The Diagnostic Potential Of Amyloidogenic Proteins Part 1

Abstract: 

Neurodegenerative disorders are a highly prevalent class of diseases, whose pathological mechanisms start before the appearance of any clear symptoms. This fact has prompted scientists to search for biomarkers that could aid early treatment. 

These currently incurable pathologies share the presence of aberrant aggregates called amyloids in the nervous system, which are composed of specific proteins. 

In this review, we discuss how these proteins, their conformations, and modifications could be exploited as biomarkers for diagnostic purposes. 

Protein is one of the important substances for maintaining good health, and it also has an important impact on memory. Protein is composed of amino acids, which are the raw materials for brain cells and neurotransmitters. Therefore, protein intake is closely related to memory.

First of all, protein is an essential substance for the body to build and repair tissues. Brain cells are rich in enzymes and regulatory substances, which require protein as a component to function normally. Too low protein content in the body will cause brain cells to not function normally, thus affecting memory. Therefore, maintaining adequate protein intake plays an important role in brain development and memory maintenance.

Secondly, protein has the effect of enhancing brain cell activity. The human body needs a large amount of brain neurotransmitters to maintain the normal operation of functions such as thinking, memory, and behavior. Brain neurotransmitters are synthesized from amino acids in the body, and the source of these amino acids is protein. Therefore, choosing foods rich in high-quality protein can increase the synthesis of brain neurotransmitters, thereby enhancing brain cell activity and helping to improve memory.

In addition, protein also helps maintain the stability of the body and prevent blood sugar fluctuations. Low blood sugar levels can cause symptoms such as inattention, dizziness, and fatigue in the body, which often affect memory performance. By maintaining a moderate intake of protein, blood sugar fluctuations can be effectively avoided, which helps maintain the stability of the body and improve memory.

In short, protein is closely related to memory. A moderate intake of protein can maintain the stability of the body, enhance the activity of brain cells, and thus improve memory. We should try to eat more high-quality protein-rich foods, such as fish, meat, beans, and dairy products, and avoid completely removing protein from the diet. This will help us stay healthy, improve our memory, and enjoy a better life. It can be seen that we need to improve our memory, and Cistanche can significantly improve memory because Cistanche is a traditional Chinese medicine with many unique effects, one of which is to improve memory. The efficacy of Cistanche comes from the various active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc., which can promote brain health in many ways.

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We focus on proteins that are associated with the most prevalent neurodegenerative disorders, including Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, and frontotemporal dementia. 

We also describe current challenges in detection, the most recent techniques with diagnostic potential,s and possible future developments in diagnosis.

Keywords: neurodegenerative diseases; biomarker; amyloid; oligomer; post-translational modification.

1. The Need for Novel Diagnostic Approaches for Neurodegeneration

Neurodegenerative diseases are fatal and incurable disorders, characterized by the progressive loss of neurons in specific regions of the nervous system. They are a highly heterogeneous group of pathologies, which include Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS). 

Currently, worldwide, more than 50 million people suffer from various forms of neurodegeneration [1]. The clinical course of neurodegenerative diseases usually spans several years and leads to progressive deficits in memory, cognition, and movement to different extents depending on the specific pathology [2]. Existing drug treatments focus on the relief of these symptoms [3]. 

Furthermore, neuropsychological assessment is still considered crucial in the diagnosis of neurodegeneration associated with dementia [4]. However, key molecular mechanisms of disease occur before the appearance of any significant symptoms. 

The development of accurate diagnostic approaches would facilitate timely therapeutic interventions to restore neuronal physiology before irreversible damage occurs. 

It would also promote the establishment of new therapeutics, and the revaluation of current ones which could be more effective if administrated at earlier stages. Despite the urgent need for diagnostic approaches for neurodegeneration, their development is still a daunting challenge, due to the limited accessibility of the brain for physical examination and the complexity of clinical tests based on cognitive abilities [2]. 

Recent technological advances have enabled the characterization of novel pathways, biomolecules, and structures in the nervous system and other regions of the body that could be used as disease markers of neurodegeneration [5,6]. 

Although the different neurodegenerative diseases have some distinct phenotypes, they also share some key molecular features. In particular, in many of these disorders, specific proteins and peptides, which would normally be soluble, undergo a self-assembly process that leads to the formation of large fibrillar aggregates, called amyloids [7,8]. 

This process also involves the generation of smaller oligomeric intermediates, which are highly toxic and currently regarded as major players in the disease mechanisms [8]. Amyloid aggregation is also linked to other aggregation processes, such as the formation of condensates [9]. However, these will not be the focus of the present review. 

Here, we describe some of the most known amyloidogenic proteins and their diagnostic relevance. 

We focus on amyloid-beta (Aβ) and tau for AD, α-synuclein (α-syn) for PD, fused in sarcoma (FUS), and the TAR DNA-binding protein 43 (TDP-43) for ALS and FTD. We also discuss state-of-the-art advancements in detection approaches to monitor the aggregation of these proteins.

2. Amyloid Aggregation as Potential Source of Biomarkers

Amyloids are insoluble fibrillar aggregates enriched in a cross-β structure, and their formation has been extensively characterized in vitro [7,8]. 

Amyloid aggregation consists of a complex network of nucleation events. Initially, soluble monomeric proteins interact and form oligomers by primary nucleation. Primary nucleation can also be triggered by the presence of other biomolecules, such as other proteins [10], nucleic acids [11,12,] and membranes [13]. 

Oligomers then convert into higher-order aggregates and, finally, into amyloid fibrils. Once a critical concentration of fibrils has formed, the surface of these fibrils catalyzes the formation of additional oligomers by secondary nucleation [7,8]. Fibrils can also elongate by the addition of monomers at their ends, and undergo fragmentation [7,8]. Amyloid fibrils are extremely stable protein species due to their rich cross β-sheet content [8,14–16]. 

On the contrary, oligomers rapidly convert into higher-order amyloid aggregates (Figure 1). The transient nature of oligomers makes them difficult to isolate and characterize at the structural level. 

Only recently, thanks to new approaches such as single-molecule fluorescence and electron microscopy [14,17,18], has it been shown that oligomers are highly heterogeneous in their physiochemical properties and structures, with varying secondary structure content [16,19,20].

Oligomers are toxic by a multitude of mechanisms including aberrant hydrophobic interactions. It has been shown that, in tissue and isolated cells, oligomers can affect membrane permeability, and ion homeostasis, and induce oxidative stress [16,21–23]. 

Oligomer-induced free radicals can then trigger protein misfolding, mitochondrial dysfunction, and eventually apoptosis [24]. Oligomers of Aβ and α-syn have also been associated with neuroinflammation [22] and synapsis loss [25,26]. 

Several factors can affect the formation of amyloid fibrils and oligomers. These include genetic mutations, cellular stress, and the presence or absence of specific biomolecules. 

Furthermore, amyloids are extensively post-translationally modified in vivo [27–29] and post-translational modifications (PTMs) significantly alter the formation and the toxicity of amyloid fibrils in vitro [27–31].

3. Amyloidogenic Proteins Involved in Neurodegeneration

In the context of neurodegeneration, disease markers can be divided into neuropsychological, neuroimaging, genetic,c, and biochemical markers [2]. In particular, biochemical markers (or biomarkers) are measurable molecules in our body (e.g., proteins, nucleic acids, metabolites), which report the stage of a disease [32]. 

Amyloidogenic proteins are promising biomarkers, as they inform on the biochemical profile of the nervous system dysfunction [2]. Below, we highlight relevant amyloidogenic proteins and their pathological modifications which can serve as biomarkers for neurodegenerative conditions.

3.1. Aβ and Tau in AD

AD is the most prevalent form of dementia. The characteristic lesions in AD brains are extracellular senile plaques composed of amyloid aggregates of Aβ and intracellular neurofibrillary tangles (NFTs) formed by paired helical amyloid filaments (PHFs) of hyperphosphorylated tau (p-tau) protein [2,27]. 

Aβ is a short peptide generated by the cleavage of a larger transmembrane precursor, called the amyloid precursor protein (APP), by the sequential cleavage of the β- and γ-secretases and released into the extracellular space [2,33]. 

This process can generate Aβ isoforms of various lengths (Table 1), which have various degrees of toxicity in the context of AD [34]. The most common Aβ isoforms are the 40- and 42-residue long ones, generally referred to as Aβ40 and Aβ42, respectively. Aβ40 is the most abundant variant in the plaques (~80% to 90%) and is also present in healthy people's brains. 

Aβ42 has a much higher propensity to aggregate, and an increase in Aβ42/Aβ40 ratios is associated with AD and other forms of dementia [30,34] (Table 2). Besides cleavage, genetic mutations (A692G, E693Q in the APP gene [35,36], Table 2) and many other PTMs of Aβ have been associated with AD, including oxidation, phosphorylation, glycosylation and isomerization [30]. 

Studies show the occurrence of acetylation (e.g., Lys16 and Lys28), phosphorylation (e.g., Ser8 and Ser26), nitration (e.g., Tyr10), pyroglutamate (e.g., Glu3 and Glu11), isomerization (e.g., Asp1 and Asp7) and racemization (e.g., Asp1, Asp23, and Ser26) in the context of disease [30,37–39] (Table 1). 

It is worth noting that cognitive decline correlates more with soluble intermediate forms of Aβ rather than the degree of amyloid deposits [16]. Tau is a major microtubule-associated protein that stabilizes the microtubules in neurons [2,40]. In human brains, tau exists as six different isoforms that carry either three or four microtubule-binding repeats (R). These isoforms are called 3R and 4R, respectively. 

It has been found that the presence of either 3R or 4R or both 3R and 4R amyloids isdisease-specificc. As an example, in AD, ALS, FT, D, and Parkinsonism, both 3R and 4R amyloids are present, while in corticobasal degenerations and Pick's disease only 4R and 3R amyloids, respectively, are found [41,42]. Tau undergoes PTMs, particularly phosphorylation [40]. 

Pathological hyperphosphorylation reduces tau affinity for microtubules and causes their detachment from microtubules, resulting in the formation of PHFs and NFTs [2]. 

To date, 85 potential phosphorylation sites of tau have been identified [40]. Furthermore, molecular and cellular studies revealed that acetylation (e.g., Lys174, Lys274 and Lys280), oxidation (e.g., Cys322), nitration (e.g., Tyr29), glycation (e.g., Lys87, Lys132 and Lys150), truncation (e.g., at Asp13 and Asp421 and Glu391) and ubiquitination (e.g., Lys48 and Lys63) also affect tau aggregation [27] (Table 1).

3.2. α-Syn in PD

Unlike AD, PD primarily affects the motor system, causing tremors, rigidity, bradykinesia, and postural instability [2]. The pathological hallmark of PD is the occurrence of cytoplasmic amyloid inclusions, known as Lewy bodies (LBs) and Lewy neurites (LNs). LBs and LNs are comprised of amyloid aggregates, whose main component is α-syn [43].

α-Syn contains 140 residues with a positively charged N-terminal region, an aggregation-prone non-amyloid-β component (NAC) central region,n,n, and a negatively charged C-terminal region [44,45]. 

Duplications or triplications on the α-syn chromosome region (4q21-23) and mutations including A53T, G51D, H50Q, E46K, and A30P in the α-syn sequence, are associated with early-onset PD [46–48] (Table 2). α-Syn belongs to a protein family which also includes β- and γ-synucleins with 55%–62% similarity. β-Synuclein has a reduced propensity to aggregate and has been discovered to suppress the aggregation of α-syn as a natural inhibitor while oxidized γ-synuclein can initiate α-syn aggregation [49,50]. 

Several PTMs are known to affect the aggregation of α-syn, are associated with PD [28,], and hold diagnostic potential [51] (Table 1). 

These include N-terminal acetylation, several truncations at the N-terminus (e.g., α-syn7-140, 14-140, 40-140, and 72–140 found in vitro, 5–140 and 68–140 found in vivo and several in both) and C-terminus (e.g., α-syn1-115, 1-119, 1-122, 1-124, 1-125, 1-129, 1-133, and 1-135), phosphorylation of Ser87 and Ser129, oxidation of Met1, Met5, Met116 and Met127, sumolyation of Lys96 and Lys102, nitration of Tyr39, Tyr125 and Tyr133, and ubiquitination of Lys6, Lys10, Lys12 Lys21, Lys23, Lys43 and Lys96 [28,52,53] (Table 1).

3.3. TDP-43 and FUS in ALS and FTD

ALS and FTD are neurodegenerative diseases with overlapping mechanisms. ALS affects upper and lower neurons, causing loss of muscle control. FTD is a form of dementia linked to the degeneration of the frontal and anterior temporal lobes [54]. 

Around 97% of ALS and 45% of FTD cases are associated with the presence of inclusions of aggregates of ubiquitinated, hyperphosphorylated and C-terminally truncated TDP-43 in the cytoplasm of neurons and glial cells [29]. 

TDP-43 is a 414 residue-long ribonucleoprotein able to form amyloid-like aggregates in vitro and condensates (i.e., stress granules) [9] under pathological conditions. It is composed of an N-terminal tract with a nuclear localization signal, two RNA recognition motifs, a nuclear export signal, and a disordered C-terminal region [29]. 

All these regions have been reported to play a critical role in the aggregation of the protein [55–57]. Several TDP-43 mutations have been identified in both sporadic and familial cases of ALS and FTD, including G294A, Q331K, M337V [58], and K181E [59] (Table 2). As with Aβ and α-syn, PTMs of TDP-43 also play a key role in the aggregation of the protein and disease progression. 

It is worth noting that the truncated 25 kDa and 35 kDa C-terminal fragments are commonly found in pathological aggregates in ALS patients [29,31,54]. Ubiquitination is also a typical modification of TDP-43 inclusions [31]. Finally, aberrant phosphorylation, acetylation, and oxidation of TDP-43 are often associated with the mislocalization and aberrant aggregation of the protein [29] (Table 1). 

ALS and FTD are also associated with another RNA/DNA-binding protein, FUS. FUS is a 526 residue-long protein made by an N-terminal transcriptional activation domain and C-terminal domain, which interacts with transcriptional factors and also includes a nuclear localization signal [60,61]. 

Both domains contain low-complexity regions and play a role in the formation of condensates and hydrogels [62]. More than 50 mutations (e.g., R521C, R521H [63]) of FUS are reported in ALS/FTD cases. 

Unlike TDP-43, FUS is generally found as a full-length protein in the aggregates [61]. Phosphorylation occurs in the prion-like domains of FUS and has been shown to affect its phase separation and aggregation pattern while mutations and PTMs (mainly methylation and phosphorylation) on its C-terminal domain are found to regulate its nuclear/cytoplasm localization [61,64]. 

Despite the clear pathological role of FUS, ALS/FTD phenotypes are less frequently associated with FUS than with TDP-43 dysfunctions [61,65]. Thus, the role of FUS as a biomarker remains to be determined.

4. Diagnostic Potential of Genetic, Structural, and Chemical Features of Amyloidogenic Proteins

Several detection approaches have been used to quantify amyloidogenic proteins in biological samples and to determine the link between these proteins and neurodegenerative diseases. 

Some of these strategies aim at quantifying the changes in the expression levels/concentration of amyloidogenic proteins regardless of their conformation or modification (Table 2). Some other approaches, instead, focus on probing specific structural (e.g., aggregated states) or chemical (i.e., PTM) properties of amyloidogenic proteins (Table 1). 

All these approaches analyze different regions of the body. Techniques, such as positron emission tomography (PET), can probe proteins directly within the central nervous system (CNS), for example, in the brain [2,44,66]. 

However, amyloidogenic proteins can be also detected in other more accessible regions of the body. In this section, we discuss findings that have been obtained from the analysis of both brain tissues and accessible body fluids, mainly using immunoassays, such as immunoblotting and enzyme-linked immunosorbent assays (ELISA). 

The fluids under consideration include the cerebrospinal fluid (CSF), which is in direct contact with the extracellular portion of the brain and, as such, is an optimal fluid for measurements of brain metabolism [44].

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