Ion Mobility Mass Spectrometry Reveals Rare Sialylated Glycosphingolipid Structures in Human Cerebrospinal Fluid.

Gangliosides (GGs) represent an important class of biomolecules associated with the central nervous system (CNS). In view of their special role at a CNS level, GGs are valuable diagnostic markers and prospective therapeutic agents. By ion mobility separation mass spectrometry (IMS MS), recently implemented by us in the investigation of human CNS gangliosidome, we previously discovered a similarity between GG profiles in CSF and the brain. Based on these findings, we developed IMS tandem MS (MS/MS) to characterize rare human CSF glycoforms, with a potential biomarker role. To investigate the oligosaccharide and ceramide structures, the ions detected following IMS MS separation were submitted to structural analysis by collision-induced dissociation (CID) MS/MS in the transfer cell. The IMS evidence on only one mobility feature, together with the diagnostic fragment ions, allowed the unequivocal identification of isomers in the CSF. Hence, by IMS MS/MS, GalNAc-GD1c(d18:1/18:1) and GalNAc-GD1c(d18:1/18:0) having both Neu5Ac residues and GalNAc attached to the external galactose were for the first time discovered and structurally characterized. The present results demonstrate the high potential of IMS MS/MS for biomarker discovery and characterization in body fluids, and the perspectives of method implementation in clinical analyses targeting the early diagnosis of CNS diseases through molecular fingerprints.

Glycoblotting method allows for rapid and efficient glycome profiling of human Alzheimer's disease brain, serum and cerebrospinal fluid towards potential biomarker discovery.

BACKGROUND: Understanding of the significance of posttranslational glycosylation in Alzheimer's disease (AD) is of growing importance for the investigation of the pathogenesis of AD as well as discovery research of the disease-specific serum biomarkers. METHODS: We designed a standard protocol for the glycoblotting combined with MALDI-TOFMS to perform rapid and quantitative profiling of the glycan parts of glycoproteins (N-glycans) and glycosphingolipids (GSLs) using human AD's post-mortem samples such as brain tissues (dissected cerebral cortices such as frontal, parietal, occipital, and temporal domains), serum and cerebrospinal fluid (CSF). RESULTS: The structural profiles of the major N-glycans released from glycoproteins and the total expression levels of the glycans were found to be mostly similar between the brain tissues of the AD patients and those of the normal control group. In contrast, the expression levels of the serum and CSF protein N-glycans such as bisect-type and multiply branched glycoforms were increased significantly in AD patient group. In addition, the levels of some gangliosides such as GM1, GM2 and GM3 appeared to alter in the AD patient brain and serum samples when compared with the normal control groups. CONCLUSION: Alteration of the expression levels of major N- and GSL-glycans in human brain tissues, serum and CSF of AD patients can be monitored quantitatively by means of the glycoblotting-based standard protocols. GENERAL SIGNIFICANCE: The changes in the expression levels of the glycans derived from the human post-mortem samples uncovered by the standardized glycoblotting method provides potential serum biomarkers in central nervous system disorders and can contribute to the insight into the molecular mechanisms in the pathogenesis of neurodegenerative diseases and future drug discovery. Most importantly, the present preliminary trials using human post-mortem samples of AD patients suggest that large-scale serum glycomics cohort by means of various-types of human AD patients as well as the normal control sera can facilitate the discovery research of highly sensitive and reliable serum biomarkers for an early diagnosis of AD. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Myelin glycosphingolipid immunoreactivity and CSF levels in multiple sclerosis.

OBJECTIVES: Patients with multiple sclerosis were reported to harbour antibodies not only against proteins and glycoproteins but also against glycolipids, including sulfatide and galactosylceramide (GalCer), the two major glycosphingolipids of myelin. However, previous results were inconsistent concerning glycosphingolipid levels, antibody type, dominance of serum or Cerebrospinal fluid compartments and relationship to the multiple sclerosis (MS) course. RESULTS: We hereby report that the cerebrospinal fluid levels of sulfatide were increased in patients with MS (n = 46) compared with controls (n = 50, P < 0.001). In addition, patients had higher serum IgM anti-glycosphingolipid titres than controls (P = 0.03 for sulfatide, <0.001 for GalCer), while the anti-glycosphingolipid IgM antibodies in the cerebrospinal fluid were essentially normal. However, in seven of 46 patients cerebrospinal fluid IgG antibodies against GalCer (P = 0.004) could be detected, which was not found in any of the control individuals, and this finding might mirror the occurrence of more specific B-cell clones behind the blood-brain barrier. CONCLUSIONS: The IgM immunoreactivity in serum did not show any relationship to the type of course or severity of MS, arguing against a phenomenon secondary to myelin damage. Thus, the IgM antibody findings are compatible with an early antigen challenge or autoimmunity associated with natural antibodies.

Neurenteric cyst of the lower clivus.

OBJECTIVE: Neurenteric cysts (NCs) typically arise as benign ventral intradural extramedullary developmental malformations of the spine which contain heterotopic epithelium resembling the intestinal or respiratory tracts. Intracerebral NCs are extremely rare, though the frequency of symptomatic reports and incidental findings is increasing, perhaps because of advances in neuroimaging. Recognition of the unique radiographic and histopathologic features of this entity is of growing importance in the treatment of cysts of the neural axis. We present an unusual case of an NC arising at the lower clivus. CLINICAL PRESENTATION: A 58-year-old man presented with occipitalgia, diplopia, a bilateral hearing deficit, and mild dysphagia. Computed tomography and magnetic resonance imaging demonstrated a 5 x 2 x 3-cm extra-axial cystic midline mass anterior to the brainstem at the lower clivus with posterior cyst wall enhancement. INTERVENTION: The patient underwent a left lateral suboccipital total macroscopic resection of the lesion. Microscopic examination and histopathologic findings were consistent with a diagnosis of NC. CONCLUSION: We describe the clinical presentation, imaging, and histopathologic characteristics, and discuss the diagnosis and surgical treatment of this rare lesion and related pathologic entities. Because of the remote possibility of delayed recurrence, even in cases of apparent total cyst wall removal, long-term serial imaging and a consideration of reoperation for recurrences is advisable.

Glycosphingolipid expression in cerebrospinal fluid of infants with neurological abnormalities: report of three cases.

The aim of this study was to analyse glycosphingolipid expression in cerebrospinal fluid (CSF) from one idiopathic West syndrome (IWS) infant, one with Reye like syndrome, and one with congenital hydrocephalus, in comparison to control group (n=7) using highly sensitive thin-layer chromatography-immunostaining methods. Gangliotetraose-series gangliosides (acidic glycosphingolipids) were not detected in CSF of infant with idiopathic West syndrome and infant with congenital hydrocephalus. CSF of infant with IWS showed traces of neolacto-tetraose ganglioside fractions, which were absent in all other CSF examined. In addition, lactosylceramide fraction, and one ceramide fraction were highly expressed only in IWS CSF These results confirmed previously described lack of gangliotetraose-series gangliosides in IWS patient and for the first time is described increased expression of neolacto-series glycosphingolipids in IWS patient. Since follow up until the age of five years showed almost normal IWS patient psychomotor development, the discribed shift of glycosphingolipid expression may implicate on transient inhibition of specific glycosyl transferases in the age of seven months.

Glycosphingolipids as potential diagnostic markers and/or antigens in neurological disorders.

Glycosphingolipids are most abundant in the nervous system within which are developmental, regional, structural and cellular differences regarding their composition. The are shedded to the cerebrospinal fluid and thus potential markers for pathogenic alterations in the brain, such as developmental abnormalities, demyelination, gliosis, neuronal cell destruction. The glycosphingolipids have also been found to be antigens in autoimmune processes involving the nervous system, in particular in peripheral neuropathies like Guillain Barré syndrome, multifocal motor neuropathy etc. The immune response might have been triggered by infectious agents with an antigen epitope which mimic the glycosphingolipid or by a primary nerve tissue damage leading to release of glycosphingolipids. There is a series of support for a clinical significance of cerebrospinal fluid glycosphingolipid determinations and the presence of anti-glycosphingolipid antibodies but this has to be further explored. This paper is a mini review of the state of the art and discuss methodological aspects and improvements that might help to explore the relevance of glycosphingolipids in neurological disorders.

Possible use of CSF glycosphingolipids for the diagnosis and therapeutic monitoring of lysosomal storage diseases.

We used a high-performance liquid chromatography method to measure CSF gangliosides, neutral glycolipids, and sulfatides in patients with lysosomal storage disorders. These measurements could be done on less than 1 milliliter of CSF. In patients with GM1 gangliosidosis, GM1 ganglioside was increased, and in GM2 gangliosidosis patients, GM2 ganglioside was increased in CSF. Sulfatides were variably increased in CSF early in the course of the disease and appeared to be a means of monitoring patients, following bone marrow transplantation. Fabry's disease patients showed an increase in globotriaosylceramide, but Krabbe's disease patients did not demonstrate an increase in galactosylceramide. This study suggests that CSF glycosphingolipid measurements may prove helpful in the diagnosis and monitoring of lysosomal storage diseases.

Glycosphingolipids in the cerebrospinal fluid of patients with multiple sclerosis.

Glycosphingolipids in cerebrospinal fluid (CSF) of individual patients with multiple sclerosis (MS) were analyzed using a glycolipid-overlay technique. The ganglioside composition of CSF of non-MS patients was characterized by an abundance of polysialo species, including GT1b and GQ1b. This pattern is completely different from that of human white or gray matter, in which mono- and disialogangliosides predominate. Increased levels of GM1, either associated with or without increases of other gangliosides, such as GD1a, were observed in 16% of the patients with MS (6 of 37 cases: 1 of 15 progressive progressive stage, 4 of 16 progressive stationary stage, and 1 of 6 relapsing stage). The concentration of GD3 was increased in 23% (3 of 13 cases), whereas 1 of 13 cases (8%) showed a dramatic increase of sulfoglucuronyl paragloboside (SGPG) associated with a high level of GD3. These changes may reflect the cellular changes associated with the known pathological lesions in MS, which are characterized by demyelination, gliosis, and/or remyelination with oligodendrocytic proliferation.

Influence of sialic acid groups on the retention of glycosphingolipids in blood plasma.

The removal of several glycosphingolipids from the circulation and their disposal in different tissue and fluid compartments was studied in adult rats. 3H-labeled dihydro analogs of several glycosphingolipids were injected intravenously and radioactivity was measured in arterial blood samples at subsequent time intervals, to obtain half life values for the labeled compound in the plasma. Half life values of less than 1 min were obtained for neutral glycosphingolipids whereas the half lives of labeled gangliosides were much longer and ranged from 3.8 to 21 h. The prompt removal of labeled neutral glycosphingolipids but not of the gangliosides indicates that sialic acid groups play a significant role in the retention of glycosphingolipids in the circulation. The results suggest that neutral glycosphingolipids are rapidly exchanged with their counterparts in a large extraplasma pool and that a major portion of this exchange could occur between plasma and liver. The detection of only a minute fraction of the injected glycosphingolipids in the cerebrospinal fluid indicates that a blood-cerebrospinal fluid barrier exists for these compounds in the rat.