What Can N-glycomics and N-glycoproteomics of Cerebrospinal Fluid Tell Us about Alzheimer Disease?

Proteomics-large-scale studies of proteins-has over the last decade gained an enormous interest for studies aimed at revealing proteins and pathways involved in disease. To fully understand biological and pathological processes it is crucial to also include post-translational modifications in the "omics". To this end, glycomics (identification and quantification of glycans enzymatically or chemically released from proteins) and glycoproteomics (identification and quantification of peptides/proteins with the glycans still attached) is gaining interest. The study of protein glycosylation requires a workflow that involves an array of sample preparation and analysis steps that needs to be carefully considered. Herein, we briefly touch upon important steps such as sample preparation and preconcentration, glycan release, glycan derivatization and quantification and advances in mass spectrometry that today are the work-horse for glycomics and glycoproteomics studies. Several proteins related to Alzheimer disease pathogenesis have altered protein glycosylation, and recent glycomics studies have shown differences in cerebrospinal fluid as well as in brain tissue in Alzheimer disease as compared to controls. In this review, we discuss these techniques and how they have been used to shed light on Alzheimer disease and to find glycan biomarkers in cerebrospinal fluid.

N-Glycomics of Cerebrospinal Fluid: Method Comparison.

Cerebrospinal fluid (CSF) contains valuable biological and neurological information. However, its glycomics analysis is hampered due to the low amount of protein in the biofluid, as has been demonstrated by other glycomics studies using a substantial amount of CSF. In this work, we investigated different N-glycan sample preparation approaches to develop a more sensitive method. These methods, one with an increased amount of buffer solution during the N-glycan release step with a lower amount of sample volume and the other with Filter-Aided N-Glycan Separation (FANGS), were compared with recent work to demonstrate their effectiveness. It was demonstrated that an increased amount of buffer solution showed higher intensity in comparison to the previously published method and FANGS. This suggested that digestion efficiency during the N-glycan release step was not in an optimal condition from the previously published method, and that there is a substantial loss of sample with FANGS when preparing N-glycans from CSF.

N-Glycan Profile of Cerebrospinal Fluids from Alzheimer's Disease Patients Using Liquid Chromatography with Mass Spectrometry.

Glycosylation, an essential post-translational protein modification, is known to be altered in a variety of diseases, including neurodegenerative diseases such as Alzheimer's disease (AD), which is one of the most common neurodegenerative disorders that results in cognitive and memory impairments. To investigate the progression of such a condition, cerebrospinal fluid (CSF), a unique biofluid that may possess significant biochemical and neurochemical changes due to the disease, is utilized. However, due to the low concentration of proteins in CSF, a large volume of the biofluid is often required to comprehensively characterize the glycome in CSF. In this work, a glycomic study of CSF was performed using as little as 10 µL of CSF. This approach was executed with permethylation of released N-glycans with minimal sample cleanup, in conjunction with an online purification system attached to liquid chromatography and a high-resolution mass spectrometer. This technique was then applied to clinical samples. Preliminary data suggest that fucosylated and bisecting GlcNAc structures were higher in abundances in females with AD, while both females and males exhibited lower abundances of high-mannose structures. Although there seems to be statistically significant differences between disease state and disease-free CSF, due to the lack of number of samples, further validation study should be conducted.

CSF N-Glycoproteomics Using MALDI MS Techniques in Neurodegenerative Diseases.

CSF diagnostics has proved to be a formidable testing ground for N-glycoproteomic analysis of neurological diseases. To characterize specific N-glycan profiles of CSF in early and advanced phases of Alzheimer's disease, as well as in lysosomal storage disorders such as Tay-Sachs disease, we set up in our lab a robust and feasible protocol by coupling bioanalytical methods and mass spectrometry analysis.Starting from a few microliters of CSF, after protein denaturation, reduction, and alkylation, N-glycans are released from glycoproteins using the peptide-N-glycosidase F (PNGase F) and purified. The analysis of permethylated N-glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS/MS allowed us to identify specific glyco-structures and also to distinguish between isobaric N-glycans.

CSF N-Glycomics Using MALDI MS Techniques in Alzheimer's Disease.

In this chapter, we present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep™ Hypercarb™ solid-phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-linked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer's disease.

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.

CSF N-glycan profile reveals sialylation deficiency in a patient with GM2 gangliosidosis presenting as childhood disintegrative disorder.

Protein N-glycosylation consists in the synthesis and processing of the oligosaccharide moiety (N-glycan) linked to a protein and it serves several functions for the proper central nervous system (CNS) development and function. Previous experimental and clinical studies have shown the importance of proper glycoprotein sialylation for the synaptic function and the occurrence of autism spectrum disorders (ASD) in the presence of sialylation deficiency in the CNS. Late-onset Tay Sachs disease (LOTSD) is a lysosomal disorder caused by mutations in the HEXA gene resulting in GM2-ganglioside storage in the CNS. It is characterized by progressive neurological impairment and high co-occurrence of psychiatric disturbances. We studied the N-glycome profile of the cerebrospinal fluid (CSF) in a 14 year-old patient with GM2-gangliosidosis (LOTSD). At the age of 4, the patient presented regressive autism fulfilling criteria for childhood disintegrative disorder (CDD). A CSF sample was obtained in the course of diagnostic work-up for the suspicion of an underlying neurodegenerative disorder. We found definite changes of CSF N-glycans due to a dramatic decrease of sialylated biantennary and triantennary structures and an increase of asialo-core fucosylated bisected N-glycans. No changes of total plasma N-glycans were found. Herein findings highlight possible relationships between the early onset psychiatric disturbance featuring CDD in the patient and defective protein sialylation in the CNS. In conclusion, the study first shows aberrant N-glycan structures of CSF proteins in LOTSD; unveils possible pathomechanisms of GM2-gangliosidosis; supports existing relationships between neuropsychiatric disorders and unproper protein glycosylation in the CNS.

A unique N-glycan on human transferrin in CSF: a possible biomarker for iNPH.

Idiopathic normal pressure hydrocephalus (iNPH) is an elderly dementia caused by abnormal metabolism in the cerebrospinal fluid (CSF). The tap test is the current basis for confirming iNPH, but it shows very low sensitivity, indicating that many patients who might be cured by a shunt operation will be missed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we found two transferrin isoforms: one had a unique N-glycan (Tf-1) whereas the other had N-glycan similar to that of serum transferrin (Tf-2). Glycan analyses revealed that Tf-1 had branching (biantennary) asialo- and agalacto-complex type N-glycans (N-acetylglucosamine [GlcNAc]-terminated glycans), which carried bisecting ß1,4-N-acetylglucosamine and core α1,6-fucose. To examine glycoform whether changes in iNPH, we introduced the Tf-2/Tf-1 ratio as a diagnostic index, which minimized blot-to-blot variations in measurement. The Tf-2/Tf-1 ratios of iNPH patients are significantly higher than those of controls (p = 0.0019) and Alzheimer's patients (p = 0.0010). This suggests that the Tf-2/Tf-1 ratio could distinguish iNPH from Alzheimer's disease, and possibly other dementias. In conclusion, glycoform analysis has diagnostic potential in neurological diseases.

Identification of N-glycosylation changes in the CSF and serum in patients with schizophrenia.

Schizophrenia is a major neuropsychiatric disorder that affects 2% of the population worldwide. No biochemical diagnostic tests are available, and patients must undergo lengthy clinical evaluation periods before an accurate diagnosis can be given. Blood and cerebrospinal fluid are candidates for the identification of potential biomarkers for this disease. We have identified several N-glycans that distinguish first onset, unmedicated schizophrenia patients from healthy individuals. This is the first report of the N-glycome from low abundance serum proteins and cerebrospinal fluid. The tetraantennary tetrasialylated glycan with a polylactosamine extension, A4G4LacS4, from low abundance serum proteins showed a 2-fold increase in serum from male schizophrenia patients. Gender specificity was also demonstrated as the triantennary trisialylated glycan containing the SLex epitope was increased significantly in male schizophrenia patients on both high and low abundance serum proteins. Levels of bisecting and sialylated glycans in the cerebrospinal fluid showed a general down-regulation in schizophrenia patients and a 95% positive predictive power for distinguishing patients from controls. These changes are consistent with the reported down-regulation of beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase III and beta-galactoside alpha-2,3/6-sialyltransferases in the prefrontal cortex from schizophrenia patients. These alterations in the N-glycosylation signature could be used potentially for early diagnosis and monitoring of patients after treatment.

[Cryptococcosis].

Cryptococcosis is a common fungal infection that caused by Cryptococcus neoformans in immunocompetent individuals as well as in immunosuppressive patients with such as the HIV infection. Measurements of the values of glucuronoxylomannan structuring capsule of C. neoformans in serum or cerebrospinal fluid are quite useful to diagnosis of pulmonary cryptococcosis and cryptococcal meningitis as major two forms of cryptococcosis. Azole anti-fungal agents including fulconazole, itraconazole for pulmonary cryptococcosis, and amphotericin B plus flucytosine for cryptococcal meningitis are recommended as first line therapy in Japanese guideline for the diagnosis and treatment of deep seated mycosis revised in 2007. The clinical appearance, diagnostic methods, treatment and prognosis of pulmonary cryptococcosis and cryptococcal meningitis are described based on the Japanese guideline in this manuscript.

Characterization of Cryptococcus neoformans var. neoformans serotype A and A/D in samples from Egypt.

The cryptococcal polysaccharide antigen was detected in 10 cerebrospinal fluid (CSF) and 23 serum samples from cryptococcal meningitis and intestinal cryptococcosis by the cryptococcal antigen latex agglutination system (CALAS). CALAS titers in CSF and serum samples of cryptococcal meningitis ranged over 8-2048 and 32-2048, respectively, while in cases of intestinal cryptococcosis, serum titers ranged over 8-2048. The isolates of yeast Cryptococcus neoformans were determined to be of serotype A or of the A/D pair. The total leukocyte count and biochemical parameters in CSF were significantly increased as indicators of microbial infection. Furthermore, the in vitro change of the teleomorph (sexual state) to the anamorph (asexual state) was also detected and the teleomorph state changed in vivo to the encapsulated anamoph state which is more virulent during infection in vivo than the yeast-like noncapsulated form. Two primers for internal transcribed spacer (ITS) regions of ribosomal DNA were used for molecular detection of C. neoformans. After PCR amplification, a DNA band of 415 bp, visualized on agarose gel, indicated the presence of C. neoformans cells in the tested CSF and serum samples. The primer sensitivity was also characterized using purified yeast chromosomal DNA as template; it was about 20 pg or more chromosomal DNA which represents about 10 cells of C. neoformans. The primers were also specific for ITS regions of C. neoformans and gave negative results with Candida albicans and E. coli chromosomal DNA templates.

Fucosylated glycans in the periventricular structures and the cerebrospinal fluid of the fetal rat forebrain. An autoradiographic and lectin binding histiotopic study.

Our autoradiographic 3H-fucose incorporation study of the brains of 20-day-old rat fetuses showed that the synthesis of fucosylated glycans is significantly higher in the ventricular germinative zone of the forebrain hemisphere than in the more superficial layers, including the cortical plate. Intense incorporation of 3H-fucose also occurred in the choroid plexus, both its epithelial and stromal component, in the primordial ependymal lining of the lateral ventricles, meninges and capillaries of the forebrain parenchyma. In the lateral ventricles, densely labeled microprecipitates of the cerebrospinal fluid (CSF) were occasionally observed. The histiotopic differences in 3H-fucose labeling were absent, or were much less expressed, in the autoradiograms prepared from unfixed cryostat sections containing mainly unincorporated isotope. This indicates that the blood-mediated supply of 3H-fucose to the studied brain compartments was essentially equal and our incorporation data reflect actual differences in the rate of fucosylation within the forebrain hemispheres. The cytochemical lectin-binding assay, carried out with Ulex europaeus and Lotus tetragonolobus agglutinins, showed that regions with a higher rate of 3H-fucose incorporation were also richer in fucose-bearing glycoconjugates. The study revealed that the periventricular regions and the CSF of fetal rat forebrain form a fucosylated glycan-enriched complex, which represents a new chemoarchitectonic feature that may be of importance for maintaining the germinative properties of the ventricular neuroepithelium and the growth of the hemispheric ventricles.

Comparison of two rapid latex agglutination tests for detection of cryptococcal capsular polysaccharide.

The Murex Cryptococcus Test was compared with the Cryptococcal Antigen Latex Agglutination System (CALAS) for detecting cryptococcal polysaccharide in 173 cerebrospinal fluid (CSF) specimens and 117 serum samples with 99% and 97% concordance, respectively. Eighteen CSF samples and 17 serum samples were positive in both assays, and 249 were negative. The sensitivity and specificity of the Murex relative to the CALAS were 90% and 100%, respectively, for CSF, and 81% and 100%, respectively, for serum. Six discrepancies were arbitrated by retesting, using a third analytic method, review of other laboratory and clinical data, or both. The reaction in 1 CSF specimen was considered false positive by the CALAS, and the reactions in 2 serum samples were false negatives by the Murex. For 3 patients with previous cryptococcal meningitis but no active disease, only the CALAS detected antigen, suggesting that the Murex has less analytic sensitivity in this context. Titer differences dictate that direct comparisons between the 2 tests are not feasible. There were no false-positive reactions in limited testing with either method using specimens from patients with concurrent noncryptococcal infections or in rheumatoid factor-positive serum samples. Infections caused by Cryptococcus neoformans serotypes A or AD were detected equally by both assays. Based on our study, we have elected to continue to use the CALAS for routine testing for cryptococcal antigen.

Cryptococcal infections of the central nervous system: an analysis of predisposing factors, laboratory findings and outcome in patients from South India with special reference to HIV infection.

The incidence of cryptococcosis in patients with AIDS is significant. Predisposing factors, laboratory findings and outcome were assessed in 60 patients with cryptococcal infections of the central nervous system over a 17.5-year period (Jan. 1978-June 1995). Predisposing factors for cryptococcal infection were identified in 36 patients, with HIV infection being the commonest (18). Cryptococcal cultures were positive in all patients. India ink staining was positive in 48 patients and cryptococcal antigen was detected in 35 of 36 patients tested. Comparison of clinical and laboratory parameters between HIV-positive and HIV-negative patients showed that CSF cell response was poorer, culture of cryptococci from non-neural sites was more frequent and mortality was higher in the HIV-positive group. Although not statistically significant, concurrent systemic infections, especially tuberculosis, were more frequent in the HIV-positive group.

Evaluation of new monoclonal antibody-based latex agglutination test for detection of cryptococcal polysaccharide antigen in serum and cerebrospinal fluid.

We evaluated the performance of CRYPTO-LEX (Trinity Laboratories, Inc., Raleigh, N. C.), a new mouse immunoglobulin M monoclonal antibody latex agglutination reagent which reacts with the capsular polysaccharide of the four serogroups of Cryptococcus neoformans. This test was compared with CALAS (Meridian Diagnostics, Cincinnati, Ohio) for the ability to detect cryptococcal antigen in serum and cerebrospinal fluid (CSF). A total of 580 clinical specimens (327 serum and 253 CSF samples), primarily from human immunodeficiency virus-infected patients, were tested in this study. Sixty-seven specimens (44 serum and 23 CSF samples) were positive for cryptococcal antigen with both tests, and 511 (282 serum and 229 CSF samples) were negative. The two latex reagents agreed for 326 of 327 serum specimens (44 positives and 282 negatives). One serum specimen with a titer of 1:2 was CALAS positive but CRYPTO-LEX negative. The titer correlation coefficient for the two tests was 0.884 when two highly discordant serum specimens were eliminated from analysis of the data. The two latex tests agreed for 252 of 253 CSF specimens (23 positives and 229 negatives). One specimen with a titer of 1:2 was positive with CALAS and negative by CRYPTO-LEX. The correlation coefficient of the two tests for CSF titers was 0.886. The sensitivity and specificity of CRYPTO-LEX were 97 and 100%, respectively, with a 99.6% correlation with CALAS. These data show that the performance of CRYPTO-LEX is comparable to that of CALAS for detection of cryptococcal antigen in serum and CSF.

Measurement of cryptococcal antigen in serum and cerebrospinal fluid: value in the management of AIDS-associated cryptococcal meningitis.

The value of monitoring titers of cryptococcal antigen in serum and cerebrospinal fluid (CSF) during therapy for AIDS-associated cryptococcal meningitis was evaluated. Baseline and final titers of antigen in serum and CSF from participants in two studies of such therapy were categorized as increased (a rise of at least two dilutions), unchanged, or decreased (a fall of at least two dilutions). There was no correlation between outcome and changes in serum titers of cryptococcal antigen during treatment for acute meningitis or during suppressive therapy. During therapy for acute infection, an unchanged or increased titer of antigen in CSF was correlated with clinical and microbiological failure to respond to treatment; the correlation was especially strong among patients whose baseline titer of antigen was > or = 1:8 (P = .01). A rise in CSF antigen titer during suppressive therapy was associated with relapse of cryptococcal meningitis (P < .001). We conclude that serial monitoring of cryptococcal antigen, as conducted in these studies, has a limited role in the management of AIDS patients with cryptococcal meningitis.

Cryptococcal antigen detection from the urine of AIDS patients.

Cryptococcal disease occurs in < or = 10% of AIDS patients. Detection of the capsular polysaccharide antigen of the yeast in spinal fluid or serum is used to establish the diagnosis. In addition, cryptococcal antigen (CAg) analysis is used to adjust treatment and evaluate recurrence of active disease. A specimen such as urine, obtained noninvasively, would be optimum for this evaluation. Urine, cerebrospinal fluid (CSF), and serum for CAg analysis, and culture of urine and CSF, were obtained for 103 sets of specimens from 92 patients. CSF and urine specimens for CAg were analyzed with and without pronase treatment; serum was analyzed with pronase only. Twenty percent (21 of 103) of specimen sets showed CAg from eight patients. In all cases, patients with positive CSF and/or serum titers also had positive urine titers. Titers were always serum > CSF > urine, with ranges of 1: 64-65000; 1: 64-6250; and 1: 2-512, respectively. Pronase treatment did not affect CSF titers, but 14 of 23 titers from urine treated with pronase were at least one dilution higher than those without treatment. No false-positive reactions were observed during the study. CSF cultures were positive from seven of eight, and urine cultures were positive from five of eight patients with CAg. These results indicate that urine can be used as a specimen for detection of CAg in AIDS patients and that use of pronase may increase its sensitivity.

Comparison of three latex agglutination kits and counterimmunoelectrophoresis for the detection of bacterial antigens in a pediatric population.

Three commercial latex agglutination kits (Bactigen, Wampole Laboratories, Cranbury, NJ; Directigen, Hynson, Westcott & Dunning, Baltimore, MD; Wellcogen, Wellcome Diagnostics, Dartford, England) used for the detection of bacterial polysaccharide antigens (Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis) were compared with counterimmunoelectrophoresis and Gram stain for the identification of systemic bacterial disease in children. Urine and (when available) cerebrospinal fluid specimens were saved for all patients. Positive blood or cerebrospinal fluid culture isolates included 36 with H. influenzae type b, 11 with S. pneumoniae, 3 with N. meningitidis and 6 with other organisms. All latex kits performed similarly for the detection of H. influenzae type b antigen with a sensitivity range of 91 to 100%. The four methods performed poorly for the detection of S. pneumoniae (23 to 50%) and N. meningitidis (0%) antigen. Gram stain of cerebrospinal fluid appeared to be equally sensitive to the antigen detection methods for patients with meningitis. The false positive rates for the latex kits and counterimmunoelectrophoresis ranged from 2.8 to 9.2%, with Wellcogen having the lowest rates. The false negative rates ranged from 6.5% to 12% with Directigen having the lowest rate.

Quantitation of Haemophilus influenzae serotype b capsular polysaccharide antigen in body fluids by enzyme immunoassay.

A heterogeneous enzyme immunoassay (EIA) has been developed to quantify capsular polysaccharide antigen of Haemophilus influenzae serotype b (Hib) polyribosyl - polyribitol -phosphate (PRP) in body fluids. Anti-Hib immunoglobulin G form rabbit adsorbed to the solid phase reacts with PRP existing in free soluble form in cerebrospinal fluid, serum or urine during Hib infection. IgG-anti-Hib labelled with horseradish peroxidase then links to PRP; the enzyme activity is measured by oxidation of the chromogenic substrate o-phenylenediamine. PRP concentrations ranged between 2 micrograms/1 to 21 mg/1 detected in acute Hib disease. The applicability of the EIA as a diagnostic aid is limited by cross-reaction of other bacterial antigens. However, quantitative measurements of PRP by enzyme immunoassay should improve studies on Hib disease.