Proteomic studies of cerebrospinal fluid biomarkers of Alzheimer’s disease: an update

Introduction: Alzheimer’s disease (AD) is a neurodegenerative disease affecting the brain. Today there are three cerebrospinal fluid (CSF) biomarkers, amyloid-β consisting of 42 amino acids (Aβ42), total-tau (t-tau) and phosphorylated-tau (p-tau), which combined have sensitivity and specificity figures around 80%. However, pathological studies have shown that comorbidity is a common feature in AD and that the three currently used CSF biomarkers do not optimally reflect the activity of the disease process. Thus, additional markers are needed.

Areas covered: In the present review, we screened PubMed for articles published the last five years (2012–2017) for proteomic studies in CSF with the criteria that AD had to be included as one of the diagnostic groups. Based on inclusion criteria, 28 papers were included reporting in total 224 biomarker-data that were altered in AD compared to control. Both mass spectrometry and multi-panel immunoassays were considered as proteomic studies.

Expert commentary: A large number of pilot studies have been reported but so far there is a lack of replicated findings and to date no CSF biomarker discovered in proteomic studies has reached the clinic to aid in the diagnostic work-up of patients with cognitive impairment.     

Features of ceruloplasmin in the cerebrospinal fluid of Alzheimer’s disease patients

The level of the apo-form of the copper enzyme ceruloplasmin (CP) is an established peripheral marker in diseases associated with copper imbalance. In view of the proposal that disturbances of copper homeostasis may contribute to neurodegeneration associated with Alzheimer’s disease (AD), the present work investigates, by Western blot and non-reducing SDS-PAGE followed by activity staining, the features of CP protein, and the copper/CP relationship in cerebrospinal fluid (CSF) and serum of AD patients. Results show that only a fraction of total copper is associated with CP in the CSF, at variance with serum, both in affected and in healthy individuals. Furthermore, a conspicuous amount of apo-ceruloplasmin and a decrease of CP oxidase activity characterize the CSF of the affected individuals, and confirm that an impairment of copper metabolism occurs in their central nervous system. In the CSF of AD patients the decrease of active CP, associated with the increase in the pool of copper not sequestered by this protein, may play a role in the neurodegenerative process.     

Rare V203I mutation in the PRNP gene of a Chinese patient with Creutzfeldt–Jakob disease

Here, we report a Chinese case of Creutzfeldt–Jakob disease (CJD) with a rare mutation in the prion protein gene (PRNP) leading to an exchange of amino acid from valine (Val) to isoleucine (I) at codon 203 (V203I). The 80-y-old male presented with sudden memory loss, rapid loss of vocabulary, inattention and slow responses, accompanied by dizziness, blurred vision and ataxia. Two weeks after admission, he exhibited tremor, myoclonus and bilateral Babinski signs. At the end of the clinical course, he developed severe akinetic mutism. The cerebrospinal fluid (CSF) was positive for 14-3-3 protein. Increased bilateral signal intensity in the frontal and parietal lobes was seen on diffusion-weighted imaging (DWI); periodic activity was recorded on an electroencephalogram (EEG). There was no family history of similar symptoms. The total clinical course was approximately two months.     

Pathological progression of genetic Creutzfeldt–Jakob disease with a PrP V180I mutation

In comparison to sporadic Creutzfeldt–Jakob disease (sCJD) with MM1-type and MM2- cortical (MM2C)-type, genetic CJD with a prion protein gene V180I mutation (V180I gCJD) is clinically characterized by onset at an older age, slower progress, and the absence of visual disturbances or cerebellar symptoms. In terms of pathological characteristics, gliosis and neuronal loss are generally milder in degree, and characteristic spongiform change can be observed at both the early and advanced stages. However, little is known on the progress of spongiform change over time or its mechanisms. In this study, to elucidate the pathological course of V180I gCJD, statistical analysis of the size and dispersion of the major diameters of vacuoles in six V180I gCJD cases was performed, with five MM1-type sCJD and MM2C-type sCJD cases as controls. As a result, V180I gCJD showed no significant difference in vacuolar diameter regardless of disease duration. In addition, the dispersion of the major diameters of vacuoles in V180I gCJD was larger than that in the MM1-type, which was smaller than that in the MM2C-type. We speculated that the absence of difference in the size of the vacuoles regardless of disease duration suggests that tissue rarefaction does not result from the expansion of vacuole size and increase in number of vacuoles in V180Ig CJD. These features were considered to be significant pathological findings of V180I gCJD.     

Proteomics-derived cerebrospinal fluid markers of autopsy-confirmed Alzheimer’s disease

The diagnostic performance of several candidate cerebrospinal fluid (CSF) protein biomarkers in neuropathologically confirmed Alzheimer’s disease (AD), non-demented (ND) elderly controls and non-AD dementias (NADD) was assessed. Candidate markers were selected on the basis of initial two-dimensional gel electrophoresis studies or by literature review. Markers selected by the former method included apolipoprotein A-1 (ApoA1), haemopexin (HPX), transthyretin (TTR) and pigment epithelium-derived factor (PEDF), while markers identified from the literature included Aβ1-40, Aβ1-42, total tau, phosphorylated tau, α-1 acid glycoprotein (A1GP), haptoglobin, zinc α-2 glycoprotein (Z2GP) and apolipoprotein E (ApoE). Ventricular CSF concentrations of the markers were measured by enzyme-linked immunosorbent assay (ELISA). The concentrations of Aβ1-42, ApoA1, A1GP, ApoE, HPX and Z2GP differed significantly among AD, ND and NADD subjects. Logistic regression analysis for the diagnostic discrimination of AD from ND found that Aβ1-42, ApoA1 and HPX each had significant and independent associations with diagnosis. The CSF concentrations of these three markers distinguished AD from ND subjects with 84% sensitivity and 72% specificity, with 78% of subjects correctly classified. By comparison, using Aβ1-42 alone gave 79% sensitivity and 61% specificity, with 68% of subjects correctly classified. For the diagnostic discrimination of AD from NADD, only the concentration of Aβ1-42 was significantly related to diagnosis, with a sensitivity of 58%, specificity of 86% and 86% correctly classified. The results indicate that for the discrimination of AD from ND control subjects, measurement of a set of markers including Aβ1-42, ApoA1 and HPX improved diagnostic performance over that obtained by measurement of Aβ1-42 alone. For the discrimination of AD from NADD subjects, measurement of Aβ1-42 alone was superior.     

Recent cerebrospinal fluid biomarker studies of Alzheimer’s disease

Alzheimer’s disease is a progressive neurodegenerative disorder and the most common form of dementia. The disease is confirmed by the presence of neuritic plaques and neurofibrillary tangles in the cerebral cortex at autopsy, but the accuracy of antemortem diagnosis, especially at the early stages of the disease, is not ideal. Thus, there is a substantial need for the discovery and validation of diagnostic biomarkers. Many Alzheimer’s disease biomarker discovery studies emphasize the analysis of cerebrospinal fluid (CSF) because of its close association with the brain. Here, we review recent mass spectrometry-based studies of Alzheimer’s disease CSF, and additionally discuss issues associated with CSF in proteomics studies.     

Secretion of N-terminal domain of α-dystroglycan in cerebrospinal fluid

α-Dystroglycan (α-DG) plays crucial roles in maintaining the stability of cells. We demonstrated previously that the N-terminal domain of α-DG (α-DG-N) is secreted by cultured cells into the culture medium. In the present study, to clarify its function in vivo, we generated a monoclonal antibody against α-DG-N and investigated the secretion of α-DG-N in human cerebrospinal fluid (CSF). Interestingly, we found that a considerable amount of α-DG-N was present in CSF. α-DG-N in CSF was a sialylated glycoprotein with both N- and O-linked glycan. These observations suggest that secreted α-DG-N may be transported via CSF and have yet unidentified effects on the nervous system.     

Lymphatic drainage of cerebrospinal fluid in mammals – are arachnoid granulations the main route of cerebrospinal fluid outflow?

The outflow of the cerebrospinal fluid (CSF) in animals was over the years the subject of detailed analysis. For a long time it was stated that arachnoid granulations of the venous sinuses play a key role in CSF circulation. However, recent studies on this subject have shown that a considerable part of the CSF is drained to the lymphatic vessels. Moreover, disorders in the CSF passage may result in severe central nervous system diseases such as e.g. hydrocephalus. In this paper, we summarize the current knowledge concerning the lymphatic drainage of the CSF in mammals. We present in detail comparative anatomy of different species taking into account cranial and spinal compartment. In addition, we clarified role of the lymphatic vessels in the CSF outflow and the relationship between impairment in this transport and central nervous system diseases. In the author’s opinion knowledge on CSF circulation is still poorly examined and therefore required comment.     

Mutant PrP prevails over wild-type PrP in the brain of V210I and R208H genetic Creutzfeldt–Jakob disease patients

Creutzfeldt–Jakob disease (CJD) is a neurodegenerative disorder characterized by the deposition of the pathological conformer (PrP^CJD) of the host encoded cellular prion protein (PrP^C). In genetic CJD associated with V210I or R208H PrP substitutions, the pathogenic role of mutant residues is still poorly understood. To understand how V210I or R208H PrP mutations facilitate the development of the disease, we determined by mass spectrometry the quantitative ratio of mutant/wild-type PrP^CJD allotypes in brains from affected subjects. We found that the mutant PrP^CJD allotypes moderately exceeds of 2- or 3-fold the amount of the wild-type counterpart suggesting that these mutations mainly exert their pathogenic effect on the onset of the pathogenic cascade.     

Proteomic profiling identifies markers for inflammation-related tumor–fibroblast interaction

Background

Cancer associated fibroblasts are activated in the tumor microenvironment and contribute to tumor progression, angiogenesis, extracellular matrix remodeling, and inflammation.

Methods

To identify proteins characteristic for fibroblasts in colorectal cancer we used liquid chromatography-tandem mass spectrometry to derive protein abundance from whole-tissue homogenates of human colorectal cancer/normal mucosa pairs. Alterations of protein levels were determined by two-sided t test with greater than threefold difference and an FDR of < 0.05. Public available datasets were used to predict proteins of stromal origin and link protein with mRNA regulation. Immunohistochemistry confirmed the localization of selected proteins.

Results

We identified a set of 24 proteins associated with inflammation, matrix organization, TGFβ receptor signaling and angiogenesis mainly originating from the stroma. Most prominent were increased abundance of SerpinB5 in the parenchyme and latent transforming growth factor β-binding protein, thrombospondin-B2, and secreted protein acidic-and-cysteine-rich in the stroma. Extracellular matrix remodeling involved collagens type VIII, XII, XIV, and VI as well as lysyl-oxidase-2. In silico analysis of mRNA levels demonstrated altered expression in the tumor and the adjacent normal tissue as compared to mucosa of healthy individuals indicating that inflammatory activation affected the surrounding tissue. Immunohistochemistry of 26 tumor specimen confirmed upregulation of SerpinB5, thrombospondin B2 and secreted protein acidic-and-cysteine-rich.

Conclusions

This study demonstrates the feasibility of detecting tumor- and compartment-specific protein-signatures that are functionally meaningful by proteomic profiling of whole-tissue extracts together with mining of RNA expression datasets. The results provide the basis for further exploration of inflammation-related stromal markers in larger patient cohorts and experimental models.

     

Metabolite profiling of small cerebrospinal fluid sample volumes with gas chromatography–mass spectrometry: application to a rat model of multiple sclerosis

Analysis of metabolites in biofluids by gas chromatography–mass spectrometry (GC–MS) after oximation and silylation is a key method in metabolomics. The GC–MS method was modified by a modified vial design and sample work-up procedure in order to make the method applicable to small volumes of cerebrospinal fluid (CSF), i.e. 10 μL, with similar coverage compared to the standard procedure using ≥100 μL of CSF. The data quality of the modified GC–MS method was assessed by analyzing a study sample set in an animal model for multiple sclerosis, including repetitively analysed quality control rat CSF samples. Automated normalization and intra- and inter-batch correction significantly improved the data quality with the majority of metabolites showing a relative standard deviation <20 %. The modified GC–MS method was successfully applied in rat model of multiple sclerosis where statistical analysis of 93 metabolites, of which 73 were (tentatively) identified, in 10 μL of rat CSF showed statistically significant differences in metabolite profiles of rats at the onset and peak of experimental autoimmune encephalomyelitis compared to rats in the control group. The modified GC–MS method presented proved to be a valid and valuable metabolomics method when only limited sample volumes are available.     

RARB and STMN2 polymorphisms are not associated with sporadic Creutzfeldt–Jakob disease (CJD) in the Korean population

Polymorphisms in the prion protein gene (PRNP) can affect the susceptibility of humans to prion diseases. Recently, aside from PRNP, single nucleotide polymorphisms (SNPs) of two candidate genes for susceptibility to human prion diseases have been identified by human genome-wide association studies (GWAS) in the British population. One SNP of retinoic acid receptor beta (RARB), which is correlated with prion disease incubation time in mice, was associated with human prion diseases such as variant and iatrogenic CJD in the British population. The other SNP of the gene that encodes SCG10 (STMN2), which is related to clinical onset of sporadic CJD, was also associated with variant CJD and kuru. In order to investigate whether two polymorphisms located in upstream of RARB and STMN2 are associated with sporadic CJD in the Korean population, we compared genotype and allele frequencies of these polymorphisms in 217 sporadic CJD patients and 216 healthy Koreans. The genotype distribution and allele frequencies in upstream of the RARB and STMN2 polymorphisms were not significantly different between healthy controls and Korean sporadic CJD patients. This finding indicates that the two SNPs are not correlated with genetic susceptibility to sporadic CJD in the Korean population. This is the first genetic association study of RARB and STMN2 with sporadic CJD in an Asian population.     

Multiple γ-secretase product peptides are coordinately increased in concentration in the cerebrospinal fluid of a subpopulation of sporadic Alzheimer's disease subjects

ABSTRACT: BACKGROUND: Alcadeinα (Alcα) is a neuronal membrane protein that colocalizes with the Alzheimer's amyloid-β precursor protein (APP). Successive cleavage of APP by β- and γ-secretases generates the aggregatable amyloid-β peptide (Aβ), while cleavage of APP or Alcα by α- and γ-secretases generates non-aggregatable p3 or p3-Alcα peptides. Aβ and p3-Alcα can be recovered from human cerebrospinal fluid (CSF). We have previously reported alternative processing of APP and Alcα in the CSF of some patients with sporadic mild cognitive impairment (MCI) and AD (SAD). RESULTS: Using the sandwich enzyme-linked immunosorbent assay (ELISA) system that detects total p3-Alcα, we determined levels of total p3-Alcα in CSF from subjects in one of four diagnostic categories (elderly controls, MCI, SAD, or other neurological disease) derived from three independent cohorts. Levels of Aβ40 correlated with levels of total p3-Alcα in all cohorts. CONCLUSIONS: We confirm that Aβ40 is the most abundant Aβ species, and we propose a model in which CSF p3-Alcα can serve as a either (1) a nonaggregatable surrogate marker for γ-secretase activity; (2) as a marker for clearance of transmembrane domain peptides derived from integral protein catabolism; or (3) both. We propose the specification of an MCI/SAD endophenotype characterized by co-elevation of levels of both CSF p3-Alcα and Aβ40, and we propose that subjects in this category might be especially responsive to therapeutics aimed at modulation of γ-secretase function and/or transmembrane domain peptide clearance. These peptides may also be used to monitor the efficacy of therapeutics that target these steps in Aβ metabolism.     

Early Appearance but Lagged Accumulation of Detergent-Insoluble Prion Protein in the Brains of Mice Inoculated with a Mouse-Adapted Creutzfeldt–Jakob Disease Agent

SUMMARY 1. To elucidate mechanisms for the generation of the detergent-insoluble, proteinase K-resistant prion protein (PrP^Sc) from the detergent-soluble, proteinase K-sensitive PrP (PrP^C) and the replication of the infectious agent in prion diseases, we followed the kinetics of detergent-insoluble PrP and PrP^Sc levels, infectious titers, and associated pathological changes in the brains of mice inoculated with a mouse-adapted Creutzfeldt–Jakob disease agent.2. PrP^Sc in brain homogenate and detergent-insoluble PrP enriched by two-cycle ultracentrifugation were detected by immunoblotting and their relative amounts were estimated according to a standard curve plotted between the amount of PrP and signal intensity on immunoblotting. The titer of infectivity was determined by the incubation periods of mice inoculated with the unfractionated homogenate on the basis of a standard curve plotted between the titer and incubation period.3. Detergent-insoluble PrP became detectable 4 weeks postinoculation (p.i.) well before the detection of PrP^Sc. The low level of detergent-insoluble PrP continued until dramatic accumulation occurred at 14 weeks p.i., correlating well with the accumulation of PrP^Sc and development of pathological changes. The infectious titer was undetectable at 4 weeks p.i. and its logarithmic increase occurred 10 weeks p.i. preceding the logarithmic accumulation of PrPs.4. The lag time of detergent-insoluble PrP accumulation and the discrepancy between infectious titers and PrPs observed during the early period after inoculation suggest a slow and rate-limiting step for the detergent-insoluble PrP to become the infectious agent-associated PrP^Sc.     

The Val-210-Ile pathogenic Creutzfeldt–Jakob disease mutation increases both the helical and aggregation propensities of a sequence corresponding to helix-3 of PrPC

A peptide corresponding to the third helical region within the PrP^C protein, from residues 198 to 218 (helix-3), was synthesised with and without the familial 210-Val to Ile Creutzfeldt–Jakob disease mutation. The NMR structure of PrP^C predicts no global variation in stability for this mutation, indicating that local sequence rather than global structural factors are involved in the pathological effects of this mutation. ¹H NMR analysis of peptides with and without this mutation indicated that it had no significant effect on local helical structure. Temperature denaturation studies monitored by CD showed that the mutation increased the helical content within this region (helical propensity), but did not stabilise the helix toward denaturation (helical stability). Aggregation data indicated that, in addition to increasing helical propensity, this mutation increased the aggregation propensity of this sequence. CD and NMR data indicate that helical interactions, stabilised by the Val-210-Ile mutation, may precede the formation of β-sheet aggregates in this peptide sequence. Therefore, this pathological mutation probably does not facilitate PrP^C to PrP^Sc conversion by directly destabilising the helical structure of PrP^C, but may preferentially stabilise PrP^Sc by facilitating β-sheet formation within this sequence region of PrP. In addition, helical interactions between helix-3 in two or more PrP^C molecules may promote conversion to PrP^Sc.     

SNAP-25 is a promising novel cerebrospinal fluid biomarker for synapse degeneration in Alzheimer’s disease

Background

Synaptic degeneration is an early pathogenic event in Alzheimer’s disease, associated with cognitive impairment and disease progression. Cerebrospinal fluid biomarkers reflecting synaptic integrity would be highly valuable tools to monitor synaptic degeneration directly in patients. We previously showed that synaptic proteins such as synaptotagmin and synaptosomal-associated protein 25 (SNAP-25) could be detected in pooled samples of cerebrospinal fluid, however these assays were not sensitive enough for individual samples.

Results

We report a new strategy to study synaptic pathology by using affinity purification and mass spectrometry to measure the levels of the presynaptic protein SNAP-25 in cerebrospinal fluid. By applying this novel affinity mass spectrometry strategy on three separate cohorts of patients, the value of SNAP-25 as a cerebrospinal fluid biomarker for synaptic integrity in Alzheimer’s disease was assessed for the first time. We found significantly higher levels of cerebrospinal fluid SNAP-25 fragments in Alzheimer’s disease, even in the very early stages, in three separate cohorts. Cerebrospinal fluid SNAP-25 differentiated Alzheimer’s disease from controls with area under the curve of 0.901 (P?<?0.0001).

Conclusions

We developed a sensitive method to analyze SNAP-25 levels in individual CSF samples that to our knowledge was not possible previously. Our results support the notion that synaptic biomarkers may be important tools for early diagnosis, assessment of disease progression, and to monitor drug effects in treatment trials.

     

Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC–MS/MS

Human synovial fluid (SF) provides nutrition and lubrication to the articular cartilage. Particularly in arthritic diseases, SF is extensively accumulating in the synovial junction. During the last decade lipids have attracted considerable attention as their role in the development and resolution of diseases became increasingly recognized. Here, we describe a capillary LC–MS/MS screening platform that was used for the untargeted screening of lipids present in human SF of rheumatoid arthritis (RA) patients. Using this platform we give a detailed overview of the lipids and lipid‐derived mediators present in the SF of RA patients. Almost 70 different lipid components from distinct lipid classes were identified and quantification was achieved for the lysophosphatidylcholine and phosphatidylcholine species. In addition, we describe a targeted LC–MS/MS lipid mediator metabolomics strategy for the detection, identification and quantification of maresin 1, lipoxin A₄ and resolvin D5 in SF from RA patients. Additionally, we present the identification of 5S,12S-diHETE as a major marker of lipoxygenase pathway interactions in the investigated SF samples. These results are the first to provide a comprehensive approach to the identification and profiling of lipids and lipid mediators present in SF and to describe the presence of key anti-inflammatory and pro-resolving lipid mediators identified in SF from RA patients.