Innovative Biomarkers for Alzheimer’s Disease: Focus on the Hidden Disease Biomarkers

Abstract

The criteria for the clinical diagnosis of AD include the analysis of biomarkers of the underlying brain disease pathology; a set of cerebrospinal fluid (CSF) tests, amyloid-β1-42 (Aβ₄₂), total-tau (t-tau), and phosphorylated tau (p-tau), are available and their performance in a clinical setting has been assessed in several studies. Thus, in dementia research, great advances have been made in the discovery of putative biomarkers; however, disappointingly, few of them have been translated into clinically applicable assays. To find biomarkers able to reliably detect AD pathology already at prodromal stages and in blood is even more important. Recent technical breakthroughs have provided ultrasensitive methods that allow the detection of brain-specific proteins in blood. In the present review, we will focus on the usefulness of ultrasensitive technologies for biomarker discovery and trace elements detection; moreover, we will review studies on circulating nano-compartments, a promising novel source of material for molecular diagnostics.

Keywords

Alzheimer’s disease biomarkers extracellular vesicles/exosomes trace elements

INTRODUCTION

The criteria for the clinical diagnosis of AD were established by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer’s Disease and Related Disorders Association (ADRDA) workgroup in 1984 [1], and then revised in 2011 by the National Institute of Aging and the Alzheimer’s Association workgroup [2 –5]; the revised criteria include the analysis of biomarkers that reflect the underlying brain disease pathology: amyloid plaques, axonal degeneration, and intraneuronal tangles, the three major alterations in the Alzheimer’s disease (AD) brain, can be monitored with the cerebrospinal fluid (CSF) biomarkers amyloid-β1-42 (Aβ₄₂), total-tau (t-tau), and phosphorylated tau (p-tau), respectively. Regarding AD classical CSF biomarkers, data collected over the past years show that 1) CSF levels of Aβ₄₂, the most investigated Aβ variant, are significantly lower in AD patients than in age-matched healthy elderly controls [3, 6]; 2) the levels of total tau and phosphorylated tau (p-tau181 and p-tau231) in AD CSF are significantly higher than those of controls [3 , 7–9]. The greatest future potential in biomarker AD diagnostics is the detection of the disease in its pre-clinical stages. Thus, reconstruction of longitudinal trajectories of CSF biomarker changes is central to understanding the sequence of events that finally lead to cognitive symptoms. In this context, the main findings are 1) Aβ₄₂ decrease is evident long before the onset of clinical symptoms in both autosomal dominant and sporadic AD and may predict cognitive decline and dementia in cognitively normal elderly individuals [10 –15]; 2) in some individuals, t-tau increase occurs during the preclinical and prodromal stages of the disease subsequent to the decrease in Aβ₄₂ levels [7, 9 , 16–19], and high total t-tau levels have been associated with fast progression from mild cognitive impairment (MCI) to AD, and with a more malignant course of AD [20, 21]. In conclusion, as recently shown by a meta-analysis, CSF Aβ₄₂, t-tau, and p-tau are biomarkers that robustly separate patients with AD from controls and that also discriminate between MCI due to AD and stable MCI [22]; in addition, the performance of those CSF core biomarkers in a clinical setting has been assessed in several studies [23, 24].

It is most important to note, however, that although Aβ_1 - 42 is an early biomarker when used alone, it is not specific for AD [25] and is non-specifically decreased in other disorders without plaque pathology [26]; in addition, levels of t-tau in CSF correlate with neuronal tissue damage and are increased in various disorders [27 –29].

CSF levels of p-tau are more specific to AD than are levels of t-tau, but we can conclude that a biomarker used alone has a limited ability to discriminate AD from other major forms of dementia.

Thus, in dementia research, great advances have been made in the discovery of putative biomarkers, however, disappointingly few of them have been translated into clinically applicable assays. The overlap in pathology between AD and other neurodegenerative disorders preclude CSF core biomarkers from achieving a specificity of 100%.

In the present review, we will focus on the usefulness of ultrasensitive technologies for biomarker discovery and trace elements detection; moreover, we will review studies on circulating nano-compartments, a promising novel source of material for molecular diagnostics (Fig. 1).

Fig.1

The figure summarizes old versus innovative technologies and old versus innovative biomarkers. ND-EVs, neuron-derived extracellular vesicles; AD-EVs, astrocyte-derived extracellular vesicles.

ULTRASENSITIVE TECHNOLOGIES FOR BIOMARKER DISCOVERY IN ALZHEIMER’S DISEASE

There is an urgent need for suitable CSF and blood biomarkers to improve diagnostic tools and treatment in various neurodegenerative diseases. Recently, Olsson and coauthors provided the most comprehensive meta-analysis of CSF and blood biomarkers for the diagnosis of AD: along with the core CSF biomarkers of neurodegeneration (i.e., Aβ₄₂, t-tau, p-tau), only CSF neurofilament light protein (NFL) and plasma t-tau were strongly associated with AD; emerging CSF biomarkers reflecting neurodegeneration (i.e., NSE, VLP-1, HFABP) and glial activation (i.e., YKL-40) were only moderately associated with AD. AD may present heterogeneity in terms of clinical presentations, neuroanatomical involvement, and neuropathological profiles mainly, but not exclusively, in the dominantly inherited forms [31, 32]. The reason why it is so difficult to find robust and reproducible AD biomarkers could be due to the molecular basis underlying AD phenotypic heterogeneity. Sporadic and familial AD cases were described to be characterized by distinct Aβ profile in brain deposits, and these different molecular AD subtypes could be recognized by CSF analysis [33, 34]. According to this view, biomarkers can help stratify patients into subgroups that share the same pathologic mechanisms and possibly individualized treatments. An alternative explanation is that disease biomarkers are still “hidden” and present at very low levels in biofluid. Thus, heterogeneity of sample population, and insufficient analytical sensitivity for the current ELISA methods could contribute to low reproducibility of the published results.

To find biomarkers able to reliably detect AD pathology already at prodromal stages and in blood is even more important. For large-scale assessments of patients in primary care settings, blood-based core biomarkers (and in general, blood-based biomarkers) are needed.

In the last few years, technological developments have given ultrasensitive measurement techniques, such as electrochemiluminescence immunoassay (ECL) and Single-molecule array/counting methods (e.g., Simoa, SMC), that allow measurement of brain-derived proteins in blood samples. A sensitive digital ELISA for measurement of Aβ₄₂ has been recently developed: the optimized assay was successfully used to quantify Aβ₄₂ in clinical samples from patients treated with the β-site amyloid precursor protein cleaving enzyme 1 inhibitor LY2886721 [35]. The same approach was employed to measure both plasma Aβ₄₂ and Aβ₄₀ in a large cohort of individuals including patients with subjective cognitive decline, MCI, and AD: levels of Aβ₄₂ and Aβ₄₀ were reduced in AD compared with all other diagnostic groups; however, during the preclinical or prodromal AD stages, plasma concentration of Aβ₄₂ was just moderately decreased, whereas Aβ₄₀ levels were unchanged [36]. These data suggest that prominent changes in Aβ metabolism occur later in the periphery (i.e., plasma) compared to the brain.

Using an ultrasensitive method, Mattsson and colleagues recently published the largest study on plasma tau in AD: they found associations between elevated plasma tau and AD hallmarks, but the associations were mild and differed between cohorts [37]. Levels of tau protein in plasma seem to be associated with neurodegeneration and cognitive function in a population-based elderly cohort [38]. By using a newly developed ultrasensitive immunoassay, Tabete and colleague, in an exploratory pilot study, reported for the first-time quantitative data on the plasma levels of p-tau₁₈₁ in patients with AD [39].

The use of platforms with high analytical sensitivity showed that NFL can be accurately measured in serum and that correlations between paired CSF and serum samples is strongest for Simoa (r = 0.88) and ECL (r = 0.78) and weaker for ELISA measurements (r = 0.38) [40]. Of note, using the Simoa NFL assay it has been demonstrated that plasma NFL levels are increased in AD and are associated with cognitive, biochemical, and imaging hallmarks of the disease [41]; since NFL plasma levels are increased also in other neurodegenerative diseases [42, 43], plasma NFL may be a valuable noninvasive tool to assess neurodegeneration and prove to be a useful outcome measure for clinical trials.

In addition, the use of ultrasensitive technologies allowed the identification of innovative biomarkers in human CSF, including synaptic and microglial markers. Specifically, it has been demonstrated that baseline levels of the synaptic marker neurogranin are increased in AD [44, 45] and are highly correlated with t-tau and p-tau₁₈₁. Moreover, CSF neurogranin concentrations 1) were higher in patients with MCI who progressed to AD than in stable MCI; 2) were predictive of progression from MCI to AD; and 3) increased over time in cognitively normal participants and predicted future cognitive impairment in controls [44, 45]. Thus, the CSF synaptic marker neurogranin seems to be a good diagnostic and prognostic marker for early AD that is comparable to the core CSF biomarkers for AD. Interestingly, using ECL immunoassay, it has been demonstrated that the CSF microglial marker soluble variant of Triggering Receptor Expressed on Myeloid Cells 2 (sTREM2) concentrations are increased in the early symptomatic phase of AD [46]; this finding reinforces the idea that the inflammatory and microglial response change during the progression of AD.

Several lines of evidence have converged to demonstrate that soluble Aβ assemblies (oligomers) are the principal neurotoxic species and exist in a complex equilibrium with fibrillar amyloid plaques [47]. Because of the important role of soluble Aβ oligomers in AD pathogenesis, interest in the detection of oligomers in CSF has gained much attention. Aβ oligomers are present in human CSF at very low levels and, therefore, their robust quantification requires the use of ultrasensitive platforms. Recently, using an SMC-based immunoassay, Yang and colleagues were able to measure Aβ oligomers in biological samples, including human CSF. Levels of soluble Aβ oligomers tended to be higher in CSF from MCI subjects than in AD or control subjects, but there was considerable overlap between all three groups [48]. In addition to SMC-based immunoassay, the Attenuated Total Reflection - Fourier Transform Infrared spectroscopy (ATR-FTIR) was successfully applied to differentiate Aβ oligomers from fibrils based on their spectral features [49]. Furthermore, an immuno-infrared-sensor able to monitor secondary structures of Aβ, especially α-helix and β-sheets, was recently developed [50]. When applied to CSF and plasma samples, this assay detected an increase of β-sheets structures—a pathological misfolding—in AD patients and in MCI converting to AD [51]. Additional studies, including the further implementation of immunoassays, are required to determine whether measurement of Aβ oligomers and monitoring of Aβ secondary structures have a diagnostic potential.

TRACE ELEMENTS IN ALZHEIMER’S DISEASE

Besides the studies reported above on protein biomarkers, meta-analysis studies focusing on metals in AD have revealed the potential of these small molecular weight compounds, present in trace amount in our body fluids, in this neurological disorder. Failure of homeostatic control of essential metals has, in fact, been reported among the putative risk factors for AD [52, 53]. Numerous studies are available for copper, zinc, and iron, although a meta-analysis of the role of manganese in AD has also recently been carried out, demonstrating a decrease of this metal in the disease [54].

The role of iron appears controversial, and indeed, either the meta-analyses in the brain [55] or those in the periphery [56, 57] report no change in iron levels between AD patients and healthy controls. Some recent studies point out to a potential role of ferritin as marker of disease progression in the early stages of AD [58, 59]. Zinc appears decreased in AD, as reported in recent meta-analyses [56 , 60].

Several meta-analyses contributed to demonstrate a copper decrease in the brain [48] and a copper increase in the periphery [56 , 61–64], as well as the expansion of the plasma component of exchangeable copper not bound to proteins classically defined as copper not bound to ceruloplasmin (Non-Cp copper, also known as ‘free’ copper) [63]. Non-Cp copper has been the focus of a number of publications demonstrating its association with the disease severity [65, 66] and with other markers of AD [67, 68] and its potential as diagnostic marker for AD [69, 70]. It has also a value in AD prognosis [68] and prediction of conversion from MCI to full dementia [71]. Recently, the existence of a copper subtype of AD, which can be distinguished by the ‘typical’ sporadic form of the disease for specific EEG and genetic features, was proposed [70 , 72–74]. Robust data demonstrate a cumulative effect of lead dose on trajectories of cognitive decline [75] and specifically the association of the tibia lead concentration with a worse cognitive performance in 50–70-year-old adults.

The most widely employed analytical methods for metal detection in serum are atomic absorption spectrometry (AAS), X-ray fluorescence spectrometry (XRF), inductively coupled plasma-mass-spectrometry (ICP-MS), and some colorimetric automated methods, as presented in a review describing the pros and cons of these techniques [76]. The growing interest of copper involvement in AD, prompted us and other groups to spend efforts in the detection of the exchangeable Non-Cp copper in serum [70, 77]. The low molecular weight plasma copper component is composed by amino oxidase, ferroxidase (II), SOD3, metallothioneins, a small quantity of metallothionein, a not well defined 45 KDa protein, along with small copper carriers (SCCs), which have a molecular weight in the range of 1-2 KDa [78], and exchanges copper with albumin (10–15%) and transcuprein (5–15%). This plasma component has the potential to play a role as hidden accelerator of the disease at least in a subpopulation of AD patients. In fact, it is toxic whenexceeding certain levels (normal ref values 0. 1–1.6μM [79]). The critical point is that in serum, if copper is not used for a catalytic function in cuproenzymes, it can enter Cu(II)/Cu(I) redox cycles and produce hydroxyl radical (OH; Fenton reaction) in a continuous manner. Table 1 reports the studies and methods developed in the latest years which attempted to isolate and measure the exchangeable fraction of copper in serum. Recently, a fluorescent method, measuring the Cu(II) specifically [70], has demonstrated the feasibility of separating the fraction of labile non-Cp Cu from total copper in serum (Table 1).

Table 1

Analytical methods for detection of Non-Cp copper

Analytical Method
Reference	Separation	Measurement	Description
Bohrer et al. [80]	Solid phase (SPE)	AAS	Retention of Copper bound to ceruloplasmin; measurements of non-Cp Cu
Squitti et al. [70]	Solid phase (SPE)	Fluorimetric	Retention of Copper bound to ceruloplasmin; measurements of non-Cp Cu complexed by a fluorescent probe
Bohrer et al. [80]	Ultrafiltration 20 KDa membrane	AAS	Ultrafiltration with membranes that retain Copper bound to Cp; measurement of non-Cp Cu
Mc Millin et al. [81]	Ultrafiltration 30 KDa	ICP-MS
El Balkhy et al. [82] El Balkhy [83]	Ultrafiltration 30 KDa	AAS
Boll et al. [84]	Ultrafiltration 3 KDa	AAS
Venelinov et al. [85]	Dyalisis	ICP-MS	Dialysis with histidine and chelex concentration to extract non-Cp Cu; measurement of non-Cp Cu
Lai et al. [86]	Ultrafiltration with membranes that retain Copper bound to Cp; measurement of non-Cp Cu	AAS
Inagaki et al. [87]	Size exclusion chromatography (SEC) and Chelex-100	ICP-MS	Albumin copper selectively removed from serum by adsorption on resin after chelating resin pretreatment; measurement of Non-Cp Cu

The experimental determination of Non-Cp copper is performed in two steps: the first consists in the separation of the copper bound to ceruloplasmin from the exchangeable copper [solid phase (SP) extraction or ultrafiltration). The second involves the measurement of copper levels, generally with AAS, ICP-MS or fluorescent probes.

CIRCULATING NANO-COMPARTMENTS: A PROMISING NOVEL SOURCE OF MATERIAL FOR MOLECULAR DIAGNOSTICS IN ALZHEIMER’S DISEASE

Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important means of intercellular communication by serving as transfer vehicles between cells of membrane and cytosolic proteins, lipids, DNA, and RNA and show a wide range of regulatory functions [88]. In the biomarker discovery field, the interest in EVs has expanded exponentially over the past decade in response to the finding that EVs are abundantly present in body fluids and carry molecular messages [89, 90]. Several pathogenic proteins that are involved in central nervous system diseases are released from cells in association with EVs [91]. Regarding AD, in 2006, Rajendran and colleagues provided the first evidence that Aβ peptides are released in association with exosomes. In addition, their finding that proteins associated with exosome (e.g., flotillins, Alix) are enriched in the amyloid plaques suggested that exosome-associated Aβ may be involved in the formation of plaques [92]. Subsequent studies demonstrated that full length AβPP, AβPP fragments, Aβ precursors, and members of the secretase complex, responsible for Aβ generation, are secreted within exosomes [93 –96]; thus, this nano-compartment seems to be actively involved in Aβ peptide generation and plaque formation. Cellular studies further supported the role of exosomes in AD pathogenesis: in murine primary neurons, we demonstrated that the over-expression of familial AD-associated presenilin 2 mutations inhibits the exosomal release of native and glycosylated cystatin C [95]. Since cystatin C is a neuroprotective growth factor and an inhibitor of Aβ aggregation and plaques deposition [97 –99], we hypothesized that a lack of exosome-mediated neuroprotection might occur in familial AD. Recent evidence indicated that exosomes carry on the surface molecules that can bind Aβ and Aβ assemblies (e.g., the cellular prion protein and glycosphingolipids) and that, by this interaction, they can influence Aβ aggregation and toxicity [100 –102]; however, whether exosomes, and in general EVs, promote or counteract Aβ toxic action remains controversial [103].

All these studies suggested that circulating EVs could represent a promising source of biomarker for molecular diagnostics in AD. The first pioneer studies were done on human CSF, a source of markers reflecting central neuropathologic features of AD. It has been reported that the concentration of microvesicles of microglial origin is increased in CSF from subjects with MCI and in AD patients. Of note, the concentration these microvesicles positively correlates with the CSF levels of t-tau and p-tau as well as with the atrophy of the hippocampus, classical markers of neurodegeneration [104, 105]. Moreover, microvesicles isolated from CSF of AD patients can promote extracellular formation of neurotoxic Aβ species [105]. In addition, the EVs purified from CSF of both AD patients and MCI subjects have elevated amounts of Aβ₄₂ (predominantly bound to the EVs surface) and can induce degeneration of cerebral cortical neurons [106].

Along with Aβ, other molecules associated with CSF-derived EVs were proposed as biomarker of AD. Tau protein is released in association with exosomes, mainly in its phosphorylated and aggregated forms [107, 108]: in human CSF-derived exosomes the percentage of p-tau₁₈₁, relative to t-tau, is increased in the very early stages of AD [107]. Moreover, the exosomes derived from human CSF of AD patients and control subjects can promote tau aggregation, and thus might contribute to the spreading of tau pathology [108]. Fatty acids were demonstrated to be compartmentalized differently in human CSF derived nanoparticles versus its soluble fraction: of interest, stage-specific changes in the fatty acid composition of EVs were reported in MCI and AD, suggesting a progression-related alteration in EVs transport of fatty acids in AD [109].

Expression profiling of microRNAs (miRNA) has emerged as a potential diagnostic tool in human disease. In AD, several studies reported an alteration of circulating and/or brain miRNA: since miRNA are secreted in cell-derived exosomes, few studies also investigated the role of exosomal miRNA as diagnostic biomarker [110]. One RNA expression study was carried out in human CSF derived-exosomes and revealed a significant abundance of miRNAs, mRNA transcripts, and long non-coding RNAs specifically present in Parkinson’s disease and AD patients [111]. An alteration of miRNA was also found in plasma-derived exosomes; specifically, the expression of seven miRNAs was highly informative in a model for predicting AD status with high accuracy [112].

There is evidence that EVs can cross the blood-brain barrier from the brain into the circulation. Thus, a method to isolate EVs enriched for neuronal origin was developed and applied for biomarker discovery in neurodegenerative diseases, and specifically in AD, where liquid phase of plasma/serum markers have often failed to show adequate sensitivity and specificity for clinical applications [113]. The application of this method allowed the identification of several potential biomarkers in exosomes isolated from blood. Levels of classical AD biomarkers (t-tau, p-tau, Aβ₄₂ in plasma/serum derived blood exosomes were nearly two orders of magnitude higher (tau and p-tau₁₈₁), or of similar in magnitude (Aβ₄₂), than the plasma/serum soluble forms. Of relevance, all biomarkers’ exosomal concentrations were higher in AD and the combination of p-tau and Aβ₄₂ could distinguish AD from controls with a sensitivity of 96% [114]. Moreover, significantly elevated exosomal levels of these proteins were detected in high-risk, but cognitively normal, subjects up to 10 years before clinical diagnosis of AD [114]. Conversely, in blood-derived exosomes several synaptic proteins were described to be reduced in AD, and their exosomal concentration was correlated with cognitive decline progression [115, 116]. The combination of exosomal classical biomarkers and synaptic biomarkers, e.g., neurogranin, can accurately predict the conversion of MCI to AD [117]. More recently, a method to enrich astrocytes-derived exosomes from blood was developed: interestingly, in these vesicles, only the level of Aβ₄₂, BACE-1, and soluble AβPP was specifically changed in AD and not in FTD [118].

Altogether these studies indicate that EVs, and specifically exosomes, are promising sources of innovative biomarkers for AD. The still-limited utility of exosomes in diagnostics is mainly due to difficulties in characterizing them using a scalable phenotyping method. In this view, we proposed a novel platform for exosomes analysis based on interferometric imaging [119]. We have developed a method that can detect individual nanovesicles on a capture surface in a microarray format and, potentially, assess their size [120]. Of note, this interferometric imaging method could capture nanoparticles, which have a size compatible with exosomes, from a very small volume (20μl) of human CSF, and thus we believe that it might accelerate the translation of exosome studies from research into clinics.

CONCLUDING REMARKS

In AD, several studies have consistently identified a specific CSF biomarker signature that reflects the neuropathological hallmarks of the disease. A set of CSF tests reflecting key aspects of AD pathology (amyloid plaques, axonal degeneration, and intraneuronal tangles) are available, and their performance in a clinical setting has been assessed in several studies.

Biomarker discovery of undetectable species because of their low level of expression is one of the biggest promises in AD research. The potential advantage of a blood-based test for biomarker discovery is obvious, since the source of the biomarker can be obtained routinely in primary care with a reduced invasiveness and increased patient acceptance [121]. Nevertheless, brain-specific proteins are present in blood at very low concentrations, making their reliable quantification using standard methods difficult. Recent technical breakthroughs have provided ultrasensitive methods that allow the dosage of brain-derived proteins in blood. In particular, plasma NFL seems to be a promising, noninvasive tool to assess neurodegeneration and a useful outcome measure for clinical trials. Regarding trace elements, plasma component of exchangeable copper not bound to proteins—which is toxic when exceeding certain levels—is expanded in AD; thus, in the last years efforts have been mainly spent on its detection in serum. Thus, important advances in our ability to detect the pathophysiological process of AD at the peripheral level have occurred. In addition, the use of ultrasensitive technologies allowed the identification of innovative biomarkers in human CSF including synaptic and microglial markers; in particular, the CSF neurogranin seems to be a good diagnostic and prognostic marker for early AD that is comparable to the core CSF biomarkers.

Recent studies have highlighted EVs as a promising source of material for molecular diagnostics. New methods have been developed to selectively capture brain derived-EVs from blood; these methods allowed the identification of several potential blood biomarkers.

A strong research effort is thus still needed to characterize new biomarkers in order to improve sensitivity and specificity of the future biological tests.

Footnotes

ACKNOWLEDGMENTS

This work was supported by the Italian Ministry of Health (Ricerca Corrente) and NET-2011-02346784 Italian Ministry of Health.

Authors’ disclosures available online ().

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