The Diagnostic Potential Of Amyloidogenic Proteins Part 2

4.1. Levels of Amyloidogenic Proteins in Neurodegeneration

So far, the most common detection strategies probe specific amyloidogenic proteins, regardless of their conformations or PTMs, providing information on the total amount of amyloidogenic protein in disease conditions. 

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These methods are particularly effective when the pathological mechanism depends on genetic mutations (e.g. when a genetic variant of a protein is specifically expressed in patients, Table 2), protein expression levels (e.g., when protein expression is significantly altered in patients), or mislocalization (e.g. when a protein accumulates in specific regions of the body in patients). 

In the case of AD, initial experiments focused on measuring the CSF concentration of total Aβ. However, no significant differences between patients and controls were found [33]. In contrast, significant differences have been observed for total tau. 

Researchers found a marked increase in total tau in the CSF of AD patients compared to healthy controls [67]. Although there is a clear link between the concentration of tau in the CSF and AD, higher concentrations of tau are also observed in acute stroke, brain trauma, and other forms of dementia [66,68], making disease-specific diagnosis challenging [44,66]. 

In the case of PD, it has been found that α-syn is present at slightly lower concentrations in the CSF of patients compared to healthy individuals in small cohort studies [68–70]. This has been observed also in plasma and saliva. 

In larger studies, CSF α-syn has a significantly lower concentration than healthy controls [69]. This lower concentration of α-syn inaccessible body fluids may be a consequence of the intracellular aggregation of the protein [69]. However, the use of α-syn alone is not sufficient as a single biomarker for PD diagnosis [69]. Due to its low concentration, TDP-43 is challenging to reliably measure in the blood [71,72]. 

However, in the CSF, it has been shown that TDP-43 concentration is higher in ALS patients (6.92 ± 3.71 ng/mL) than in healthy subjects (5.31 ± 0.94 ng/mL) and people affected by other conditions, for example, PD, multiple sclerosis, and Guillain-Barré syndrome [73]. 

Recent studies have suggested that TDP-43 present in the CSF could be a biomarker for the diagnosis of ALS, ALS/FTD but not for FTD alone [74,75]. In particular, it should be noted that proteins, including TDP-43, can diffuse from the blood into the CSF. 

Furthermore, TDP-43 is also abundant in other parts of the body besides the brain [54,76]. Thus, TDP-43 in the CSF may originate from other body regions and may not adequately represent the brain pathology in FTD. Currently, research efforts are focusing on monitoring TDP-43 contained in CSF exosomes, which are brain-derived and thus better represent brain pathology [77]. 

Overall, so far, despite compelling evidence of the fact that protein expression and concentrations are altered in disease, probing total protein concentration alone has not yielded a widely adopted diagnostic approach.

4.2. PTMs Associated with Neurodegeneration

As previously mentioned, PTMs affect the aggregation propensity of amyloidogenic proteins and play a critical role in the pathological mechanism of neurodegenerative diseases [27,28]. 

Furthermore, PTMs are also found in biological samples from patients [27–29]. Aberrant PTMs may occur well before clinically observable symptoms. This observation makes PTMs potentially suitable to be used as biomarkers for early diagnosis (Table 1).

4.2.1. Fragmentation and Cleavage

Protein cleavage is a PTM commonly associated with amyloid aggregation and has been extensively investigated both in vitro and in vivo. In AD, for example, several APP fragments have been linked to the disease, Aβ42 and Aβ40 being the most extensively investigated ones. However, other forms of Aβ also hold a diagnostic potential, including Aβ37, Aβ38, Aβ39, and Aβ43 [32]. 

It has been found that the CSF concentration of Aβ42 is lower in AD patients than in controls in approximately 50% of cases [78]. 

However, currently, the CSF concentration ratio Aβ42/Aβ40 is considered diagnostically more accurate, as it takes into account the physiological inter-individual protein expression fluctuations [78,103]. 

Besides Aβ40 and Aβ42, the concentration of Aβ43 is lower in the CSF of patients with early-onset AD, but also in patients with mild cognitive impairment and other forms of dementia [79]. 

On the contrary, Aβ38 is present at higher concentrations in AD patients and specifically discriminates AD from other forms of dementia [66]. Thus, the ratio Aβ42/Aβ38 may correlate with different types of dementia and be used in combined detection diagnostic approaches [103,104]. 

Despite α-syn and TDP-43 also being able to undergo fragmentation, which plays a role in their pathological mechanisms, very few studies had detected truncated α-syn and TDP-43 isoforms in body fluids, and their roles as potential biomarkers remained unknown [28,54,105,106].

4.2.2. Phosphorylation

In Aβ, the phosphorylation of Ser8 and Ser26 is believed to increase the abundance and stability of toxic aggregates. Enrichment of these modifications in oligomeric Aβ species, compared to monomers, has been shown using phosphor-specific mono- or polyclonal antibodies [80,81]. 

As a consequence of hyper-phosphorylation, tau dissociates from the microtubule network and aggregates. Therefore, tau phosphorylation is considered a key event in the formation of NFT [27]. 

In recent years, several antibodies have been developed to specifically recognize p-tau and a variety of ELISA protocols have been developed for different phosphorylated epitopes. 

Recently, p-tau at Thr217 (p-tau217) was shown to be a robust plasma [86] and CSF [107] biomarker for AD as it distinguished AD from other neurodegenerative diseases including other tauopathies. Similarly, α-syn is also phosphorylated at multiple residues. Noteworthy to mention is the variant of α-syn carrying the phosphorylation of Ser129 (p-α-syn129). 

This modification occurs by multiple kinases and affects the aggregation and the toxicity of α-syn both in vitro [95,96] and in vivo [108]. Mass spectrometry (MS) and antibody-based experiments have found that LBs isolated from PD patients' brains are enriched in p-α-syn129. 

They have also shown that the concentration of p-α-syn129 in body fluids is higher in PD patients and correlates with the disease severity [109,110]. The quantification of p-α-syn129, along with total α-syn, in the CSF was suggested to aid the modeling of PD and potentially the differential diagnosis of PD from other Parkinsonisms [110,111]. 

Several antibodies that are specific for phosphorylated TDP-43 (e.g., at Ser409 and Ser410) have been developed. 

However, TDP-43 undergoes several other modifications, and probing only the phosphorylation state of the protein does not accurately describe the disease state. Detection approaches currently under development involve the combined use of different antibodies targeting different modifications and conformations of TDP-43 [75,77].

4.2.3. Acetylation

Acetylation is another major PTM involved in AD. It has been reported that the overall acetylation levels of Aβ42- and tau- aggregates are significantly higher in AD hippocampus compared to healthy subjects [112]. 

Furthermore, several studies have shown that the acetylation of Lys16 and Lys28 of Aβ both alter the aggregation of the peptide in vitro [38]. In particular, the acetylation of Lys16 leads to the formation of amorphous aggregates instead of amyloids [38,39]. 

Tau undergoes acetylation at various residues. In particular, acetylation at Lys174, Lys274, and Lys280 have received the most attention as these were shown to also regulate the function of the protein [87]. 

Among these modifications, Lys280 was specifically found acetylated in transgenic mice and patients' hippocampus tissues [87]. In the case of PD and dementia with LBs (DLB), it has been reported that brain tissues (temporal cortex and dorsolateral prefrontal cortex) from patients contain soluble and aggregated N-terminally acetylated α-syn [113]. 

N-terminal acetylation has been found to significantly alter the structure of monomeric α-syn in vitro. This PTM induces the helical folding of the N-terminus of α-syn [97], which has been suggested to promote the pathological association of the protein to membranes [114]. TDP-43 is another protein that undergoes acetylation. 

This PTM abrogates RNA binding and promotes the aggregation of the protein. In particular, acetylated TDP-43 at Lys145 could be detected in lesions in ALS but not FTD patients, suggesting that TDP-43 acetylation was linked to ALS pathogenesis and could be used for discriminating ALS from FTD, which is helpful as a biomarker if easily detectable in body fluids [29].

4.2.4. Oxidation and Nitration

Oxidative stress has been described as a contributing factor in neurodegenerative diseases [115]. In AD, oxidation of Aβ at Met35 was detected post-mortem in the occipital cortex in AD patients [116]. 

However, this modification was shown to slow down the aggregation of the protein in vitro [30,82] and further investigations will be needed to clarify the exact role in the disease. Instead of targeting oxidized amyloidogenic proteins, current diagnostic approaches mainly focus on measuring oxidative stress biomarkers (e.g., oxidation products and specific metabolites) [115]. 

However, these biomarkers are not specific for neurodegeneration, as oxidative stress is also induced by inflammation and in the context of various pathologies, including cancer [115]. The development of antibodies directed against oxidized amyloidogenic proteins, such as tau and TDP-43, coupled with highly sensitive approaches may help diagnosis. 

Nitration of Tyr residues has been found in several amyloidogenic proteins from patients' samples (e.g., Tyr10 in Aβ, Tyr29 in tau, Tyr39 in α-syn) [28,30,37,89]. 

In particular, it has been found that nitrated α-syn levels in peripheral blood cells (monocytes and erythrocytes) are specifically higher in PD patients. Thus, this modification is currently regarded as a potential diagnostic biomarker, although larger-scale studies are required [99,117]. 

Nitrosative stress molecules are currently assessed as potential biomarkers. However, also in this case, they are not specific for neurodegeneration, and further investigation is needed [118].

4.2.5. Glycosylation

Studies found short Aβ O-glycopeptides (Aβ1-15 to 20) in AD CSF samples and suggested that they could be useful biomarkers, and the glycopeptide profiles and concentrations need to be confirmed in much larger prospective studies [119] (Table 1). 

Tau has also been shown to undergo N- and O-glycosylation. Interestingly, O-glycosylation plays a protective mechanism against hyperphosphorylation and PHF formation [91]. Lower levels of O-glycosylated tau are found in AD brains compared to healthy controls [91]. 

In contrast, N-glycosylation was shown to facilitate tau hyperphosphorylation, thus promoting its accumulation in PHF. N-glycosylation has been found in human AD brains, but not in control brains, which could be a relevant modification for diagnosis [83]. 

In PD, O-linked glycosylated with N-acetylglucosamine of α-syn was also found in vivo [100]. 

Diagnostic approaches based on the detection of glycosylated amyloidogenic proteins are still largely unexplored and new techniques are under development. The assessment of glycosylation patterns in patients includes N-glycome profiling using glycoblotting and MS [120,121].

4.2.6. Ubiquitination

Ubiquitin labels misfolded proteins and targets them for degradation through the ubiquitin-proteasome system. It is believed that abnormal accumulation of amyloids might result from the alteration of the ubiquitin-proteasome pathway [122]. 

It has been found that ubiquitin is present at higher concentrations in AD patients' cortical tissues compared to healthy controls [122,123]. Although early studies have shown that the majority of the ubiquitination occurs when tau is aggregated in the tangles, ubiquitin has also been found in soluble tau, for example, via Lys63 conjugation [92,93]. 

Notably, Lys48 and, particularly, Lys63-linked polyubiquitination, as well as monoubiquitin modifications were found to contribute to the biogenesis of the tangles. 

Further analyses could focus on the detection of Lys63 ubiquitinated tau to evaluate its diagnostic value [94]. α-Syn is found, mono-, di- and tri-ubiquitinated, in LBs from the cortical tissues of autopsied DLB patients' brains [124]. 

While ubiquitination at Lys6, Lys12, Lys21, Lys43, and Lys96 has been shown to have an inhibitory effect on fibril formation, ubiquitination at Lys10 and Lys23 has the opposite effect and could have a pathological role. 

However, their presence in body fluids and connection with PD pathology remain unknown [28]. Ubiquitinated TDP-43 is a major component of the aggregates in about 50% of FTD cases and 97% of ALS cases, and appears to be downstream of phosphorylation. Nevertheless, the effects of ubiquitination on TDP-43 aggregation still need to be further clarified [29]. 

Currently, potential diagnostic approaches focus on measuring free nonconjugated ubiquitin rather than ubiquitinated proteins in the CSF [125]. It has been observed that the amount of ubiquitin in the CSF reflects the number of protein aggregates in the brain and could serve as a biomarker [125,126].

4.3. Amyloid Aggregates and Amyloid-Sensitive Probes

Currently, increasing research efforts are focusing on generating biomolecular tools to target specific aggregate conformations (Figure 2). The rationale underlying this strategy consists of the fact that amyloidogenic proteins are generally poorly toxic when monomeric, while their toxicity significantly increases when they are aggregated [13]. 

Recent evidence shows that oligomers are particularly toxic [13], thus, to monitor the progression and severity of neurodegenerative diseases, one could specifically quantify the concentration of the amyloids and/or of the oligomers (Table 2, Figure 3). 

In this context, several probes for amyloids and amyloid-like aggregates have been developed, including small molecule fluorescent probes, conformation-selective antibodies, and aptamers [127–130]. 

In particular, single-molecule studies using amyloid-sensitive fluorescent molecules were able to probe individual soluble aggregates present in the body fluids of patients. 

To do so, small molecules, such as thioflavin T (ThT), which increase their fluorescence upon binding to the cross-β-sheet component of the amyloids, have been used. Using this approach, researchers managed to quantify soluble amyloid-like aggregates in AD and PD CSF samples [131,132]. 

Besides ThT, other molecules with similar fluorescent properties have been developed, including molecular rotors [133,134], such as thioflavin X [135].

In addition to these small molecule probes, high-specific biomolecules have been developed, such as antibody fragments and aptamers able to probe oligomers of different amyloidogenic proteins [127,136,137]. 

Recently, super-resolution microscopy with amyloid-specific aptamer and DNA point accumulation in nanoscale topography was able to detect aggregated α-syn and Aβ in samples from PD patients without the need for a conjugated fluorophore [138]. 

Traditionally, antibodies have been generated using well-established techniques in vivo by immunization and hybridoma technologies [139,140] or in vitro using display technologies [141]. 

The emergence of novel in silico antibody discovery methods has allowed the generation of antibodies in a cost- and time-effective manner [142]. AbDesign, a structure-based algorithm that utilizes combinatorial and energy-based design, has been successful in designing antibody fragments that target insulin and mycobacterial acyl carrier protein [143]. 

Similar successes have been made using structure-based procedures such as OptMAVEn [144] and Rosetta antibody design [145]. More recently, the Cascade method [128] has been developed to design complementary peptides to a target epitope that can be grafted to the complementarity-determining region of an antibody scaffold. 

The Cascade method has previously been employed in designing conformation-specific antibodies to target Aβ42 amyloids and oligomers [127,146].

4.4. PET Neuroimaging of Amyloids in the Brain

PET imaging utilizes radiotracers to screen the presence of amyloids in the brain in vivo [147]. The radiotracers are characterized by high lipophilicity, which allows them to pass across the blood-brain barrier. 

Pittsburgh compound B (11C-PiB) is a fluorescent analog of ThT and was first used to trace amyloid fibrils using PET in 2004. 11C-PiB is currently among the most used radioligands for PET imaging of cerebral Aβ pathology [148] (Table 2). 

Amyloid-PET can distinguish dementia from mild cognitive impairment and normal aging. However, it cannot discriminate against different forms of dementia. Furthermore, these techniques are expensive, and current clinically used radiotracers only reflect the level of insoluble aggregates in the brain. 

In contrast, novel antibody-based radioligands are under development to detect soluble oligomeric and protofibrillar Aβ forms and have been tested in transgenic mice [149,150].

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