Biomarkers for Alzheimer's Disease – An Overview

Alzheimer's disease (AD) is a complex neurodegenerative disorder with a significant global impact on public health. The emergence of atypical clinical phenotypes challenges traditional diagnostic approaches, necessitating a deeper exploration of biomarkers for accurate identification. The US Food and Drug Administration (FDA) classification of biomarkers and their integration into different stages of AD provide a structured framework for their application in research and clinical settings. Within the context of AD drug development, biomarkers are essential for participant selection, target engagement evaluation, and assessment of pathological hallmarks, including Aβ and tau protein abnormalities. The incorporation of nanoparticles with a biodegradable approach introduces innovative strategies to address the complexities of AD. This paper extensively discusses biomarkers associated with synaptic dysfunction, neuroinflammation, and glial activation, recognizing their significance in elucidating disease mechanisms. Common pathologies such as synuclein and TDP-43 further underscore the multifaceted nature of AD. Current biomarkers for AD diagnosis, encompassing cerebral spinal fluid (CSF) biomarkers and various imaging modalities, reflect the ongoing efforts to enhance early detection and monitoring. Intriguingly, novel biomarkers continue to emerge, offering promising avenues for improved understanding and intervention. Current review provides a comprehensive survey of biomarkers for AD, elucidating their diverse roles across different aspects of the disease. By highlighting their contributions to diagnosis, drug development, and mechanistic insights, this overview underscores the importance of biomarker research in the pursuit of effective AD management and treatment strategies.     

Detection of Aβ Monomers and Oligomers: Early Diagnosis of Alzheimer's Disease

Alzheimer's disease (AD), as the most common progressive neurodegenerative disorder, is pathologically characterized by deposition of extracellular plaque composed of amyloid‐β peptide (Aβ). Different assembled states of Aβ have been considered as both important biomarkers and drug targets for the diagnosis and therapy of AD. Recent studies demonstrate that small, diffusible Aβ oligomers formed by aggregation of Aβ monomers are the major toxic agents in AD. Therefore, the development of reliable assays for Aβ (both monomers and oligomers) will be important for the early differential diagnosis of dementia, predicting the progression of AD, as well as monitoring the effectiveness of novel anti‐Aβ drugs for AD. In this review, we summarize the recent progress made in the development of techniques for detection of Aβ monomers and oligomers. In particular, the principles governing the design of these sensors are classified and summarized. Moreover, the advantages and disadvantages of the assays are evaluated. This review also discusses the improvements and challenges for application of these assays in the early diagnosis of AD.     

Tacrines as Therapeutic Agents for Alzheimer's Disease. V. Recent Developments

Herein we have reviewed our recent developments for the identification of new tacrine analogues for Alzheimer's disease (AD) therapy. Tacrine, the first cholinesterase inhibitor approved for AD treatment, did not stop the progression of AD, producing only some cognitive improvements, but exhibited secondary effects mainly due to its hepatotoxicity. Thus, the drug was withdrawn from the clinics administration. Since then, many publications have described non‐hepatotoxic tacrines, and in addition, important efforts have been made to design multitarget tacrines by combining their cholinesterase inhibition profile with the modulation of other biological targets involved in AD.     

基于质谱检测的相关色谱技术在阿尔茨海默病诊断中的应用

随着世界老年人口的急速增长,阿尔茨海默病发病人数也逐年增多,已成为继心脑血管疾病和恶性肿瘤之后威胁人类健康的“第三大杀手”。疾病的诊断和治疗同等重要,阿尔茨海默病诊断通常依靠典型的临床特征、神经影像技术以及检测疾病相关的生物标志物等。近些年来蛋白质组学和质谱技术迅速发展,可以利用这些技术寻找到与疾病相关的特异性的蛋白质分子作为早期诊断的生物标志物。本文就此进行了综述,主要包括基于蛋白质组学的诊断标志物的筛选和基于质谱检测的色谱技术在阿尔茨海默病诊断中的应用,引用文献34篇。     

Analysis of chiral amino acids in cerebrospinal fluid samples linked to different stages of Alzheimer disease

Chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC-LIF) was used to investigate D- and L-amino acid contents in cerebrospinal fluid (CSF) samples related to different Alzheimer disease (AD) stages. CSF samples were taken from (i) control subjects (S1 pool), (ii) subjects showing a mild cognitive impairment who remained stable (S2 pool), (iii) subjects showing an mild cognitive impairment that progressed to AD (S3 pool) and (iv) subjects diagnosed with AD (S4 pool). The optimized procedure only needed 10 μL of CSF and it included sample cleaning, derivatization with FITC and chiral-MEKC-LIF separation. Eighteen standard amino acids were baseline separated with efficiencies up to 703,000 plates/m, high sensitivity (LODs in the nM range) and good resolution (values ranging from 2.6 to 9.5). Using this method, L-Arg, L-Leu, L-Gln, γ-aminobutyric acid, L-Ser, D-Ser, L-Ala, Gly, L-Lys, L-Glu and L-Asp were detected in all the CSF samples. S3 and S4 samples (i.e. AD subjects) showed significant lower amounts of L-Arg L-Lys, L-Glu and L-Asp compared to the non-AD S1 and S2 samples, showing in the S4 group the lowest amounts of L-Arg L-Lys, L-Glu and L-Asp. Moreover, γ-aminobutyric acid was significantly higher in AD subjects with the highest amount also found for S4. No significant differences were observed for the rest of amino acids including D-Ser. Based on the obtained chiral-MEKC-LIF data, it was possible to correctly classify all the samples into the four groups. These results demonstrate that the use of enantioselective procedures as the one developed in this work can provide some new light on the investigations of AD, including the discovery of new biomarkers related to different stages of AD.     

Liquid chromatography–mass spectrometry methods for urinary biomarker detection in metabonomic studies with application to nutritional studies

The effects of sample preparation and chromatographic method differences on the classification and recovery of metabolic biomarkers from UPLC‐MS measurements on urine samples of humans exposed to different dietary interventions have been investigated. Eight volunteers consumed three high‐fat meals (rich in saturated, monounsaturated and polyunsaturated fatty acids, respectively) in randomized order with a washout period in between. For each participant, urine samples were obtained prior to and at three timed intervals after each meal. Samples were processed either by dilution (1 : 4) or by liquid–liquid extraction and then run under two different gradient conditions. For each analysis method, a total of 96 observations (eight participants, four time points, three diets) were measured. The total ion count chromatograms were analyzed using partial‐least‐squares discriminant analysis. All three dietary classes could be discriminated irrespective of sample preparation and chromatographic method. However, the main discriminating metabolites varied according to sample preparation, indicating that sample treatment and chromatographic conditions influence the ability to extract biomolecular information. Diluted samples showed higher m/z compounds (ca 400 u) while liquid–liquid extraction samples showed low m/z at the same retention time span. Optimized methods for metabolite identification (e.g. organic acids) were statistically inferior to global screening for mixed compound identification, confirming that multiple compound class‐based metabolic profiles are likely to give superior metabonomic (diagnostic) classification, although great care has to be taken in the interpretation in relation to matrix effects. Copyright © 2009 John Wiley & Sons, Ltd.     

Detecting Alzheimer's disease biomarkers: From antibodies to new bio-mimetic receptors and their application to established and emerging bioanalytical platforms – A critical review

The failure of therapeutic treatment of Alzheimer's disease (AD) patients can be related to the late onset of symptoms and, consequently, to a delayed pharmacological aid to counteract neurodegenerative progression. This is coupled to the fact that the diagnosis based on clinical criteria alone introduces high misdiagnosis rate. The availability of assessed biomarkers is therefore of crucial importance not only to counteract late diagnosis, but also to manage patients at high risk of AD development eligible for novel therapies. At the present time, amyloid-β peptides (Aβ_1-40 and Aβ_1-42 isoforms), alone or in combination with Tau protein (total and phosphorylated forms (p-tau)) constitute reliable AD biomarkers and result highly predictive of progression to AD dementia in patients with mild cognitive impairment (MCI), the earliest clinical presentation of AD. Improvement of existing diagnostic tools must take advantage of innovative bioanalytical approaches. In this review, starting from commercially available diagnostic platforms based on antibodies as recognition elements, we intended to provide a double point of view on the issue: 1) progresses achieved on innovative bioanalytical platforms (mainly sensors and biosensors) by using antibodies as consolidated receptors; 2) advance on promising bio-mimetic receptors alternative to antibodies in AD research, and their applications on conventional or innovative analytical platforms. In particular, we first focused on optical- (Propagating and Localized Surface Plasmon Resonance, named here SPR and LSPR) and electrochemical (voltammetric and impedimetric) transduction principles. Together with bioanalytical assays for AD biomarkers quantification, works aimed to investigate and understand their behavior, characteristics, and roles will also be considered in the discussion.     

Elevated serum cytokine levels in mesothelioma patients who have undergone pleurectomy or extrapleural pneumonectomy and adjuvant intraoperative photodynamic therapy

Patients treated on a Phase-I clinical trial of photodynamic therapy (PDT) developed a systemic capillary leak syndrome that constituted the dose-limiting toxicity. We examined serum samples from patients treated at the maximally tolerated dose level for evidence of a systemic, cytokine-mediated inflammatory response. Patients underwent pleurectomy or extrapleural pneumonectomy (EPP) followed by intraoperative PDT of the thorax using Foscan at a dose of 0.1 mg/kg 6 days before surgery and 652 nm red light at a dose of 10 J/cm2. Levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, IL-8, IL-10 and IL-12 were assayed before Foscan administration; after anesthetic induction, surgical resection and light delivery; in postoperative recovery and the day after the surgery. Of the analyzed patients, eight underwent a pleurectomy and one an EPP followed by PDT. IFN-gamma, TNF-alpha and IL-12 showed no elevation, but IL-1beta, IL-6, IL-8 and IL-10 levels were elevated after surgery and PDT. IL-1beta showed a statistically significant variation from baseline after surgery and IL-6, after PDT. The results suggest a systemically mediated inflammatory response resulting from thoracic surgery followed by PDT. Further investigation of specific mechanisms is warranted.     

Chemometric evaluation of nine alcohol biomarkers in a large population of clinically-classified subjects: pre-eminence of ethyl glucuronide concentration in hair for confirmatory classification

An important goal of forensic and clinical toxicology is to identify biological markers of ethanol consumption that allow an objective diagnosis of chronic alcohol misuse. Blood and head hair samples were collected from 175 subjects—objectively classified as non-drinkers (N = 65), social drinkers (N = 51) and active heavy drinkers (N = 59)—and analyzed to determine eight traditional indirect biomarkers of ethanol consumption [aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γ-GT), alkaline phosphatase (ALP), mean corpuscular volume (MCV), carbohydrate-deficient transferrin (CDT), and cholesterol and triglycerides in blood] and one direct biomarker [ethyl glucuronide (EtG) in head hair]. The experimental values obtained from these determinations were submitted to statistical evaluations. In particular, Kruskal–Wallis, Mann–Whitney and ROC curve analyses, together with principal component analysis (PCA), allowed the diagnostic performances of the various biomarkers to be evaluated and compared consistently. From these evaluations, it was possible to deduce that EtG measured in head hair is the only biomarker that can conclusively discriminate active heavy drinkers from social and non-drinkers, using a cut-off value of 30 pg/mg. In contrast, a few indirect biomarkers such as ALP, cholesterol, and triglycerides showed extremely low diagnostic abilities and may convey misleading information. AST and ALT proved to be highly correlated and exhibited quite low sensitivity and specificity. Consequently, either of these parameters can be discarded without compromising the classification efficiency. Among the indirect biomarkers, γ-GT provided the highest diagnostic accuracy, while CDT and MCV yielded high specificity but low sensitivity. It was therefore concluded that EtG in head hair is the only biomarker capable of supporting a confirmatory diagnosis of chronic alcohol abuse in both forensic and clinical practice, while it was found that γ-GT, CDT, MCV, and AST—whether used alone or in combination—do not allow the conclusive classification of subjects according to ethanol consumption. However, a diagnostic strategy combining these four parameters could be formulated in order to create a multivariate model capable of screening suspected active heavy drinkers.     

Chiral separation of disease biomarkers with 2‐hydroxycarboxylic acid structure

Chiral 2‐hydroxycarboxylic acids are compounds that have been linked to particular diseases and are putative biomarkers with some diagnostic potential. The importance of identifying whether a particular enantiomer is related to certain diseases has been encouraged recently. However, in many cases it has not yet been elucidated whether there are stereochemical implications with respect to these biomarkers and whether their enantioselective analysis provides new insights and diagnostic potential. In this study 13 disease‐related chiral 2‐hydrocarboxylic acids were studied for their chiral separation by high‐performance liquid chromatography on three cinchona alkaloid‐derived chiral stationary phases. From a subgroup of eight 2‐hydroxymonocarboxylic acids, baseline resolution could be achieved and inversion of elution order by exchanging tert‐butylcarbamoyl quinidine chiral stationary phase (Chiralpak QD‐AX) for the corresponding quinine analogue (Chiralpak QN‐AX) is shown for seven of them. Furthermore, conditions for chiral separation of the 2‐hydroxydicarboxylic acids, citramalic acid, 2‐isopropylmalic acid, and 2‐hydroxyadipic acid are reported and compared to the previous reported conditions for 2‐hydroxyglutaric acid and malic acid.     

Identification of novel biomarker candidates by differential peptidomics analysis of cerebrospinal fluid in Alzheimer's disease

The objective of this work was the application of peptidomics technologies for the detection and identification of reliable and robust biomarkers for Alzheimer's disease (AD) contributing to facilitate and further improve the diagnosis of AD. Using a new method for the comprehensive and comparative profiling of peptides, the differential peptide display (DPD), 312 cerebrospinal fluid (CSF) samples from AD patients, cognitively unimpaired subjects and from patients suffering from other primary dementia disorders were analysed as four independent analytical sets. By combination with a cross validation procedure, candidates were selected from a total of more than 6,000 different peptide signals based on their discriminating power. Twelve candidates were identified using mass-spectrometric techniques as fragments of the possibly neuroprotective neuroendocrine protein VGF and another one as the complement factor C3 descendent C3f. The combination of peptide profiling and cross validation resulted in the detection of novel potential biomarkers with remarkable robustness and a close relation to AD pathophysiology.     

Single-cell RNA sequencing reveals B cell–related molecular biomarkers for Alzheimer’s disease

In recent years, biomarkers have been integrated into the diagnostic process and have become increasingly indispensable for obtaining knowledge of the neurodegenerative processes in Alzheimer’s disease (AD). Peripheral blood mononuclear cells (PBMCs) in human blood have been reported to participate in a variety of neurodegenerative activities. Here, a single-cell RNA sequencing analysis of PBMCs from 4 AD patients (2 in the early stage, 2 in the late stage) and 2 normal controls was performed to explore the differential cell subpopulations in PBMCs of AD patients. A significant decrease in B cells was detected in the blood of AD patients. Furthermore, we further examined PBMCs from 43 AD patients and 41 normal subjects by fluorescence activated cell sorting (FACS), and combined with correlation analysis, we found that the reduction in B cells was closely correlated with the patients’ Clinical Dementia Rating (CDR) scores. To confirm the role of B cells in AD progression, functional experiments were performed in early-stage AD mice in which fibrous plaques were beginning to appear; the results demonstrated that B cell depletion in the early stage of AD markedly accelerated and aggravated cognitive dysfunction and augmented the Aβ burden in AD mice. Importantly, the experiments revealed 18 genes that were specifically upregulated and 7 genes that were specifically downregulated in B cells as the disease progressed, and several of these genes exhibited close correlation with AD. These findings identified possible B cell-based AD severity, which are anticipated to be conducive to the clinical identification of AD progression.Subject terms: Cell death in the nervous system, Neurotrophic factors     

Blood-based Alzheimer's disease diagnosis using fluorescent peptide nanoparticle arrays

There is a critical need to diagnose and monitor the progression of Alzheimer’s disease(AD) using bloodbased biomarkers. At present, it is believed that tau biomarkers can be utilized to reliably detect AD.Multimodal techniques are highly sought after for AD diagnosis and progression monitoring. For this purpose, we developed a fluorescent peptide nanoparticles(f-PNPs) arrays that is capable of detecting multiple signals simultaneously. The concentration, aggregation stages, and Young’s modulus ...     

Effects of intranasal phototherapy on nasal mucosa in patients with allergic rhinitis

RATIONALE: Rhinophototherapy has been shown to be effective in the treatment of allergic rhinitis. Considering that phototherapy with ultraviolet light (UV) induces DNA damage, it is of outstanding importance to evaluate the damage and repair process in human nasal mucosa. METHODS: We have investigated eight patients undergoing intranasal phototherapy using a modified Comet assay technique and by staining nasal cytology samples for cyclobutane pyrimidine dimers (CPDs), which are UV specific photoproducts. RESULTS: Immediately after last treatment Comet assay of nasal cytology samples showed a significant increase in DNA damage compared to baseline. Ten days after the last irradiation a significant decrease in DNA damage was observed compared to data obtained immediately after finishing the treatment protocol. Difference between baseline and 10 days after last treatment was not statistically significant. Two months after ending therapy, DNA damage detected by Comet assay in patients treated with intranasal phototherapy was similar with that of healthy individuals. None of the samples collected before starting intranasal phototherapy stained positive for CPDs. In all samples collected immediately after last treatment strong positive staining for CPDs was detected. The number of positive cells significantly decreased 10 days after last treatment, but residual positive staining was present in all the examined samples. This finding is consistent with data reported in skin samples after UV irradiation. Cytology samples examined two months after ending therapy contained no CPD positive cells. CONCLUSION: Our results suggest that UV damage induced by intranasal phototherapy is efficiently repaired in nasal mucosa.     

Dielectrophoretic isolation of DNA and nanoparticles from blood

The ability to effectively detect disease-related DNA biomarkers and drug delivery nanoparticles directly in blood is a major challenge for viable diagnostics and therapy monitoring. A DEP method has been developed which allows the rapid isolation, concentration and detection of DNA and nanoparticles directly from human and rat whole blood. Using a microarray device operating at 20 V peak-to-peak and 10 kHz, a wide range of high molecular weight (HMW)-DNA and nanoparticles were concentrated into high-field regions by positive DEP, while the blood cells were concentrated into the low-field regions by negative DEP. A simple fluidic wash removes the blood cells while the DNA and nanoparticles remain concentrated in the DEP high-field regions where they can be detected by fluorescence. HMW-DNA could be detected at 260 ng/mL, which is a detection level suitable for analysis of disease-related cell-free circulating DNA biomarkers. Fluorescent 40 nm nanoparticles could be detected at 9.5 × 10(9) particles/mL, which is a level suitable for monitoring drug delivery nanoparticles. The ability to rapidly isolate and detect DNA biomarkers and nanoparticles from undiluted whole blood will benefit many diagnostic applications by significantly reducing sample preparation time and complexity.     

UPLC-TOF MS-Based Metabonomic Study on Coadministration of Huperzine A and Ligustrazine Phosphate for Treatment of Alzheimer��s Disease

This work was designed to assess the holistic efficacy of concurrent treatment with huperzine A (HA) and ligustrazine phosphate (LP) in the animal model, and to interpret the metabonomics characteristics of the poly-therapy in treating Alzheimer??s disease (AD). Metabolic profiling of the brain was performed using ultra performance liquid chromatography coupled to time-of-flight mass spectroscopy (UPLC-TOF MS). Principal component analysis and partial least squares discriminant analysis were utilized to classify and reveal the differences among the control, model and treatment groups. Ten potential biomarkers have been finally gained. The metabonomic results revealed that the coadministration of HA and LP might slow down the progression of AD through synergistic therapeutic efficacies, exhibiting better effect on amnesia compared with the mono-drug therapy. Importantly, the UPLC-TOF MS-based metabonomic approach would be a useful technique for the diagnosis of AD, the discovery of metabolic biomarkers and the evaluation of the treatment effects.     

UPLC-TOF MS-Based Metabonomic Study on Coadministration of Huperzine A and Ligustrazine Phosphate for Treatment of Alzheimer’s Disease

This work was designed to assess the holistic efficacy of concurrent treatment with huperzine A (HA) and ligustrazine phosphate (LP) in the animal model, and to interpret the metabonomics characteristics of the poly-therapy in treating Alzheimer’s disease (AD). Metabolic profiling of the brain was performed using ultra performance liquid chromatography coupled to time-of-flight mass spectroscopy (UPLC-TOF MS). Principal component analysis and partial least squares discriminant analysis were utilized to classify and reveal the differences among the control, model and treatment groups. Ten potential biomarkers have been finally gained. The metabonomic results revealed that the coadministration of HA and LP might slow down the progression of AD through synergistic therapeutic efficacies, exhibiting better effect on amnesia compared with the mono-drug therapy. Importantly, the UPLC-TOF MS-based metabonomic approach would be a useful technique for the diagnosis of AD, the discovery of metabolic biomarkers and the evaluation of the treatment effects.

     

Rapid Supercritical Fluid Chromatography Method for Separation of Chlorthalidone Enantiomers

Supercritical fluid chromatography employing chiral stationary phases is a popular separation technique to perform enantioselective separations. The main advantages of supercritical fluid chromatography are low analysis time, low consumption of organic modifiers, and therefore lower costs and higher environmental friendliness. A novel method for the separation of chlorthalidone enantiomers, widely used diuretic drug, is reported that clearly demonstrates the advantages of supercritical fluid chromatography. The effects of the amount and type of organic modifiers, temperature, and back pressure on enantioselectivity and resolution of the enantiomers were evaluated. The baseline separation was achieved in less than 2.5 min in the optimized system composed of Chiralpak AD column, mobile phase CO₂/MeOH 50/50 (v/v), temperature 40°C, a flow rate of 4.0 mL/min, and 120 bar back pressure. Moreover, enantiomers of chlorthalidone were determined in two commercially available pharmaceuticals. The proposed method may be easily transferred to a semi-preparative scale.     

Effects of Citicoline,Homotaurine, and Vitamin E on Contrast Sensitivity and Visual-Related Quality of Life in Patients with Primary Open-Angle Glaucoma: A Preliminary Study

The aim of the present study was to evaluate the effects of supplementation with a fixed combination of citicoline 500 mg, homotaurine 50 mg, and vitamin E 12 mg (CIT/HOMO/VITE) on contrast sensitivity and visual-related quality of life in patients with primary open-angle glaucoma (POAG) in mild stage. This was a multicenter, observational, cross-over, short-term, pilot study on POAG patients with stable controlled intraocular pressure (IOP). Patients were randomly assigned to Group 1 (current topical therapy for 4 months and then current topical therapy plus CIT/HOMO/VITE for 4 months) or Group 2 (CIT/HOMO/VITE in addition to current topical therapy for 4 months and then topical therapy alone for 4 months). Best-corrected visual acuity, IOP, visual field, and the Spaeth/Richman contrast sensitivity (SPARCS) test score were recorded at baseline and after 4 and 8 months. The Glaucoma Quality of Life-15 (GQL-15) questionnaire was administered at each check time. Forty-four patients were assigned to Group 1 and 65 to Group 2. Over the follow-up period, there were no significant changes in IOP or visual field findings, whereas SPARCS and GQL-15 findings significantly varied from baseline, both being improved in subjects treated with CIT/HOMO/VITE fixed combination. These results demonstrate that a daily intake of a fixed combination of citicoline, homotaurine, and vitamin E in addition to the topical medical treatment significantly increased the total score of the contrast sensitivity test and the quality of life in patients with POAG.     

Computer‐aided prediction of toxicity with substructure pattern and random forest

Toxicity of chemicals induced by different factors is an important consideration, especially during the drug research and development process. Thus, there is urgent need to develop computationally effective models that can predict the toxicity or adverse effects of chemicals for a specific class of chemicals. In this study, random forest (RF) was used to classify five toxicity data sets from Distributed Structure‐Searchable Toxicity database network, using substructure fingerprints calculated directly from simple molecular structure. Three model validation approaches, out‐of‐bag validation incorporated in RF, fivefold cross‐validation, and an independent validation set, were used for assessing the prediction capability of our models. The chemical space analysis of data sets was explored by multidimensional scaling plots, and outlying molecules were also detected by the proximity measure in RF. At the same time, the important substructure fingerprints, recognized by the RF technique, gave some insights into the structure features related to toxicity of chemicals. The results obtained showed that these in silico classification models with substructure patterns and RF are applicable for potential toxicity prediction of chemical compounds. Copyright © 2012 John Wiley & Sons, Ltd.