A novel unbiased proteomic approach to detect the reactivity of cerebrospinal fluid in neurological diseases

Neurodegenerative diseases, such as multiple sclerosis represent global health issues. Accordingly, there is an urgent need to understand the pathogenesis of this and other central nervous system disorders, so that more effective therapeutics can be developed. Cerebrospinal fluid is a potential source of important reporter molecules released from various cell types as a result of central nervous system pathology. Here, we report the development of an unbiased approach for the detection of reactive cerebrospinal fluid molecules and target brain proteins from patients with multiple sclerosis. To help identify molecules that may serve as clinical biomarkers for multiple sclerosis, we have biotinylated proteins present in the cerebrospinal fluid and tested their reactivity against brain homogenate as well as myelin and myelin-axolemmal complexes. Proteins were separated by two-dimensional gel electrophoresis, blotted onto membranes and probed separately with biotinylated unprocessed cerebrospinal fluid samples. Protein spots that reacted to two or more multiple sclerosis-cerebrospinal fluids were further analyzed by matrix assisted laser desorption ionization-time-of-flight time-of-flight mass spectrometry. In addition to previously reported proteins found in multiple sclerosis cerebrospinal fluid, such as αβ crystallin, enolase, and 14-3-3-protein, we have identified several additional molecules involved in mitochondrial and energy metabolism, myelin gene expression and/or cytoskeletal organization. These include aspartate aminotransferase, cyclophilin-A, quaking protein, collapsin response mediator protein-2, ubiquitin carboxy-terminal hydrolase L1, and cofilin. To further validate these findings, the cellular expression pattern of collapsin response mediator protein-2 and ubiquitin carboxy-terminal hydrolase L1 were investigated in human chronic-active MS lesions by immunohistochemistry. The observation that in multiple sclerosis lesions phosphorylated collapsin response mediator protein-2 was increased, whereas Ubiquitin carboxy-terminal hydrolase L1 was down-regulated, not only highlights the importance of these molecules in the pathology of this disease, but also illustrates the use of our approach in attempting to decipher the complex pathological processes leading to multiple sclerosis and other neurodegenerative diseases.     

Leukocyte-derived microparticles and scanning electron microscopic structures in two fractions of fresh cerebrospinal fluid in amyotrophic lateral sclerosis: a case report

ABSTRACT: INTRODUCTION: Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder characterized by degeneration of motoneuron cells in anterior spinal horns. There is a need for early and accurate diagnosis with this condition. In this case report we used two complementary methods: scanning electron microscopy and fluorescence-activated cell sorting. This is the first report to our knowledge of microparticles in the cerebrospinal fluid of a patient with amyotrophic lateral sclerosis. CASE PRESENTATION: An 80-year-old Swedish man of Caucasian ethnicity presented to our facility with symptoms of amyotrophic lateral sclerosis starting a year before his first hospital examination, such as muscle weakness and twitching in his right hand progressing to arms, body and leg muscles. Electromyography showed classical neurophysiological findings of amyotrophic lateral sclerosis. Routine blood sample results were normal. A lumbar puncture was performed as a routine investigation and his cerebrospinal fluid was normal with regard to cell count and protein levels, and there were no signs of inflammation. However, scanning electron microscopy and fluorescence-activated cell sorting showed pronounced abnormalities compared to healthy controls. Flow cytometry analysis of two fractions of cerebrospinal fluid from our patient with amyotrophic lateral sclerosis was used to measure the specific binding of antibodies to phosphatidylserine (lactadherin), CD42a, CD144 and CD45. Our patient displayed over 100 times more phosphatidylserine-positive microparticles and over 400 times more cell-derived microparticles of leukocyte origin in his cerebrospinal fluid compared to healthy control subjects. The first cerebrospinal fluid fraction contained about 50% more microparticles than the second fraction. The scanning electron microscopy filters used with cerebrospinal fluid from our patient were filled with compact aggregates of spherical particles of lipid appearance, sticking together in a viscous batter. The quantitative increase in scanning electron microscopy findings corresponded to the flow cytometry result of an increase in leukocyte-derived microparticles. CONCLUSIONS: Microparticles represent subcellular arrangements that can influence the pathogenesis of amyotrophic lateral sclerosis and may serve as biomarkers for underlying cellular disturbances. The increased number of leukocyte-derived microparticles with normal cell counts in cerebrospinal fluid may contribute to the amyotrophic lateral sclerosis inflammatory process by formation of immune complexes of prion-like propagation, possibly due to misfolded proteins. The two complementary methods used in this report may be additional tools for revealing the etiology of amyotrophic lateral sclerosis, for early diagnostic purposes and for evaluation of clinical trials, long-term follow-up studies and elucidating the pathophysiology in amyotrophic lateral sclerosis.     

Anti-galactocerebroside antibodiesin human cerebrospinal fluids determined by enzyme-linked immunosorbent assay (ELISA)

The standard ELISA technique was improved for the detection of antigalactocerebroside antibody in biological fluid. Mouse monoclonal antigalactocerebroside antibody was used to demonstrate specificity and sensitivity of the technique. After optimization of the assay, the usefulness of this measurement for the evaluation of patients with multiple sclerosis was assessed. The presence of antigalactocerebroside antibodies in the cerebrospinal fluid of 20 patients with multiple sclerosis, 10 with other neurological diseases and 10 normal individuals was determined. All the CSF samples from normal individuals were negative. In patients with multiple sclerosis 14 of the 20 samples had elevated levels of antigalactocerebroside antibody, whereas with other neurological diseases 5 out of 10 were positive. Antigalactocerebroside levels were lower in samples from patients during an acute relapse than in those from more chronic cases. These results indicate that the presence of anti-galactocerebroside antibody in cerebrospinal fluid is not specific to MS but may reflect previous damage to myelin.Abbreviations and trivial names used ELISA Enzyme-Linked Immunosorbent Assay - CSF cerebrospinal fluid; galacto- or glucocerebroside, ceramide-1-0-beta-galactoside or-glucoside     

Effects of Ferrofluid and Phytoalexin Spirobrassinin on Thioflavin-T-Based Fluorescence in Cerebrospinal Fluid of the Elderly and Multiple Sclerosis Patients

It is well known that misfolded peptides/proteins can play a role in processes of normal ageing and in the pathogenesis of many diseases including Alzheimer’s disease. Previously, we evaluated samples of cerebrospinal fluid from patients with Alzheimer’s disease and multiple sclerosis by means of thioflavin-T-based fluorescence. We observed attenuated effects of magnetite nanoparticles operated via anti-aggregation actions on peptides/proteins from patients with Alzheimer’s disease but not from those with multiple sclerosis when compared to age-related controls. In this study, we have evaluated the in vitro effects of anti-aggregation operating ferrofluid and phytoalexin spirobrassinin in the cerebrospinal fluid of patients with multiple sclerosis and Alzheimer’s disease. We have found significant differences in native fluorescence (λ excitation = 440 nm, λ emission = 485 nm) of samples among particular groups (young controls < multiple sclerosis, Alzheimer’s disease < old controls). Differences among groups were observed also in thioflavin-T-based fluorescence (young controls = multiple sclerosis < Alzheimer’s disease < old controls) and the most marked change from native to thioflavin-T-based fluorescence was found in young controls (28–40 years old people). Both ferrofluid and spirobrassinin evoked drops in thioflavin-T-based fluorescence; however, ferrofluid was more efficient in old controls (54–75 years old people) and spirobrassinin in multiple sclerosis patients, both compared to young controls. The results are discussed especially in relation to aggregated peptides/proteins and liposoluble fluorescent products of lipid peroxidation. Based on the significant effect of spirobrassinin in vitro, we suggest that spirobrassinin may be of therapeutic value in multiple sclerosis.     

Proteomics of human cerebrospinal fluid: discovery and verification of biomarker candidates in neurodegenerative diseases using quantitative proteomics

There is an urgent need for novel biomarkers that can be used to improve the diagnosis, predict the disease progression, improve our understanding of the pathology or serve as therapeutic targets for neurodegenerative diseases. Cerebrospinal fluid (CSF) is in direct contact with the CNS and reflects the biochemical state of the CNS under different physiological and pathological settings. Because of this, CSF is regarded as an excellent source for identifying biomarkers for neurological diseases and other diseases affecting the CNS. Quantitative proteomics and sophisticated computational software applied to analyze the protein content of CSF has been fronted as an attractive approach to find novel biomarkers for neurological diseases. This review will focus on some of the potential pitfalls in biomarker studies using CSF, summarize the status of the field of CSF proteomics in general, and discuss some of the most promising proteomics biomarker study approaches. A brief status of the biomarker discovery efforts in multiple sclerosis, Alzheimer's disease, and Parkinson's disease is also given.     

Cerebrospinal Fluid IL-12p40, CXCL13 and IL-8 as a Combinatorial Biomarker of Active Intrathecal Inflammation

Diagnosis and management of the neuroinflammatory diseases of the central nervous system (CNS) are hindered by the lack of reliable biomarkers of active intrathecal inflammation. We hypothesized that measuring several putative inflammatory biomarkers simultaneously will augment specificity and sensitivity of the biomarker to the clinically useful range. Based on our pilot experiment in which we measured 18 inflammatory biomarkers in 10-fold concentrated cerebrospinal fluid (CSF) derived from 16 untreated patients with highly active multiple sclerosis (MS) we selected a combination of three CSF biomarkers, IL-12p40, CXCL13 and IL-8, for further validation.Concentrations of IL-12p40, CXCL13 and IL-8 were determined in a blinded fashion in CSF samples from an initial cohort (n = 72) and a confirmatory cohort (n = 167) of prospectively collected, untreated subjects presenting for a diagnostic work-up of possible neuroimmunological disorder. Diagnostic conclusion was based on a thorough clinical workup, which included laboratory assessment of the blood and CSF, neuroimaging and longitudinal follow-up. Receiver operating characteristic (ROC) curve analysis in conjunction with principal component analysis (PCA), which was used to combine information from all three biomarkers, assessed the diagnostic value of measured biomarkers.Each of the three biomarkers was significantly increased in MS and other inflammatory neurological disease (OIND) in comparison to non-inflammatory neurological disorder patients (NIND) at least in one cohort. However, considering all three biomarkers together improved accuracy of predicting the presence of intrathecal inflammation to the consistently good to excellent range (area under the ROC curve = 0.868–0.924).Future clinical studies will determine if a combinatorial biomarker consisting of CSF IL-12p40, CXCL13 and IL-8 provides utility in determining the presence of active intrathecal inflammation in diagnostically uncertain cases and in therapeutic development and management.     

Little change in cerebrospinal fluid amino acids in subtypes of multiple sclerosis compared with acute polyradiculoneuropathy.

Levels of free amino acids were determined in randomised, blinded samples of cerebrospinal fluid (CSF) from patients with relapsing-remitting or chronic progressive multiple sclerosis (MS), all in the active phase of disease. The levels were compared with amino acid amounts in patients with an acute polyradiculoneuropathy (Guillain-Barré syndrome (GBS)) and a control population of patients with no known neurological disease or deficit. The data did not indicate any significant changes in amino acid levels between MS subgroups. The only significant differences between MS patients and controls were a modest reduction in glutamate and a slight increase in taurine, but the changes were so small that the biological relevance is dubious. These results contrasted with the marked increases for many amino acids in CSF from patients with acute polyradiculoneuropathy compared with controls. The amino acid profile in cerebrospinal fluid (CSF) does not appear to provide evidence of differential pathology in multiple sclerosis (MS). The increase in hydrophobic amino acids and lysine in CSF from patients with acute polyradiculoneuropathy is consistent with transudation over the blood-CSF barrier following an infection. The increases in glutamine and alanine may reflect increased nitrogen removal from brain.     

Immunoglobulin Levels in the Cerebrospinal Fluid

The immunoglobulins G, A, M, and D have been measured in the cerebrospinal fluid of 207 patients with neurological disease. Raised levels of IgG, expressed as a percentage of total cerebrospinal fluid (C.S.F.) protein, were found in 62% of 45 cases of multiple sclerosis compared with 14% of 160 cases with various other neurological disorders. Thus measurement of the IgG level is probably a useful confirmatory investigation in multiple sclerosis. IgA and IgM were found only in the C.S.F. of patients with a raised protein level, and IgD was not detected.     

Analysis of cerebrospinal fluid cell populations with monoclonal antibodies

Sixty-five samples of cerebrospinal fluid (CSF) were evaluated using an automated cytoflow method with the CD-Sapphire hematology analyzer in order to investigate possible relationships between cell population patterns and diagnostic groups and better understand the biology of neurological disease. A basic panel of CD markers, including CD3/4/8/19/138/HLA-DR, was used to analyze CSF samples from clinical and laboratory confirmed cases of multiple sclerosis, neuroborreliosis, viral and bacterial neuroinfective diseases, malignant infiltrations of meninges and scavenger macrophagic reactions of the central nervous system. The principles of immune response and the contribution of cytological 'disease-related patterns' for these nosological entities are described. The distinct patterns of lymphocyte subpopulations in neuroborreliosis appear to be characteristic and could possibly serve as diagnostic indicators. Further verification and research will be necessary to clarify the significance and nature of CD4+ CD8+ positive subset in cerebrospinal fluid.     

An endogenous pentapeptide acting as a sodium channel blocker in inflammatory autoimmune disorders of the central nervous system

Reversible blockade of sodium channels by endogenous substances has been claimed to account for the fast exacerbations and relapses commonly seen in demyelinating autoimmune diseases. Evidence has been provided that in the cerebrospinal fluid of patients with multiple sclerosis or Guillain-Barré syndrome, a sodium-channel-blocking factor exists that has properties of local anesthetic agents. This factor could contribute to the nerve conduction block and paresis seen in these disorders. We describe here a previously unknown endogenous substance in human cerebrospinal fluid with distinct channel-blocking properties even at very low (0.00001 M) concentrations. The pentapeptide with the sequence Gln-Tyr-Asn-Ala-Asp exerted its blocking action by shifting the steady-state inactivation curve of the sodium channels to more-negative potentials, as most local anesthetics do. In the cerebrospinal fluid of healthy individuals, its concentration was about 3 microM, whereas in patients with multiple sclerosis and Guillain-Barré syndrome, it increased 300-1,400%. At these concentrations, the peptide's blocking efficacy was higher than that of 50 microM lidocaine. At a concentration of 10 microM, lidocaine is able to 'unmask' subclinical lesions in multiple sclerosis; thus, the endogenous pentapeptide may well contribute to the fast changes of symptoms. Furthermore, it may become valuable as a marker of disease activity.     

A rapid method for preparation of the cerebrospinal fluid proteome

The cerebrospinal fluid (CSF) proteome is of great interest for investigation of diseases and conditions involving the CNS. However, the presence of high‐abundance proteins (HAPs) can interfere with the detection of low‐abundance proteins, potentially hindering the discovery of new biomarkers. Therefore, an assessment of the CSF subproteome composition requires depletion strategies. Existing methods are time consuming, often involving multistep protocols. Here, we present a rapid, accurate, and reproducible method for preparing the CSF proteome, which allows the identification of a high number of proteins. This method involves acetonitrile (ACN) precipitation for depleting HAPs, followed by immediate trypsination. As an example, we demonstrate that this method allows discrimination between multiple sclerosis patients and healthy subjects.     

Characterization of the human ventricular cerebrospinal fluid proteome obtained from hydrocephalic patients

The continuing expansion of proteomic technology has been fueled by the potential for discovering novel biomarkers that may be used for the early detection of disease. It has been proposed that human cerebrospinal fluid (CSF), which surrounds and protects the brain and spinal cord from traumatic injury, may be a valuable target for the diagnosis of a variety of conditions such as Alzheimer's disease, traumatic brain injury, amyotrophic lateral sclerosis and Parkinson's disease. The immense complexity of biofluids, however, still requires that considerable development be made in the analytical techniques used so that comprehensive coverage of the proteins present in such samples is achieved. Using a simple separation strategy the protein complement of human ventricular cerebrospinal fluid obtained from patients with hydrocephalus was evaluated. The study resulted in the identification of over 1500 unique proteins that were found within all nine CSF samples that were analyzed. Comparison with the HUPO serum proteome database demonstrated that human ventricular CSF contains a large array of proteins that may be unique to CSF. This analysis greatly increases our knowledge of the protein content of this clinically important biofluid.     

Proteomic profiling of cerebrospinal fluid identifies biomarkers for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons. We tested the hypothesis that proteomic analysis will identify protein biomarkers that provide insight into disease pathogenesis and are diagnostically useful. To identify ALS specific biomarkers, we compared the proteomic profile of cerebrospinal fluid (CSF) from ALS and control subjects using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). We identified 30 mass ion peaks with statistically significant (p < 0.01) differences between control and ALS subjects. Initial analysis with a rule-learning algorithm yielded biomarker panels with diagnostic predictive value as subsequently assessed using an independent set of coded test subjects. Three biomarkers were identified that are either decreased (transthyretin, cystatin C) or increased (carboxy-terminal fragment of neuroendocrine protein 7B2) in ALS CSF. We validated the SELDI-TOF-MS results for transthyretin and cystatin C by immunoblot and immunohistochemistry using commercially available antibodies. These findings identify a panel of CSF protein biomarkers for ALS.     

The repetitive use of samples to measure multiple cytokines: the sequential ELISA

The inexpensive and highly effective enzyme-linked immunosorbant assay (ELISA) is widely used for the quantification of biomarkers in a variety of biological samples. The applicability of the standard ELISA is difficult when experiments yield low volume samples. In such studies, the capacity of sample collection system does not meet the sample volume requirements to measure multiple different cytokines by the traditional ELISA protocol. In the modified methodology of the sequential ELISA, samples are re-used in multiple successive cycles, dramatically increasing the number of biomarkers which may be measured. Although the protocols presented to date were developed for quantification of cytokines in either blood plasma or cerebrospinal fluid, the sequential ELISA protocol has wide potential for further uses. When only limited quantities of samples are available for analysis, the sequential ELISA technique based on commercially available antibody pairs can be an attractive alternative to more advanced, costly multiplex methods. Additionally, any laboratory that currently runs traditional ELISAs has all the necessary equipment and reagents to perform the sequential ELISA.     

An improved radioimmunoassay for myelin basic protein-like immunoreactive material in cerebrospinal fluid

A modified radioimmunoassay protocol is described which can measure elevated levels of myelin basic protein-like immunoreactive material in the cerebrospinal fluid of some patients with multiple sclerosis or head injury and in rats developing an acute demyelinating form of experimental allergic encephalomyelitis. The assay uses synthetic peptides that differ in their sequences from natural myelin basic protein for both standard and radioligand, but in apparent contrast to previously published assays serial dilutions of samples produce the expected dose estimates when interpolated from the standard curve. A second radioimmunoassay was produced with high sensitivity and specificity for myelin basic protein peptides with a carboxyl terminus at phenylalanine 89. This assay was used in attempts to detect the myelin basic protein-like immunoreactivity recently reported to occur in human urine. This radioimmunoassay failed to detect specific immunoreactivity in urine samples from control and multiple sclerosis donors. The specificities of both assays were studied using a wide range of synthetic peptides and the importance of this information to the design of future assays is addressed. Our work reinforces that of others in suggesting the complex situation involved in the design of assays for MBP fragments in body fluids. We are willing to distribute the reagents for use in our CSF assay to researchers who request them.     

Lumbar cerebrospinal fluid proteome in multiple sclerosis: characterization by ultrafiltration, liquid chromatography, and mass spectrometry

Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M.S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC-ESI-MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M.S. and non-M.S. patients using ELISA.     

CSF-PR 2.0: An Interactive Literature Guide to Quantitative Cerebrospinal Fluid Mass Spectrometry Data from Neurodegenerative Disorders

The rapidly growing number of biomedical studies supported by mass spectrometry based quantitative proteomics data has made it increasingly difficult to obtain an overview of the current status of the research field. A better way of organizing the biomedical proteomics information from these studies and making it available to the research community is therefore called for. In the presented work, we have investigated scientific publications describing the analysis of the cerebrospinal fluid proteome in relation to multiple sclerosis, Parkinson's disease and Alzheimer's disease. Based on a detailed set of filtering criteria we extracted 85 data sets containing quantitative information for close to 2000 proteins. This information was made available in CSF-PR 2.0 (http://probe.uib.no/csf-pr-2.0), which includes novel approaches for filtering, visualizing and comparing quantitative proteomics information in an interactive and user-friendly environment. CSF-PR 2.0 will be an invaluable resource for anyone interested in quantitative proteomics on cerebrospinal fluid.     

Human herpesvirus 8 DNA in the cerebrospinal fluid of a patient with sensory impairment.

This note reports the finding of human herpes virus 8 DNA in the cerebrospinal fluid of a woman with sensory impairment correlated with a basal disease suspected to be multiple sclerosis.