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Background Increasing evidence implicates dysfunctional proteostasis and the involvement of the autophagic and endo-lysosomal system and the ubiquitin-proteasome system in neurodegenerative diseases. In Alzheimer’s disease (AD), there is an accumulation of autophagic vacuoles within the neurons. In Parkinson’s disease (PD), susceptibility has been linked to genes encoding proteins involved in autophagy and lysosomal function, as well as mutations causing lysosomal disorders. Furthermore, both diseases are characterized by the accumulation of protein aggregates. Methods Proteins associated with endocytosis, lysosomal function, and the ubiquitin-proteasome system were identified in the cerebrospinal fluid (CSF) and targeted by combining solid-phase extraction and parallel reaction monitoring mass spectrometry. In total, 50 peptides from 18 proteins were quantified in three cross-sectional cohorts including AD (N = 61), PD (N = 21), prodromal AD (N = 10), stable mild cognitive impairment (N = 15), and controls (N = 68). Results A pilot study, including subjects selected based on their AD CSF core biomarker concentrations, showed increased concentrations of several targeted proteins in subjects with core biomarker levels indicating AD pathology compared to controls. Next, in a clinically characterized cohort, lower concentrations in CSF of proteins in PD were found compared to subjects with prodromal AD. Further investigation in an additional clinical study again revealed lower concentrations in CSF of proteins in PD compared to controls and AD. Conclusion In summary, significantly different peptide CSF concentrations were identified from proteins AP2B1, C9, CTSB, CTSF, GM2A, LAMP1, LAMP2, TCN2, and ubiquitin. Proteins found to have altered concentrations in more than one study were AP2B1, CTSB, CTSF, GM2A, LAMP2, and ubiquitin. Interestingly, given the genetic implication of lysosomal function in PD, we did identify the CSF concentrations of CTSB, CTSF, GM2A, and LAMP2 to be altered. However, we also found differences in proteins associated with endocytosis (AP2B1) and the ubiquitin-proteasome system (ubiquitin). No difference in any peptide CSF concentration was found in clinically characterized subjects with AD compared to controls. In conclusion, CSF analyses of subjects with PD suggest a general lysosomal dysfunction, which resonates well with recent genetic findings, while such changes are minor or absent in AD.

Parkinson's disease (PD) is characterized by intraneuronal inclusions of aggregated α-synuclein protein (so-called Lewy bodies) in distinct brain regions. Multiple posttranslational modifications may affect the structure and function of α-synuclein. Mass spectrometry-based analysis may be useful for the characterization and quantitation of α-synuclein forms, but has proven challenging, mainly due to the insolubility of Lewy bodies in aqueous buffer. In the present study, we developed a novel method by combining differential solubilization with immunoprecipitation and targeted proteomics using liquid chromatography and tandem mass spectrometry. Brain tissue homogenization and sample preparation were modified to facilitate analysis of soluble, detergent-soluble, and detergent-insoluble protein fractions (Lewy body-enriched). The method was used to compare α-synuclein forms from cingulate cortex (affected) and occipital cortex (unaffected) in two study sets of PD patients and controls. We identified ∼20 modified α-synuclein variants, including species with N-terminal acetylation and C-terminal truncations at amino acids 103 and 119. The levels of α-synuclein forms Ac-α-syn1-6, α-syn13-21, α-syn35-43, α-syn46-58, α-syn61-80, and α-syn81-96 except α-syn103-119 were significantly increased in PD cingulate region compared to controls in the Lewy body-enriched α-synuclein fraction. In the soluble fraction, only Ac-α-syn1-6 was significantly increased in PD compared to controls. None of the detected α-synuclein variants were Lewy body-specific, but acetylated forms should be examined further as potential biomarkers for abnormal α-synuclein accumulation.

BackgroundNeurogranin (Ng) is a neuron-specific and postsynaptic protein that is abundantly expressed in the brain, particularly in the dendritic spine of the hippocampus and cerebral cortex. The enzymatic cleavage of Ng produces fragments that are released into cerebrospinal (CSF), which have been shown to be elevated in Alzheimer’s disease (AD) patients and predict cognitive decline. Thus, quantification of distinctive cleavage products of Ng could elucidate different features of the disease.MethodsIn this study, we developed novel ultrasensitive single molecule array (Simoa) assays for measurement of full-length neurogranin (FL-Ng) and C-terminal neurogranin (CT-Ng) fragments in CSF. The Ng Simoa assays were evaluated in CSF samples from AD patients (N = 23), mild cognitive impairment due to AD (MCI-AD) (N = 18), and from neurological controls (N = 26).ResultsThe intra-assay repeatability and inter-assay precision of the novel methods had coefficients of variation below 7% and 14%, respectively. CSF FL-Ng and CSF CT-Ng median concentrations were increased in AD patients (6.02 ng/L,P P = 0.00001, respectively) and in patients with MCI-AD (5.69 ng/L,P P P = 0.008) and in patients with MCI-AD (ratio = 115,P = 0.016) compared to neurological controls (ratio = 180). CSF of FL-Ng, CT-Ng, and ratio of CT-Ng/FL-Ng could each significantly differentiate AD patients from controls (FL-Ng, AUC = 0.907; CT-Ng, AUC = 0.913; CT-Ng/FL-Ng, AUC = 0.775) and patients with MCI-AD from controls (FL-Ng, AUC = 0.937; CT-Ng, AUC = 0.963; CT-Ng/FL-Ng, AUC = 0.785).ConclusionsAssessments of the FL-Ng and CT-Ng levels in CSF with the novel sensitive immunoassays provide a high separation of AD from controls, even in early phase of the disease. The novel Ng assays are robust and highly sensitive and may be valuable tools to study synaptic alteration in AD, as well as to monitor the effect on synaptic integrity of novel drug candidates in clinical trials.

Background: Abnormal α-synuclein aggregation and deposition is the pathological hallmark of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), but is also found in Alzheimer disease (AD). Therefore, there is a gaining interest in α-synuclein in cerebrospinal fluid (CSF) as potential biomarker for these neurodegenerative diseases. To broaden the available choices of α-synuclein measurement in CSF, we developed and validated a new assay for detecting total α-synuclein. Methods: This novel ELISA uses commercially available antibodies and is based on electrochemiluminescence technology. The assay protocol is straightforward, with short and simple incubation steps, and requires only small amounts of CSF. We validated this assay for precision, parallelism, dilution linearity, specificity, and spike recovery. We further compared it to the newly validated α-synuclein assay from BioLegend by analyzing a set of 50 CSF samples with both assays. Results: The new assay quantifies α-synuclein in CSF with a lower limit of detection of 36.3 pg/mL and shows no cross-reactivity with human β- and γ-synuclein. Results of dilution linearity, parallelism, spike recovery, and precision classify this assay as well suited for α-synuclein detection in human CSF samples. Conclusions: We present a novel assay based on freely available components to quantify total α-synuclein in CSF as an additional method for α-synuclein as a biomarker in neurodegenerative diseases. The assay convinces with its simple and convenient protocol paired with high sensitivity.

Background: Microglia-mediated proteolysis of the triggering receptor expressed on myeloid cells-2 (TREM-2) produces soluble TREM-2 (sTREM-2) that can be measured in cerebrospinal fluid (CSF) samples. Loss-of-function mutations in TREM2 or in the gene encoding its adaptor protein cause the rare Nasu–Hakola disease (NHD). Multiple sclerosis (MS) is an autoimmune disease that in common with NHD is characterized by demyelination and microglial activation. Objective: To investigate the potential utility of sTREM-2 as a biomarker for MS and to follow treatment effects. Methods: sTREM-2 was analyzed in CSF samples from subjects with MS ( N = 59); relapsing-remitting MS (RRMS) ( N = 36), secondary progressive MS (SPMS) ( N = 20) and primary progressive MS (PPMS) ( N = 3), and controls ( N = 27). CSF levels of sTREM-2 were also assessed before and after treatment of patients with natalizumab or mitoxantrone. Results: CSF levels of sTREM-2 were significantly increased in patients with RRMS, SPMS, and PPMS compared with controls. After natalizumab treatment, the levels of sTREM-2 were normalized to control levels. The levels of sTREM-2 were also reduced after mitoxantrone treatment. Conclusion: Increased CSF levels of sTREM-2, a new marker of microglial activation, in MS and normalization upon treatment with either natalizumab or mitoxantrone support a role for microglial activation in active MS.

Here we describe a method using mass spectrometry to characterize and quantify immuno-enriched α-synuclein forms from biochemically fractionated brain tissue.

The purpose of this study was to investigate whether cerebrospinal fluid (CSF) levels of tau, phosphorylated tau, β-amyloid42 , α-synuclein, neurofilament light, and YKL-40 change over time and if changes correlate with motor progression and/or cognitive decline in patients with PD and controls.

The ability to discriminate between Alzheimer’s disease (AD), subcortical vascular disease, and other cognitive disorders is crucial for diagnostic purposes and clinical trial outcomes. Patients with primarily subcortical vascular disease are unlikely to benefit from treatments targeting the AD pathogenic mechanisms and vice versa. The Gothenburg mild cognitive impairment (MCI) and dementia studies are prospective, observational, single-center cohort studies suitable for both cross-sectional and longitudinal analysis that outline the cognitive profiles and biomarker characteristics of patients with AD, subcortical vascular disease, and other cognitive disorders. The studies, the first of which started in 1987, comprise inpatients with manifest dementia and patients seeking care for cognitive disorders at an outpatient memory clinic. This article gives an overview of the major published papers (neuropsychological, imaging/physiology, and neurochemical) of the studies including the ongoing Gothenburg MCI study. The main findings suggest that subcortical vascular disease with or without dementia exhibit a characteristic neuropsychological pattern of mental slowness and executive dysfunction and neurochemical deviations typical of white matter changes and disturbed blood–brain barrier function. Our findings may contribute to better healthcare for this underrecognized group of patients. The Gothenburg MCI study has also published papers on multimodal prediction of dementia, and cognitive reserve.

There is a need for increased nosological knowledge to enable rational trials in Alzheimer’s disease (AD) and related disorders. The ongoing Gothenburg mild cognitive impairment (MCI) study is an attempt to conduct longitudinal in-depth phenotyping of patients with different forms and degrees of cognitive impairment using neuropsychological, neuroimaging, and neurochemical tools. Particular attention is paid to the interplay between AD and subcortical vascular disease, the latter representing a disease entity that may cause or contribute to cognitive impairment with an effect size that may be comparable to AD. Of 664 patients enrolled between 1999 and 2013, 195 were diagnosed with subjective cognitive impairment (SCI), 274 with mild cognitive impairment (MCI), and 195 with dementia, at baseline. Of the 195 (29%) patients with dementia at baseline, 81 (42%) had AD, 27 (14%) SVD, 41 (21%) mixed type dementia (=AD + SVD = MixD), and 46 (23%) other etiologies. After 6 years, 292 SCI/MCI patients were eligible for follow-up. Of these 292, 69 (24%) had converted to dementia (29 (42%) AD, 16 (23%) SVD, 15 (22%) MixD, 9 (13%) other etiologies). The study has shown that it is possible to identify not only AD but also incipient and manifest MixD/SVD in a memory clinic setting. These conditions should be taken into account in clinical trials.

Introduction Cerebrospinal fluid (CSF) total α-synuclein is considered a potential biomarker for Parkinson's disease (PD), but little is known about the evolution of this marker during the course of the disease. Our objective was to investigate whether CSF total α-synuclein concentrations change over time and are associated with motor and cognitive function in PD. Methods CSF total α-synuclein concentrations were quantified in 56 longitudinally followed PD patients, 27 of whom provided CSF repeatedly 2 and/or 4 years later. Another 18 subjects were included as controls. The samples were analyzed using two independent, validated ELISA methods: our recently developed and validated in-house ELISA and a commercial kit from BioLegend. Results CSF total α-synuclein levels did not distinguish PD patients from controls, displayed no substantial changes during a period of up to 4 years, and did not predict subsequent motor or cognitive decline. These findings were consistent for both analytical methods. Conclusion Our findings do not support the clinical utility of total α-synuclein as a single diagnostic or prognostic biomarker in PD.

Synaptic dysfunction is an early event in Alzheimer's disease (AD) pathogenesis and directly related to cognitive impairment. Consequently, synaptic biomarkers may be valuable tools for both early diagnosis and disease stage. Neurogranin (Ng) is a postsynaptic protein involved in memory consolidation.We developed three monoclonal anti-Ng antibodies. Mass spectrometry and a novel enzyme-linked immunosorbent assay were used to analyze cerebrospinal fluid (CSF) Ng in three independent clinical cohorts including patients with AD dementia (n = 100 in total), mild cognitive impairment patients (MCI), (n = 40) and controls (n = 80 in total).We show in three independent clinical cohorts a marked increase in CSF Ng levels in AD dementia (P.001 in all studies). In addition, high CSF Ng levels at the MCI stage predicted progression to dementia due to AD with a hazard ratio of 12.8 (95% confidence interval 1.6-103.0, P = .02). In amyloid-positive MCI patients, high CSF Ng correlated with a more rapid change in cognition during clinical follow-up (P = .03).These results suggest that CSF Ng is a novel AD biomarker that may be used to monitor synaptic degeneration, and correlates with the rate of cognitive decline in prodromal AD.

Parkinson's disease (PD) is a progressive, multi-focal neurodegenerative disease for which there is no effective disease modifying treatment. A critical requirement for designing successful clinical trials is the development of robust and reproducible biomarkers identifying PD in preclinical stages.To investigate the potential for a cluster of biomarkers visualized with multiple analytical platforms to provide a clinically useful tool.Gas Chromatography-Mass Spectrometry (GC-TOFMS) based metabolomics and immunoassay-based protein/peptide analyses on samples from patients with PD diagnosed in Northern Sweden. Low molecular weight compounds from both plasma and cerebrospinal fluid (CSF) from 20 healthy subjects (controls) and 20 PD patients at the time of diagnosis (baseline) were analyzed.In plasma, we found a significant increase in several amino acids and a decrease in C16-C18 saturated and unsaturated fatty acids in patients as compared to control subjects. We also observed an increase in plasma levels of pyroglutamate and 2-oxoisocaproate (ketoleucine) that may be indicative of increased metabolic stress in patients. In CSF, there was a generally lower level of metabolites in PD as compared to controls, with a specific decrease in 3-hydroxyisovaleric acid, tryptophan and creatinine. Multivariate analysis and modeling of metabolites indicates that while the PD samples can be separated from control samples, the list of detected compounds will need to be expanded in order to define a robust predictive model. CSF biomarker immunoassays of candidate peptide/protein biomarkers revealed a significant decrease in the levels of Aβ-38 and Aβ-42, and an increase in soluble APPα in CSF of patients. Furthermore, these peptides showed significant correlations to each other, and positive correlations to the CSF levels of several 5- and 6-carbon sugars. However, combining these metabolites and proteins/peptides into a single model did not significantly improve the statistical analysis.Together, this metabolomics study has detected significant alterations in plasma and CSF levels of a cluster of amino acids, fatty acids and sugars based on clinical diagnosis and levels of known protein and peptide biomarkers.

Objective: Cerebral accumulation of amyloid beta (A beta) is a pathological hallmark of Alzheimer's disease (AD). Proteolytic processing of amyloid precursor protein (APP) by alpha- or beta-secretase results in two soluble metabolites, sAPP alpha and sAPP beta, respectively. However, previous data have shown that both alpha- and beta-secretase have multiple cleavage sites. The aim of this study was to characterize the C-termini of sAPP alpha and sAPP beta in cerebrospinal fluid (CSF) by mass spectrometry (MS) and to evaluate whether different combinations of these fragments better separate between AD patients and controls by comparing two different sAPP immunoassays. Methods: Using immunoprecipitation and high resolution MS, the APP species present in CSF were investigated. CSF levels of sAPP alpha and sAPP beta from patients with AD (n=43) and from non-demented controls (n=44) were measured using AlphaLISA and MSD immunoassays that employ different antibodies for C-terminal recognition of sAPP alpha. Results: Four different C-terminal forms of sAPP were identified, sAPP beta-M671, sAPP beta-Y681, sAPP alpha-Q686, and 5APP alpha-K687 (APP770 numbering). Neither immunoassay for the sAPP species could separate the two patient groups. The correlation (R-2) between the two immunoassays was 0.41 for sAPP alpha and 0.45 for sAPP beta. Conclusion: Using high resolution MS, we show here for the first time that sAPP alpha in CSF ends at Q686 and K687. The findings also support the conclusion from several previous studies that sAPP alpha and sAPP beta levels are unaltered in AD. (C) 2013 Elsevier B.V. All rights reserved. (Less)

Background: The well-established core biomarkers used to identify Alzheimer's disease (AD) overlap with other dementia disorders such as dementia with Lewy bodies (DLB) and Parkinson's disease with dementia (PDD). This study aimed to evaluate whether the cerebrospinal fluid (CSF) amyloid-β (Aβ)1-42/Aβ1-40 ratio, measured by a novel method, could improve the differential diagnosis of AD, DLB and PDD. Method: CSF levels of Aβ1-40 and Aβ1-42 in patients with PDD, DLB, AD, Parkinson's disease and controls were analyzed using an amplified luminescent proximity homogenous immunoassay along with conventional immunoassays. Results: The CSF Aβ1-42/Aβ1-40 ratio increased discrimination of AD from PDD and DLB compared with either of the two Aβ biomarkers individually. Conclusion: The use of the Aβ1-42/Aβ1-40 ratio could improve the differentiation of AD from PDD and DLB.

To be able to live a good, independent life cognitive functions need to be intact. Dementia, stroke and neuropsychiatric disorders are the major disorders underlying disability. Stroke is usually a consequence of an underlying vessel wall disease that has lasted for a longer period. This vessel wall disease is commonly silent or without prominent symptoms. Damage to the small penetrating arterioles of the brain, arteriolosclerosis, induced by aging and hypertension, as well as other factors such as diabetes and genetic vulnerability, plays an important role in the origin of white matter changes. The pathological vascular wall process leads to lumen constriction, impaired ability to change lumen diameter according to metabolic needs and possible ischemic-hypoxic tissue damage in the vulnerable vascular architectural terminal areas of the long penetrating arteries. The arteriolosclerotic blood vessels are associated with inflammation and remodelling of the extracellular matrix. Enzymes connected to this process have also been found to be involved in demyelination and blood brain barrier opening but also in the repair process of angiogenesis and neurogenesis. Biochemical changes reflecting these processes might be early indicators of small vessel disease and hence increase the knowledge about the disease characteristic mechanisms. Moreover, monitoring disease modifying treatment effects can be an important application for small vessel disease specific biochemical markers.

Background: Novel biomarkers are important for identifying as well as differentiating subcortical vascular dementia (SVD) and Alzheimer’s disease (AD) at an early stage in the disease process. Methods: In two independent cohorts, a multiplex immunoassay was utilized to analyze 90 proteins in cerebrospinal fluid (CSF) samples from dementia patients and patients at risk of developing dementia (mild cognitive impairment). Results: The levels of several CSF proteins were increased in SVD and its incipient state, and in moderate-to-severe AD compared with the control group. In contrast, some CSF proteins were altered in AD, but not in SVD. The levels of heart-type fatty acid binding protein (H-FABP) were consistently increased in all groups with dementia but only in some of their incipient states. Conclusions: In summary, these results support the notion that SVD and AD are driven by different pathophysiological mechanisms reflected in the CSF protein profile and that H-FABP in CSF is a general marker of neurodegeneration.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder of the central nervous system. Two pathological hallmarks in the brain of AD patients are neurofibrillary tangles and senile plaques. The plaques consist mainly of beta-amyloid (Abeta) peptides that are produced from the amyloid precursor protein (APP), by sequential cleavage by beta- and gamma-secretase. Most previous studies have been focused on the C-terminal fragments of APP, where the Abeta sequence is localized. The purpose of this study was to search for N-terminal fragments of APP in cerebrospinal fluid (CSF) using mass spectrometry (MS). By using immunoprecipitation (IP) combined with matrix-assisted laser desorption/ionization time-of-flight MS as well as nanoflow liquid chromatography coupled to high resolution tandem MS we were able to detect and identify six novel N-terminal APP fragments [APP((18-119)), APP((18-121)), APP((18-122)), APP((18-123)), APP((18-124)) and APP((18-126))], having molecular masses of approximately 12 kDa. The presence of these APP derivatives in CSF was also verified by Western blot analysis. Two pilot studies using either IP-MS or Western blot analysis indicated slightly elevated levels of N-terminal APP fragments in CSF from AD patients compared with controls, which are in need of replications in independent and larger patient materials.

The association of alpha-synuclein (alpha-syn) neuropathology with Parkinson's disease (PD) and several related disorders has led to an intense research effort to develop cerebrospinal fluid (CSF)- or blood-based alpha-syn biomarkers for these types of diseases. Recent studies show that alpha-syn is present in CSF and possible to measure using enzyme-linked immunosorbent assay (ELISA). Here, we describe a novel ELISA that allows for quantification of alpha-syn in CSF down to 50pg/mL. The diagnostic value of the test was assessed using CSF samples from 66 Alzheimer's disease (AD) patients, 15PD patients, 15 patients with dementia with Lewy bodies (DLB) and 55 cognitively normal controls. PD and DLB patients and controls displayed similar CSF alpha-syn levels. AD patients had significantly lower alpha-syn levels than controls (median [inter-quartile range] 296 [234-372] and 395 [298-452], respectively, p

We characterized the inflammatory response after a single dose of 8 Gy to the brains of postnatal day 9 rats. Affymetrix gene chips revealed activation of multiple inflammatory mechanisms in the acute phase, 6 h after irradiation. In the subacute phase, 7 days after irradiation, genes related to neurogenesis and cell cycle were down-regulated, but glial fibrillary acidic protein (GFAP) was up-regulated. The concentrations of 14 different cytokines and chemokines were measured using a microsphere-based xMAP technology. CCL2, Gro/KC and IL-1alpha were the most strongly up-regulated 6 h after irradiation. CCL2 was expressed in astrocytes and microglia in the dentate gyrus and the subventricular zone (SVZ). Hypertrophy, but not hyperplasia, of astrocytes was demonstrated 7 days after irradiation. In summary, we found transient activation of multiple inflammatory mechanisms in the acute phase (6 h) after irradiation and activation of astrocytes in the subacute phase (7 days) after irradiation. It remains to be elucidated whether these transient changes are involved in the persistent effects of radiation observed on neurogenesis and cognition in rodents.

In Pisum sativum, the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). Expression of two of these genes (SAD-A and -C) in Escherichia coli or Pichia pastoris resulted in full-length soluble proteins. Purified SAD-A was used as antigen for antibody production in rabbits. With these antibodies the recombinant SAD-C protein (which was most highly expressed of the two isoforms) was shown to be a tetramer consisting of a dimer of dimers. The SAD genes are transiently expressed in plants by short exposures to ultraviolet-B radiation (UV-B), as judged by northern blotting. In turn, mRNA accumulation leads to formation of SAD protein in leaf and stem tissue upon prolonged UV-B irradiation.

Background The discovery that heterozygous missense mutations in the gene encoding triggering receptor expressed on myeloid cells 2 (TREM2) are risk factors for Alzheimer’s disease (AD), with only the apolipoprotein E (APOE) ε4 gene allele conferring a higher risk, has led to increased interest in immune biology in the brain. TREM2 is expressed on microglia, the resident immune cells of the brain and has been linked to phagocytotic clearance of amyloid β (Aβ) plaques. Soluble TREM2 (sTREM2) has previously been measured in cerebrospinal fluid (CSF) by ELISA but in our hands commercial kits have proved unreliable, suggesting that other methods may be required. We developed a mass spectrometry method using selected reaction monitoring for the presence of a TREM2 peptide, which can be used to quantify levels of sTREM2 in CSF. Findings We examined CSF samples from memory clinics in Sweden and the UK. For all samples the following were available: clinical diagnosis, age, sex, and measurements of the CSF AD biomarkers Aβ42, T-tau and P-tau181. AD patients (n = 37) all met biomarker (IWG2) criteria for AD. Control individuals (n = 22) were cognitively normal without evidence for AD in CSF. We found significantly higher sTREM2 concentration in AD compared to control CSF. There were significant correlations between CSF sTREM2 and T-tau as well as P-tau181. CSF sTREM2 increase in AD was replicated in a second, independent cohort consisting of 24 AD patients and 16 healthy volunteers. Conclusion CSF concentrations of sTREM2 are higher in AD than in controls, and correlate with markers of neurodegeneration. CSF sTREM2 may be used to quantify glial activation in AD. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0071-x) contains supplementary material, which is available to authorized users.

BACKGROUND: Alzheimer's disease biomarkers are important for early diagnosis in routine clinical practice and research. Three core CSF biomarkers for the diagnosis of Alzheimer's disease (Aβ42, T-tau, and P-tau) have been assessed in numerous studies, and several other Alzheimer's disease markers are emerging in the literature. However, there have been no comprehensive meta-analyses of their diagnostic performance. We systematically reviewed the literature for 15 biomarkers in both CSF and blood to assess which of these were most altered in Alzheimer's disease. METHODS: In this systematic review and meta-analysis, we screened PubMed and Web of Science for articles published between July 1, 1984, and June 30, 2014, about CSF and blood biomarkers reflecting neurodegeneration (T-tau, NFL, NSE, VLP-1, and HFABP), APP metabolism (Aβ42, Aβ40, Aβ38, sAPPα, and sAPPβ), tangle pathology (P-tau), blood-brain-barrier function (albumin ratio), and glial activation (YKL-40, MCP-1, and GFAP). Data were taken from cross-sectional cohort studies as well as from baseline measurements in longitudinal studies with clinical follow-up. Articles were excluded if they did not contain a cohort with Alzheimer's disease and a control cohort, or a cohort with mild cognitive impairment due to Alzheimer's disease and a stable mild cognitive impairment cohort. Data were extracted by ten authors and checked by two for accuracy. For quality assessment, modified QUADAS criteria were used. Biomarker performance was rated by random-effects meta-analysis based on the ratio between biomarker concentration in patients with Alzheimer's disease and controls (fold change) or the ratio between biomarker concentration in those with mild cognitive impariment due to Alzheimer's disease and those with stable mild cognitive impairment who had a follow-up time of at least 2 years and no further cognitive decline. FINDINGS: Of 4521 records identified from PubMed and 624 from Web of Science, 231 articles comprising 15 699 patients with Alzheimer's disease and 13 018 controls were included in this analysis. The core biomarkers differentiated Alzheimer's disease from controls with good performance: CSF T-tau (average ratio 2·54, 95% CI 2·44-2·64, p

Objective: To investigate whether certain CSF biomarkers at baseline can predict future progression of motor symptoms and cognitive decline in patients with Parkinson disease (PD). Methods: Patients and controls were recruited from hospitals in southern Sweden as part of the prospective and longitudinal Swedish BioFinder Study. In the present study, we included 42 patients with PD and 69 controls who had clinical assessment and lumbar puncture at baseline. Baseline CSF samples were analyzed for α-synuclein (αSyn), β-amyloid 1–42 (Aβ 42 ), tau, phosphorylated tau, and neurofilament light. Associations between CSF markers at baseline and change in clinical characteristics after 2 years of follow-up were investigated using multivariate models adjusting for age, sex, disease duration, and levodopa-equivalent daily dose. Results: Higher levels of αSyn within the PD group were associated with progression of motor symptoms and cognitive decline over 2 years, indicated by significant relationships between αSyn and change in Hoehn and Yahr (β = 0.394, p = 0.043), Unified Parkinson9s Disease Rating Scale, Part III (UPDRS-III) (β = 0.449, p = 0.013), Timed Up and Go (β = 0.406, p = 0.023), and A Quick Test of Cognitive Speed (β = 0.423, p = 0.018). Lower levels of Aβ 42 were associated with worsening of performance on delayed memory recall ( F = 5.834, p = 0.022). Finally, high levels of phosphorylated tau were associated with worsening in motor symptoms (UPDRS-III, β = 0.350, p = 0.045; Hoehn and Yahr, β = 0.366, p = 0.038). Conclusion: We found evidence of a link between higher levels of αSyn at baseline and worsening of motor symptoms and cognitive speed over 2 years in PD. Increased αSyn might be a marker of more intense synaptic degeneration in PD. The results indicate that cortical amyloid pathology (low CSF Aβ 42 ) is associated with memory decline.

© 2015 Elsevier Inc. All rights reserved., Decreased levels of alpha-synuclein (aSyn) in cerebrospinal fluid (CSF) in Parkinson's 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., This is an EU Joint Program – Neurodegenerative Disease Research (JPND) project. The project is supported through the following funding organizations under the aegis of JPND–www.jpnd.eu: The Alzheimer Research Foundation (SAO-FRA), Belgium (grant number S#12018); Danish Council for Strategic Research, Denmark (grant number 0603-00511B); Bundesministerium für Bildung und Forschung (BMBF), Germany (grant number OIED1203C); General Secretary of Research and Technology, Greece; Italian Ministry of Health (Ricerca Corrente), Italy; The Fundação para a Ciência e a Tecnologia, Portugal (grant numbers JPND/0004/2011, IMM/BPD/51-2013, IMM/BI/7-2013, JPND/0005/2011); Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain (grant numbers PI11/03026, PI11/03035; cofinanced by the Fondo Europeo de Desarrollo Regional); The Ministry of Education, Research and Sport of the Slovak Republic, Slovakia; The Slovenian Research Agency, Slovenia (grant number A3-0001); The Swedish Research Council, Sweden; Ministry of Education for Netherlands: ZonMw (The Netherlands Organisation for Health Research and Development), The Netherlands (grant number 629000002); The Scientific and Technological Research Council of Turkey (grant number 112S335).

Alzheimer's disease (AD) is a progressive brain amyloidosis that injures brain regions involved in memory consolidation and other higher brain functions. Neuropathologically, the disease is characterized by accumulation of a 42 amino acid peptide called amyloid β (Aβ42) in extracellular senile plaques, intraneuronal inclusions of hyperphosphorylated tau protein in neurofibrillary tangles, and neuronal and axonal degeneration and loss. Biomarker assays capturing these pathologies have been developed for use on cerebrospinal fluid samples but there are additional molecular pathways that most likely contribute to the neurodegeneration and full clinical expression of AD. One way of learning more about AD pathogenesis is to identify novel biomarkers for these pathways and examine them in longitudinal studies of patients in different stages of the disease. Here, we discuss targeted proteomic approaches to study AD and AD-related pathologies in closer detail and explorative approaches to discover novel pathways that may contribute to the disease. This article is part of a Special Issue entitled: Neuroproteomics: Applications in neuroscience and neurology.

Annika € Ohrfelt, Gunnar Brinkmalm, William G. Honer, Lutz Fr€olich, Lucrezia Hausner, Lennart Minthon, Oskar Hansson, Anders Wallin, Henrik Zetterberg, Kaj Blennow, Ann Brinkmalm, Sahlgrenska Academy at University of Gothenburg, M€olndal, Sweden; 2 Sahlgrenska Academy at University of Gothenburg, Molndal, Sweden; Department of Psychiatry, M€olndal, Sweden; Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; 5 Central Institute of Mental Health, Mannheim, Germany; 6 Department of Clinical Sciences, Malm€o, Sweden; Depatment of Neurology, Sk ane University Hospital, Malm€o, Sweden; University of Gothenburg, M€olndal, Sweden; Sahlgrenska Academy, University of Gothenburg, M€olndal, Sweden; 10 Sahlgrenska Academy, University of Gothenburg, M€olndal, Sweden. Contact e-mail: annika. ohrfelt@neuro.gu.se

We report a novel strategy for studying synaptic pathology by concurrently measuring levels of four SNARE complex proteins from individual brain tissue samples. This method combines affinity purification and mass spectrometry and can be applied directly for studies of SNARE complex proteins in multiple species or modified to target other key elements in neuronal function. We use the technique to demonstrate altered levels of presynaptic proteins in Alzheimer disease patients and prion-infected mice.

INTRODUCTION: The objective was to study whether α-synuclein oligomers are altered in the cerebrospinal fluid (CSF) of patients with dementia, including Parkinson disease with dementia (PDD), dementia with Lewy bodies (DLB), and Alzheimer disease (AD), compared with age-matched controls. METHODS: In total, 247 CSF samples were assessed in this study, including 71 patients with DLB, 30 patients with PDD, 48 patients with AD, and 98 healthy age-matched controls. Both total and oligomeric α-synuclein levels were evaluated by using well-established immunoassays. RESULTS: The levels of α-synuclein oligomers in the CSF were increased in patients with PDD compared with the controls (P

To assess the ability of 5 cerebrospinal fluid(CSF) biomarkers to differentiate between common dementia and parkinsonian disorders.A cross-sectional, clinic-based study.Cerebrospinal fluid samples (N=453) were obtained from healthy individuals serving as controls and from patients with Parkinson disease (PD), PD with dementia(PDD), dementia with Lewy bodies (DLB), Alzheimer disease (AD), progressive supranuclear palsy(PSP), multiple system atrophy (MSA), or corticobasal degeneration (CBD).Neurology and memory disorder clinics.Cerebrospinal fluid biomarker levels in relation to clinical diagnosis.Cerebrospinal fluid levels of -synuclein were decreased in patients with PD, PDD, DLB, and MSA but increased in patients with AD. Cerebrospinal fluid levels of α-amyloid 1-42 were decreased in DLB and even further decreased in AD. Cerebrospinal fluid levels of total tau and hyperphosphorylated tau were increased in AD. Multivariate analysis revealed that these biomarkers could differentiate AD from DLB and PDD with an area under the curve of 0.90, with -synuclein and total tau contributing most to the model. Cerebrospinal fluid levels of neurofilament light chain were substantially increased in atypical parkinsonian disorders (ie, PSP, MSA,and CBD), and multivariate analysis revealed that the level of neurofilament light chain alone could differentiate PD from atypical parkinsonian disorders, with an area under the curve of 0.93.Ascertainment of the -synuclein level in CSF somewhat improves the differential diagnosis of AD vs DLB and PDD when combined with established AD biomarkers.The level of neurofilament light chain alone may differentiate PD from atypical parkinsonian disorders.

Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative diseases that are characterized by intra-neuronal inclusions of Lewy bodies in distinct brain regions. These inclusions consist mainly of aggregated α-synuclein (α-syn) protein. The present study used immunoprecipitation combined with nanoflow liquid chromatography (LC) coupled to high resolution electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) to determine known and novel isoforms of α-syn in brain tissue homogenates. N-terminally acetylated full-length α-syn (Ac-α-syn1–140) and two N-terminally acetylated C-terminally truncated forms of α-syn (Ac-α-syn1–139 and Ac-α-syn1–103) were found. The different forms of α-syn were further studied by Western blotting in brain tissue homogenates from the temporal cortex Brodmann area 36 (BA36) and the dorsolateral prefrontal cortex BA9 derived from controls, patients with DLB and PD with dementia (PDD). Quantification of α-syn in each brain tissue fraction was performed using a novel enzyme-linked immunosorbent assay (ELISA).

Alterations in cerebrospinal fluid (CSF) have been found in Parkinson disease (PD) and in PD dementia (PDD), but the prognostic importance of such changes is not well known. In vivo biomarkers for disease processes in PD are important for future development of disease-modifying therapies.To assess the diagnostic and prognostic value of a panel of CSF biomarkers in patients with early PD and related disorders.Regional population-based, prospective cohort study of idiopathic parkinsonism that included patients diagnosed between January 1, 2004, and April 30, 2009, by a movement disorder team at a university hospital that represented the only neurology clinic in the region. Participants were 128 nondemented patients with new-onset parkinsonism (104 with PD, 11 with multiple system atrophy, and 13 with progressive supranuclear palsy) who were followed up for 5 to 9 years. At baseline, CSF from 30 healthy control participants was obtained for comparison.Cerebrospinal fluid concentrations of neurofilament light chain protein, Aβ1-42, total tau, phosphorylated tau, α-synuclein, and heart fatty acid-binding protein were quantified by 2 blinded measurements (at baseline and after 1 year). Follow-up included an extensive neuropsychological assessment. As PD outcome variables, mild cognitive impairment and incident PDD were diagnosed based on published criteria.Among the 128 study participants, the 104 patients with early PD had a different CSF pattern compared with the 13 patients with progressive supranuclear palsy (baseline area under the receiver operating characteristic curve, 0.87; P .0001) and the 30 control participants (baseline area under the receiver operating characteristic curve, 0.69; P = .0021). A CSF biomarker pattern associated with the development of PDD was observed. In PD, high neurofilament light chain protein, low Aβ1-42, and high heart fatty acid-binding protein at baseline were related to future PDD as analyzed by Cox proportional hazards regression models. Combined, these early biomarkers predicted PDD with high accuracy (hazard ratio, 11.8; 95% CI, 3.3-42.1; P = .0001) after adjusting for possible confounders.The analyzed CSF biomarkers have potential usefulness as a diagnostic tool in patients with parkinsonism. In PD, high neurofilament light chain protein, low Aβ1-42, and high heart fatty acid-binding protein were related to future PDD, providing new insights into the etiology of PDD.

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

Purpose: Dysfunctional proteostasis, with decreased protein degradation and an accumulation of ubiquitin into aggregated protein inclusions, is a feature of neurodegenerative diseases. Identifying new potential biomarkers in cerebrospinal fluid (CSF) reflecting this process could contribute important information on pathophysiology. Experimental design: A developed method combining SPE and PRM-MS is employed to monitor the concentration of ubiquitin in CSF from subjects with Alzheimer's disease (AD), Parkinson's disease (PD), and progressive supranuclear palsy (PSP). Four independent cross-sectional studies are conducted, studies 1–4, including controls (n = 86) and participants with AD (n = 60), PD (n = 15), and PSP (n = 11). Results: The method shows a repeatability and intermediate precision not exceeding 6.1 and 7.9%, respectively. The determined LOD is 0.1 nm and the LOQ range between 0.625 and 80 nm. The CSF ubiquitin concentration is 1.2–1.5-fold higher in AD patients compared with controls in the three independent AD-control studies (Study 1, p < 0.001; Study 2, p < 0.001; and Study 3, p = 0.003). In the fourth study, there is no difference in PD or PSP, compared to controls. Conclusion and clinical relevance: CSF ubiquitin may reflect dysfunctional proteostasis in AD. The described method can be used for further exploration of ubiquitin as a potential biomarker in neurodegenerative diseases. (Less)