Protein Composition Of Cerebrospinal Fluid Research Articles

O15: DISRUPTION OF THE BLOOD-SPINAL CORD BARRIER PREDICTS PERMANENT PARAPLEGIA AFTER THORACOABDOMINAL AORTIC ANEURYSM REPAIR

Abstract Introduction Paraplegia post-thoracoabdominal aortic aneurysm (TAAA) repair remains both a devastating and poorly understood complication. We related temporal changes in cellular and protein composition of cerebrospinal fluid (CSF) to neurological outcomes after TAAA repair to gain mechanistic insights driving paraplegia. Method Patients undergoing TAAA repair (open or endovascular) with a CSF drain were prospectively recruited between 2016-2018. CSF was collected pre-operatively and 24-hourly until removal. Daily neurological examinations were performed by blinded neurologists to the study. CSF cell content was characterised by flow cytometry and proteome analysed by tandem-mass-tag proteomics. An in-vivo rat model was modified using 15 minutes of aortic occlusion to produce consistent paraplegia. Rats were analysed neuro-behaviourally and histologically. Result CSF was analysed from 52 patients (age: 70.27+/-11.4; 66% male; open (n=9), endovascular (n=43)). 12 developed paraplegia of whom 5 remained permanently-paraplegic. Demographics were comparable between paraplegics, those who recovered and without post-op neurology. Permanent paraplegia was associated with a significant infiltration of CSF CD45+ leucocytes (P<0.0001). Levels of ADVS-1 was >3-fold higher in permanent-paraplegics CSF versus those who recovered (P=0.0008). ADVS-1 >15ng/ml predicted permanent paraplegia with 100% specificity. Pre-treatment with ADVS-1 inhibition significantly improved walking (<0.001) and increased astrocytic staining in the lateral corticospinal, reticulospinal and rubrospinal tracts versus controls (P=0.03, 0.04, 0.04 respectively). Conclusion Permanent paraplegia is associated with shedding of ADVS-1 from parenchymal cord into CSF and blood/spinal-cord barrier disruption leading to cord oedema/leucocyte infiltration. Pre-treatment with ADVS-1 inhibition led to neurobehavioural and histological improvements offering translational hope for this devastating complication. Take-home message ADVS-1 is a novel biomarker of paraplegia where accurate biomarkers have proven challenging but more importantly it has proven a therapeutic target with genuine translational potential.

Proteome profiling in cerebrospinal fluid reveals novel biomarkers of Alzheimer's disease.

Neurodegenerative diseases are a growing burden, and there is an urgent need for better biomarkers for diagnosis, prognosis, and treatment efficacy. Structural and functional brain alterations are reflected in the protein composition of cerebrospinal fluid (CSF). Alzheimer's disease (AD) patients have higher CSF levels of tau, but we lack knowledge of systems‐wide changes of CSF protein levels that accompany AD. Here, we present a highly reproducible mass spectrometry (MS)‐based proteomics workflow for the in‐depth analysis of CSF from minimal sample amounts. From three independent studies (197 individuals), we characterize differences in proteins by AD status (> 1,000 proteins, CV < 20%). Proteins with previous links to neurodegeneration such as tau, SOD1, and PARK7 differed most strongly by AD status, providing strong positive controls for our approach. CSF proteome changes in Alzheimer's disease prove to be widespread and often correlated with tau concentrations. Our unbiased screen also reveals a consistent glycolytic signature across our cohorts and a recent study. Machine learning suggests clinical utility of this proteomic signature.

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

CSF-Based Analysis for Identification of Potential Serum Biomarkers of Neural Tube Defects.

The protein composition of cerebrospinal fluid (CSF) in neural tube defects (NTDs) remains unknown. We investigated the protein composition of CSF from 9 infants with NTDs using isobaric tags for relative and absolute quantitation (iTRAQ). We identified 568 proteins in the CSF of infants with spina bifida, which is the most common type of NTD. Among these, 18 proteins were associated with neural tube closure in the CSF during human embryonic neurulation and 5 were involved in NTDs. Based on these results, an animal model was further utilized to investigate early serum biomarkers for NTDs. We found that the myristoylated alanine-rich C-kinase substrate, Kunitz-type protease inhibitor 2, and apolipoprotein B-100 protein levels were decreased in both embryos and the sera of pregnant Sprague-Dawley rats carrying embryos with NTDs. CSF proteins may be useful in the discovery of potential serum biomarkers for NTDs.

Sex-Related Differences in Rat Choroid Plexus and Cerebrospinal Fluid: AcDNA Microarray and Proteomic Analysis.

The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood and the cerebrospinal fluid (CSF), which is mostly produced by the CP itself. Because the CP transcriptome is regulated by the sex hormone background, the present study compared gene/protein expression profiles in the CP and CSF from male and female rats aiming to better understand sex-related differences in CP functions and brain physiology. We used data previously obtained by cDNA microarrays to compare the CP transcriptome between male and female rats, and complemented these data with the proteomic analysis of the CSF of castrated and sham-operated males and females. Microarray analysis showed that 17128 and 17002 genes are expressed in the male and female CP, which allowed the functional annotation of 141 and 134 pathways, respectively. Among the most expressed genes, canonical pathways associated with mitochondrial dysfunctions and oxidative phosphorylation were the most prominent, whereas the most relevant molecular and cellular functions annotated were protein synthesis, cellular growth and proliferation, cell death and survival, molecular transport, and protein trafficking. No significant differences were found between males and females regarding these pathways. Seminal functions of the CP differentially regulated between sexes were circadian rhythm signalling, as well as several canonical pathways related to stem cell differentiation, metabolism and the barrier function of the CP. The proteomic analysis identified five down-regulated proteins in the CSF samples from male rats compared to females and seven proteins exhibiting marked variation in the CSF of gonadectomised males compared to sham animals, whereas no differences were found between sham and ovariectomised females. These data clearly show sex-related differences in CP gene expression and CSF protein composition that may impact upon neurological diseases.

High Blood Pressure Effects on the Blood to Cerebrospinal Fluid Barrier and Cerebrospinal Fluid Protein Composition: A Two-Dimensional Electrophoresis Study in Spontaneously Hypertensive Rats

The aim of the present work is to analyze the cerebrospinal fluid proteomic profile, trying to find possible biomarkers of the effects of hypertension of the blood to CSF barrier disruption in the brain and their participation in the cholesterol and β-amyloid metabolism and inflammatory processes. Cerebrospinal fluid (CSF) is a system linked to the brain and its composition can be altered not only by encephalic disorder, but also by systemic diseases such as arterial hypertension, which produces alterations in the choroid plexus and cerebrospinal fluid protein composition. 2D gel electrophoresis in cerebrospinal fluid extracted from the cistern magna before sacrifice of hypertensive and control rats was performed. The results showed different proteomic profiles between SHR and WKY, that α-1-antitrypsin, apolipoprotein A1, albumin, immunoglobulin G, vitamin D binding protein, haptoglobin and α-1-macroglobulin were found to be up-regulated in SHR, and apolipoprotein E, transthyretin, α-2-HS-glycoprotein, transferrin, α-1β-glycoprotein, kininogen and carbonic anhidrase II were down-regulated in SHR. The conclusion made here is that hypertension in SHR produces important variations in cerebrospinal fluid proteins that could be due to a choroid plexus dysfunction and this fact supports the close connection between hypertension and blood to cerebrospinal fluid barrier disruption.

Blood to cerebrospinal fluid barrier in spontaneous hypertension and ventricular dilation

Background Arterial hypertension produces ventricular dilation, CSF protein composition variations as well as alterations in the circumventricular organ and choroid plexus (CP). Cerebrospinal fluid (CSF) has an important relationship with the blood and the brain, and encephalic pathology could alter the connections between the blood, brain and CSF, and consequently the protein composition of the CSF. Furthermore, certain proteins could be used as markers such as transthyretin and S-100β, which are extravasated when central nervous system (CNS) barriers are breached. The S-100β is primarily synthesized in the brain by the astrocytes and is released into the blood when the blood brain barrier (BBB) is disrupted. Transthyretin is a protein primarily located in the brain in choroid plexus (CP), subcommissural organ (SCO) and, as soluble monomer, in the CSF, where it has a positive gradient for extravasation in peripheral blood, when the blood to CSF barrier (BCB) is altered. The aim of the present work is to analyze the CNS barriers in the arterial hypertension using antibodies against S-100β, transthyretin monomer (TTRm), TA-p73 and Reissner fibre (AFRU). Materials and methods Brain, cerebrospinal fluid and serum were extracted from control Wistar-Kioto (WKY) rats and spontaneously hypertensive rats (SHR) of 10 months of age. Brain sections containing the SCO and the CP were processed by immunohistochemistry using the following as primary antibodies: anti-S-100β, anti-TTRm anti-TA-p73 and AFRU. Western blot and 2-D electrophoresis of CSF, serum and extracts of SCO and CP were performed.

Proteomic analysis of cerebrospinal fluid discriminates malignant and nonmalignant disease of the central nervous system and identifies specific protein markers

CNS diseases are often accompanied by changes in the protein composition of cerebrospinal fluid (CSF). SELDI-TOF-MS provides an approach for identifying specific protein markers of disease in biological fluids. We compared the CSF proteomes from patients with neoplastic and reactive/inflammatory CNS diseases to identify potential biomarkers. SELDI-TOF-MS was performed on CSF derived from lumbar puncture of 32 patients, including 10 with CNS malignancies, 12 with inflammatory or reactive conditions, and 10 with unknown CNS disease. Using the SAX-2 (strong anionic exchange) chip, we uncovered three conserved protein peak ranges within each disease category. For neoplastic diseases, we identified conserved peaks at 7.5-8.0 kDa (9/10 samples), 15.1-15.9 kDa (8/10 samples), and 30.0-32.0 kDa (5/10 samples). In reactive/inflammatory diseases, conserved peaks were found at 6.7-7.1 kDa (10/12 samples), 11.5-11.9 kDa (12/12 samples), and 13.3-13.7 kDa (9/12 samples). A protein from the 30.0 to 32.0 kDa peak range found in neoplastic CSF was identified by MALDI analysis as carbonic anhydrase, a protein overexpressed in many malignancies including high-grade gliomas. Similarly, cystatin C was identified in the 13.3-13.7 kDa peak range in non-neoplastic CSF and was most prominent in inflammatory conditions. Our approach provides a rational basis for identifying biomarkers that could be used for detection, diagnosis, and monitoring of CNS diseases.

Protein Analysis in Human Cerebrospinal Fluid: Physiological Aspects, Current Progress and Future Challenges

The introduction of lumbar puncture into clinical medicine over 100 years ago marks the beginning of the study of central nervous system diseases using the human cerebrospinal fluid (CSF). Ever since, CSF has been analyzed extensively to elucidate the physiological and biochemical bases of neurological disease. The proximity of CSF to the brain makes it a good target for studying the pathophysiology of brain functions, but the barrier function of the CSF also impedes its diagnostic value. Today, measurements to determine alterations in the composition of CSF are central in the differential diagnosis of specific diseases of the central nervous system (CNS). In particular, the analysis of the CSF protein composition provides crucial information in the diagnosis of CNS diseases. This enables the assessment of the physiology of the blood-CSF barrier and of the immunology of intrathecial responses. Besides those routine measurements, protein compositional studies of CSF have been extended recently to many other proteins in the expectation that comprehensive analysis of lower abundance CSF proteins will lead to the discovery of new disease markers. Disease marker discovery by molecular profiling of the CSF tissue has the enormous potential of providing many new disease relevant molecules. New developments in protein profiling techniques hold promise for the discovery and validation of relevant disease markers. In this review, we summarize the current efforts and progress in CSF protein profiling measurements using conventional and current protein analysis tools. We also discuss necessary development in methodology in order to have the highest impact on the study of the molecular composition of CSF proteins.

Proteomics Analysis of Phosphotyrosyl-Proteins in Human Lumbar Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a secretion product of several different central nervous system (CNS) structures, including the choroid plexus in the ventricles. Pathological CNS processes are reflected in the protein composition of CSF. To elucidate the molecular events that occur in the homeostatic and pathological processes of the CNS, the high-throughput characterization of differentially expressed proteins, and of post-translationally modified proteins, is needed for proteomics studies of CSF. Among the post-translational modifications of proteins, phosphorylation is the most common and important mechanism for the reversible regulation of protein function. In this study, CSF phosphotyrosyl (p-Tyr)-proteins were detected with antibodies and were analyzed with proteomics methods. Three different combination methods--1D gel electrophoresis and Western blotting, immunoprecipitation and 2D gel electrophoresis, and 2D gel electrophoresis and Western blotting--were used to detect p-Tyr-proteins in human lumbar CSF samples. Six protein spots, representing four proteins on a 2D Western blot, were identified as p-Tyr-proteins with the 2D gel electrophoresis and Western blotting method. Those four p-Tyr-proteins are kallikrein-6 precursor, complement C4 gamma-chain, gelsolin, and ceruloplasmin precursor. Additionally, four other nonphosphorylated CSF proteins--beta-2-glycoprotein I precursor, fibulin-1 precursor, EGF-containing fibulin-like extracellur matrix protein 1 precursor, and angiotensinogen precursor--were characterized for the first time.

Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry.

Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain-specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two-dimensional gel electrophoresis (2-DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid-phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO-Ruby-stained 2-D gels, with final protein identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). To optimise the focusing of the protein spots on the 2-D gel, the ampholyte concentration in liquid-phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2-D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2-D gels from prefractionated CSF compared with direct 2-DE separations of CSF. Several proteins, including cystatin C, IgM-kappa, hemopexin, acetyl-coenzyme A carboxylase-alpha, and alpha-1-acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. alpha-1-acid glycoprotein and alpha-2-HS glycoprotein, can be enriched, thus better resolved and detected on the 2-D gel. Liquid-phase IEF, as a prefractionation step prior to 2-DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI-MS analysis.

Proteomics in molecular medicine: applications in central nervous systems disorders.

Bodily fluids such as cerebrospinal fluid (CSF) and serum can be analysed at the time of presentation and throughout the course of the disease. Changes in the protein composition of CSF may be indicative of altered CNS protein expression pattern with a causative or diagnostic disease link. These findings can be strengthened through subsequent proteomic analysis of specific brain areas implicated in the pathology. New isolation strategies of clinically relevant cellular material such as laser capture microdissection, protein enrichment procedures and proteomic approaches to neuropeptide and neurotransmitter analysis give us the opportunity to map out complex cellular interaction at an unprecedented level of detail. In neurological disorders multiple underlying pathogenic mechanisms as well as an acute and a chronic CNS disease components may require a selective repertoire of molecular targets and biomarkers rather than an individual protein to better define a complex disease. The resulting proteome database bypasses many ambiguities of experimental models and may facilitate pre- and clinical development of more specific disease markers and new selective fast acting therapeutics.

Protein composition of cerebrospinal fluid in the developing chick embryo

Proteins from cerebrospinal fluid of chick embryos at 4–19 days of development were qualitative and quantitatively analyzed. The total protein concentration and the relative concentration of each protein fraction were calculated for each day. The main proteins found along development were immunoglobulin G, transferrin, α-fetoprotein, serum albumin, ovalbumin, prealbumin, and an unidentified component. α-Fetoprotein was found to be the major protein at 4–16 days, and serum albumin at 17–19 days of development. The unidentified fraction was present from 4 to 11 days; at day 5 it represented 28% of the total cerebrospinal fluid protein concentration.

Localization of human prealbumin in choroid plexus epithelium.

Cerebrospinal fluid (CSF) is generally considered to be derived from plasma through a combined process of ultrafiltration and secretion by the choroid plexus. However, the mechanisms ultimately responsible for achieving the final protein composition of CSF are uncertain. Some proteins, in particular prealbumin, are present in quantities not easily explained by usual physicochemical considerations. To investigate the possibility of de novo synthesis by choroid epithelium, we have examined human choroid plexus an ependyma for the presence of prealbumin. Using the peroxidase-antiperoxidase method at the light and electron microscopic level, as well as immunofluorescence, we localized prealbumin in choroid epithelial cytoplasm on the endoplasmic reticulum and in association with the Golgi apparatus. Ependymal cells and stroma did not reveal immunocytochemical labeling. These findings indicate that the choroid plexus epithelium contributes to the final CSF composition by de novo protein synthesis.

Further Studies on the Modified Takata Reaction (M.T.R.) on the Cerebrospinal Fluids of Neurological and Psychiatric Patients

Ucko (1935) showed that the reaction time of the Takata test on serum can be reduced from 24 hours to 90 minutes by modifying the concentration and volume of the reagents. These reagents were applied with a further slight modification in the amount of the fluid tested and as a one-tube method for cerebrospinal fluid (Fleischhacker, 1938). 177 cerebrospinal fluids were examined, and the conclusion was drawn that the M.T.R. will indicate even very fine disturbances in the protein composition of cerebrospinal fluid. Later the test, together with others, was applied to another 165 C.S.Fs. from neurological and psychiatric patients and was found to give weak positive results in many psychoses of the so-called “non-organic” type (especially typical schizophrenics), while it remained negative in manic depressives and neurotic disorders (Fleischhacker, 1943).