The Diagnostic Potential Of Amyloidogenic Proteins Part 3

4.5. Combined Detection

Neurodegenerative diseases share many symptoms and pathological mechanisms. In particular, aggregates of the same amyloidogenic protein can exist in people affected by different forms of neurodegeneration [151]. 

As people age, their physical health gradually shows signs of decline and aging. Among them, neurodegenerative disease is a serious disease that affects people's physical functions, intelligence level, and memory ability. However, we don't need to be frustrated and afraid because there are many ways to delay the occurrence of neurodegenerative diseases. At the same time, it is also very important to adjust our mentality and maintain a positive attitude towards life. This article will explore the relationship between neurodegenerative diseases and memory, and provide some effective ways to enhance memory so that we can have a healthy, happy, and fulfilling life.

Neurodegenerative diseases are diseases involving the nervous system, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. These diseases usually affect people's memory, manifested as forgetting recent things, repeating the same tasks and gradually losing control of daily affairs. To avoid the occurrence of these symptoms, we need to take some active measures, such as:

1. Keep good health. Good physical condition is one of the best ways to prevent neurodegenerative diseases. Doing moderate exercise can enhance physical function and maintain blood and oxygen supply to the brain. In addition, healthy eating habits are also very important, which can provide enough nutrition and energy to keep the body healthy.

2. Actively exercise the brain. If we want to maintain our memory, we need to exercise our brains regularly. This can include learning new things, games, and activities that challenge the brain's cognitive abilities, or even stimulating the brain by participating in social activities. These activities can promote the formation of new neural connections and keep the brain flexible and resilient.

3. Maintain a positive attitude towards life. Psychological factors also play a key role in the long-term prevention of neurodegenerative diseases. A joyful, positive, and optimistic person is often less affected by negative factors and is more likely to maintain good physical and mental health. A positive attitude towards life can promote new ways of paying attention and thinking, and help enhance memory.

In short, preventing neurodegenerative diseases is a long-term task that requires regular attention to one's physical and brain health. By maintaining a healthy diet, moderate exercise, and a positive way of thinking, we can enhance our inner strength and resistance and effectively prevent the occurrence of neurodegenerative diseases. At the same time, we should also maintain an optimistic, positive, and happy attitude and enjoy a healthy, happy, and fulfilling life. It can be seen that we need to improve our memory, and Cistanche can significantly improve memory because Cistanche can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche deserticola can improve blood flow and promote oxygen delivery, which can ensure that the brain obtains adequate nutrition and energy, thereby improving brain vitality and endurance.

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For example, Aβ and p-tau aggregates can also be found in patients with DLB, and α-syn aggregates in patients with AD. The co-occurrence of Aβ, α-syn, and tau suggests an overlap between AD, tauopathies, and synucleinopathies [152]. 

Amyloid aggregates can also be present in people who do not show any disease symptoms [151]. These observations lead to the conclusion that, in some cases, monitoring one individual amyloidogenic protein alone may not be sufficient for delivering an accurate diagnosis. 

Various studies have assessed the diagnostic performance of monitoring the levels of different amyloidogenic proteins together (Table 2). For example, the quantification of the CSF concentrations of both p-tau181, carrying the phosphorylated Thr181, and the ratio Aβ42/Aβ38 was able to differentiate AD from other neurodegenerative diseases [66,104]. 

A recent study conducted on a cohort of 4444 participants over 14 years was able to associate the plasma levels of NFTs and Aβ42 with the risk of developing AD and all-cause dementia [153]. Furthermore, the analysis of the CSF levels of both total α-syn and total tau could help the identification of synucleinopathies over other neurodegenerative diseases [68]. 

Recent work has shown that the determination of the ratio of oligomericα-syn/total α-syn, p-α-syn129, and p-tau181 in the CSF was able to identify PD patients from controls [154]. 

It has also been shown that an increase in glycation, α-syn Tyr 39 nitration, and pTyr125, and a decrease in SUMO-1 levels in blood samples were associated with PD [117]. 

Finally, it has been found that the concentration in the CSF of total TDP-43 and the ratio total tau/pThr181-tau discriminate ALS/FTD patients from healthy controls [155]. In Table 2, we summarize the main proteins which can be monitored in combination for diagnostic purposes.

5. Recent Advances in Detection Technology

Given their affordable costs and the possibility of easily making them high-throughput, immunoassays, such as ELISA and immunoblotting, have been the most commonly used techniques to quantify amyloidogenic proteins' concentrations in body fluids and tissues. 

Other approaches are also in use, including PET, MS, and microscopy, and novel detection technologies with ultra-sensitivity are emerging. An important example is provided by MS. 

A recently established capillary isotachophoresis–-electrospray ionization MS could detect picomolar concentrations of Aβ [157]. In another study, an automated clinical mass spectrometer could detect different Aβ variants in the CSF in a multiplex manner [158]. Noteworthy, both approaches were antibody-free and did not require an immuno-enrichment step. 

Aβ42/Aβ40 ratios in CSF could also be determined by LC-MS/MS assay with a high clinical sensitivity [159]. The benefits of MS include a small sample size, fast turnaround time, broad applicability, and sensitivity. 

Single-particle analysis of amyloids can be performed by microscopic methods including fluorescence, atomic force, and electron microscopy. Notably, fluorescence-based methods provide ultrasensitive detection of individual amyloid fibrils and oligomers in neurodegenerative diseases. Furthermore, super-resolution methods offer insight into structural properties and surface hydrophobicity [17]. 

Nanopore sensing is a non-optical technique that has recently been demonstrated to allow single-molecule analysis of polymeric proteins and could be extended to amyloids and oligomers [160,161]. 

In nanopore sensing, a biomolecule is translocated through a nanopore embedded within a thin dielectric membrane separating two chambers with electrolytes. Distinct conformations of the biomolecule can be characterized upon its translocation through the nanopore by the analysis of the change in the ionic conductance of the pore [162,163]. 

Other recent methods include amyloid seeding assays, such as protein misfolding cyclic amplification [164], and real-time quaking-induced conversion (RT-QuIC) [165]. In these assays, the composition and number of amyloids in biological samples are determined by the ability of these samples to induce the aggregation of a recombinant monomeric amyloidogenic protein using ThT-based aggregation measurements. 

These assays were established for several amyloidogenic proteins. Especially, the RT-QuIC assay for α-syn showed high diagnostic sensitivity for PD and DLB [165]. Both MS and microscopic techniques require sophisticated instrumentation. 

ELISA kits for several biomarkers including Aβ42, p-tau, total-tau, and α-syn are commercially available. However, ELISA can be labor-intensive to set up and with confined sensitivity. Various high-sensitivity ELISA techniques have been developed. For instance, researchers have measured total α-syn concentration in body fluids using immobilized lipids [166]. 

Also, ELISA has been coupled with other detection technologies, such as novel plate-based electrochemiluminescence [167]. This approach achieved markedly shortened processing time, with smaller sample volume requirements and simultaneous processing of multiple biomarkers [167]. Besides electrochemiluminescence, digital ELISA has also been developed with single molecule array (Simoa) technology. 

This method is reported to have increased sensitivity for Aβ42 detection in the human plasma (in the pM range) [168]. Moreover, a surface-based fluorescence intensity distribution analysis (sFIDA) assay was established resembling a sandwich ELISA where Aβ oligomers were immobilized on the functionalized glass surface via antibodies, imaged by high-resolution fluorescence microscopy [169]. 

The Multi-Analyte Profiling (xMAP) platform stands out from a wide range of approaches based on its multiplexing capability. 

Simultaneous quantification of up to 100 samples in a single assay could be achieved on a semi-automated assay. Studies provided that xMAP data for total tau, p-tau, Aβ40, and Aβ42 correlated well with research-based ELISA values with higher sensitivity and specificity [170].

Immuno-polymerase chain reaction (I-PCR) utilizes real-time PCR (also known as quantitative PCR) to combine nucleic acid amplification with antibody-based assays to increase the 10 to 109-fold sensitivity of conventional immune assays. 

Researchers quantified multiple phosphorylated tau epitopes using I-PCR in CSF [171] and developed a nano-I-PCR approach that utilized gold nanoparticles functionalized with a tau-specific monoclonal antibody for total tau quantification in CSF samples [172]. The level of total Aβ40 present in microdissected neurons could also be quantitated using I-PCR with high sensitivity and detection range [173]. 

This technique is suitable for small sample volumes, provides rapid time to results, and can be amenable to multiplexing. Point-of-care (POC) diagnosis is, undoubtedly, an emerging trend. 

This approach allows conventional ELISA, Luminex xMAP, and qPCR to be developed into inexpensive, portable, and easy-to-use POC devices [174]. Paper-based ELISA is the simplest option for POC diagnostics.

6. Conclusions and Potential Future Directions

Disease biomarkers represent an essential requirement for the development of accurate diagnostic approaches. In this review, we have discussed how amyloidogenic proteins hold potential as biomarkers for neurodegenerative diseases and described detection technologies to assess their concentrations in the body (Figure 3). 

Besides genetic mutations, many PTMs and specific conformations of amyloidogenic proteins are associated with disease and are emerging as potential biomarkers (Tables 1 and 2). In this context, noteworthy among all aggregated conformations are the oligomers, which are highly toxic and regarded as major players in the disease onset and progression. 

The use of amyloidogenic proteins as biomarkers comes with challenges. Firstly, neurodegenerative diseases share some key pathological mechanisms, including the formation of aggregates by the same amyloidogenic protein (e.g., Aβ and p-tau deposits can be found in patients with DLB) [152]. 

This makes it difficult to distinguish one form of neurodegeneration from another based on the detection of one specific amyloidogenic protein alone. Furthermore, amyloidogenic proteins are difficult to access within the CNS and their concentrations in inaccessible body fluids have noticeable fluctuations, particularly at the early stages of the disease. 

In our opinion, promising diagnostic strategies that may overcome these issues are those based on the detection of multiple amyloidogenic proteins or protein features (Section 4.5). 

Additionally, amyloidogenic proteins could be monitored in conjunction with other types of biomarkers, such as metabolites. Recent investigations have shown that metabolic pathways are affected by neurodegeneration, and detection platforms have been developed for metabolic profiling [175]. 

For example, nuclear magnetic resonance and MS have been successfully employed to determine metabolic changes in cellular systems [176], post-mortem brain samples [177], and CSF from patients [178]. 

Combined approaches could also involve neuropsychological assessment and neuroimaging. Furthermore, amyloidogenic proteins can also be detected in regions of the body besides the CNS and body fluids. 

For example, aggregated forms of α-syn have been found in the digestive system of PD patients [179]. The diagnostic relevance of this finding is twofold: it shows that other parts of the body can be examined for potential biomarkers of neurodegeneration [180]; it also implies that patients may show symptoms/disorders that are unrelated to the neurodegenerative condition but instead could be used for early diagnosis. 

Sensitivity and specificity are important attributes of detection technologies for amyloidogenic proteins. In Sections 4.3 and 5, we described several promising approaches which are currently under development. 

These are based on biosensors, single-molecule detection, and molecular probes (e.g., antibodies). In our view, antibody-based approaches in particular hold great potential as they allow detection in complex mixtures. Moreover, antibodies can be developed to target different protein features, including PTMs and conformations (e.g., the oligomers).

In conclusion, amyloidogenic proteins are appealing potential biomarkers of neurodegeneration. Their diagnostic success is intertwined with the development of combined detection strategies, involving other types of biomarkers, organ systems, and ultra-sensitive technologies.

Author Contributions: Writing-original draft preparation, Y.J., D.M.V., D.G., Y.G., R.T., J.T.W., F.A.A.; writing-review and editing, Y.J., D.M.V., D.G., Y.G., R.T., J.T.W., F.A.A. All authors have read and agreed to the published version of the manuscript.

Funding: We thank UK Research and Innovation (Future Leaders Fellowship MR/S033947/1), the Alzheimer's Society, UK (Grant 511), and Alzheimer's Research UK (ARUK-PG2019B-020) for support.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Acknowledgments: Figures were created with ChemDraw v19.1 and Biorender.com (accessed on 5 April 2021).

Conflicts of Interest: F.A.A. is an inventor of patent PCT/GB2020/051965.

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