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Background: Established psychosocial risk factors increase the risk for experimentation among Mexican origin youth. Now, we comprehensively investigate the added contribution of select polymorphisms in candidate genetic pathways associated with sensation seeking, risk taking, and smoking phenotypes to predict experimentation.Methods: Participants (N = 1,118 Mexican origin youth) recruited from a large population-based cohort study in Houston, TX, provided prospective data on cigarette experimentation over 3 years. Psychosocial data were elicited twice—baseline and final follow-up. Participants were genotyped for 672 functional and tagging variants in the dopamine, serotonin, and opioid pathways.Results: After adjusting for gender and age, with a Bayesian False Discovery Probability set at 0.8 and prior probability of 0.05, six gene variants were significantly associated with risk of experimentation. After controlling for established risk factors, multivariable analyses revealed that participants with six or more risk alleles were 2.25 [95% confidence interval (CI), 1.62–3.13] times more likely to have experimented since baseline than participants with five or fewer. Among committed never-smokers (N = 872), three genes (OPRM1, SNAP25, HTR1B) were associated with experimentation as were all psychosocial factors. Among susceptible youth (N = 246), older age at baseline, living with a smoker, and three different genes (HTR2A, DRD2, SLC6A3) predicted experimentation.Conclusions: Our findings, which have implications for development of culturally specific interventions, need to be validated in other ethnic groups.Impact: These results suggest that variations in select genes interact with a cognitive predisposition toward smoking. In susceptible adolescents, the impact of the genetic variants appears to be larger than committed never-smokers. Cancer Epidemiol Biomarkers Prev; 21(1); 228–38. ©2011 AACR.

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The postnatal neural stem cell (NSC) pool hosts quiescent and activated radial glia-like NSCs contributing to neurogenesis throughout adulthood. However, the underlying regulatory mechanism during the transition from quiescent NSCs to activated NSCs in the postnatal NSC niche is not fully understood. Lipid metabolism and lipid composition play important roles in regulating NSC fate determination. Biological lipid membranes define the individual cellular shape and help maintain cellular organization and are highly heterogenous in structure and there exist diverse microdomains (also known as lipid rafts), which are enriched with sugar molecules, such as glycosphingolipids. An often overlooked but key aspect is that the functional activities of proteins and genes are highly dependent upon their molecular environments. We previously reported that ganglioside GD3 is the predominant species in NSCs and that the reduced postnatal NSC pools are observed in global GD3-synthase knockout (GD3S-KO) mouse brains. The specific roles of GD3 in determining the stage and cell-lineage determination of NSCs remain unclear, since global GD3S-KO mice cannot distinguish if GD3 regulates postnatal neurogenesis or developmental impacts. Here we show that inducible GD3 deletion in postnatal radial glia-like NSCs promotes the NSC activation, resulting in the loss of the long-term maintenance of the adult NSC pools. The reduced neurogenesis in the subventricular zone (SVZ) and the dentate gyrus (DG) of GD3S-conditional-knockout mice led to impaired olfactory and memory functions. Thus, our results provide convincing evidence that postnatal GD3 maintains the quiescent state of radial glia-like NSCs in the adult NSC niche.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting the body and mind of millions of people in the world. As PD progresses, bradykinesia, rigidity, and tremor worsen. These motor symptoms are associated with the neurodegeneration of dopaminergic neurons in the substantia nigra. PD is also associated with non-motor symptoms, including loss of smell (hyposmia), sleep disturbances, depression, anxiety, and cognitive impairment. This broad spectrum of non-motor symptoms is in part due to olfactory and hippocampal dysfunctions. These non-motor functions are suggested to be linked with adult neurogenesis. We have reported that ganglioside GD3 is required to maintain the neural stem cell (NSC) pool in the subventricular zone (SVZ) of the lateral ventricles and the subgranular layer of the dentate gyrus (DG) in the hippocampus. In this study, we used nasal infusion of GD3 to restore impaired neurogenesis in A53T alpha-synuclein-expressing mice (A53T mice). Intriguingly, intranasal GD3 administration rescued the number of bromodeoxyuridine + (BrdU +)/Sox2 + NSCs in the SVZ. Furthermore, the administration of gangliosides GD3 and GM1 increases doublecortin (DCX)-expressing immature neurons in the olfactory bulb, and nasal ganglioside administration recovered the neuronal populations in the periglomerular layer of A53T mice. Given the relevance of decreased ganglioside on olfactory impairment, we discovered that GD3 has an essential role in olfactory functions. Our results demonstrated that intranasal GD3 infusion restored the self-renewal ability of the NSCs, and intranasal GM1 infusion promoted neurogenesis in the adult brain. Using a combination of GD3 and GM1 has the potential to slow down disease progression and rescue dysfunctional neurons in neurodegenerative brains.

Patients with nervous system disorders suffer from impaired cognitive, sensory and motor functions that greatly inconvenience their daily life and usually burdens their family and society. It is difficult to achieve functional recovery for the damaged central nervous system (CNS) because of its limited ability to regenerate. Glycosphingolipids (GSLs) are abundant in the CNS and are known to play essential roles in cell-cell recognition, adhesion, signal transduction, and cellular migration, that are crucial in all phases of neurogenesis. Despite intense investigation of CNS regeneration, the roles of GSLs in neural regeneration remain unclear. Here we focus on the respective potentials of glycolipids to promote regeneration and repair of the CNS. Mice lacking glucosylceramide, lactosylceramide or gangliosides show lethal phenotypes. More importantly, patients with ganglioside deficiencies exhibit severe clinical phenotypes. Further, neurodegenerative diseases and mental health disorders are associated with altered GSL expression. Accumulating studies demonstrate that GSLs not only delimit physical regions but also play central roles in the maintenance of the biological functions of neurons and glia. We anticipate that the ability of GSLs to modulate behavior of a variety of molecules will enable them to ameliorate biochemical and neurobiological defects in patients. The use of GSLs to treat such defects in the human CNS will be a paradigm-shift in approach since GSL-replacement therapy has not yet been achieved in this manner clinically.

Gangliosides, the major sialic-acid containing glycosphingolipids in the mammalian brain, play important roles in brain development and neural functions. Here, we show that the b-series ganglioside GD3 and its biosynthetic enzyme, GD3-synthase (GD3S), were up-regulated predominantly in the microglia of mouse hippocampus from 2 to 7 days following global cerebral ischemia (GCI). Interestingly, GD3S knockout (GD3S-KO) mice exhibited decreased hippocampal neuronal loss following GCI, as compared to wild-type (WT) mice. While comparable levels of astrogliosis and microglial proliferation were observed between WT and GD3S-KO mice, the phagocytic capacity of the GD3S-KO microglia was significantly compromised after GCI. At 2 and 4 days following GCI, the GD3S-KO microglia demonstrated decreased amoebic morphology, reduced neuronal material engulfment, and lower expression of the phagolysosome marker CD68, as compared to the WT microglia. Finally, by using a microglia-primary neuron co-culture model, we demonstrated that the GD3S-KO microglia isolated from mouse brains at 2 days after GCI are less neurotoxic to co-cultured hippocampal neurons than the WT-GCI microglia. Moreover, the percentage of microglia with engulfed neuronal elements in the co-cultured wells was also significantly decreased in the GD3S-KO mice after GCI. Interestingly, the impaired phagocytic capacity of GD3S-KO microglia could be partially restored by pre-treatment with exogenous ganglioside GD3. Altogether, this study provides functional evidence that ganglioside GD3 regulates phagocytosis by microglia in an ischemic stroke model. Our data also suggest that the GD3-linked microglial phagocytosis may contribute to the mechanism of delayed neuronal death following ischemic brain injury.

HPV vaccination is highly effective at preventing several types of cancer; however, vaccine uptake is suboptimal. The COVID-19 pandemic has affected participation in cancer prevention measures such as HPV vaccination. To assess changes and barriers to HPV vaccination during the COVID-19 pandemic, we conducted a statewide cross-sectional survey of health-care professionals (HCPs) in Texas. Specifically, we evaluated changes observed by HCPs regarding HPV vaccination during the COVID-19 pandemic: 1) hesitancy, 2) refusal, and 3) uptake. Decreased HPV vaccination uptake were reported by 19.3% of HCPs, whereas increased HPV vaccination hesitancy and refusal were reported by 17.1% and 14.8% of HCPs in Texas, respectively. The COVID-19 pandemic had a negative impact on HPV vaccination. Our study identified barriers to HPV vaccination that are unique to the COVID-19 pandemic.

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

Ganglioside GD3, a major ganglioside species in neural stem cells, plays a crucial role in maintenance of the self-renewal capacity of these cells. However, its bioactivity in postnatally differentiated neurons in the neurogenic regions of adult brains has not been elucidated. Here, we describe for the first time that deletion of GD3 not only impairs neurotrophin-induced stem cell proliferation, but also alters the dendritic structure as well as the number of synapses of nascent neurons in the dentate gyrus of adult brain. When examining the behavioral phenotypes, GD3 synthase-knockout (GD3S-KO) mice displayed impairment in hippocampus-dependent memory function. To further gain insight into its cellular function, we examined GD3-binding partners from mouse brain extract using a GD3-specific monoclonal antibody, R24, followed by LC-MS/MS analysis and identified a mitochondrial fission protein, the dynamin-related protein-1 (Drp1), as a novel GD3-binding protein. Biochemical and imaging analyses revealed mitochondrial fragmentation in GD3-depleted dentate gyrus neurons, suggesting that GD3 is essential for the mitochondrial Drp1 turnover that is required for efficient mitochondrial fission. These results suggest that GD3 is required for proper dendritic and spine maturation of newborn neurons in adult brain through the regulation of mitochondrial dynamics.

Parkinson's disease (PD) is characterized by Lewy bodies (composed predominantly of alpha-synuclein [aSyn]) and loss of pigmented midbrain dopaminergic neurons comprising the nigrostriatal pathway. Most PD patients show significant deficiency of gangliosides, including GM1, in the brain, and GM1 ganglioside appears to keep dopaminergic neurons functioning properly. Thus, supplementation of GM1 could potentially provide some rescuing effects. In this study, we demonstrate that intranasal infusion of GD3 and GM1 gangliosides reduces intracellular aSyn levels. GM1 also significantly enhances expression of tyrosine hydroxylase (TH) in the substantia nigra pars compacta of the A53T aSyn overexpressing mouse, following restored nuclear expression of nuclear receptor related 1 (Nurr1, also known as NR4A2), an essential transcription factor for differentiation, maturation, and maintenance of midbrain dopaminergic neurons. GM1 induces epigenetic activation of the TH gene, including augmentation of acetylated histones and recruitment of Nurr1 to the TH promoter region. Our data indicate that intranasal administration of gangliosides could reduce neurotoxic proteins and restore functional neurons via modulating chromatin status by nuclear gangliosides.

The traditional methods to study lipid rafts and their association with membrane proteins are based mainly on the isolation of a detergent-resistant membrane by biochemical fractionation. However, the use of detergents may induce lipid segregation and/or redistribution of membrane proteins during the process of sample preparation. Here, we describe a detergent-free method to study the glycolipid and growth factor receptor interaction and their association with lipid rafts. This method combines the biochemical and immunoblotting tools with confocal microscopic imaging, which allows for evaluation and verification of the membrane protein interaction and association with the lipid rafts components in a multifaceted manner.

We previously reported that ganglioside GD3 is the predominant species in neural stem cells (NSCs) and reduced postnatal NSC pools are observed in both the subventricular zone and dentate gyrus (DG) of GD3-synthase knockout (GD3S-KO) mouse brains. Specifically, deficiency of GD3 in GD3S-KO animals revealed a dramatic reduction in cellularity in the DG of the hippocampus of the developing mouse brain, resulting in severe behavioral deficits in these animals. To further evaluate the functional role of GD3 in postnatal brain, we performed rescue experiments by intracerebroventricular infusion of ganglioside GD3 in adult GD3S-KO animals and found that it could restore the NSC pools and enhance the NSCs for self-renewal. Furthermore, 5xFAD mouse model was utilized, and GD3 restored NSC numbers and GM1 promoted neuronal differentiation. Our results thus demonstrate that exogenously administered gangliosides are capable to restore the function of postnatal NSCs. Since ganglioside expression profiles are associated not only with normal brain development but also with pathogenic mechanisms of diseases, such as Alzheimer’s disease, we anticipate that the administration of exogenous gangliosides, such as GD3 and GM1, may represent a novel and effective strategy for promoting adult neurogenesis in damaged brain for disease treatment.

The central nervous system is generated from progenitor cells that are recognized as neural stem cells (NSCs). NSCs are defined as undifferentiated neural cells that are characterized by the capacity for self-renewal and multipotency. Throughout neural development, NSCs undergo proliferation, migration, and cellular differentiation, and dynamic changes are observed in the composition of carbohydrate-rich molecules, including gangliosides. Gangliosides are sialic acid-containing glycosphingolipids with essential and multifaceted functions in brain development and NSC maintenance, which reflects the complexity of brain development. Our group has pioneered research on the importance of gangliosides for growth factor receptor signaling and epigenetic regulation of ganglioside biosynthesis in NSCs. We found that GD3 is the predominant ganglioside species in NSCs (>80%) and modulates NSC proliferation by interacting with epidermal growth factor receptor signaling. In postnatal brain, GD3 is required for long-term maintenance of NSCs. Deficiency in GD3 leads to developmental and behavioral deficits, such as depression. The synthesis of GD3 is switched to the synthesis of complex, brain-type gangliosides, namely, GM1, GD1a, GD1b, and GT1b, resulting in terminal differentiation and loss of “stemness” of NSCs. In this process, GM1 is augmented by a novel GM1-modulated epigenetic gene regulation mechanism of glycosyltransferases at a later differentiation stage. Consequently, our research suggests that stage-specific gangliosides play specific roles in maintaining NSC activities and in cell fate determination.

Gangliosides are sialic acid-containing glycosphingolipids that are most abundant in the nerve tissues. The quantity and expression pattern of gangliosides in brain change drastically throughout development and are mainly regulated through stage-specific expression of glycosyltransferase (ganglioside synthase) genes. We previously demonstrated that acetylation of histones H3 and H4 on the N-acetylgalactosaminyltransferase I (GalNAcT, GA2/GM2/GD2/GT2-synthase) gene promoter resulted in recruitment of trans-activation factors. In addition, we reported that epigenetic activation of the GalNAcT gene was also detected as accompanied by an apparent induction of neuronal differentiation in neural stem cells responding to an exogenous supplement of ganglioside GM1. Here, we present evidence supporting the concept that nuclear GM1 is associated with gene regulation in neuronal cells. We found that nuclear GM1 binds acetylated histones on the promoters of the GalNAcT and NeuroD1 genes in differentiated neurons. Our study demonstrates for the first time that GM1 interacts with chromatin via acetylated histones at the nuclear periphery of neuronal cells.

The maintenance of a neural stem cell (NSC) population in mammalian postnatal and adult life is crucial for continuous neurogenesis and neural repair. However, the molecular mechanism of how NSC populations are maintained remains unclear. Gangliosides are important cellular membrane components in the nervous system. We previously showed that ganglioside GD3 plays a crucial role in the maintenance of the self-renewal capacity of NSCsin vitro. Here, we investigated its role in postnatal and adult neurogenesis in GD3-synthase knock-out (GD3S-KO) and wild-type mice. GD3S-KO mice with deficiency in GD3 and the downstream b-series gangliosides showed a progressive loss of NSCs both at the SVZ and the DG of the hippocampus. The decrease of NSC populations in the GD3S-KO mice resulted in impaired neurogenesis at the granular cell layer of the olfactory bulb and the DG in the adult. In addition, defects of the self-renewal capacity and radial glia-like stem cell outgrowth of postnatal GD3S-KO NSCs could be rescued by restoration of GD3 expression in these cells. Our study demonstrates that the b-series gangliosides, especially GD3, play a crucial role in the long-term maintenance NSC populations in postnatal mouse brain. Moreover, the impaired neurogenesis in the adult GD3S-KO mice led to depression-like behaviors. Thus, our results provide convincing evidence linking b-series gangliosides deficiency and neurogenesis defects to behavioral deficits, and support a crucial role of gangliosides in the long-term maintenance of NSCs in adult mice.

Removal of 9-O-acetyl residues from the cell surface N-acetylneuraminic acid makes ALL cells drug sensitive., The development of resistance to chemotherapy is a major cause of relapse in acute lymphoblastic leukemia (ALL). Though several mechanisms associated with drug resistance have been studied in detail, the role of carbohydrate modification remains unexplored. Here, we investigated the contribution of 9-O-acetylated N-acetylneuraminic acid (Neu5Ac) to survival and drug resistance development in ALL cells. A strong induction of 9-O-acetylated Neu5Ac including 9-O-acetyl GD3 was detected in ALL cells that developed resistance against vincristine or nilotinib, drugs with distinct cytotoxic mechanisms. Removal of 9-O-acetyl residues from Neu5Ac on the cell surface by an O-acetylesterase made ALL cells more vulnerable to such drugs. Moreover, removal of intracellular and cell surface–resident 9-O-acetyl Neu5Ac by lentiviral transduction of the esterase was lethal to ALL cells in vitro even in the presence of stromal protection. Interestingly, expression of the esterase in normal fibroblasts or endothelial cells had no effect on their survival. Transplanted mice induced for expression of the O-acetylesterase in the ALL cells exhibited a reduction of leukemia to minimal cell numbers and significantly increased survival. This demonstrates that Neu5Ac 9-O-acetylation is essential for survival of these cells and suggests that Neu5Ac de-O-acetylation could be used as therapy to eradicate drug-resistant ALL cells.

Journal of Lipid Research Volume 54, 2013 1 Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc. Chuck Sweeley died in Lansing, Michigan, on September 21, 2012, at the age of 82 after a long battle with a rare form of bladder cancer. He was born in Williamsport, Pennsylvania on April 15, 1930, where he grew up and attended primary and secondary school. He received a B.S. in Chemistry from the University of Pennsylvania in 1952 and a Ph.D. in Biochemistry in 1955 at the University of Illinois, Urbana-Champaign, working under the direction of Professor Herbert Carter. After further training with Evan Horning at the National Institutes of Health, Chuck took a position in the Department of Biochemistry and Nutrition at the University of Pittsburgh in 1960. He was promoted to Professor in 1966. He moved to the Department of Biochemistry at Michigan State University in 1968 and spent the rest of his career there. He served as Chairperson of the department at MSU from 1979 to 1985 and was a University Distinguished Professor at Michigan State University when he retired in 1992.( 1 ) During his career, he made major scientifi c contributions to the fi elds of sphingolipids and biomedical mass spectrometry. Here, we summarize and highlight some of his accomplishments, which are described in greater detail in a recent review ( 2 ). Chuck’s illustrious research career began during his Ph.D. training. His Ph.D. thesis was on the chemistry of antibiotics, one of the major interests of the Carter laboratory. After graduation, Chuck switched his interest to the chemistry and biochemistry of sphingolipids and glycosphingolipids, stating that “It might seem odd to some that I would devote much of my professional career studying the biochemistry of sphingolipids... because ...my doctoral thesis was entitled “Studies on Streptolidine, a Degradation Product of Streptothricin”....Carter had two laboratories, one for students working on antibiotics and one for students working on sphingosine and sphingolipids. Little did I know that the research being done in the “sphingolipids lab” would stick to me In Memoriam: Charles (Chuck) Crawford Sweeley, Jr. (1930–2012)

The structural diversity and localization of cell surface glycosphingolipids (GSLs), including gangliosides, in glycolipid-enriched microdomains (GEMs, also known as lipid rafts) render them ideally suited to play important roles in mediating intercellular recognition, interactions, adhesion, receptor function, and signaling. Gangliosides, sialic acid-containing GSLs, are most abundant in the nerve tissues. The quantity and expression pattern of gangliosides in brain change drastically throughout development and these changes are mainly regulated through stage-specific expression of glycosyltransferase genes. We previously demonstrated for the first time that efficient histone acetylation of the glycosyltransferase genes in mouse brain contributes to the developmental alteration of ganglioside expression. We further demonstrated that acetylation of histones H3 and H4 on the N-acetylgalactosaminyltransferase I (GalNAcT, GA2/GM2/GD2/GT2-synthase; B4galnt1) gene promoter resulted in recruitment of trans-activation factors. In addition, we showed that epigenetic activation of the GalNAcT gene was detected and accompanied by an apparent induction of neuronal differentiation of neural stem cells (NSCs) responding to an exogenous supplement of ganglioside GM1. Most recently, we found that nuclear GM1 binds with acetylated histones on the promoters of the GalNAcT as well as on the NeuroD1 genes in differentiated neurons. Here, we will introduce epigenetic regulation of ganglioside synthase genes in neural development and neuronal differentiation of NSCs.

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into brain-forming cells. Several signaling pathways have been shown to be involved in the fate determination process of NSCs, but the molecular mechanisms underlying the maintenance of neural cell stemness remain largely unknown. Our previous study showed that human natural killer carbohydrate epitopes expressed specifically by mouse NSCs modulate the Ras-MAPK pathway, raising the possibility of regulatory roles of glycoprotein glycans in the specific signaling pathways involved in NSC fate determination. To address this issue, we performed comparative N-glycosylation profiling of NSCs before and after differentiation in a comprehensive and quantitative manner. We found that Lewis X-carrying N-glycans were specifically displayed on undifferentiated cells, whereas pauci-mannose-type N-glycans were predominantly expressed on differentiated cells. Furthermore, by knocking down a fucosyltransferase 9 with short interfering RNA, we demonstrated that the Lewis X-carrying N-glycans were actively involved in the proliferation of NSCs via modulation of the expression level of Musashi-1, which is an activator of the Notch signaling pathway. Our findings suggest that Lewis X carbohydrates, which have so far been characterized as undifferentiation markers, actually operate as activators of the Notch signaling pathway for the maintenance of NSC stemness during brain development.

Background: Measurement of anti-GM1 IgM antibodies in multifocal motor neuropathy (MMN) sera is confounded by relatively low sensitivity that limits clinical usefulness. Combinatorial assay methods, in which antibodies react to heteromeric complexes of two or more glycolipids, are being increasingly applied to this area of diagnostic testing. Methods: A newly developed combinatorial glycoarray able to identify antibodies to 45 different heteromeric glycolipid complexes and their 10 individual glycolipid components was applied to a randomly selected population of 33 MMN cases and 57 normal or disease controls. Comparison with an enzyme-linked immunosorbent assay (ELISA) was conducted for selected single glycolipids and their complexes. Results: By ELISA, 22/33 MMN cases had detectable anti-GM1 IgM antibodies, whereas 19/33 MMN samples were positive for anti-GM1 antibodies by glycoarray. Analysis of variance (anova) revealed that of the 55 possible single glycolipids and their 1:1 complexes, antibodies to the GM1:galactocerebroside (GM1:GalC) complex were most significantly associated with MMN, returning 33/33 MMN samples as positive by glycoarray and 29/33 positive by ELISA. Regression analysis revealed a high correlation in absolute values between ELISA and glycoarray. Receiver operator characteristic analysis revealed insignificantly different diagnostic performance between the two methods. However, the glycoarray appeared to offer slightly improved sensitivity by identifying antibodies in four ELISA-negative samples. Conclusions: The use of combinatorial glycoarray or ELISA increased the diagnostic sensitivity of anti-glycolipid antibody testing in this cohort of MMN cases, without significantly affecting specificity, and may be a useful assay modification for routine clinical screening. © 2012 The Author(s) European Journal of Neurology © 2012 EFNS.

Cycad seed consumption by the native islanders of Guam is frequently associated with high rates of amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS/PDC); furthermore, accompanying pathological examination often exhibits α-synuclein inclusions in the neurons of the affected brain. Acylated steryl-β-glucoside (ASG) contained in cycad seeds is considered as causative environmental risk factor. We aimed to investigate whether ASG influences aggregation and cell toxicity of α-synuclein. To understand whether ASG is a causative factor in the development of ALS/PDC, soybean-derived ASG was tested for its effect on in vitro aggregation of α-synuclein using Thioflavin-T. ASG was also tested to determine whether it modulates α-synuclein cytotoxicity in yeast cells. In addition, we determined whether an interaction between ASG and α-synuclein occurs in the plasma membrane or cytoplasm using three factors: GM1 ganglioside, small unilamellar vesicles, and ATP. In the present study, we found that ASG-mediated acceleration of α-synuclein aggregation is influenced by the presence of ATP, but not by the presence of GM1. ASG accelerated the α-synuclein aggregation in the cytoplasm. ASG also enhanced α-synuclein-induced cytotoxicity in yeast cells. This study demonstrated that ASG directly enhances aggregation and cytotoxicity of α-synuclein, which are often observed in patients with ALS/PDC. These results, using assays that replicate cytoplasmic conditions, are consistent with the molecular mechanism that cytotoxicity is caused by intracellular α-synuclein fibril formation in neuronal cells.