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Abstract The glymphatic system is a crucial component in preserving brain homeostasis by facilitating waste clearance from the central nervous system (CNS). Aquaporin-4 (AQP4) water channels facilitate the continuous interchange between cerebrospinal fluid and brain interstitial fluid by convective flow movement. This flow is responsible for guiding proteins and metabolites away from the CNS. Proteinopathies are neurological conditions characterized by the accumulation of aggregated proteins or peptides in the brain. In Alzheimer’s disease (AD), the deposition of amyloid-β (Aβ) peptides causes the formation of senile plaques. This accumulation has been hypothesized to be a result of the imbalance between Aβ production and clearance. Recent studies have shown that an extended form of AQP4 increases Aβ clearance from the brain. In this mini-review, we present a summary of these findings and explore the potential for future therapeutic strategies aiming to boost waste clearance in AD.

Abstract Introduction The exclusion of affected populations from Alzheimer's disease (AD) clinical research limits our understanding of disease heterogeneity and its impact on clinical care. While micro sampling with dried plasma spots (DPS) can promote inclusivity by enabling sample collection in remote areas, current techniques lack the sensitivity required for the quantification of phosphorylated tau at Thr181 (pTau‐181) in DPS extracts. Methods We developed an assay for pTau‐181 with reduced bead count and improved bead read efficiency (BRE) using a prototype Simoa instrument. This novel assay's performance was evaluated against standard pTau‐181 assays on two Simoa platforms, and DPS extracts were tested for pTau‐181 quantification feasibility. Results The novel assay quantifies pTau‐181 at concentrations up to 16x lower than traditional pTau‐181 assays on HD‐X and SR‐X platforms. DPS extracts tested with our low‐bead assay were quantified considerably above the lower limit of quantification (LLOQ), indicating the suitability of this assay for future DPS extract measurements. Discussion Implementing DPS sampling and pTau‐181 quantification could increase participation from underrepresented groups in AD research. However, additional assay optimization and an in‐depth study of preanalytical sample stability are essential for the transition to clinical applicability.

Abstract Following retirement from sport, the chronic consequences of prior‐recurrent contact are evident and retired rugby union players may be especially prone to accelerated cognitive decline. The present study sought to integrate molecular, cerebrovascular and cognitive biomarkers in retired rugby players with concussion history. Twenty retired rugby players aged 64 ± 5 years with three (interquartile range (IQR), 3) concussions incurred over 22 (IQR, 6) years were compared to 21 sex‐, age‐, cardiorespiratory fitness‐ and education‐matched controls with no prior concussion history. Concussion symptoms and severity were assessed using the Sport Concussion Assessment Tool. Plasma/serum nitric oxide (NO) metabolites (reductive ozone‐based chemiluminescence), neuron specific enolase, glial fibrillary acidic protein and neurofilament light‐chain (ELISA and single molecule array) were assessed. Middle cerebral artery blood velocity (MCAv, doppler ultrasound) and reactivity to hyper/hypocapnia (CVRCO2hyper/CVRCO2hypo) were assessed. Cognition was determined using the Grooved Pegboard Test and Montreal Cognitive Assessment. Players exhibited persistent neurological symptoms of concussion (U = 109(41), P = 0.007), with increased severity compared to controls (U = 77(41), P

Abstract Background Alzheimer’s disease (AD) is a complex neurodegenerative disorder with β-amyloid pathology as a key underlying process. The relevance of cerebrospinal fluid (CSF) and brain imaging biomarkers is validated in clinical practice for early diagnosis. Yet, their cost and perceived invasiveness are a limitation for large-scale implementation. Based on positive amyloid profiles, blood-based biomarkers should allow to detect people at risk for AD and to monitor patients under therapeutics strategies. Thanks to the recent development of innovative proteomic tools, the sensibility and specificity of blood biomarkers have been considerably improved. However, their diagnosis and prognosis relevance for daily clinical practice is still incomplete. Methods The Plasmaboost study included 184 participants from the Montpellier’s hospital NeuroCognition Biobank with AD (n = 73), mild cognitive impairments (MCI) (n = 32), subjective cognitive impairments (SCI) (n = 12), other neurodegenerative diseases (NDD) (n = 31), and other neurological disorders (OND) (n = 36). Dosage of β-amyloid biomarkers was performed on plasma samples using immunoprecipitation-mass spectrometry (IPMS) developed by Shimadzu (IPMS-Shim Aβ42, Aβ40, APP669–711) and Simoa Human Neurology 3-PLEX A assay (Aβ42, Aβ40, t-tau). Links between those biomarkers and demographical and clinical data and CSF AD biomarkers were investigated. Performances of the two technologies to discriminate clinically or biologically based (using the AT(N) framework) diagnosis of AD were compared using receiver operating characteristic (ROC) analyses. Results The amyloid IPMS-Shim composite biomarker (combining APP669–711/Aβ42 and Aβ40/Aβ42 ratios) discriminated AD from SCI (AUC: 0.91), OND (0.89), and NDD (0.81). The IPMS-Shim Aβ42/40 ratio also discriminated AD from MCI (0.78). IPMS-Shim biomarkers have similar relevance to discriminate between amyloid-positive and amyloid-negative individuals (0.73 and 0.76 respectively) and A−T−N−/A+T+N+ profiles (0.83 and 0.85). Performances of the Simoa 3-PLEX Aβ42/40 ratio were more modest. Pilot longitudinal analysis on the progression of plasma biomarkers indicates that IPMS-Shim can detect the decrease in plasma Aβ42 that is specific to AD patients. Conclusions Our study confirms the potential usefulness of amyloid plasma biomarkers, especially the IPMS-Shim technology, as a screening tool for early AD patients.

Abstract Synaptic degeneration is an early event closely associated with the course of Alzheimer’s disease (AD). The identification of synaptic blood biomarkers is, therefore, of great interest and clinical relevance. The levels of most synaptic proteins are increased in the cerebrospinal fluid (CSF) of patients with AD, but their detection in blood is hitherto either unavailable or not very informative. This paradigm is related to their low concentration, their peripheral origin, or the presence of highly abundant blood proteins that hinder detection. In recent years, significant progress has been made in detecting the presynaptic protein β-synuclein. This mini-review summarizes the results that highlight the role of β-synuclein as a candidate blood marker for synaptic degeneration in AD.

Neurofilament light chain (NfL) is a potential diagnostic and prognostic plasma biomarker for numerous neurological diseases including Alzheimer’s disease (AD). In this study, we investigated the relationship between baseline plasma concentration of Nfl and Mild Cognitive Impairment in participants who did and did not have a clinically determined diagnosis of dementia by the end of the three-year study. Additionally, we explored the connection between baseline plasma concentration of NfL and AD dementia patients, considering their demographics, clinical features, and cognitive profiles. A total of 350 participants from the Biomarker of AmyLoid pepTide and AlZheimer’s diseAse Risk (BALTAZAR) multicenter prospective study were investigated: 161 AD dementia participants and 189 MCI participants (of which 141 had amnestic MCI and 48 non-amnestic MCI). Plasma biomarkers were measured at baseline and the progression of clinical and cognitive profiles was followed over the three years of follow-up. Baseline plasma NfL concentration increased across the Alzheimer’s disease continuum with a mean NfL value of 17.1 ng/mL [SD = 6.1] in non-amnestic MCI, 20.7 ng/mL [SD = 12.0] in amnestic MCI, and 23.1 ng/mL [SD = 22.7] in AD dementia patients. Plasma NfL concentration correlated with age, body mass index (BMI), and global cognitive performance and decline, as measured by the Mini-Mental State Examination (MMSE). MMSE scores decreased in parallel with increasing plasma NfL concentration, independently of age and BMI. However, NfL concentration did not predict MCI participants’ conversion to dementia within three years. Discussion: Baseline plasma NfL concentration is associated with cognitive status along the AD continuum, suggesting its usefulness as a potential informative biomarker for cognitive decline follow-up in patients.

Abstract Background Cerebrospinal fluid (CSF) Aβ1–42 levels and the Aβ1–42/Aβ1–40 ratio are markers of amyloid pathology, but previous studies suggest that their levels might be influenced by additional pathophysiological processes. Aims To compare Aβ1–42 and the Aβ1–42/Aβ1–40 ratio in CSF in different neurodegenerative disorders and study their association with other biomarkers (tTau, pTau181, and NfL) and with cognitive and functional progression. Methods We included all participants from the Sant Pau Initiative on Neurodegeneration (SPIN) with CSF Aβ1–42 and Aβ1–42/Aβ1–40. Participants had diagnoses of Alzheimer’s disease (AD), dementia with Lewy bodies, frontotemporal lobar degeneration-related syndromes, non-neurodegenerative conditions, or were cognitively normal. We classified participants as “positive” or “negative” according to each marker. We compared CSF levels of tTau, pTau181, and NfL between concordant and discordant groups through ANCOVA and assessed differences in cognitive (MMSE, FCSRT) and functional (GDS, CDR-SOB) progression using Cox regression and linear-mixed models. Results In the 1791 participants, the agreement between Aβ1–42 and Aβ1–42/Aβ1–40 was 78.3%. The Aβ1–42/Aβ1–40 ratio showed a stronger correlation with tTau and pTau181 than Aβ1–42 and an agreement with tTau and pTau181 of 73.1% and 77.1%, respectively. Participants with a low Aβ1–42/Aβ1–40 ratio showed higher tTau and pTau181 and worse cognitive and functional prognosis, regardless of whether they were positive or negative for Aβ1–42. The results were consistent across stages, diagnostic categories, and use of different cutoffs. Conclusion Although Aβ1–42 and Aβ1–42/Aβ1–40 are considered markers of the same pathophysiological pathway, our findings provide evidence favoring the use of the Aβ1–42/Aβ1–40 ratio in clinical laboratories in the context of AD.

Neurofilament-light chain (Nf-L) is a non-specific early-stage biomarker widely studied in the context of neurodegenerative diseases (NDD) and traumatic brain injuries (TBI), which can be measured in biofluids after axonal damage. Originally measured by enzyme-linked immunosorbent assay (ELISA) in cerebrospinal fluid (CSF), Nf-L can now be quantified in blood with the emergence of ultrasensitive assays. However, to ensure successful clinical implementation, reliable clinical thresholds and reference measurement procedures (RMP) should be developed. This includes establishing and distributing certified reference materials (CRM). As a result of the complexity of Nf-L and the number of circulating forms, a clear definition of what is measured when immunoassays are used is also critical to achieving standardization to ensure the long-term success of those assays. The use of powerful tools such as mass spectrometry for developing RMP and defining the measurand is ongoing. Here, we summarize the current methods in use for quantification of Nf-L in biofluid showing potential for clinical implementation. The progress and challenges in developing RMP and defining the measurand for Nf-L standardization of diagnostic tests are addressed. Finally, we discuss the impact of pathophysiological factors on Nf-L levels and the establishment of a clinical cut-off.

Translation involves the biosynthesis of a protein sequence following the decoding of the genetic information embedded in a messenger RNA (mRNA). Typically, the eukaryotic mRNA was considered to be inherently monocistronic, but this paradigm is not in agreement with the translational landscape of cells, tissues, and organs. Recent ribosome sequencing (Ribo-seq) and proteomics studies show that, in addition to currently annotated reference proteins (RefProt), other proteins termed alternative proteins (AltProts), and microproteins are encoded in regions of mRNAs thought to be untranslated or in transcripts annotated as non-coding. This experimental evidence expands the repertoire of functional proteins within a cell and potentially provides important information on biological processes. This review explores the hitherto overlooked alternative proteome in neurobiology and considers the role of AltProts in pathological and healthy neuromolecular processes.

Neurological biomarkers are particularly valuable to clinicians as they can be used for diagnosis, prognosis, or response to treatment. This field of neurology has evolved considerably in recent years with the improvement of analytical methods, allowing the detection of biomarkers not only in cerebrospinal fluid (CSF) but also in less invasive fluids like blood. These advances greatly facilitate the repeated quantification of biomarkers, including at asymptomatic stages of the disease. Among the various informative biomarkers of neurological disorders, neurofilaments (NfL) have proven to be of particular interest in many contexts, such as neurodegenerative diseases, traumatic brain injury, multiple sclerosis, stroke, and cancer. Here we discuss these different pathologies and the potential value of NfL assay in the management of these patients, both for diagnosis and prognosis. We also describe the added value of NfL compared to other biomarkers currently used to monitor the diseases described in this review.

Plasma tau phosphorylated at threonine 181 (p-tau181) predicts Alzheimer’s disease (AD) pathology. Here, the authors investigated whether plasma ptau181 could be a potential biomarker of AD in individuals with Down syndrome (DS) and find plasma p-tau181 can detect AD in DS adults.

Blood microsampling combined with large panels of clinically relevant tests are of major interest for the development of home sampling and predictive medicine. The aim of the study was to demonstrate the practicality and medical utility of microsamples quantification using mass spectrometry (MS) in a clinical setting by comparing two types of microsamples for multiplex MS protein detection. In a clinical trial based on elderly population, we compared 2 µL of plasma to dried blood spot (DBS) with a clinical quantitative multiplex MS approach. The analysis of the microsamples allowed the quantification of 62 proteins with satisfactory analytical performances. A total of 48 proteins were significantly correlated between microsampling plasma and DBS (p < 0.0001). The quantification of 62 blood proteins allowed us to stratify patients according to their pathophysiological status. Apolipoproteins D and E were the best biomarker link to IADL (instrumental activities of daily living) score in microsampling plasma as well as in DBS. It is, thus, possible to detect multiple blood proteins from micro-samples in compliance with clinical requirements and this allows, for example, to monitor the nutritional or inflammatory status of patients. The implementation of this type of analysis opens new perspectives in the field of diagnosis, monitoring and risk assessment for personalized medicine approaches.

Abstract Peripheral inflammation mechanisms involved in Alzheimer's disease (AD) have yet to be accurately characterized and the identification of blood biomarker profiles could help predict cognitive decline and optimize patient care. Blood biomarkers described to date have failed to provide a consensus signature, which is mainly due to the heterogeneity of the methods used or the cohort. The present work aims to describe the potential informativity of peripheral inflammation in AD, focusing in particular on the potential association between the level of plasma neurofilament light (NFL), peripheral inflammation (by quantifying IL-1β, IL-6, TNFα, CCL5, TNF-R1, sIL-6R, TIMP-1, IL-8 in blood) and cognitive decline (assessed by the MMSE and ADAScog scales) through a 2-year follow-up of 40 AD patients from the Cytocogma cohort (CHU Poitiers, Pr M. Paccalin). Our results show for the first time a strong correlation between plasma NFL and TNF-R1 at each time of follow-up (baseline, 12 and 24 months), thus opening an interesting perspective for the prognosis of AD patients.

Abstract Background Anemia is a significant problem in patients on ICU. Its commonest cause, iron deficiency (ID), is difficult to diagnose in the context of inflammation. Hepcidin is a new marker of ID. We aimed to assess whether hepcidin levels would accurately guide treatment of ID in critically ill anemic patients after a prolonged ICU stay and affect the post-ICU outcomes. Methods In a controlled, single-blinded, multicenter study, anemic (WHO definition) critically ill patients with an ICU stay ≥ 5 days were randomized when discharge was expected to either intervention by hepcidin treatment protocol or control. In the intervention arm, patients were treated with intravenous iron (1 g of ferric carboxymaltose) when hepcidin was

Abstract Unlike variant Creutzfeldt–Jakob disease prions, sporadic Creutzfeldt–Jakob disease prions have been shown to be difficult to amplify in vitro by protein misfolding cyclic amplification (PMCA). We assessed PMCA of pathological prion protein (PrPTSE) from 14 human sCJD brain samples in 3 substrates: 2 from transgenic mice expressing human prion protein (PrP) with either methionine (M) or valine (V) at position 129, and 1 from bank voles. Brain extracts representing the 5 major clinicopathological sCJD subtypes (MM1/MV1, MM2, MV2, VV1, and VV2) all triggered seeded PrPTSE amplification during serial PMCA with strong seed- and substrate-dependence. Remarkably, bank vole PrP substrate allowed the propagation of all sCJD subtypes with preservation of the initial molecular PrPTSE type. In contrast, PMCA in human PrP substrates was accompanied by a PrPTSE molecular shift during heterologous (M/V129) PMCA reactions, with increased permissiveness of V129 PrP substrate to in vitro sCJD prion amplification compared to M129 PrP substrate. Combining PMCA amplification sensitivities with PrPTSE electrophoretic profiles obtained in the different substrates confirmed the classification of 4 distinct major sCJD prion strains (M1, M2, V1, and V2). Finally, the level of sensitivity required to detect VV2 sCJD prions in cerebrospinal fluid was achieved.

Abstract Background Amyloid pathology, which is one of the characteristics of Alzheimer’s disease (AD), results from altered metabolism of the beta-amyloid (Aβ) peptide in terms of synthesis, clearance, or aggregation. A decrease in cerebrospinal fluid (CSF) level Aβ1–42 is evident in AD, and the CSF ratio Aβ42/Aβ40 has recently been identified as one of the most reliable diagnostic biomarkers of amyloid pathology. Variations in inter-individual levels of Aβ1–40 in the CSF have been observed in the past, but their origins remain unclear. In addition, the variation of Aβ40 in the context of AD studied in several studies has yielded conflicting results. Methods Here, we analyzed the levels of Aβ1–40 using multicenter data obtained on 2466 samples from six different cohorts in which CSF was collected under standardized protocols, centrifugation, and storage conditions. Tau and p-tau (181) concentrations were measured using commercially available in vitro diagnostic immunoassays. Concentrations of CSF Aβ1–42 and Aβ1–40 were measured by ELISA, xMAP technology, chemiluminescence immunoassay (CLIA), and mass spectrometry. Statistical analyses were calculated for parametric and non-parametric comparisons, linear regression, correlation, and odds ratios. The statistical tests were adjusted for the effects of covariates (age, in particular). Results Regardless of the analysis method used and the cohorts, a slight but significant age-independent increase in the levels of Aβ40 in CSF was observed in AD. We also found a strong positive correlation between the levels of Aβ1–40 and p-tau (181) in CSF, particularly in control patients. Conclusions These results indicate that an increase in the baseline level of amyloid peptides, which are associated with an increase in p-tau (181), may be a biological characteristic and possibly a risk factor for AD. Further studies will be needed to establish a causal link between increased baseline levels of Aβ40 and the development of the disease.

Parkinson’s disease is a complex neurodegenerative disorder resulting in a multifaceted clinical presentation which includes bradykinesia combined with either rest tremor, rigidity, or both, as well as many non-motor symptoms. The motor features of the disorder are associated with the pathological form of alpha synuclein aggregates and fibrils in Lewy bodies and loss of dopaminergic neurons in the substantia nigra. Parkinson’s disease is increasingly considered as a group of underlying disorders with unique genetic, biological, and molecular abnormalities that are likely to respond differentially to a given therapeutic approach. For this reason, it is clinically challenging to treat and at present, no therapy can slow down or arrest the progression of Parkinson’s disease. There is a clear unmet clinical need to develop reliable diagnostic and prognostic biomarkers. When disease-modifying treatments become available, prognostic biomarkers are required to support a definitive diagnosis and clinical intervention during the long prodromal period as no clinical implications or symptoms are observed. Robust diagnostic biomarkers would also be useful to monitor treatment response. Potential biomarkers for the sporadic form of Parkinson’s disease have mostly included synuclein species (monomer, oligomer, phosphorylated, Lewy Body enriched fraction and isoforms). In this review, we consider the analysis of synuclein and its proteoforms in biological samples using proteomics techniques (immunoassay and mass spectrometry) applied to neurodegenerative disease research.

Abstract Background Cerebrospinal fluid biomarker profiles characterized by decreased amyloid-beta peptide levels and increased total and phosphorylated tau levels at threonine 181 (pT181) are currently used to discriminate between Alzheimer’s disease and other neurodegenerative diseases. However, these changes are not entirely specific to Alzheimer’s disease, and it is noteworthy that other phosphorylated isoforms of tau, possibly more specific for the disease process, have been described in the brain parenchyma of patients. The precise detection of these isoforms in biological fluids remains however a challenge. Methods In the present study, we used the latest quantitative mass spectrometry approach, which achieves a sensitive detection in cerebrospinal fluid biomarker of two phosphorylated tau isoforms, pT181 and pT217, and first analyzed a cohort of probable Alzheimer’s disease patients and patients with other neurological disorders, including tauopathies, and a set of cognitively normal controls. We then checked the validity of our results on a second cohort comprising cognitively normal individuals and patients with mild cognitive impairments and AD stratified in terms of their amyloid status based on PiB-PET imaging methods. Results In the first cohort, pT217 but not pT181 differentiated between Alzheimer’s disease patients and those with other neurodegenerative diseases and control subjects much more specificity and sensitivity than pT181. T217 phosphorylation was increased by 6.0-fold in patients with Alzheimer’s disease whereas T181 phosphorylation was only increased by 1.3-fold, when compared with control subjects. These results were confirmed in the case of a second cohort, in which the pT217 cerebrospinal fluid levels marked out amyloid-positive patients with a sensitivity and a specificity of more than 90% (AUC 0.961; CI 0.874 to 0.995). The pT217 concentrations were also highly correlated with the PiB-PET values (correlation coefficient 0.72; P

Abstract The cerebrospinal fluid (CSF) biochemical markers (biomarkers) Amyloidβ 42 (Aβ42), total Tau (T-tau) and Tau phosphorylated at threonine 181 (P-tau181) have proven diagnostic accuracy for mild cognitive impairment and dementia due to Alzheimer’s Disease (AD). In an effort to improve the accuracy of an AD diagnosis, it is important to be able to distinguish between AD and other types of dementia (non-AD). The concentration ratio of Aβ42 to Aβ40 (Aβ42/40 Ratio) has been suggested to be superior to the concentration of Aβ42 alone when identifying patients with AD. This article reviews the available evidence on the use of the CSF Aβ42/40 ratio in the diagnosis of AD. Based on the body of evidence presented herein, it is the conclusion of the current working group that the CSF Aβ42/40 ratio, rather than the absolute value of CSF Aβ42, should be used when analysing CSF AD biomarkers to improve the percentage of appropriately diagnosed patients.

Abstract Background Metastatic breast cancer (MBC) prognosis is variable, depending on several clinical and biological factors. A better prediction of a patient’s outcome could allow for a more accurate choice of treatments. The role of serum biomarkers in predicting outcome remains unclear in this setting. Tau, a microtubule-associated protein, is a neuronal marker that is also expressed in normal breast epithelial cells and cancer cells. Its tissue expression is associated with prognosis in MBC. However, the prognostic value of Tau serum levels in these patients is unknown. We aimed at evaluating the prognostic value of Tau (and other classical biomarkers) in MBC patients, and to assess its association with the presence of brain metastases (BM). Methods 244 MBC patients treated at our institution (2007–2015) were retrospectively selected. The usual MBC clinical and pathological variables were collected, altogether with CA15–3, CEA and HER2 extra-cellular domain (ECD) serum levels. Tau serum levels were measured with a novel immunoassay (digital ELISA) using Single Molecule Array (Simoa) technology. Overall survival (OS) was estimated with the Kaplan-Meier method. To investigate prognostic factors, a multivariate analysis was performed. Cut-offs were set using the Youden index method associated with receiver-operating characteristics (ROC) curves to evaluate the accuracy of biomarkers to identify patients with BM. Results With a median follow-up of 40.8 months, median OS was 15.5 months (95%CI 12.4–20.2). Elevated serum levels of Tau were independently associated with a poor outcome in the whole population as well as in patients with (n = 86) and without BM (n = 158). Median serum Tau levels tended to be higher in patients with BM (p = 0.23). In univariate analysis, patients with BM had an increased risk of serum Tau > 3.17 pg/mL (OR = 2.2, p = 0.049). In multivariate analysis, high values of Tau (OR = 3.98, p = 0.034) accurately identified patients with BM in our cohort. Conclusions Tau is a new biomarker of interest in MBC. Its serum level could represent an independent prognostic factor in these patients (both with and without BM). It also seems to be associated with the presence of BM. A validation of these results in an independent set of MBC patients is necessary to confirm these findings.

Within the neurovascular unit (NVU), the blood–brain barrier (BBB) operates as a key cerebrovascular interface, dynamically insulating the brain parenchyma from peripheral blood and compartments. Increased BBB permeability is clinically relevant for at least two reasons: it actively participates to the etiology of central nervous system (CNS) diseases, and it enables the diagnosis of neurological disorders based on the detection of CNS molecules in peripheral body fluids. In pathological conditions, a suite of glial, neuronal, and pericyte biomarkers can exit the brain reaching the peripheral blood and, after a process of filtration, may also appear in saliva or urine according to varying temporal trajectories. Here, we specifically examine the evidence in favor of or against the use of protein biomarkers of NVU damage and BBB permeability in traumatic head injury, including sport (sub)concussive impacts, seizure disorders, and neurodegenerative processes such as Alzheimer's disease. We further extend this analysis by focusing on the correlates of human extreme physiology applied to the NVU and its biomarkers. To this end, we report NVU changes after prolonged exercise, freediving, and gravitational stress, focusing on the presence of peripheral biomarkers in these conditions. The development of a biomarker toolkit will enable minimally invasive routines for the assessment of brain health in a broad spectrum of clinical, emergency, and sport settings.

BackgroundThe ε4 allele of apolipoprotein E (APOE) gene and increasing age are two of the most important known risk factors for developing Alzheimer disease (AD). The diagnosis of AD based on clinical symptoms alone is known to have poor specificity; recently developed diagnostic criteria based on biomarkers that reflect underlying AD neuropathology allow better assessment of the strength of the associations of risk factors with AD. Accordingly, we examined the global and age-specific association between APOE genotype and AD by using the A/T/N classification, relying on the cerebrospinal fluid (CSF) levels of β-amyloid peptide (A, β-amyloid deposition), phosphorylated tau (T, pathologic tau), and total tau (N, neurodegeneration) to identify patients with AD.Methods and findingsThis case-control study included 1,593 white AD cases (55.4% women; mean age 72.8 [range = 44-96] years) with abnormal values of CSF biomarkers from nine European memory clinics and the American Alzheimer's Disease Neuroimaging Initiative (ADNI) study. A total of 11,723 dementia-free controls (47.1% women; mean age 65.6 [range = 44-94] years) were drawn from two longitudinal cohort studies (Whitehall II and Three-City), in which incident cases of dementia over the follow-up were excluded from the control population. Odds ratio (OR) and population attributable fraction (PAF) for AD associated with APOE genotypes were determined, overall and by 5-year age categories. In total, 63.4% of patients with AD and 22.6% of population controls carried at least one APOE ε4 allele. Compared with non-ε4 carriers, heterozygous ε4 carriers had a 4.6 (95% confidence interval 4.1-5.2; p < 0.001) and ε4/ε4 homozygotes a 25.4 (20.4-31.2; p < 0.001) higher OR of AD in unadjusted analysis. This association was modified by age (p for interaction < 0.001). The PAF associated with carrying at least one ε4 allele was greatest in the 65-70 age group (69.7%) and weaker before 55 years (14.2%) and after 85 years (22.6%). The protective effect of APOE ε2 allele for AD was unaffected by age. Main study limitations are that analyses were based on white individuals and AD cases were drawn from memory centers, which may not be representative of the general population of patients with AD.ConclusionsIn this study, we found that AD diagnosis based on biomarkers was associated with APOE ε4 carrier status, with a higher OR than previously reported from studies based on only clinical AD criteria. This association differs according to age, with the strongest effect at 65-70 years. These findings highlight the need for early interventions for dementia prevention to mitigate the effect of APOE ε4 at the population level.

ABSTRACT To date, approximately 500 iatrogenic Creutzfeldt-Jakob disease cases have been reported worldwide, most of them resulting from cadaveric dura mater graft and from the administration of prion-contaminated human growth hormone. The unusual resistance of prions to decontamination processes, their large tissue distribution, and the uncertainty about the prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the general population lead to specific recommendations regarding identification of tissue at risk and reprocessing of reusable medical devices, including the use of dedicated treatment for prion inactivation. We previously described an in vitro assay, called Surf-PMCA, which allowed us to classify prion decontamination treatments according to their efficacy on vCJD prions by monitoring residual seeding activity (RSA). Here, we used a transgenic mouse line permissive to vCJD prions to study the correlation between the RSA measured in vitro and the in vivo infectivity. Implantation in mouse brains of prion-contaminated steel wires subjected to different decontamination procedures allows us to demonstrate a good concordance between RSA measured by Surf-PMCA (in vitro) and residual infectivity (in vivo). These experiments emphasize the strength of the Surf-PMCA method as a rapid and sensitive assay for the evaluation of prion decontamination procedures and also confirm the lack of efficacy of several marketed reagents on vCJD prion decontamination. IMPORTANCE Creutzfeldt-Jakob diseases are neurodegenerative disorders for which transmission linked to medical procedures have been reported in hundreds of patients. As prion diseases, they are characterized by an unusual resistance to conventional decontamination processes. Moreover, their large tissue distribution and the ability of prions to attach to many surfaces raised the risk of transmission in health care facilities. It is therefore of major importance that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated for prion inactivation. We previously described an in vitro assay, which allowed us to classify accurately prion decontamination treatments according to their efficacy on variant Creutzfeldt-Jakob disease. The significance of this study is in demonstrating the concordance between previous in vitro results and infectivity studies in transgenic mice. Furthermore, commercial reagents currently used in hospitals were tested by both protocols, and we observed that most of them were ineffective on human prions.

Top-Down approaches have an extremely high biological relevance, especially when it comes to biomarker discovery, but the necessary pre-fractionation constraints are not easily compatible with the robustness requirements and the size of clinical sample cohorts. We have demonstrated that intact protein profiling studies could be run on UHR-Q-ToF with limited pre-fractionation (Schmit et al., 2017) [1]. The dataset presented herein is an extension of this research.Proteoforms known to play a role in the pathophysiology process of Alzheimer's disease were identified as candidate biomarkers. In this article, mass spectrometry performance of these candidates are demonstrated. Keywords: Top-Down label-free proteoform profiling, Clinical proteomics, Alzheimer disease, Ultra-high resolution Q-Tof

Abstract Body fluid biomarkers have great potential for different clinical purposes, including diagnosis, prognosis, patient stratification and treatment effect monitoring. This is exemplified by current use of several excellent biomarkers, such as the Alzheimer’s disease cerebrospinal fluid (CSF) biomarkers, anti-neuromyelitis optica antibodies and blood neurofilament light. We still, however, have a strong need for additional biomarkers and several gaps in their development and implementation should be filled. Examples of such gaps are i) limited knowledge of the causes of neurological diseases, and thus hypotheses about the best biomarkers to detect subclinical stages of these diseases; ii) the limited success translating discoveries obtained by e.g. initial mass spectrometry proteomic low-throughput studies into immunoassays for widespread clinical implementation; iii) lack of interaction among all stakeholders to optimise and adapt study designs throughout the biomarker development process to medical needs, which may change during the long period needed for biomarker development. The Society for CSF Analysis and Clinical Neurochemistry (established in 2015) has been founded as a concerted follow-up of large standardisation projects, including BIOMARKAPD and SOPHIA, and the BioMS-consortium. The main aims of the CSF society are to exchange high level international scientific experience, to facilitate the incorporation of CSF diagnostics into clinical practice and to give advice on inclusion of CSF analysis into clinical guidelines. The society has a broad scope, as its vision is that the gaps in development and implementation of biomarkers are shared among almost all neurological diseases and thus they can benefit from the activities of the society.

Abstract Introduction Cerebrospinal fluid α‐synuclein level is increased in sporadic Creutzfeldt‐Jakob disease cases. However, the clinical value of this biomarker remains to be established. In this study, we have addressed the clinical validation parameters and the interlaboratory reproducibility by using an electrochemiluminescent assay. Methods Cerebrospinal fluid α‐synuclein was quantified in a total of 188 sporadic Creutzfeldt‐Jakob disease and non–Creutzfeldt‐Jakob‐disease cases to determine sensitivity and specificity values and lot‐to‐lot variability. Two round robin tests with 70 additional cases were performed in six independent laboratories. Results A sensitivity of 93% and a specificity of 96% were achieved in discriminating sporadic Creutzfeldt‐Jakob disease. No differences were detected between lots. The mean interlaboratory coefficient of variation was 23%, and the intralaboratory coefficient of variations ranged 2.70%–11.39%. Overall, 97% of samples were correctly diagnosed. Discussion The herein validated α‐synuclein assay is robust, accurate, and reproducible in identifying Creutzfeldt‐Jakob disease cases. Thus, it is ready for implementation in the clinical practice to support the diagnosis of Creutzfeldt‐Jakob disease.

Discovered more than a century ago, Alzheimer’s disease (AD) is not only still present in our societies but has also become the most common dementia, with 50 million people worldwide affected by the disease. This number is expected to double in the next generation, and no cure is currently available to slow down or stop the disease progression. Recently, some advances were made due to the approval of the aducanumab treatment by the American Food and Drug Administration. The etiology of this human-specific disease remains poorly understood, and the mechanisms of its development have not been completely clarified. Several hypotheses concerning the molecular mechanisms of AD have been proposed, but the existing studies focus primarily on the two main markers of the disease: the amyloid β peptides, whose aggregation in the brain generates amyloid plaques, and the abnormally phosphorylated tau proteins, which are responsible for neurofibrillary tangles. These protein aggregates induce neuroinflammation and neurodegeneration, which, in turn, lead to cognitive and behavioral deficits. The challenge is, therefore, to create models that best reproduce this pathology. This review aims at gathering the different existing AD models developed in vitro, in cellulo, and in vivo. Many models have already been set up, but it is necessary to identify the most relevant ones for our investigations. The purpose of the review is to help researchers to identify the most pertinent disease models, from the most often used to the most recently generated and from simple to complex, explaining their specificities and giving concrete examples.

A patient with a heterozygous variant of Creutzfeldt-Jakob disease (CJD) with a methionine/valine genotype at codon 129 of the prion protein gene was recently reported. Using an ultrasensitive and specific protein misfolding cyclic amplification–based assay for detecting variant CJD prions in cerebrospinal fluid, we discriminated this heterozygous case of variant CJD from cases of sporadic CJD.

Proteomic technologies have been recently adapted to the new field of clinical proteomics. The origin of errors and biases has been well-identified in the pre-analytical steps, leading to the measurement of clinical analytes. One possible source of inadequacy in clinical proteomics is linked to sample pooling. This practice is usually related to low sample availability, variability, experiment time/cost. In this study, we first asked whether sample pooling in top–down proteomics is suitable to obtain a relevant biological average. Our second objective was to identify inflammatory biomarkers of outlier samples in our population of Creutzfeldt-Jakob disease patients. Our results demonstrated that, in a proteomics study, sample pooling as well as the inflammation status was an important source of errors: missed detection of biomarkers and false identification of others. Pooled samples were not equivalent to the average of biological values. In addition, this procedure reduced the statistical value of the identified biomarkers due to a stabilization of their standard deviation and rendered outlier samples difficult to detect. We identified serum amyloid A as a candidate biomarker of outlier samples. The presence of this protein, which could be explained by inflammatory processes, induced major modifications in the sample profiles.

A combination of low cerebrospinal fluid (CSF) Amyloid β1–42 (Aβ1–42) and high Total-Tau (T-Tau) and Phosphorylated-Tau (P-Tau) occurs at a prodromal stage of Alzheimer’s disease (AD) and recent findings suggest that network abnormalities and interneurons dysfunction contribute to cognitive deficits. Somatostatin (SOM) and Neuropeptide Y (NPY) are two neuropeptides which are expressed in GABAergic interneurons with different fates in AD the former only being markedly affected. The aim of this study was to analyze CSF SOM, NPY and CSF Aβ1–42; T-Tau, P-Tau relationships in 43 elderly mild cognitively impairment (MCI) participants from the Biomarker of AmyLoïd pepTide and AlZheimer’s disease Risk (BALTAZAR) cohort. In these samples, CSF SOM and CSF Aβ1–42 on the one hand, and CSF NPY and CSF T-Tau and P-Tau on the other hand are positively correlated. CSF SOM and NPY concentrations should be further investigated to determine if they can stand for early AD biomarkers.Clinical Trial Registration: www.ClinicalTrials.gov, identifier #NCT01315639.

Abstract Introduction Cerebrospinal fluid (CSF) biomarker ratios were never evaluated in late‐onset (>65 years) behavioral variant of frontotemporal lobar degeneration (bvFTLD) versus Alzheimer's disease (AD). Methods A retrospective monocentric study on 44 clinically suspected amnestic AD or bvFTLD patients with onset after 65 years and available CSF and clinical data. Results The final clinical diagnosis was AD (n = 28; 64%), late‐onset bvFTLD (n = 14; 32%), and others (n = 2; 4%). Applying the CSF cutoff total‐tau/Aβ1–42 of 1.06, all the bvFTLD were in the FTLD range (1.06, AD/AD) or in the FTLD range (

BackgroundBefore implementation in clinical practice, biomarker assays need to be thoroughly analytically validated. There is currently a strong interest in implementation of the ratio of amyloid-β peptide 1-42 and 1-40 (Aβ42/Aβ40) in clinical routine. Therefore, in this study, we compared the analytical performance of six assays detecting Aβ40 in cerebrospinal fluid (CSF) in six laboratories according to a recently standard operating procedure (SOP) developed for implementation of ELISA assays for clinical routine.MethodsAβ40 assays of six vendors were validated in up to three centers per assay according to recently proposed international consensus validation protocols. The performance parameters included sensitivity, precision, dilutional linearity, recovery, and parallelism. Inter-laboratory variation was determined using a set of 20 CSF samples. In addition, test results were used to critically evaluate the SOPs that were used to validate the assays.ResultsMost performance parameters of the different Aβ40 assays were similar between labs and within the predefined acceptance criteria. The only exceptions were the out-of-range results of recovery for the majority of experiments and of parallelism by three laboratories. Additionally, experiments to define the dilutional linearity and hook-effect were not executed correctly in part of the centers. The inter-laboratory variation showed acceptable low levels for all assays. Absolute concentrations measured by the assays varied by a factor up to 4.7 for the extremes.ConclusionAll validated Aβ40 assays appeared to be of good technical quality and performed generally well according to predefined criteria. A novel version of the validation SOP is developed based on these findings, to further facilitate implementation of novel immunoassays in clinical practice.

From its discovery, hepcidin has generated many hopes in terms of diagnosis and management of a wide variety of iron-related diseases. However, in clinical use its accurate quantification remains a challenge due to the limited sensitivity, specificity or reproducibility of the techniques described. In this work, we adapted a highly specific and quantitative mass spectrometry method based on selected reaction monitoring (SRM) to measure hepcidin. Our objective was to adapt the feasibility and reproducibility of the workflow to a clinical environment. Analytical validation was performed according to ISO 15189 norms for determining the limit of detection (LOD, 2 ng/mL), limit of quantification (LOQ, 6 ng/mL), repeatability, reproducibility and linearity (up to 200 ng/mL). Using the serum of patients with various iron-related diseases we compared our SRM detection method to the well-characterized competitive ELISA (cELISA) test. The two methods were commutable (Bland–Altman plot) and we found a positive and significant correlation (r2 = 0.96, Pearson correlation coefficient p

Increasing age is the most important risk factor for developing Alzheimer's disease (AD). The aim of this study was to investigate the relationships between age and cerebrospinal fluid (CSF) levels of β-amyloid (Aβ 1–42), total Tau and phosphorylated Tau (pTau-181), in AD and non-AD patients explored for cognitive disorders. 966 patients (AD, n=528; non-AD, n=438) were included between January 2008 and December 2010 (mean age, 69.5 years; mean MMSE, 20.2) from three French memory centers. Multivariable linear regression models were used to study the relationship between CSF biomarker levels and age in AD and non-AD patients. The capacity of each CSF biomarker in discriminating patients was evaluated using the area under the receiver-operating characteristic (ROC) curves by quartile of distribution of age. In AD patients, older age was associated with higher CSF Aβ 1–42 and lower Tau levels. Conversely, in non-AD patients, age was associated with lower CSF Aβ 1–42, higher Tau, and higher pTau-181 levels. In sex-stratified analysis, these relationships were significant only in women. Using ROC curve analysis, CSF AD biomarkers were more discriminant in younger patients than in older ones. In this clinically-based study, younger patients with AD had exacerbated CSF anomalies compared to older patients with AD. CSF biomarkers were more discriminant in younger patients than in older ones for the diagnosis of AD, especially in women. These results support the idea of an overlap in AD neuropathological lesions in oldest subjects with or without AD.

The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10-8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.

One of the main challenges for neurodegenerative disorders that are principally incurable is the development of new therapeutic strategies, which raises important medical, scientific and societal issues. Creutzfeldt-Jakob diseases are rare neurodegenerative fatal disorders which today remain incurable. The objective of this study was to evaluate the efficacy of the down-regulation of the prion protein (PrP) expression using siRNA delivered by, a water-in-oil microemulsion, as a therapeutic candidate in a preclinical study. After 12 days rectal mucosa administration of Aonys/PrP-siRNA in mice, we observed a decrease of about 28% of the brain PrP(C) level. The effect of Aonys/PrP-siRNA was then evaluated on prion infected mice. Several mice presented a delay in the incubation and survival time compared to the control groups and a significant impact was observed on astrocyte reaction and neuronal survival in the PrP-siRNA treated groups. These results suggest that a new therapeutic scheme based an innovative delivery system of PrP-siRNA can be envisioned in prion disorders.

Prion diseases are irreversible progressive neurodegenerative diseases, leading to severe incapacity and death. They are characterized in the brain by prion amyloid deposits, vacuolisation, astrocytosis, neuronal degeneration, and by cognitive, behavioural and physical impairments. There is no treatment for these disorders and stem cell therapy therefore represents an interesting new approach. Gains could not only result from the cell transplantation, but also from the stimulation of endogenous neural stem cells (NSC) or by the combination of both approaches. However, the development of such strategies requires a detailed knowledge of the pathology, particularly concerning the status of the adult neurogenesis and endogenous NSC during the development of the disease. During the past decade, several studies have consistently shown that NSC reside in the adult mammalian central nervous system (CNS) and that adult neurogenesis occurs throughout the adulthood in the subventricular zone of the lateral ventricle or the Dentate Gyrus of the hippocampus. Adult NSC are believed to constitute a reservoir for neuronal replacement during normal cell turnover or after brain injury. However, the activation of this system does not fully compensate the neuronal loss that occurs during neurodegenerative diseases and could even contribute to the disease progression. We investigated here the status of these cells during the development of prion disorders. We were able to show that NSC accumulate and replicate prions. Importantly, this resulted in the alteration of their neuronal fate which then represents a new pathologic event that might underlie the rapid progression of the disease.

BACKGROUND:So far, all clinical cases of new variant Creutzfeldt-Jakob disease (vCJD), thought to result from the Bovine Spongiform Encephalopathy (BSE) prion agent, have shown Methionine-Methionine (M/M) homozygosity at the M129V polymorphism of the PRNP gene. Although established, this relationship is still not understood. In both vCJD and experimental BSE models prion agents do reach the bloodstream, raising concerns regarding disease transmission through blood transfusion. METHODOLOGY/PRINCIPAL FINDINGS:We investigated the impact of the M129V polymorphism on the expression and processing of the prion protein in human peripheral blood mononuclear cells (PBMCs) from three blood donor populations with Methionine-Methionine (M/M), Valine-Valine (V/V) and M/V genotypes. Using real-time PCR, ELISA and immunoblot assays we were unable to find differences in prion protein expression and processing relating to the M129V polymorphism. CONCLUSIONS/SIGNIFICANCE:These results suggest that in PBMCs, the M129V PrP polymorphism has no significant impact on PrP expression, processing and the apparent glycoform distribution. Prion propagation should be investigated further in other cell types or tissues.