Justyna M.C. Bahl

Characterization of the Human Cerebrospinal Fluid Phosphoproteome by Titanium Dioxide Affinity Chromatography and Mass Spectrometry

Biomarkers in the cerebrospinal fluid (CSF) may be important for the diagnosis of chronic degenerative disorders in the central nervous system including dementia. Existing CSF biomarkers for dementia, however, are relatively nonspecific. More specific markers may be found by targeting investigations based on knowledge of the molecular pathology of the disease in question. In Alzheimers disease, hyperphosphorylation of the tau protein is a characteristic feature and thus a comprehensive characterization of the phosphoproteome of the CSF may be pursued to obtain a complete picture of phosphorylation aberrations in health and disease. Toward that goal we here describe a method for a comprehensive isolation and identification of phosphorylated tryptic peptides derived from CSF proteins using a simple sample preparation step and titanium dioxide-affinity chromatography followed by MALDI-TOF or LC-MS/MS linear ion-trap-FT mass spectrometry. Whereas not all previously reported phosphoproteins were found in normal CSF, we detected 56 putative novel phosphorylation sites in 38 proteins in addition to known sites. The approach seems to be a promising foundation for the discovery of new biomarkers embedded in the CSF phosphoproteome.

The diagnostic efficiency of biomarkers in sporadic Creutzfeldt-Jakob disease compared to Alzheimer's disease.

Laboratory markers have a prominent place among the diagnostic criteria for sporadic Creutzfeldt-Jakob disease (sCJD). Here we investigate the capability of protein 14-3-3, total-tau (t-tau), threonin-181-phosphorylated tau (p-tau), and neuron-specific enolase (NSE) in cerebrospinal fluid (CSF) together with the prion protein gene genotype to discriminate patients with sCJD (n=21) from neurological controls (n=164) and Alzheimers disease (AD) patients (n=49). Low p-tau/t-tau ratio was the best single marker for sCJD with 90% specificity against neurological controls at 86% sensitivity whilst NSE was the least accurate with 79% sensitivity at 90% specificity. Many of the sCJD patients had extremely elevated t-tau values but normal values of the AD-marker p-tau. Protein 14-3-3 was very sensitive (95%) although the specificity was relatively low (75%). A combination of elevated t-tau concentration with the presence of 14-3-3 protein in CSF gave the best test specificity of 96% at 84% sensitivity. We conclude that the combination of more than one CSF marker for neurodegeneration can improve the diagnostic test accuracy for sCJD against neurological controls including patients with other dementias.

Chronic administration of the selective P2X3, P2X2/3 receptor antagonist, A-317491, transiently attenuates cancer-induced bone pain in mice

The purinergic P2X3 and P2X2/3 receptors are in the peripheral nervous system almost exclusively confined to afferent sensory neurons, where they are found both at peripheral and central synapses. The P2X3 receptor is implicated in both neuropathic and inflammatory pain. However, the role of the P2X3 receptor in chronic cancer-induced bone pain is less known. Here we investigated the effect of systemic acute and chronic administration of the selective P2X3, P2X2/3 receptor antagonist (5-[[[(3-Phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl]-1,2,4-benzenetricarboxylic acid sodium salt hydrate) (A-317491) in a murine model of cancer-induced bone pain. Chronic administration of A-317491 (30 μmol/kgs.c., b.i.d.) resulted in a transient attenuation of pain related behaviours in the early stage of the bone cancer model, but had no effect in the late and more progressed stage of bone cancer. Also, acute administration of A-317491 (100 μmol/kgs.c.) had no effect in the progressed stage of the bone cancer pain model. Thus, systemically administered A-317491 did not demonstrate a robust effect in the present mouse model of cancer-induced bone pain.

Pre-analytical factors influencing the stability of cerebrospinal fluid proteins

Cerebrospinal fluid (CSF) is a potential source for new biomarkers due to its proximity to the brain. This study aimed to clarify the stability of the CSF proteome when undergoing pre-analytical factors. We investigated the effects of repeated freeze/thaw cycles, protease inhibitors and delayed storage for 4h, 24h or 14 days at -20°C, 4°C and room temperature (RT) after centrifugation compared with our standard practice of two hours at RT before placing the samples in an -80°C environment. The results were obtained using immunoassays for amyloid-beta 1-42 (Aβ42), tau, phosphorylated tau (P-tau) and cystatin C and using surface-enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) mass spectrometry for proteomic profiling. Tau and P-tau were susceptible to repeated freeze/thaw cycles while SELDI-TOF analysis produced eight significant peaks and additional artefact peaks from samples with added protease inhibitors. Delayed storage for different durations and in different temperatures produced six significant SELDI-TOF peaks. Aβ42 and tau were susceptible to increased temperatures and the duration before storage, whereas P-tau and cystatin C were not. Transthyretin and several of its isoforms were found using SELDI-TOF and were susceptible to freeze/thaw cycles and to increased temperature and length of time prior to storage. We recommend that CSF should be collected and centrifuged immediately after sampling and prior to storage at -80°C without the addition of protease inhibitors. Freeze/thawing should be avoided because of the instability of tau, P-tau and transthyretin. Standardised CSF sampling, handling and storage for biomarker research are essential for accurately comparing the results obtained by different studies and institutions.

Use of amyloid-PET to determine cutpoints for CSF markers: A multicenter study.

Objectives: To define CSF β-amyloid 1–42 (Aβ42) cutpoints to detect cortical amyloid deposition as assessed by 11C-Pittsburgh compound B ([11C]PiB)-PET and to compare these calculated cutpoints with cutpoints currently used in clinical practice. Methods: We included 433 participants (57 controls, 99 with mild cognitive impairment, 195 with Alzheimer disease [AD] dementia, and 82 with non-AD dementia) from 5 European centers. We calculated for each center and for the pooled cohort CSF Aβ42 and Aβ42/tau ratio cutpoints for cortical amyloid deposition based on visual interpretation of [11C]PiB-PET images. Results: Amyloid-PET–based calculated CSF Aβ42 cutpoints ranged from 521 to 616 pg/mL, whereas existing clinical-based cutpoints ranged from 400 to 550 pg/mL. Using the calculated cutpoint from the pooled sample (557 pg/mL), concordance between CSF Aβ42 and amyloid-PET was 84%. Similar concordance was found when using a dichotomized Aβ42/tau ratio. Exploratory analysis showed that participants with a positive amyloid-PET and normal CSF Aβ42 levels had higher CSF tau and phosphorylated tau levels and more often had mild cognitive impairment or AD dementia compared with participants who had negative amyloid-PET and abnormal CSF Aβ42 levels. Conclusions: Amyloid-PET–based CSF Aβ42 cutpoints were higher and tended to reduce intercenter variability compared with clinical-based cutpoints. Discordant participants with normal CSF Aβ42 and a positive amyloid-PET may be more likely to have AD-related amyloid pathology than participants with abnormal CSF Aβ42 and a negative amyloid-PET. Classification of evidence: This study provides Class II evidence that an amyloid-PET–based CSF Aβ42 cutpoint identifies individuals with amyloid deposition with a sensitivity of 87% and specificity of 80%.

Characterization and stability of transthyretin isoforms in cerebrospinal fluid examined by immunoprecipitation and high-resolution mass spectrometry of intact protein.

Post-translational modifications (PTMs) contribute significantly to the complexity of proteins. PTMs may vary in certain patterns according to diseases and microenviroments making them potential markers for pathological processes. Human transthyretin (TTR) is a transporter of thyroxine and retinol in blood and cerebrospinal fluid (CSF). A single free cysteine thiol group in TTR possesses the ability to form mixed disulfides potentially related to diseases such as TTR amyloidosis and Alzheimers disease (AD). Additionally, TTR-Cys10 S-thiolations might mirror the oxidative stress and redox balance of CSF. Here we describe a quick and gentle method for immunoprecipitating (IP) TTR from CSF with minimal introduction of sample-handling artifacts. A high-resolution mass spectrometer (LTQ-Orbitrap XL) was used in a simple setup with direct infusion that generates data suitable for confident assignment of TTR isoforms and validation of the protocol. Moreover, we demonstrate how simple storage of CSF at 4°C induces major oxidative modifications of TTR. Using the optimized method, we show data from a limited number of mild cognitive impairment (MCI) and AD patients. The protocol controls and minimizes the introduction of sample-handling artifacts during purification of TTR isoforms for high-resolution MS analysis.

Amyloid-β and α-Synuclein Decrease the Level of Metal-Catalyzed Reactive Oxygen Species by Radical Scavenging and Redox Silencing

The formation of reactive oxygen species (ROS) is linked to the pathogenesis of neurodegenerative diseases. Here we have investigated the effect of soluble and aggregated amyloid-β (Aβ) and α-synuclein (αS), associated with Alzheimer’s and Parkinson’s diseases, respectively, on the Cu2+-catalyzed formation of ROS in vitro in the presence of a biological reductant. We find that the levels of ROS, and the rate by which ROS is generated, are significantly reduced when Cu2+ is bound to Aβ or αS, particularly when they are in their oligomeric or fibrillar forms. This effect is attributed to a combination of radical scavenging and redox silencing mechanisms. Our findings suggest that the increase in ROS associated with the accumulation of aggregated Aβ or αS does not result from a particularly ROS-active form of these peptides, but rather from either a local increase of Cu2+ and other ROS-active metal ions in the aggregates or as a downstream consequence of the formation of the pathological amyloid structures.

Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study

Decreased levels of alpha-synuclein (aSyn) in cerebrospinal fluid (CSF) in Parkinsons disease and related synucleinopathies have been reported, however, not consistently in all cross-sectional studies. To test the performance of one recently released human-specific enzyme-linked immunosorbent assay (ELISA) for the quantification of aSyn in CSF, we carried out a round robin trial with 18 participating laboratories trained in CSF ELISA analyses within the BIOMARKAPD project in the EU Joint Program - Neurodegenerative Disease Research. CSF samples (homogeneous aliquots from pools) and ELISA kits (one lot) were provided centrally and data reported back to one laboratory for data analysis. Our study showed that although factors such as preanalytical sample handling and lot-to-lot variability were minimized by our study design, we identified high variation in absolute values of CSF aSyn even when the same samples and same lots of assays were applied. We further demonstrate that although absolute concentrations differ between laboratories the quantitative results are comparable. With further standardization this assay may become an attractive tool for comparing aSyn measurements in diverse settings. Recommendations for further validation experiments and improvement of the interlaboratory results obtained are given.

Altered α‐synuclein, parkin, and synphilin isoform levels in multiple system atrophy brains

Together with Parkinsons disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α‐synucleinopathies. Previously, it has been shown that α‐synuclein, parkin, and synphilin‐1 display disease‐specific transcription patterns in frontal cortex in PD, dementia with Lewy bodies, and MSA, and thus may mediate the development of α‐synucleinopathies. In this study, the differential expression of α‐synuclein isoforms on transcriptional and translational levels was ascertained in MSA patients in comparison with PD cases and normal controls using isoform‐specific primers and exon‐specific antibodies in substantia nigra, striatum, cerebellar cortex, and nucleus dentatus. These regions are severely affected by α‐synuclein pathology and neurodegeneration. Furthermore, we have also investigated transcript levels for parkin and synphilin‐1 isoforms. In MSA brains, α‐synuclein140 and α‐synuclein 112 isoform levels were significantly increased, whereas levels of the α‐synuclein 126 isoform were decreased in the substantia nigra, striatum, cerebellar cortex, and nucleus dentatus versus controls. Moreover, in MSA cases, we showed increased levels of parkin isoforms lacking the N‐terminal ubiquitin‐like domain and an aggregation‐prone synphilin‐1A isoform that causes neuronal toxicity in MSA. In PD brains, parkin transcript variant 3, 7, and 11 were significantly and specifically over‐expressed in the striatum and cerebellar cortex, together with synphilin‐1A and 1C. The changes of isoform expression profiles in neurodegenerative diseases suggest alterations in the regulation of transcription and/or splicing events, leading to regional/cellular events that may be important for the highly increased aggregation of α‐synuclein in the brain.

Comparison of Different Matrices as Potential Quality Control Samples for Neurochemical Dementia Diagnostics

BACKGROUND Assay-vendor independent quality control (QC) samples for neurochemical dementia diagnostics (NDD) biomarkers are so far commercially unavailable. This requires that NDD laboratories prepare their own QC samples, for example by pooling leftover cerebrospinal fluid (CSF) samples. OBJECTIVE To prepare and test alternative matrices for QC samples that could facilitate intra- and inter-laboratory QC of the NDD biomarkers. METHODS Three matrices were validated in this study: (A) human pooled CSF, (B) Aβ peptides spiked into human prediluted plasma, and (C) Aβ peptides spiked into solution of bovine serum albumin in phosphate-buffered saline. All matrices were tested also after supplementation with an antibacterial agent (sodium azide). We analyzed short- and long-term stability of the biomarkers with ELISA and chemiluminescence (Fujirebio Europe, MSD, IBL International), and performed an inter-laboratory variability study. RESULTS NDD biomarkers turned out to be stable in almost all samples stored at the tested conditions for up to 14 days as well as in samples stored deep-frozen (at - 80°C) for up to one year. Sodium azide did not influence biomarker stability. Inter-center variability of the samples sent at room temperature (pooled CSF, freeze-dried CSF, and four artificial matrices) was comparable to the results obtained on deep-frozen samples in other large-scale projects. CONCLUSION Our results suggest that it is possible to replace self-made, CSF-based QC samples with large-scale volumes of QC materials prepared with artificial peptides and matrices. This would greatly facilitate intra- and inter-laboratory QC schedules for NDD measurements.