Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.     

Proteomics-based technologies in the discovery of biomarkers for multiple sclerosis in the cerebrospinal fluid

Multiple Sclerosis is the most common non-traumatic disorder of the central nervous system and is generally regarded as an immune-mediated disorder that occurs in young adults. Since cerebrospinal fluid is in close contact with the extracellular surface of the brain, it is of great interest to examine possible biomarkers for multiple sclerosis. Proteomic studies of cerebrospinal fluid samples represent an important step towards a better understanding of the disease and may lead to the identification of clinically useful markers. Methodological advances in proteomics allowed the comparison of the protein content in different cerebrospinal fluid samples, using gel or liquid-based approaches coupled with mass spectrometry. In this paper, we discuss the advantages and limitations of the strategies employed and the potential biomarkers for multiple sclerosis identified so far using proteomics-based approaches.     

Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease

So far only the detection of 14-3-3 proteins in cerebrospinal fluid (CSF) is included in the diagnostic criteria for sporadic Creutzfeldt-Jakob disease (sCJD). However, this assay cannot be used for screening because of the high rate of false positive results in sCJD, and often negative results in variant CJD. To facilitate the differential diagnosis of CJD, we applied 2-D differential gel-electrophoresis (2-D DIGE) as a quantitative proteomic screening system for CSF proteins. We compared 36 patients suffering from sCJD with 30 patients suffering from other neurodegenerative diseases. Sample preparation was optimized in consideration of the fact that CSF is composed of blood- and brain-derived proteins, and an improved 2-D DIGE protocol was established. Using this method in combination with protein identification by MALDI-TOF-MS, several known surrogate markers of sCJD like 14-3-3 protein, neuron-specific enolase, and lactate dehydrogenase were readily identified. Moreover, a not yet identified protein with an approximate molecular mass of 85 kDa was found as marker for sCJD with high diagnostic specificity and sensitivity. We conclude that our proteomic approach is useful to differentiate CJD from other neurodegenerative diseases and expect that CSF-optimized 2-D DIGE will find broad application in the search for other brain derived proteins in CSF.     

A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease,Type C1

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.     

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

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

Profiles of Extracellular miRNA in Cerebrospinal Fluid and Serum from Patients with Alzheimer's and Parkinson's Diseases Correlate with Disease Status and Features of Pathology

The discovery and reliable detection of markers for neurodegenerative diseases have been complicated by the inaccessibility of the diseased tissue- such as the inability to biopsy or test tissue from the central nervous system directly. RNAs originating from hard to access tissues, such as neurons within the brain and spinal cord, have the potential to get to the periphery where they can be detected non-invasively. The formation and extracellular release of microvesicles and RNA binding proteins have been found to carry RNA from cells of the central nervous system to the periphery and protect the RNA from degradation. Extracellular miRNAs detectable in peripheral circulation can provide information about cellular changes associated with human health and disease. In order to associate miRNA signals present in cell-free peripheral biofluids with neurodegenerative disease status of patients with Alzheimer''s and Parkinson''s diseases, we assessed the miRNA content in cerebrospinal fluid and serum from postmortem subjects with full neuropathology evaluations. We profiled the miRNA content from 69 patients with Alzheimer''s disease, 67 with Parkinson''s disease and 78 neurologically normal controls using next generation small RNA sequencing (NGS). We report the average abundance of each detected miRNA in cerebrospinal fluid and in serum and describe 13 novel miRNAs that were identified. We correlated changes in miRNA expression with aspects of disease severity such as Braak stage, dementia status, plaque and tangle densities, and the presence and severity of Lewy body pathology. Many of the differentially expressed miRNAs detected in peripheral cell-free cerebrospinal fluid and serum were previously reported in the literature to be deregulated in brain tissue from patients with neurodegenerative disease. These data indicate that extracellular miRNAs detectable in the cerebrospinal fluid and serum are reflective of cell-based changes in pathology and can be used to assess disease progression and therapeutic efficacy.     

Proteomic analysis of cerebrospinal fluid from multiple sclerosis patients

Multiple sclerosis is an autoimmune inflammatory demyelinating disease of the central nervous system. Disease mechanisms in multiple sclerosis at the molecular level remain poorly understood and no reliable proteinaceous disease markers are available yet. The goal of the present study is the construction of a protein database of two-dimensional gel electrophoresis (2-DE) separated cerebrospinal fluid (CSF) proteins from multiple sclerosis patients. By means of liquid chromatography tandem mass spectrometry 65 different proteins were identified from 300 spots. Eighteen of these proteins have not been reported previously on 2-DE gels of CSF. Here we report on the identification of these proteins and discuss their potential relation to multiple sclerosis.     

Proteomics identification of proteins in human cortex using multidimensional separations and MALDI tandem mass spectrometer

It is essential to characterize the proteome of various regions of human brain because most, if not all, neurodegenerative diseases are region-specific. Here we report an in-depth proteomics identification of proteins extracted from the frontal cortex, a region playing a critical role in cognitive function. The integrated proteomics analytical flow consisted of biochemical fractionation, strong cation exchange chromatography, reverse phase liquid chromatography, and MALDI-TOF/TOF mass spectrometric analysis. In total, 812 proteins were confidently identified with two or more peptides. These proteins demonstrated diverse isoelectric points and molecular weights and are involved in several molecular functions, including protein binding, catalytic activity, transport, structure, and signal transduction. A number of proteins known to be associated with neurodegenerative diseases were also identified. Detailed characterization of these proteins will supply the necessary information to appropriately interpret proteins associated with aging and/or age-related neurodegenerative diseases. Finally 140 proteins found in the cortical proteome were present in the proteome of cerebrospinal fluid, providing tissue-specific candidates for biomarker discovery in body fluid.     

Alterations in protein regulators of neurodevelopment in the cerebrospinal fluid of infants with posthemorrhagic hydrocephalus of prematurity

Neurological outcomes of preterm infants with posthemorrhagic hydrocephalus are among the worst in newborn medicine. There remains no consensus regarding the diagnosis or treatment of posthemorrhagic hydrocephalus, and the pathological pathways leading to the adverse neurological sequelae are poorly understood. In the current study, we developed an innovative approach to simultaneously identify potential diagnostic markers of posthemorrhagic hydrocephalus and investigate novel pathways of posthemorrhagic hydrocephalus-related neurological disability. Tandem multi-affinity fractionation for specific removal of plasma proteins from the hemorrhagic cerebrospinal fluid samples was combined with high resolution label-free quantitative proteomics. Analysis of cerebrospinal fluid obtained from infants with posthemorrhagic hydrocephalus demonstrated marked differences in the levels of 438 proteins when compared with cerebrospinal fluid from age-matched control infants. Amyloid precursor protein, neural cell adhesion molecule-L1, neural cell adhesion molecule-1, brevican and other proteins with important roles in neurodevelopment showed profound elevations in posthemorrhagic hydrocephalus cerebrospinal fluid compared with control. Initiation of neurosurgical treatment of posthemorrhagic hydrocephalus resulted in resolution of these elevations. The results from this foundational study demonstrate the significant promise of tandem multi-affinity fractionation-proteomics in the identification and quantitation of protein mediators of neurodevelopment and neurological injury. More specifically, our results suggest that cerebrospinal fluid levels of proteins such as amyloid precursor protein or neural cell adhesion molecule-L1 should be investigated as potential diagnostic markers of posthemorrhagic hydrocephalus. Notably, dysregulation of the levels these and other proteins may directly affect ongoing neurodevelopmental processes in these preterm infants, providing an entirely new hypothesis for the developmental disability associated with posthemorrhagic hydrocephalus.     

Two dimensional difference gel electrophoresis analysis of cerebrospinal fluid in tuberculous meningitis patients

Tuberculous meningitis (TBM) is a serious complication of tuberculosis that affects the central nervous system. Present methods to diagnose TBM are not suitable for early diagnosis. Molecular markers and sensitive methods to identify them in the early stage of infection of TBM are critically needed for efficient management. We have done the proteomic analysis of TBM cerebrospinal fluid (n=20) with 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 11 human proteins and 8 mycobacterial proteins with changed expression levels in comparison to controls. Arachidonate 5-lipoxygenase and glial fibrillary acidic protein, two of the identified proteins, were validated with western blot technique on a larger set of disease and control samples (n=40). These two proteins were also analyzed in fungal meningitis samples. We suggest that arachidonate 5-lipoxygenase can be considered for validation as a potential marker for diagnosis of TBM.     

Proteomic identification of biomarkers in the cerebrospinal fluid (CSF) of astrocytoma patients

The monitoring of changes in the protein composition of the cerebrospinal fluid (CSF) can be used as a sensitive indicator of central nervous system (CNS) pathology, yet its systematic application to analysis of CNS neoplasia has been limited. There is a pressing need for both a better understanding of gliomagenesis and the development of reliable biomarkers of the disease. In this report, we used two proteomic techniques, two-dimensional gel electrophoresis (2-DE), and cleavable Isotope-Coded Affinity Tag (cICAT) to compare CSF proteomes to identify tumor- and grade-specific biomarkers in patients bearing brain tumors of differing histologies and grades. Retrospective analyses were performed on 60 samples derived from astrocytomas WHO grade II, III, and IV, schwannomas, metastastic brain tumors, inflammatory samples, and non-neoplastic controls. We identified 103 potential tumor-specific markers of which 20 were high-grade astrocytoma-specific. These investigations allowed us to identify a spectrum of signature proteins that could be used to distinguish CSF derived from control patients versus those with low- (AII) or high-grade (AIV) astrocytoma. These proteins may represent new diagnostic, prognostic, and disease follow-up markers when used alone or in combination. These candidate biomarkers may also have functional properties that play a critical role in the development and malignant progression of human astrocytomas, thus possibly representing novel therapeutic targets for this highly lethal disease.     

The proteome of human brain microdialysate

BACKGROUND: Cerebral microdialysis has been established as a monitoring tool in neurocritically ill patients suffering from severe stroke. The technique allows to sample small molecules in the brain tissue for subsequent biochemical analysis. In this study, we investigated the proteomic profile of human cerebral microdialysate and if the identified proteins might be useful predictors for disease characteristics in stroke for tissue at risk in the contralateral hemisphere. We analysed cerebral protein expression in microdialysate from three stroke patients sampled from the hemisphere contralateral to the lesion. Using a proteomic approach based on two-dimensional gel electrophoresis and subsequent mass spectrometry, we created a protein map for the global protein expression pattern of human microdialyste. RESULTS: We found an average of 158 +/- 24 (N = 18) protein spots in the human cerebral microdialysate and could identify 95 spots, representing 27 individual proteins. Most of these have been detected in human cerebrospinal fluid before, but 10 additional proteins mainly of cerebral intracellular origin were identified exclusively in the microdialysate. CONCLUSIONS: The 10 proteins found exclusively in human cerebral microdialysate, but not in cerebrospinal fluid, indicate the possibility to monitor the progression of the disease towards deterioration. The correlation of protein composition in the human cerebral microdialysate with the patients' clinical condition and results of cerebral imaging may be a useful approach to future applications for neurological stroke diagnosis, prognosis, and treatment.     

Identification of protein bands in polyacrylamide gel by protein printing

Previously described methods for identification of proteins separated in cylindrical polyacrylamide gels have been found to be costly in time and antiserum and difficult to apply to small amounts of protein as are found in cerebrospinal fluid. We describe a method which involves printing of the proteins on the cut surface of the gel onto nitrocellulose paper. The protein bands of the imprint can then be identified using labelled antibodies. We have found this to be economical and quick, and it has permitted sensitive and reliable identification of proteins in unconcentrated cerebrospinal fluid and aqueous humour.     

Development of electrochemiluminescence-based singleplex and multiplex assays for the quantification of α-synuclein and other proteins in cerebrospinal fluid

The need for improved diagnostic accuracy and markers of progression in neurodegenerative diseases motivates the identification of objective biomarkers as well as optimized assays for their quantification. Several potential marker candidates for Parkinson's disease (PD) in cerebrospinal fluid have been identified. These include α-synuclein, a major constituent of the intracellular aggregates. We give a general overview and details of our experience in converting established enzyme-linked immunoabsorbent assays (for α-synuclein and other proteins) onto an electrochemiluminescence-based platform as well as considerations on multiplexing different assays for PD.     

Towards a high resolution separation of human cerebrospinal fluid

Human cerebrospinal fluid is an ultrafiltrate of plasma that is largely produced by the choroid plexus. It consists of a mixture of anorganic salts, various sugars, lipids and proteins from the surrounding brain tissues. The predominant proteins in cerebrospinal fluid are isoforms of serum albumin, transferrin and immunoglobulins, representing more than 70% of the total protein amount. A rough overview of the protein compounds of human cerebrospinal fluid including their respective concentrations is given by Blennow et al. [Eur. Neurol. 33 (1993) 129]. In contrast, the aim of this work is to display the detailed protein composition of CSF by two-dimensional gel electrophoresis and to identify both high and low concentrated proteins using different mass spectrometry techniques. This extensive overview of proteins in human cerebrospinal fluid will be highly relevant for clinical research. Furthermore, the comparison of 2D gels will help to analyze the standard protein variability in CSF of healthy persons and detect specific protein variations of patients with various neurological diseases (e.g., Alzheimer's disease, Huntington's chorea). Sample preparation for two-dimensional gel electrophoresis must include concentration and desalting steps such as precipitation and ultrafiltration due to the high amount of salts, sugars and lipids and the low total amount of protein of 0.3-0.7 microg/microl present in human CSF. Up to now we were able to identify more than 480 spots from suchlike generated 2D gels using MALDI- and ESI-mass spectrometry.     

Modeling of pathological traits in Alzheimer's disease based on systemic extracellular signaling proteome

The study of chronic brain diseases including Alzheimer's disease in patients is typically limited to brain imaging or psychometric testing. Given the epidemic rise and insufficient knowledge about pathological pathways in sporadic Alzheimer's disease, new tools are required to identify the molecular changes underlying this disease. We hypothesize that levels of specific secreted cellular signaling proteins in cerebrospinal fluid or plasma correlate with pathological changes in the Alzheimer's disease brain and can thus be used to discover signaling pathways altered in the disease. Here we measured 91 proteins of this subset of the cellular communication proteome in plasma or cerebrospinal fluid in patients with Alzheimer's disease and cognitively normal controls to mathematically model disease-specific molecular traits. We found small numbers of signaling proteins that were able to model key pathological markers of Alzheimer's disease, including levels of cerebrospinal fluid β-amyloid and tau, and classify disease in independent samples. Several of these factors had previously been implicated in Alzheimer's disease supporting the validity of our approach. Our study also points to proteins which were previously unknown to be associated with Alzheimer's disease thereby implicating novel signaling pathways in this disorder.     

Phosphotyrosine profiling of human cerebrospinal fluid

Background

Cerebrospinal fluid (CSF) is an important source of potential biomarkers that affect the brain. Biomarkers for neurodegenerative disorders are needed to assist in diagnosis, monitoring disease progression and evaluating efficacy of therapies. Recent studies have demonstrated the involvement of tyrosine kinases in neuronal cell death. Thus, neurodegeneration in the brain is related to altered tyrosine phosphorylation of proteins in the brain and identification of abnormally phosphorylated tyrosine peptides in CSF has the potential to ascertain candidate biomarkers for neurodegenerative disorders.

Methods

In this study, we used an antibody-based tyrosine phosphopeptide enrichment method coupled with high resolution Orbitrap Fusion Tribrid Lumos Fourier transform mass spectrometer to catalog tyrosine phosphorylated peptides from cerebrospinal fluid. The subset of identified tyrosine phosphorylated peptides was also validated using parallel reaction monitoring (PRM)-based targeted approach.

Results

To date, there are no published studies on global profiling of phosphotyrosine modifications of CSF proteins. We carried out phosphotyrosine profiling of CSF using an anti-phosphotyrosine antibody-based enrichment and analysis using high resolution Orbitrap Fusion Lumos mass spectrometer. We identified 111 phosphotyrosine peptides mapping to 66 proteins, which included 24 proteins which have not been identified in CSF previously. We then validated a set of 5 tyrosine phosphorylated peptides in an independent set of CSF samples from cognitively normal subjects, using a PRM-based targeted approach.

Conclusions

The findings from this deep phosphotyrosine profiling of CSF samples have the potential to identify novel disease-related phosphotyrosine-containing peptides in CSF.

     

Proteomic analysis of astrocytic secretion in the mouse. Comparison with the cerebrospinal fluid proteome

Astrocytes, the most abundant cell type in the central nervous system, are intimately associated with synapses. They play a pivotal role in neuronal survival and the brain inflammatory response. Some astrocytic functions are mediated by the secretion of polypeptides. Using a proteomic approach, we have identified more than 30 proteins released by cultured astrocytes. These include proteases and protease inhibitors, carrier proteins, and antioxidant proteins. Exposing astrocytes to brefeldin A, which selectively blocks secretory vesicle assembly, suppressed the release of some of these proteins. This indicates that astrocytes secrete these proteins by a classic vesicular mechanism and others by an alternative pathway. Astrocytes isolated from different brain regions secreted a similar pattern of proteins. However, the secretion of some of them, including metalloproteinase inhibitors and apolipoprotein E, was region-specific. In addition, pro-inflammatory treatments modified the profile of astrocytic protein secretion. Finally, more than two thirds of the proteins identified in the astrocyte-conditioned medium were detectable in the mouse cerebrospinal fluid, suggesting that astrocytes contribute to the cerebrospinal fluid protein content. In conclusion, this study provides the first unbiased characterization of the major proteins released by astrocytes, which may play a crucial role in the modulation of neuronal survival and function.     

Proteome analysis of chick embryonic cerebrospinal fluid

During early stages of embryo development, the brain cavity is filled with embryonic cerebrospinal fluid (E-CSF), a complex fluid containing different protein fractions that contributes to the regulation of the survival, proliferation and neurogenesis of the neuroectodermal stem cells. Using 2-DE, protein sequencing and database searches, we identified and analyzed the proteome of the E-CSF from chick embryos (Gallus gallus). We identified 26 different gene products, including proteins related to the extracellular matrix, proteins associated with the regulation of osmotic pressure and metal transport, proteins related to cell survival, MAP kinase activators, proteins involved in the transport of retinol and vitamin D, antioxidant and antimicrobial proteins, intracellular proteins and some unknown proteins. Most of these gene products are involved in the regulation of developmental processes during embryogenesis in systems other than E-CSF. Interestingly, 14 of them are also present in adult human CSF proteome, and it has been reported that they are altered in the CSF of patients suffering neurodegenerative diseases and/or neurological disorders. Understanding these molecules and the mechanisms they control during embryonic neurogenesis is a key contribution to the general understanding of CNS development, and may also contribute to greater knowledge of these human diseases.