Search

Using the Olink Explore 1536 platform, we measured 1,463 unique proteins in 303 cerebrospinal fluid (CSF) specimens from four clinical centers contributed by uninfected controls and 12 groups of people living with HIV-1 infection representing the spectrum of progressive untreated and treated chronic infection. We present three initial analyses of these measurements: an overview of the CSF protein features of the sample; correlations of the CSF proteins with CSF HIV-1 RNA and neurofilament light chain protein (NfL) concentrations; and comparison of CSF proteins in HIV-associated dementia (HAD) and neurosymptomatic CSF escape (NSE). These reveal a complex but coherent picture of CSF protein changes with highest concentrations of many proteins during CNS injury in the HAD and NSE groups and variable protein changes across the course of systemic HIV-1 progression that included two common patterns, designated as lymphoid and myeloid patterns, related to principal involvement of their underlying inflammatory cell lineages. Antiretroviral therapy reduced CSF protein perturbations, though not always to control levels. The dataset of these CSF protein measurements, along with background clinical information, is posted online. Extended studies of this unique dataset will supplement this report to provide more detailed characterization of the dynamic impact of HIV-1 infection on the CSF proteome across the spectrum of HIV-1 infection, advancing the mechanistic understanding of HIV-1-related CNS pathobiology., Competing Interests: HZ has served at scientific advisory boards and/or as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZPath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures in symposia sponsored by Alzecure, Biogen, Cellectricon, Fujirebio, Lilly, Novo Nordisk, and Roche, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (outside submitted work). These organizations and companies had no role in the study design, data collection, decision to publish, or publication of the manuscript. MG has received research grants from Gilead Sciences and honoraria as speaker, DSMB committee member and/or scientific advisor from Amgen, AstraZeneca, Biogen, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline/ViiV, Janssen-Cilag, MSD, Novocure, Novo Nordic, Pfizer and Sanofi. These organizations and companies had no role in the study design, data collection, decision to publish, or publication of the manuscript., (Copyright: © 2024 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The analysis of protein dynamics or turnover in patients has the potential to reveal altered protein recycling, such as in Alzheimer's disease, and to provide informative data regarding drug efficacy or certain biological processes. The observed protein dynamics in a solid tissue or a fluid is the net result of not only protein synthesis and degradation but also transport across biological compartments. We report an accurate 3-biological compartment model able to simultaneously account for the protein dynamics observed in blood plasma and the cerebrospinal fluid (CSF) including a hidden central nervous system (CNS) compartment. We successfully applied this model to 69 proteins of a single individual displaying similar or very different dynamics in plasma and CSF. This study puts a strong emphasis on the methods and tools needed to develop this type of model. We believe that it will be useful to any researcher dealing with protein dynamics data modeling.

Cerebrospinal fluid (CSF) biomarkers reflect brain pathophysiology and are used extensively in translational research as well as in clinical practice for diagnosis of neurological diseases, e.g., Alzheimer's disease (AD). However, CSF biomarker concentrations may be influenced by non-disease related inter-individual variability. Here we use a data-driven approach to demonstrate the existence of inter-individual variability in mean standardized CSF protein levels. We show that these non-disease related differences cause many commonly reported CSF biomarkers to be highly correlated, thereby producing misleading results if not accounted for. To adjust for this inter-individual variability, we identified and evaluated high-performing reference proteins which improved the diagnostic accuracy of key CSF AD biomarkers. Our reference protein method attenuates the risk for false positive findings, and improves the sensitivity and specificity of CSF biomarkers, with broad implications for both research and clinical practice., (© 2024. The Author(s).)

ADAM10 is the main α-secretase acting in the non-amyloidogenic processing of APP. We hypothesized that certain rare ADAM10 variants could increase the risk for AD by conferring the age-related downregulation of α-secretase. The ADAM10 gene was sequenced in 103 AD cases (82% familial) and 96 cognitively preserved nonagenarians. We examined rare variants (MAF < 0.01) and determined their potential association in the AD group with lower CSF protein levels, as analyzed by means of ELISA, and Western blot (species of 50 kDa, 55 kDa, and 80 kDa). Rare variants were found in 15.5% of AD cases (23% early-onset, 8% late-onset) and in 12.5% of nonagenarians, and some were group-specific. All were intronic variants except Q170H, found in three AD cases and one nonagenarian. The 3'UTR rs74016945 (MAF = 0.01) was found in 6% of the nonagenarians (OR 0.146, p = 0.057). Altogether, ADAM10 total levels or specific species were not significantly different when comparing AD with controls or carriers of rare variants versus non-carriers (except a Q170H carrier exhibiting low levels of all species), and did not differ according to the age at onset or APOE genotype. We conclude that ADAM10 exonic variants are uncommon in AD cases, and the presence of rare intronic variants (more frequent in early-onset cases) is not associated with decreased protein levels in CSF.

Objectives: To present a normal range of cerebrospinal fluid (CSF) protein levels in a community-based population and to evaluate factors that contribute to CSF protein level variability., Patients and Methods: Samples of CSF protein were obtained from participants aged 32 to 95 years who underwent lumbar puncture (LP) between November 1, 2007, and October 1, 2017, as part of the Mayo Clinic Study of Aging, a longitudinal, population-based study of residents of Olmsted County, Minnesota., Results: A total of 633 participants (58.1% male; 99.1% White; mean ± SD age, 70.9±11.6 years) underwent LP with recorded CSF protein level. Mean ± SD CSF protein level was 52.2±18.4 mg/dL (to convert to mg/L, multiply by 10), with a 95% reference interval of 24.0 to 93.4 mg/dL (range, 14.0-148.0 mg/dL). Spinal stenosis and arterial hypertension were associated with higher CSF protein levels on univariable analysis (P<.001). Increasing age, male sex, and diabetes were all independently associated with higher CSF protein levels on multivariable analysis (P<.001). In the 66 participants with repeated LPs within 2.5 years, the coefficient of repeatability was 26.1 mg/dL. Eleven participants (16.7%) had a CSF protein level difference of 20 mg/dL or more between serial LPs, and 4 (6.1%) had a difference of 25 mg/dL or more. There was a trend toward greater CSF protein level variability in patients with spinal stenosis (P=.054)., Conclusion: This large population-based study showed that CSF protein level can vary significantly among individuals. Elevated CSF protein level was independently associated with older age, male sex, and diabetes and is higher than listed in many laboratories. These findings emphasize the necessity of evidence-based reevaluation and standardization of CSF protein metrics., (Copyright © 2022 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

Background: Pediatric-onset multiple sclerosis (POMS) represents the earliest stage of disease pathogenesis. Investigating the cerebrospinal fluid (CSF) proteome in POMS may provide novel insights into early MS processes., Objective: To analyze CSF obtained from children at time of initial central nervous system (CNS) acquired demyelinating syndrome (ADS), to compare CSF proteome of those subsequently ascertained as having POMS versus monophasic acquired demyelinating syndrome (mADS)., Methods: Patients were selected from two prospective pediatric ADS studies. Liquid chromatography-mass spectrometry (LC-MS) was performed in a Dutch discovery cohort (POMS n = 28; mADS n = 39). Parallel reaction monitoring-mass spectrometry (PRM-MS) was performed on selected proteins more abundant in POMS in a combined Dutch and Canadian validation cohort (POMS n = 48; mADS n = 106)., Results: Discovery identified 5580 peptides belonging to 576 proteins; 58 proteins were differentially abundant with ⩾2 peptides between POMS and mADS, of which 28 more abundant in POMS. Fourteen had increased abundance in POMS with ⩾8 unique peptides. Five selected proteins were all confirmed within validation. Adjusted for age, 2 out of 5 proteins remained more abundant in POMS, that is, Carboxypeptidase E (CPE) and Semaphorin-7A (SEMA7A) ., Conclusion: This exploratory study identified several CSF proteins associated with POMS and not mADS, potentially reflecting neurodegeneration, compensatory neuroprotection, and humoral response in POMS. The proteins associated with POMS highly correlated with age at CSF sampling.

Molecular markers derived from cerebrospinal fluid (CSF) represent an accessible means of exploring the pathobiology of Huntington's disease (HD) in vivo. The endo-lysosomal/autophagy system is dysfunctional in HD, potentially contributing to disease pathogenesis and representing a potential target for therapeutic intervention. Several endo-lysosomal proteins have shown promise as biomarkers in other neurodegenerative diseases; however, they have yet to be fully explored in HD. We performed parallel reaction monitoring mass spectrometry analysis (PRM-MS) of multiple endo-lysosomal proteins in the CSF of 60 HD mutation carriers and 20 healthy controls. Using generalised linear models controlling for age and CAG, none of the 18 proteins measured displayed significant differences in concentration between HD patients and controls. This was affirmed by principal component analysis, in which no significant difference across disease stage was found in any of the three components representing lysosomal hydrolases, binding/transfer proteins and innate immune system/peripheral proteins. However, several proteins were associated with measures of disease severity and cognition: most notably amyloid precursor protein, which displayed strong correlations with composite Unified Huntington's Disease Rating Scale, UHDRS Total Functional Capacity, UHDRS Total Motor Score, Symbol Digit Modalities Test and Stroop Word Reading. We conclude that although endo-lysosomal proteins are unlikely to have value as disease state CSF biomarkers for Huntington's disease, several proteins demonstrate associations with clinical severity, thus warranting further, targeted exploration and validation in larger, longitudinal samples., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: AJL, FBR, LMB, RT, HZ and EJW are University College London employees. FBR has provided consultancy services to GLG and F. Hoffmann-La Roche Ltd. EJW reports grants from Medical Research Council, CHDI Foundation, and F. Hoffmann-La Roche Ltd during the conduct of the study; personal fees from Hoffman La Roche Ltd, Triplet Therapeutics, PTC Therapeutics, Shire Therapeutics, Wave Life Sciences, Mitoconix, Takeda, Loqus23. All honoraria for these consultancies were paid through the offices of UCL Consultants Ltd., a wholly owned subsidiary of University College London. HZ has served at scientific advisory boards for Denali, Roche Diagnostics, Wave, Samumed and CogRx, has given lectures in symposia sponsored by Fujirebio, Alzecure and Biogen, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Cerebrospinal fluid (CSF) is a circulatory fluid of the central nervous system and it can reflect the biochemical changes occurring in the brain. Although CSF retrieval through lumbar puncture is invasive, it remains the most commonly used fluid in exploring brain pathology as it is less complex and contains a higher concentration of brain-derived proteins than plasma (Reiber, H. Clin. Chim. Acta 2001, 310, 173-186; Macron et al. J. Proteome Res. 2018, 17, 4315-4319). We hypothesize that proteins produced by the brain will have diagnostic significance for brain pathologies. Hence, we expanded the previously in-house-developed 31-protein panel with more proteins classified as brain-specific by the Human Protein Atlas (HPA). Using the HPA, we selected 76 protein coding genes and screened CSF using liquid chromatography-mass spectrometry (LC-MS) and narrowed the protein list to candidates identified endogenously in CSF. Next, we developed a parallel reaction monitoring (PRM) assay for the 21 new proteins and merged it with the 31-protein assay developed earlier. In the process, we evaluated different screening strategies and optimized MS collision energies and ion isolation windows to achieve the highest possible analyte signal resulting in the PRM assay with an average linear dynamic range of 4.3 × 10 3 . We also assessed the extent of Asn (N)-Gln (Q) deamidation, N-terminal pyro-Glu (E) conversion, and Met (M) oxidation and found that deamidation can be misassigned without high mass accuracy and high-resolution settings. We also assessed how many of these proteins could be reliably measured in 10 individual patient CSF samples. Our approach allows us to measure the relative levels of 52 brain-derived proteins in CSF by a single LC-MS method. This new assay may have important applications in discovering CSF biomarkers for various neurological diseases.

Cerebrospinal fluid (CSF) is a vital liquid, providing nutrients and signaling molecules and clearing out toxic by-products from the brain. The CSF is produced by the choroid plexus (ChP), a protective epithelial barrier that also prevents free entry of toxic molecules or drugs from the blood. Here, we establish human ChP organoids with a selective barrier and CSF-like fluid secretion in self-contained compartments. We show that this in vitro barrier exhibits the same selectivity to small molecules as the ChP in vivo and that ChP-CSF organoids can predict central nervous system (CNS) permeability of new compounds. The transcriptomic and proteomic signatures of ChP-CSF organoids reveal a high degree of similarity to the ChP in vivo. Finally, the intersection of single-cell transcriptomics and proteomic analysis uncovers key human CSF components produced by previously unidentified specialized epithelial subtypes., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Meningitis patient can present with various manifestation including hydrocephalus due to multiple reason. Diagnosis of meningitis mainly rely on CSF analysis which is usually obtained from lumbar puncture. In case of hydrocephalus CSF can be obtain from ventricles during VP shunt operation. Sometimes ventricular CSF can be normal in meningitis patient while lumbar CSF shows abnormality. Possible mechanisms behind this phenomenon are discussed here. Patients who present with hydrocephalus and have normal Ventricular CSF should investigated with lumbar CSF analysis in a view of delay in diagnosis and treatment., (Copyright © 2020 Tuberculosis Association of India. Published by Elsevier B.V. All rights reserved.)

The brain ventricular system is a series of connected cavities, filled with cerebrospinal fluid (CSF), that forms within the vertebrate central nervous system (CNS). The hollow neural tube is a hallmark of the chordate CNS, and a closed neural tube is essential for normal development. Development and function of the ventricular system is examined, emphasizing three interdigitating components that form a functional system: ventricle walls, CSF fluid properties, and activity of CSF constituent factors. The cellular lining of the ventricle both can produce and is responsive to CSF. Fluid properties and conserved CSF components contribute to normal CNS development. Anomalies of the CSF/ventricular system serve as diagnostics and may cause CNS disorders, further highlighting their importance. This review focuses on the evolution and development of the brain ventricular system, associated function, and connected pathologies. It is geared as an introduction for scholars with little background in the field., (© 2020 WILEY Periodicals, Inc.)

Background: Alzheimer's disease (AD) is an inflammatory neurodegenerative disease that may be associated with prior bacterial infections. Microbial "old friends" can suppress exaggerated inflammation in response to disease-causing infections or increase clearance of pathogens such as Mycobacterium tuberculosis, which causes tuberculosis (TB). One such "old friend" is Mycobacterium vaccae NCTC 11659, a soil-derived bacterium that has been proposed either as a vaccine for prevention of TB, or as immunotherapy for the treatment of TB when used alongside first line anti-TB drug treatment., Objective: The goal of this study was to use a hypothesis generating approach to explore the effects of M. vaccae on physiological changes in the plasma and cerebrospinal fluid (CSF)., Methods: Liquid chromatography-tandem mass spectrometry-based proteomics were performed in plasma and CSF of adult male rats after immunization with a heat-killed preparation of M. vaccae NCTC 11659 or borate-buffered saline vehicle. Gene enrichment analysis and analysis of protein-protein interactions were performed to integrate physiological network changes in plasma and CSF. We used RT-qPCR to assess immune and metabolic gene expression changes in the hippocampus., Results: In both plasma and CSF, immunization with M. vaccae increased proteins associated with immune activation and downregulated proteins corresponding to lipid (including phospholipid and cholesterol) metabolism. Immunization with M. vaccae also increased hippocampal expression of interleukin-4 (IL-4) mRNA, implicating anti-inflammatory effects in the central nervous system., Conclusion: M. vaccae alters host immune activity and lipid metabolism. These data are consistent with the hypothesis that microbe-host interactions may protect against possible infection-induced, inflammation-related cognitive impairments.

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.

Cerebrospinal fluid (CSF) protein values decline over the first few months of life as the infant's blood-CSF barrier matures. However, published studies have not reported CSF protein reference values of Chinese infants and differ in the reported rate, timing, and magnitude of this decline. The objective of this study was to determine reference intervals for CSF protein using available data of children in southern China. This retrospective study included infants who had a lumbar puncture (LP) performed in the Department of Pediatrics of Southern Medical University Affiliated Maternal and Child Health Hospital of Foshan of an urban tertiary care children's hospital between January 1, 2008 and May 31, 2018. Infants with conditions suspected or known to cause elevated CSF protein concentrations were excluded. Of 3712 infants undergoing LP, 1043 (28.1%) met inclusion criteria. Results showed that there is an age-related decline in CSF protein concentration. The median CSF protein value was 62 mg/dL [interquartile range (IQR): 47-81 mg/dL] in infants aged 0 to 56 days (group 1). The 95th percentile values were 116 mg/dL for infants 0 to 28 days and 80 mg/dL for infants 29 to 56 days. The 95th percentile values by age category were as follows: ages 0 to 14 days, 117 mg/dL; ages 15 to 28 days, 107 mg/dL; ages 29 to 42 days, 96 mg/dL; and ages 43 to 56 days, 74 mg/dL. The median CSF protein value was 21 mg/dL (IQR: 16-31 mg/dL) in infants aged 2 months to <3 years (group 2). The 95th percentile values were 57 mg/dL for infants 2 to <6 months and 34 mg/dL for infants 6 to ≤24 months. The 95th percentile values by age category were as follows: ages 2 to <3 months, 66 mg/dL; ages 3 to <4 months, 52 mg/dL; ages 4 to <5 months, 53 mg/dL; and ages 5 to <6 months, 42 mg/dL. We quantify the age-related decline in CSF protein concentrations among infants 2 years of age and younger and provide age-specific reference values. The values reported here can be used to interpret the results of LP in infants ≤2 years of age.

Background: Froin syndrome is characterized by xanthochromia and hypercoagulability of the cerebrospinal fluid (CSF) due to elevated protein levels. This entity results from blockage of the spinal canal by a mass lesion leading to an isolated caudal CSF space., Case Description: A 48-year-old male, who developed spasticity after a C6 spinal cord injury (SCI) 20 years earlier, presented with subobstruction of his intrathecal baclofen pump. A catheter access port aspiration revealed an extremely high protein concentration (38 g/L) with no signs of infection. Froin syndrome was confirmed when magnetic resonance imaging showed a complete obstruction of the spinal canal at the SCI level., Conclusions: We report the first case of Froin syndrome after SCI. Froin syndrome can impact intrathecal drug delivery and CSF diagnostics., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fatigue in multiple sclerosis is a very common and cumbersome symptom, but its aetiology is poorly understood. Proteomics is increasingly implemented in multiple sclerosis research, but has not yet been used to study the neurobiological basis of fatigue in multiple sclerosis. To identify potential cerebrospinal fluid biomarkers of fatigue in multiple sclerosis, we collected cerebrospinal fluid of 20 patients with multiple sclerosis with fatigue (MS+), 20 patients with multiple sclerosis without fatigue (MS-), and 20 control subjects without multiple sclerosis and without fatigue (HC). We used a shotgun proteomics approach and label-free quantitative proteomics to analyse the protein content in cerebrospinal fluid. Selected proteins with differential abundance were further validated by immunoblotting. Out of 591 detected cerebrospinal fluid proteins, the abundance of nine proteins differed between the three groups, and seven additional proteins differed between MS+ and MS- patients. Using immunoblot or slot-blot techniques, we confirmed decreased levels of protein kinase C-binding protein NELL2, neural cell adhesion molecule L1-like protein, and reelin in MS+ patients. In conclusion, cerebrospinal fluid proteomics may provide insight into the neurobiological basis of fatigue in multiple sclerosis. The proteins identified to be decreased in MS+ are involved in synaptic plasticity and energy homeostasis, and thus appear as plausible biomarkers of this common symptom., (© 2018 European Sleep Research Society.)

Ischemic stroke is one of the most common causes of death worldwide and is a major cause of acquired disability in adults. However, there is still a need for an effective drug for its treatment. Buyang Huanwu decoction (BHD), a traditional Chinese medicine (TCM) prescription, has long been used clinically to aid neurological recovery after stroke. To establish potential clinical indicators of BHD efficacy in stroke treatment and prognosis, we conducted a combined proteomic and metabolomic analysis of cerebrospinal fluid (CSF) samples in a mouse stroke model. CSF samples were obtained from male mice with acute ischemic stroke induced by middle cerebral ischemic/reperfusion (CI/R) injury, some of which were then treated with BHD. Label-free quantitative proteomics was conducted using nano-LC-MS/MS on an LTQ Orbitrap mass and metabolomic analysis was performed using nanoprobe NMR and UHPLC-QTOF-MS. The results showed that several proteins and metabolites were present at significantly different concentrations in the CSF samples from mice with CI/R alone and those treated with BHD. These belonged to pathways related to energy demand, inflammatory signaling, cytoskeletal regulation, Wnt signaling, and neuroprotection against neurodegenerative diseases. In conclusion, our in silico data suggest that BHD treatment is not only protective but can also ameliorate defects in pathways affected by neurological disorders. These data shed light on the mechanism whereby BHD may be effective in the treatment and prevention of stroke-related neurodegenerative disease., Competing Interests: The authors have declared that no competing interests exist.

Proteo-peptidomic profiling of biofluids is used to identify disease biomarkers and to study molecular mechanisms of pathology development. Previously, we studied changes in cerebrospinal fluid (CSF) and blood plasma associated with Guillain-Barre syndrome (GBS)-a rare and severe disorder of the peripheral nervous system with an unknown etiology. Here, we describe the workflow for the analysis of endogenous peptides from CSF. The procedure covers sample preparation, liquid chromatography-mass spectrometry (LC-MS) analysis, and bioinformatics analysis and allows identification of more than 1100 peptides from 181 protein groups in ~3 h from a single CSF sample derived from non-neurological, non-oncological patients.

Brain proteomics has become a method of choice that allows zooming-in where neuropathophysiological alterations are taking place, detecting protein mediators that might eventually be measured in cerebrospinal fluid (CSF) as potential neuropathologically derived biomarkers. Following this hypothesis, mass spectrometry-based neuroproteomics has emerged as a powerful approach to profile neural proteomes derived from brain structures and CSF in order to map the extensive protein catalog of the human brain. This chapter provides a historical perspective on the Human Brain Proteome Project (HBPP), some recommendation to the experimental design in neuroproteomic projects, and a brief description of relevant technological and computational innovations that are emerging in the neurobiology field thanks to the proteomics community. Importantly, this chapter highlights recent discoveries from the biology- and disease-oriented branch of the HBPP (B/D-HBPP) focused on spatiotemporal proteomic characterizations of mouse models of neurodegenerative diseases, elucidation of proteostatic networks in different types of dementia, the characterization of unresolved clinical phenotypes, and the discovery of novel biomarker candidates in CSF.

Recent advance in high-throughput proteome analysis has enabled genome-wide and proteome-wide analyses of associations between single nucleotide polymorphisms and protein expression levels. Protein quantitative trait locus (pQTL) studies using cerebrospinal fluid (CSF) and DNA samples may provide valuable insights into the genetic basis and molecular mechanisms regulating protein expression in the central nervous system. In this chapter, we describe a step-by-step procedures of CSF collection and pQTL analysis, by using PLINK and R software.

In the field of proteomics, the emerging "top-down" MS-based proteomics approach can be used to obtain a bird's eye view of all intact proteoforms present in a sample. This alternative to the "bottom-up" approach based on tryptic protein digestion processes has some unique advantages for assessing PTMs and sequence variations. However, it requires some dedicated tools for sample preparation and LC-MS analysis, which makes it more complex to handle than the bottom-up approach. In this study, a simple methodology is presented for characterizing intact proteins in biological fluid. This method yields quantitative information using an MS1 profiling approach and makes it possible to identify the proteins regulated under various clinical conditions.

The field of neurological diseases strongly needs biomarkers for early diagnosis and optimal stratification of patients in clinical trials or to monitor disease progression. Cerebrospinal fluid (CSF) is one of the main sources for the identification of novel protein biomarkers for neurological diseases. Despite the enormous efforts employed to identify novel CSF biomarkers, the high variability observed across different studies has hampered their validation and implementation in clinical practice. Such variability is partly caused by the effect of different pre-analytical confounding factors on protein stability, highlighting the importance to develop and comply with standardized operating procedures. In this chapter, we describe the international consensus pre-analytical guidelines for CSF processing and biobanking that have been established during the last decade, with a special focus on the influence of pre-analytical confounders on the global CSF proteome. In addition, we provide novel results on the influence of different delayed storage and freeze/thaw conditions on the CSF proteome using two novel large multiplex protein arrays (SOMAscan and Olink). Compliance to consensus guidelines will likely facilitate the successful development and implementation of CSF protein biomarkers in both research and clinical settings, ultimately facilitating the successful development of disease-modifying therapies.

In the field of neurodegeneration, it is important to identify biomarkers that enable early disease prediction, since these disorders start decades before clinical symptoms manifest. Cerebrospinal fluid (CSF) is considered an excellent source for biomarker discovery since it is in direct contact with the extracellular space of the brain and directly reflects disease-specific changes.While the liquor drainage is no major risk factor for patients, it is still not as easy and popular as simple blood sampling and less liquid can be collected. Especially when a variety of experiments for one cohort is planned, the volume of CSF can be a limiting factor. Therefore, it is essential that extraction and analytical methods are adapted to low amounts of liquor. If in follow-up studies, additional replicates to increase statistical significance or different extraction approaches are planned, the required amounts have to be minimized.With this extraction method, a combined proteomics and metabolomics approach is possible. This opportunity implies a variety of advantages. First, a classification matrix based on the comprehensive data set has a potentially higher accuracy even without a deeper understanding of the biological meaning of the different omics changes. If the proteome and metabolome differences can be linked to each other, this approach can conceivably open so far unknown doors regarding the cause or progression of different diseases like Alzheimer's or Parkinson's disease.

Nowadays, diagnosis of neurodegenerative disorders is mainly based on neuroimaging and clinical symptoms, although postmortem neuropathological confirmation remains the gold standard diagnostic technique. Therefore, cerebrospinal fluid (CSF) proteome is considered a valuable molecular repository for diagnosing and targeting the neurodegenerative process. It is well known that olfactory dysfunction is among the earliest features of synucleinopathies such as Parkinson's disease (PD). Consequently, we consider that the application of tissue proteomics in primary olfactory structures is an ideal approach to explore early pathophysiological changes, detecting olfactory proteins that might be tested in CSF as potential biomarkers. Data mining of mass spectrometry-generated datasets has revealed that 30% of the olfactory bulb (OB) proteome is also localized in CSF. In this chapter, we describe a method that utilizes label-free quantitative proteomics and computational analysis to characterize human OB proteomes and potential cerebrospinal fluid (CSF) biomarkers associated with neurodegenerative syndromes. For that, we applied peptide fractionation methods, followed by tandem mass spectrometry (nanoLC-MS/MS), in silico analysis, and semi-quantitative orthogonal techniques in OB derived from PD subjects. After obtaining the differential OB proteome across Lewy-type alpha-synucleinopathy (LTS) stages and further validating the method, this workflow was applied to probe changes in NEGR1 (neuronal growth regulator 1) and GNPDA2 (glucosamine-6-phosphate deaminase 2) protein levels in CSF derived from parkinsonian subjects with respect to controls, observing an inverse correlation between both proteins and α-synuclein, the principal component analysis of Lewy pathology.

The exploration of the cerebrospinal fluid (CSF) through proteomics techniques might help in the search of molecular biomarkers relevant to neurological pathologies. Aiming this, we describe here a commercially available multiplexed proteomics technology based on the use of modified aptamers (SOMAscan™ assay). From our experience in exploring the rat CSF proteome, we detail the basic principles of this oligonucleotide-based proteomics approach, as well as the main data-processing steps to obtain relative quantitative values for proteins that could discriminate among different brain conditions, as an attempt in the search of neurological biomarkers. Finally, we give some tips on performing the SOMAscan assay and key recommendations on the verification analyses of the resulting candidate biomarkers.

Human cerebrospinal fluid (CSF) is a sample of choice in the study of brain disorders. This biological fluid circulates in the brain and the spinal cord and contains tissue-specific proteins, indicative of health and disease conditions. Despite its potential as a valid source of biological markers, CSF remains largely understudied as compared to blood, in particular due to its more invasive way of sampling.Challenges remain when performing proteomic analysis in clinical research studies. State-of-the-art mass spectrometry (MS) enables deep characterization of the human proteome. But some technical limitations are cardinal to be addressed, such as the capacity to routinely analyze large cohorts of samples. Importantly, a trade-off still needs to be made between the proteome coverage depth and the number of measured samples. In this context, we developed a scalable automated proteomic pipeline for the analysis of CSF. Because of its versatility, this workflow can be adapted to accommodate proteome coverage and/or sample throughput. It allows us to prepare and quantitatively analyze hundreds to thousands of CSF samples; it can also allow identification of more than 3000 proteins in a CSF sample when coupled with isoelectric focusing fractionation.In this chapter, we describe an end-to-end pipeline for the proteomic analysis of CSF. The main steps of the sample preparation comprise spiking of a standard, protein digestion, isobaric labeling, and purification; these are performed in a 96-well plate format enabling automation. Depending on the targeted depth of the CSF proteome, optional analytical steps can be included, such as the removal of abundant proteins and sample pre-fractionation. Liquid chromatography tandem MS as well as data processing and analysis complete the pipeline.

The embryonic cerebrospinal fluid (eCSF) influences neuroepithelial cell behavior, affecting proliferation, differentiation, and survival. One major question to resolve in the field is to precisely describe the eCSF molecules responsible and to understand how these molecules interact in order to exert their functions. Here we describe an in vitro protocol to analyze the influence of eCSF components on neuroepithelium development.

Cerebrospinal fluid (CSF) is a source of valuable information concerning brain disorders. The technical advances of high-throughput omics platforms to analyze body fluids can generate a huge amount of data, whose translation to biological meaning is a challenge. Several bioinformatic tools have emerged to help handling this data into systems biology comprehensively. Herein, we describe a step-by-step tutorial for CSF proteome data analysis in the set of neurodegenerative diseases using (1) ClueGO+CluePedia tool to perform cluster-based analysis envisioning the characterization of the biological processes dysregulated in neurodegenerative diseases including Alzheimer's and Parkinson's diseases; (2) Cytoscape to map disease-specific proteins; (3) SecretomeP to inquire the secretion pathway of CSF proteins; and (4) STRING to identify biological processes modulated by secreted CSF proteins based on protein-protein interaction analysis. This step-by-step guide might help researchers to better characterize disease pathogenesis and to identify putative disease biomarkers.

To study changes in neurological diseases and to identify disease-related mechanisms or biomarkers for diagnosis, cerebrospinal fluid (CSF) is frequently used for proteomic-based discovery. In the last years, development and application of mass spectrometry (MS) techniques have made essential contributions to proteomic studies including protein identification as well as quantification. Until recently, biomarker discovery studies were performed through bottom-up proteomics utilizing data-dependent acquisition. However, drawbacks like stochastic selection of precursor ions cause the exclusion of low-abundant ions from fragmentation as well as from data analysis leading to technical variances among different samples and result in inconsistent data sets. In contrast, data-independent acquisition (DIA) enables almost complete and reproducible quantitative analysis gaining more and more interest as a method for reliable MS-based protein quantification. Besides the utilization of a proper analysis platform, a prerequisite for biomarker studies is the selection of suitable samples and sample processing strategies. Especially for CSF, blood contamination has tremendous impact on the quantitative analysis. In addition, complex processing methods such as protein or peptide fractionation prior to MS analysis can lead to variabilities that affect the reliability of the quantitative results. Here we present methods to evaluate in a first step the CSF quality in regard to blood contamination for the subsequent MS-based sample preparation and finally a DIA method for the analysis of CSF.

Every year, a large number of published studies present biomarkers for various neurological disorders. Many of these studies are based on mass spectrometry proteomics data and describe comparison of the abundance of proteins in cerebrospinal fluid between two or more disease groups. As the number of such studies is growing, it is no longer straightforward to obtain an overview of which specific proteins are increased or decreased between the numerous relevant diseases and their many subcategories, or to see the larger picture or trends between related diseases. To alleviate this situation, we therefore mined the literature for mass spectrometry-based proteomics studies including quantitative protein data from cerebrospinal fluid of patients with multiple sclerosis, Alzheimer's disease, and Parkinson's disease and organized the extracted data in the Cerebrospinal Fluid Proteome Resource (CSF-PR). CSF-PR is freely available online at http://probe.uib.no/csf-pr , is highly interactive, and allows for easy navigation, visualization, and export of the published scientific data. This chapter will guide the user through some of the most important features of the tool and show examples of the suggested use cases.

Cerebrospinal fluid is investigated in biomarker studies for various neurological disorders of the central nervous system due to its proximity to the brain. Currently, only a limited number of biomarkers have been validated in independent studies. The high variability in the protein composition and protein abundance of cerebrospinal fluid between as well as within individuals might be an important reason for this phenomenon. To evaluate this possibility, we investigated the inter- and intraindividual variability in the cerebrospinal fluid proteome globally, with a specific focus on disease biomarkers described in the literature. Cerebrospinal fluid from a longitudinal study group including 12 healthy control subjects was analyzed by label-free quantification (LFQ) via LC-MS/MS. Data were quantified via MaxQuant. Then, the intra- and interindividual variability and the reference change value were calculated for every protein. We identified and quantified 791 proteins, and 216 of these proteins were abundant in all samples and were selected for further analysis. For these proteins, we found an interindividual coefficient of variation of up to 101.5% and an intraindividual coefficient of variation of up to 29.3%. Remarkably, these values were comparably high for both proteins that were published as disease biomarkers and other proteins. Our results support the hypothesis that natural variability greatly impacts cerebrospinal fluid protein biomarkers because high variability can lead to unreliable results. Thus, we suggest controlling the variability of each protein to distinguish between good and bad biomarker candidates, e.g., by utilizing reference change values to improve the process of evaluating potential biomarkers in future studies., Competing Interests: The authors have declared that no competing interests exist.

Objective: Epilepsy is common in individuals with mutations in POLG, the gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma. Early recognition and aggressive seizure management are crucial for patient survival. Disruption of the blood-brain barrier (BBB) is implicated in various neurological disorders including epilepsy. The aim of this study was to assess whether POLG-related disease is associated with BBB dysfunction and what clinical implications this has for patients., Methods: Our retrospective study used data from 83 patients with pathogenic POLG mutations from 4 countries--Norway, Sweden, Finland, and the United Kingdom. Data were collected using a structured questionnaire. We used the presence of raised cerebrospinal fluid (CSF) protein and a raised CSF/serum ratio of albumin (Q-alb) to evaluate the integrity of the blood-CSF barrier., Results: Raised CSF protein was found in 70% of patients (n = 58/83) and appeared to be associated with the most severe phenotypes. In those in whom it was measured, the Q-alb ratio was markedly elevated (n = 18). The majority of those with epilepsy (n = 50/66, 76%) had raised CSF protein, and this preceded seizure debut in 75% (n = 15/20). The median survival time from symptom onset for those with raised CSF protein was decreased (13 months) compared to those with normal CSF protein (32 months)., Significance: Our results indicate that there is disruption of the BBB in POLG-related disease, as evidenced by a raised CSF protein and Q-alb ratio. We also find that raised CSF protein is a common finding in patients with POLG disease. Our data suggest that the presence of BBB dysfunction predicts a poorer outcome, and elevated CSF protein may therefore be an additional biomarker both for early diagnosis and to identify those at high risk of developing epilepsy., (Wiley Periodicals, Inc. © 2018 International League Against Epilepsy.)

In this study, we tried to describe the characteristics of pain and explore the association between the incidence of pain and abnormal laboratory test results in patients during the acute phase of Guillain-Barré syndrome (GBS).This retrospective cohort study enrolled 252 patients with GBS who were in the acute phase of the disease. We collected data regarding the location and type of pain, the onset time, clinical variables and laboratory tests, including the levels of uric acid (UA), albumin, cerebrospinal fluid protein (CSFP), cerebrospinal fluid glucose (CSFG), fasting glucose upon admission, and blood creatinine. The pain descriptors were compared to the severity of disease and laboratory examination results.Around 34.5% of the patients reported pain during the acute phase of GBS. Pain was negatively correlated with the disease severity during the acute phase. In total, 29 of the 87 (33.3%) patients reported pain during the 2 weeks preceding the onset of weakness. The concentration of CSFP was positively associated with the incidence of pain, while the concentrations of UA and albumin were not correlated with the incidence of pain.We found that 33.3% of the GBS patients experienced pain within 2 weeks of onset, and the pain was positively associated with CSFP concentration but was not correlated with disease severity.

Introduction: Hydrocephalus occurs in 85% of patients with tuberculous meningitis (TBM). Ventriculoperitoneal shunt (VPS) insertion is first-line treatment for relieving increased intercranial pressure. VPS obstruction secondary to increased protein levels in cerebrospinal fluid (CSF) is a known complication., Objective: To ascertain if there is a difference in protein levels 1) between cranial and lumbar CSF, and to quantify levels associated with VPS obstruction, and 2) obtained from lumbar puncture vs. ventricular CSF., Method: A 30-year retrospective analysis was undertaken. CSF protein levels were statistically analysed to determine correlation between these levels and VPS obstruction., Results: Of 214 children and 376 adults who underwent VPS insertion for TBM, respectively 27.5% and 25.5% sustained blocked VPS. The mean protein level in CSF collected from the non-obstructed group was 1.76 g/l, compared to 2.94 g/l in the obstructed group. The mean CSF protein level from intraoperative ventricular vs. lumbar puncture samples in the VPS obstruction group was respectively 2.471 g/l and 2.474 g/l., Conclusion: Patients with increased protein levels in CSF are at a high risk of VPS blockage. In these patients, temporary measures should be employed until CSF protein levels decrease.

Aim: To determine the microbiological etiology in critically ill neurosurgical patients with nosocomial meningitis (NM) and to show the impact of Gram-negative rods and the differences between patient characteristics and the clinical and prognostic measures in Gram-negative and Gram-positive meningitis., Material and Methods: In this prospective, single-center study, we reviewed all adult patients hospitalized during a 12-year period and identified pathogens isolated from post-neurosurgical cases of NM. Demographic, clinical, and treatment characteristics were noted from the medical records., Results: Of the 134 bacterial NM patients, 78 were male and 56 were female, with a mean age of 46±15.9 and a median age of 50 (18-80) years. One hundred and forty-one strains were isolated; 82 (58.2%) were Gram-negative, 59 (41.8%) were Grampositive. The most commonly isolated microorganism was Acinetobacter baumannii (34.8%). Comparison of mortality data shows that the patients who have meningitis with Gram-negative pathogens have higher mortality than with Gram-positives (p=0.034). The duration between surgery and meningitis was shorter in Gram-negative meningitis cases compared to others (p=0.045) but the duration between the diagnosis and death was shorter in Gram-positive meningitis cases compared to Gram-negatives (p=0.017). Cerebrospinal fluid protein and lactate levels were higher and glucose level was lower in cases of NM with Gram-negatives (p values were respectively, 0.022, 0.039 and 0.049)., Conclusion: In NM, Gram-negative pathogens were seen more frequently; A. baumanni was the predominant pathogen; and NM caused by Gram-negatives had worse clinical and laboratory characteristic and prognostic outcome than Gram-positives.

Objective: Biomarkers aid diagnosis, allow inexpensive screening of therapies, and guide selection of patient-specific therapeutic regimens in most internal medicine disciplines. In contrast, neurology lacks validated measurements of the physiological status, or dysfunction(s) of cells of the central nervous system (CNS). Accordingly, patients with chronic neurological diseases are often treated with a single disease-modifying therapy without understanding patient-specific drivers of disability. Therefore, using multiple sclerosis (MS) as an example of a complex polygenic neurological disease, we sought to determine whether cerebrospinal fluid (CSF) biomarkers are intraindividually stable, cell type-, disease- and/or process-specific, and responsive to therapeutic intervention., Methods: We used statistical learning in a modeling cohort (n = 225) to develop diagnostic classifiers from DNA-aptamer-based measurements of 1,128 CSF proteins. An independent validation cohort (n = 85) assessed the reliability of derived classifiers. The biological interpretation resulted from in vitro modeling of primary or stem cell-derived human CNS cells and cell lines., Results: The classifier that differentiates MS from CNS diseases that mimic MS clinically, pathophysiologically, and on imaging achieved a validated area under the receiver operating characteristic curve (AUROC) of 0.98, whereas the classifier that differentiates relapsing-remitting from progressive MS achieved a validated AUROC of 0.91. No classifiers could differentiate primary progressive from secondary progressive MS better than random guessing. Treatment-induced changes in biomarkers greatly exceeded intraindividual and technical variabilities of the assay., Interpretation: CNS biological processes reflected by CSF biomarkers are robust, stable, disease specific, or even disease stage specific. This opens opportunities for broad utilization of CSF biomarkers in drug development and precision medicine for CNS disorders. Ann Neurol 2017;82:795-812., (Published 2017. This article is a US Government work and is in the public domain in the USA.)

Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.

We sought to determine the molecular composition of human cerebrospinal fluid (CSF) and identify the biochemical pathways represented in CSF to understand the potential for untargeted screening of inborn errors of metabolism (IEMs). Biochemical profiles for each sample were obtained using an integrated metabolomics workflow comprised of four chromatographic techniques followed by mass spectrometry. Secondarily, we wanted to compare the biochemical profile of CSF with those of plasma and urine within the integrated mass spectrometric-based metabolomic workflow. Three sample types, CSF (N=30), urine (N=40) and EDTA plasma (N=31), were analyzed from retrospectively collected pediatric cohorts of equivalent age and gender characteristics. We identified 435 biochemicals in CSF representing numerous biological and chemical/structural families. Sixty-three percent (273 of 435) of the biochemicals detected in CSF also were detected in urine and plasma, another 32% (140 of 435) were detected in either plasma or urine, and 5% (22 of 435) were detected only in CSF. Analyses of several metabolites showed agreement between clinically useful assays and the metabolomics approach. An additional set of CSF and plasma samples collected from the same patient revealed correlation between several biochemicals detected in paired samples. Finally, analysis of CSF from a pediatric case with dihydropteridine reductase (DHPR) deficiency demonstrated the utility of untargeted global metabolic phenotyping as a broad assessment to screen samples from patients with undifferentiated phenotypes. The results indicate a single CSF sample processed with an integrated metabolomics workflow can be used to identify a large breadth of biochemicals that could be useful for identifying disrupted metabolic patterns associated with IEMs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Cerebrospinal fluid (CSF) protein analysis is an important element in the diagnostic chain for various central nervous system (CNS) pathologies. Among multiple existing approaches to interpreting measured protein levels, the Reiber diagram is particularly robust with respect to physiologic inter-individual variability, as it uses multiple subject-specific anchoring values. Beyond reliable identification of abnormal protein levels, the Reiber diagram has the potential to elucidate their pathophysiologic origin. In particular, both reduction of CSF drainage from the cranio-spinal space as well as blood-CNS barrier dysfunction have been suggested ρas possible causes of increased concentration of blood-derived proteins. However, there is disagreement on which of the two is the true cause., Methods: We designed two computational models to investigate the mechanisms governing protein distribution in the spinal CSF. With a one-dimensional model, we evaluated the distribution of albumin and immunoglobulin G (IgG), accounting for protein transport rates across blood-CNS barriers, CSF dynamics (including both dispersion induced by CSF pulsations and advection by mean CSF flow) and CSF drainage. Dispersion coefficients were determined a priori by computing the axisymmetric three-dimensional CSF dynamics and solute transport in a representative segment of the spinal canal., Results: Our models reproduce the empirically determined hyperbolic relation between albumin and IgG quotients. They indicate that variation in CSF drainage would yield a linear rather than the expected hyperbolic profile. In contrast, modelled barrier dysfunction reproduces the experimentally observed relation., Conclusions: High levels of albumin identified in the Reiber diagram are more likely to originate from a barrier dysfunction than from a reduction in CSF drainage. Our in silico experiments further support the hypothesis of decreasing spinal CSF drainage in rostro-caudal direction and emphasize the physiological importance of pulsation-driven dispersion for the transport of large molecules in the CSF.

Cerebrospinal fluid (CSF) is an excellent source of biological information regarding the nervous system, once it is in close contact and accurately reflects alterations in this system. Several studies have analyzed differential protein profiles of CSF samples between healthy and diseased human subjects. However, the pathophysiological mechanisms and how CSF proteins relate to diseases are still poorly known. By applying bioinformatics tools, we attempted to provide new insights on the biological and functional meaning of proteomics data envisioning the identification of putative disease biomarkers. Bioinformatics analysis of data retrieved from 99 mass spectrometry (MS)-based studies on CSF profiling highlighted 1985 differentially expressed proteins across 49 diseases. A large percentage of the modulated proteins originate from exosome vesicles, and the majority are involved in either neuronal cell growth, development, maturation, migration, or neurotransmitter-mediated cellular communication. Nevertheless, some diseases present a unique CSF proteome profile, which were critically analyzed in the present study. For instance, 48 proteins were found exclusively upregulated in the CSF of patients with Alzheimer's disease and are mainly involved in steroid esterification and protein activation cascade processes. A higher number of exclusively upregulated proteins were found in the CSF of patients with multiple sclerosis (76 proteins) and with bacterial meningitis (70 proteins). Whereas in multiple sclerosis, these proteins are mostly involved in the regulation of RNA metabolism and apoptosis, in bacterial meningitis the exclusively upregulated proteins participate in inflammation and antibacterial humoral response, reflecting disease pathogenesis. The exploration of the contribution of exclusively upregulated proteins to disease pathogenesis will certainly help to envision potential biomarkers in the CSF for the clinical management of nervous system diseases.

Cerebrospinal fluid (CSF) is an important biofluid for diagnosis of and research on neurological diseases. However, in-depth metabolomic profiling of CSF remains an analytical challenge due to the small volume of samples, particularly in small animal models. In this work, we report the application of a high-performance chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) workflow for CSF metabolomics in Gastrodia elata and Uncaria rhynchophylla water extract (GUW)-treated experimental cerebral ischemia model of rat. The GUW is a commonly used Traditional Chinese Medicine (TCM) for hypertension and brain disease. This study investigated the amine- and phenol-containing biomarkers in the CSF metabolome. After GUW treatment for 7 days, the neurological deficit score was significantly improved with infarct volume reduction, while the integrity of brain histological structure was preserved. Over 1957 metabolites were quantified in CSF by dansylation LC-MS. The analysis of this comprehensive list of metabolites suggests that metabolites associated with oxidative stress, inflammatory response, and excitotoxicity change during GUW-induced alleviation of ischemic injury. This work is significant in that (1) it shows CIL LC-MS can be used for in-depth profiling of the CSF metabolome in experimental ischemic stroke and (2) identifies several potential molecular targets (that might mediate the central nervous system) and associate with pharmacodynamic effects of some frequently used TCMs.

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS and has a varying disease course as well as variable response to treatment. Biomarkers may therefore aid personalized treatment. We tested whether in vitro activation of MS patient-derived CD4+ T cells could reveal potential biomarkers. The dynamic gene expression response to activation was dysregulated in patient-derived CD4+ T cells. By integrating our findings with genome-wide association studies, we constructed a highly connected MS gene module, disclosing cell activation and chemotaxis as central components. Changes in several module genes were associated with differences in protein levels, which were measurable in cerebrospinal fluid and were used to classify patients from control individuals. In addition, these measurements could predict disease activity after 2 years and distinguish low and high responders to treatment in two additional, independent cohorts. While further validation is needed in larger cohorts prior to clinical implementation, we have uncovered a set of potentially promising biomarkers., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

The aims of the study were to: (i) identify differentially regulated proteins in cerebrospinal fluid (CSF) between multiple sclerosis (MS) patients and non-MS controls; (ii) examine the effect of matching the CSF samples on either total protein amount or volume, and compare four protein normalization strategies for CSF protein quantification. CSF from MS patients (n = 37) and controls (n = 64), consisting of other noninflammatory neurological diseases (n = 50) and non neurological spinal anesthetic subjects (n = 14), were analyzed using label-free proteomics, quantifying almost 800 proteins. In total, 122 proteins were significantly regulated (p < 0.05), where 77 proteins had p-value <0.01 or AUC value >0.75. Hierarchical clustering indicated that there were two main groups of MS patients, those with increased levels of inflammatory response proteins and decreased levels of proteins involved in neuronal tissue development (n = 30), and those with normal protein levels for both of these protein groups (n = 7). The main subgroup of controls clustering with the MS patients showing increased inflammation and decreased neuronal tissue development were patients suffering from chronic fatigue. Our data indicate that the preferable way to quantify proteins in CSF is to first match the samples on total protein amount and then normalize the data based on the median intensities, preferably from the CNS-enriched proteins., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study describes our efforts to study some of the mechanistic aspects of the earlier established on-surface enzymatic digestion (oSED) method. In a multitude of application areas, it has become important to be able to fully characterize and understand selective protein adsorption to biomaterial surfaces for various applications, including biomedicine (implants), nanotechnology (microchip surfaces and sensors) and materials sciences. Herein, the investigation of the mechanistic aspects was based on microdialysis catheter tubes that were flushed with controlled protein solutions mimicking the extracellular fluid of the brain. The protein adsorption properties were monitored using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) with a targeted method. The temporally resolved results show that most proteins stay adsorbed onto the surface during the entire digestion process and are only cut away piece by piece, whereas smaller proteins and peptides seem to desorb rather easily from the surface. This information will simplify the interpretation of data generated using the oSED method and can also be used for the characterization of the physicochemical properties controlling the adsorption of individual proteins to specific surfaces.

Purpose: To report a rare case of bilateral optic disc edema as presentation of an obstructing spinal plasmacytoma., Methods: Case report., Results: A 41-year-old healthy man presented with distortion of his peripheral vision for 9 months. He denied headaches or neurologic symptoms. Examination showed bilateral optic disc swelling, radial disc hemorrhages, and absent spontaneous venous pulsations. Brain magnetic resonance imaging (MRI) and magnetic resonance venography were unrevealing. Lumbar puncture showed a normal opening pressure of 19 cm cerebrospinal fluid (CSF). CSF protein was significantly elevated at 3.22 g/L (0.10-0.45 g/L). Spinal MRI with contrast revealed a tumor in the T9 vertebral body extending through the disc spaces into T8 and T10, compressing the spinal cord. Computed tomography-guided biopsy confirmed a spinal plasmacytoma. He received radiotherapy to the spine T7-T11 to reduce spinal cord compression followed by a 6-month course of chemotherapy with dexamethasone, cyclophosphamide, and thalidomide. Disc swelling improved on starting radiotherapy with complete resolution 8 months posttreatment. MRI spine showed reduction of tumor., Conclusions: This case highlights the importance of spinal imaging in patients with normal cranial scans and raised CSF protein levels who lack the typical idiopathic intracranial hypertension phenotype.

Diagnosing asymptomatic neurosyphilis (ANS) in HIV-infected patients is difficult. A recent report suggested that CXCL13 is a promising diagnostic marker for neurosyphilis in HIV-positive patients. However, whether CXCL13 can be a diagnostic marker for ANS in HIV-infected patients remains unknown. The purpose of our study was to determine the role of CXCL13 in diagnosing ANS in HIV-infected patients. This study comprised two study and three control groups. Two study groups included 12 HIV-infected patients with ANS and 25 patients with syphilis and HIV co-infection (without ANS). Three control groups included 9 patients with ANS without HIV infection, 25 HIV-infected patients without syphilis and 10 healthy volunteers. Concentrations of CSF CXCL13 were measured before and after neurosyphilis therapy. Our results showed that CSF CXCL13 concentrations were significantly increased in all of the HIV-infected patients with ANS, the 25 HIV patients with syphilis and the 9 ANS patients without HIV, but not in the patients of the other two control groups. CSF CXCL13 concentrations declined in the two study groups of patients following neurosyphilis therapy. Therefore, CSF CXCL13 concentrations could improve the diagnosis of ANS in HIV-infected patients., (© The Author(s) 2015.)

Proteomics offers the simultaneous detection of a large number of proteins in a single experiment and can provide important information regarding crucial aspects of specific proteins, particularly post-translational modifications (PTMs). Investigations of oxidative PTMs are currently performed using focused redox proteomics techniques, which rely on gel electrophoresis separations of intact proteins with the final detection of oxidative PTMs being performed by mass spectrometry (MS) analysis. The application of this technique to human biofluids is being subject of increasing investigation and is expected to provide new insights on the oxidative status of the peripheral proteome in neurological diseases such as Alzheimer's disease, towards purposes of early diagnosis and prognosis. This chapter describes all the experimental steps to perform redox proteomics analysis of cerebrospinal fluid and plasma/serum samples.

Rationale: Intravenous administration of stable isotope labeled amino acid ((13)C6-leucine) to humans recently made it possible to study the metabolism of specific biomarkers in cerebrospinal fluid (CSF) using targeted mass spectrometry (MS). This labeling approach could be of great interest for monitoring many leucine-containing peptides in parallel, using high-resolution MS. This will make it possible to quantify the rates of synthesis and clearance of a large range of proteins in humans with a view to obtaining new insights into protein metabolism processes and the pathophysiology of diseases such as Alzheimer's disease., Methods: Proteins from human lumbar and ventricular CSF samples collected at different times after intravenous (13)C6-leucine infusion were digested enzymatically with LysC/trypsin after being denatured, reduced and alkylated. Desalted tryptic peptides were fractionated using Strong Cation eXchange chromatography (SCX) and analyzed using nanoflow liquid chromatography (nano-LC) coupled to a QTOF Impact II (Bruker Daltonics) mass spectrometer. Data-dependent acquisition (DDA) mode was used to identify and quantify light and heavy (13)C6-leucine peptides. The ratios of (13)C6-leucine incorporation were calculated using the Skyline software program in order to determine the rates of appearance and clearance of proteins in the CSF., Results: After SCX fractionation and quadrupole time-of-flight (QTOF) analysis, 4528 peptides containing leucine were identified in five fractions prepared from 40 μL of CSF. Upon analyzing one of these fractions, 66 peptides (2.7%) corresponding to 61 individual proteins had significant and reproducible rate of (13)C6-leucine incorporation at various time points. The plots of the light-to-heavy peptide ratios showed the existence of proteins with different patterns of appearance and clearance in the CSF., Conclusions: The Stable Isotope Labeling Amino acid in Vivo (SILAV) method presented here, which yields unprecedented information about protein metabolism in humans, constitutes a promising new approach which certainly holds great potential in the field of clinical proteomics., (Copyright © 2015 John Wiley & Sons, Ltd.)