findings provide novel insights into the molecular alterations associated with GM3SD and establish the HEK293-T KO model as a promising platform for evaluating potential therapeutic strategies. Full article

Supported lipid bilayers (SLBs) that model neuronal membranes are needed to explore the role of membrane lipids in the misfolding and aggregation of amyloid proteins associated with neurodegenerative diseases, including Parkinson’s and Alzheimer’s disease. The neuronal membranes include not only phospholipids, but also significant amounts of cholesterol, sphingomyelin, and gangliosides, which are critical to its biological function. In this study, we explored the conditions for the formation of an SLB, for the five-component lipid mixture composed of zwitterionic 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), anionic 1,2-dioleoyl- sn-glycero-3-phospho-L-serine (DOPS), nonionic cholesterol (Chol), zwitterionic sphingomyelin (SM), and anionic ganglioside (GM), using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, by varying experimental parameters such as pH, buffer type, temperature, vesicle size, and osmotic stress. SLB formation from this multicomponent lipid system was found challenging because the vesicles adsorbed intact on the quartz crystal and failed to rupture. For most of the variables tested, other than osmotic stress, we found no or only partial vesicle rupture leading to either a supported layer of vesicles or a partial SLB that included unruptured vesicles. When osmotic stress was applied to the vesicles already adsorbed on the surface, by having a different salt concentration in the rinse buffer that follows vesicle flow compared to that of the dilution buffer during vesicle flow and adsorption, vesicle rupture increased, but it remained incomplete. In contrast, when osmotic stress was applied during vesicle flow and adsorption on the surface, by having different salt concentrations in the dilution buffer in which vesicles flowed compared to the hydration buffer in which vesicles were prepared, complete vesicle rupture and successful formation of a rigid SLB was demonstrated. The robustness of this approach to form SLBs by applying osmotic stress during vesicle adsorption was found to be independent of the number of lipid components, as shown by SLB formation from the 1-, 2-, 3-, 4-, and 5-component lipid systems. Full article

Amyloid-β (Aβ) plaque deposits in the brain are a hallmark of Alzheimer’s disease (AD) neuropathology. Plaques consist of complex mixtures of peptides like Aβ_1–42 and characteristic lipids such as gangliosides, and they are targeted by reactive microglia and astrocytes. Background: In pharmaceutical research and development, it is a formidable challenge to contextualize the different biomolecular classes and cell types of the Aβ plaque microenvironment in a coherent experimental workflow on a single tissue section and on a benchtop imaging reader. Methods: Here, we developed a workflow that combines lipid MALDI mass spectrometry imaging using a vacuum-stable matrix with histopathology stains and with the MALDI HiPLEX immunohistochemistry of plaques and multiple protein markers on a benchtop imaging mass spectrometer. The three data layers consisting of lipids, protein markers, and histology could be co-registered and evaluated together. Results: Multimodal data analysis suggested the extensive co-localization of Aβ plaques with the peptide precursor protein, with a defined subset of lipids and with reactive glia cells on a single brain section in APPPS1 mice. Plaque-associated lipids like ganglioside GM2 and phosphatidylinositol PI38:4 isoforms were readily identified using the tandem MS capabilities of the mass spectrometer. Conclusions: Altogether, our data suggests that complex pathology involving multiple lipids, proteins and cell types can be interrogated by this spatial multiomics workflow on a user-friendly benchtop mass spectrometer. Full article

DHDDS (dehydrodolichol diphosphate synthetase) and NgBR (Nogo-B Receptor) collectively form an enzymatic complex important for the synthesis of dolichol, a key component of protein N-glycosylation. Mutations in DHDDS and the gene encoding NgBR (NUS1) are associated with neurodevelopmental disorders that clinically present with epilepsy, motor impairments, and developmental delay. Previous work has demonstrated both DHDDS and NgBR can also interact with NPC2 (Niemann-Pick C (NPC) type 2), a protein which functions to traffic cholesterol out of the lysosome and, when mutated, can cause a lysosomal storage disorder (NPC disease) characterised by an accumulation of cholesterol and glycosphingolipids. Abnormal cholesterol accumulation has also been reported in cells from both individuals and animal models with mutations in NUS1, and suspected lipid storage has been shown in biopsies from individuals with mutations in DHDDS. Our findings provide further evidence for overlap between NPC2 and DHDDS disorders, showing that DHDDS patient fibroblasts have increased lysosomal volume, store cholesterol and ganglioside GM1, and have altered lysosomal Ca²⁺ homeostasis. Treatment of DHDDS cells, with the approved NPC small molecule therapy, miglustat, improves these disease-associated phenotypes, identifying a possible therapeutic option for DHDDS patients. These data suggest that treatment options currently approved for NPC disease may be translatable to DHDDS/NUS1 patients. Full article

N-Glycolylneuraminic acid (Neu5Gc) is a sialic acid variant commonly found in most mammals but not synthesized by humans due to an inactivating mutation in the CMP-Neu5Ac hydroxylase (CMAH) gene. Despite this, Neu5Gc-containing molecules are consistently detected in human tissues, particularly in malignant tumors. However, the mechanisms underlying Neu5Gc accumulation and its role in cancer development remain poorly understood. Objectives: This review aims to analyze clinical and experimental evidence regarding the presence of Neu5Gc-containing glycoconjugates in both tumor and non-tumor human tissues, exploring potential mechanisms of the Neu5Gc expression and evaluating its contribution to tumor biology, with a particular focus on the Neu5Gc-GM3 ganglioside. Methods: A comprehensive review of the literature was conducted, integrating findings from immunohistochemistry, chromatography, and molecular studies to assess the expression and implications of Neu5Gc in cancer biology. Results: Neu5Gc-containing glycoconjugates were found to preferentially accumulate in various malignant tumors, while their presence in normal tissues was restricted to cells with high turnover rates. This accumulation is potentially mediated by dietary uptake, hypoxic conditions, and metabolic alterations in cancer cells. Additionally, Neu5Gc-containing molecules were associated with the activation of oncogenic pathways. Conclusion: Neu5Gc-containing glycoconjugates play a multifaceted role in cancer progression and present potential as prognostic markers and therapeutic targets. Full article

Sandhoff disease (SD) is a progressive neurodegenerative lysosomal storage disorder characterized by GM2 ganglioside accumulation as a result of mutations in the HEXB gene, which encodes the β-subunit of the enzyme β-hexosaminidase. Lysosomal storage of GM2 triggers inflammation in the CNS and periphery. The NLRP3 inflammasome is an important coordinator of pro-inflammatory responses, and we have investigated its regulation in murine SD. The NLRP3 inflammasome requires two signals, lipopolysaccharide (LPS) and ATP, to prime and activate the complex, respectively, leading to IL-1β secretion. Peritoneal, but not bone-marrow-derived, macrophages from symptomatic SD mice, but not those from pre-symptomatic animals, secrete the cytokine following priming with LPS with no requirement for activation with ATP, suggesting that such NLRP3 deregulation is related to the extent of glycosphingolipid storage. Dysregulated production of IL-1β was dependent upon caspase activity but not cathepsin B. We investigated the role of IL-1β in SD pathology using two approaches: the creation of hexb^−/−Il1r1^−/− double knockout mice or by treating hexb^−/− animals with anakinra, a recombinant form of the IL-1 receptor antagonist, IL-1Ra. Both resulted in modest but significant extensions in lifespan and improvement of neurological function. These data demonstrate that IL-1β actively participates in the disease process and provides proof-of-principle that blockade of the pro-inflammatory cytokine IL-1β may provide benefits to patients. Full article

In pediatric and intensive care units, propofol is widely used for general anesthesia and sedation procedures as a short-acting anesthetic. Multiple studies have revealed that propofol causes hippocampal injury and cognitive dysfunction in developing animals. As is known, GM1, a type of ganglioside, plays a crucial role in promoting nervous system development. Consequently, this study explored whether GM1 mitigated neurological injury caused by propofol during developmental stages and investigated its underlying mechanisms. Seven-day-old SD rats or PC12 cells were used in this study for histopathological analyses, a Morris water maze test, a lactate dehydrogenase release assay, Western blotting, and an ELISA. Furthermore, LY294002 was employed to explore the potential neuroprotective effect of GM1 via the PI3K/AKT signaling cascade. The results indicated that GM1 exerted a protective effect against hippocampal morphological damage and pyroptosis as well as behavioral abnormalities following propofol exposure by increasing p-PI3K and p-AKT expression while decreasing p-p65 expression in developing rats. Nevertheless, the inhibitor LY294002, which targets the PI3K/AKT cascade, attenuated the beneficial effects of GM1. Our study provides evidence that GM1 confers neuroprotection and attenuates propofol-induced developmental neurotoxicity, potentially involving the PI3K/AKT/NF-κB signaling cascade. Full article

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by mutations in the methyl-CpG binding protein 2 (MECP2) gene. Despite advancements in research, no cure exists due to an incomplete understanding of the molecular effects of MeCP2 deficiency. Previous studies have identified impaired tropomyosin receptor kinase (Trk) neurotrophin (NTP) signaling and mitochondrial redox imbalances as key drivers of the pathology. Moreover, altered glycosphingolipid metabolism has been reported in RTT. GM1 ganglioside is a known regulator of the nervous system, and growing evidence indicates its importance in maintaining neuronal homeostasis via its oligosaccharide chain, coded as GM1-OS. GM1-OS directly interacts with the Trk receptors on the cell surface, triggering neurotrophic and neuroprotective pathways in neurons. In this study, we demonstrate that GM1-OS ameliorates RTT deficits in the Mecp2-null model. GM1-OS restored synaptogenesis and reduced mitochondrial oxidative stress of Mecp2-knock-out (ko) cortical neurons. When administered in vivo, GM1-OS mitigated RTT-like symptoms. Our findings indicate that GM1-OS effects were mediated by Trk receptor activation on the neuron’s plasma membrane. Overall, our results highlight GM1-OS as a promising candidate for RTT treatment. Full article

This study aimed to determine the expression and distribution of gangliosides in specific regions of the brains of rainbow trout (Oncorhynchus mykiss Walbaum) and common carp (Cyprinus carpio L.) with regard to seasonal temperature changes. Seasonal changes in ganglioside expression and distribution within the species were expected. The natural ecosystems of these fishes differ significantly due to their distinct habitat preferences, geographic distributions, and environmental requirements. Based on the fact that the common carp is eurythermic and adapts to a wide range of temperatures, while the rainbow trout is stenothermic and thrives in a narrower temperature range, it was expected that these species would exhibit distinct patterns of ganglioside modification as part of their adaptive response to temperature fluctuations. Immunohistochemistry using specific antibodies for the major brain gangliosides (GM1, GD1a, GD1b, GT1b), along with the Svennerholm method for quantifying sialic acid bound to gangliosides, revealed that cold acclimatization led to an increase in polysialylated gangliosides in the common carp brain and an increase in trisialogangliosides in the rainbow trout brain. Immunohistochemical analysis also identified region-specific changes in ganglioside expression, suggesting specific functional roles in neuronal adaptation. These results supported the hypothesis that the composition and distribution of brain gangliosides change in response to seasonal thermal shifts as part of the adaptive response. The results underscore the importance of gangliosides in neuronal function and adaptation to environmental stimuli, with implications for understanding fish resilience to temperature changes. This study offers valuable insights into species’ temperature adaptation, with implications for physiological and ecological management and improved aquaculture practices. Future research could expand the species scale, study molecular mechanisms and regulatory pathways in ganglioside metabolism, and examine ganglioside interactions with membrane proteins and lipids for a deeper understanding of thermal adaptation. Full article

Gangliosides are glycosphingolipids composed of a sialylated glycan head group and a ceramide backbone. These anionic lipids form lipid rafts and play crucial roles in regulating various proteins involved in signal transduction, adhesion, and cell–cell recognition. Neuroblastoma, a pediatric cancer of the sympathetic nervous system, is treated with intensive chemotherapy, radiation, and an antibody targeting the GD2 ganglioside. Gangliosides are critical in neuroblastoma development and serve as therapeutic targets, making it essential to establish a reliable, rapid, and cost-effective method for profiling gangliosides, particularly one capable of isomeric separation of intact species. In this study, liquid chromatography–mass spectrometry (LC-MS) was optimized using standard gangliosides, followed by the optimization of sphingolipid extraction methods from cell lines by comparing Folch and absolute methanol extraction techniques. Percent recovery and the number of identified sphingolipids were used to evaluate the analytical merits of these methods. A standard gangliosides calibration curve demonstrated excellent linearity (R² = 0.9961–0.9975). The ZIC-HILIC column provided the best separation of ganglioside GD1 isomers with a 25 min runtime. GD1a elutes before GD1b on the ZIC-HILIC column. Absolute methanol yielded better percent recovery (96 ± 7) and identified 121 different sphingolipids, the highest number between the two extraction methods. The optimized method was applied to profile gangliosides in neuroblastoma (COG-N-683), pancreatic cancer (PSN1), breast cancer (MDA-MB-231BR), and brain tumor (CRL-1620) cell lines. The ganglioside profile of the neuroblastoma cell line COG-N-683 showed an inverse relationship between GD1 and GD2. Ceramide, Hex1Cer, GM1, and GM3 were highly abundant in CRL-1620, PSN1, and MDA-MB-231BR, respectively. These results suggest that our method provides a sensitive, reliable, and high-throughput workflow for ganglioside profiling across different cell types. Full article