Proteopathic Seed Amplification Assays for Neurodegenerative Disorders

INTRODUCTION

In order to function properly, proteins typically must fold and assume defined native three-dimensional structures. However, disruption of native protein folding may allow abnormal aggregation into self-propagating assemblies (seeds) that can grow by recruiting, and misfolding, additional monomers. Such protein aggregates can be more toxic ^1,2 or more infectious ³when relatively small (oligomeric), but continued growth can result in the accumulation of highly ordered amyloid fibrils, and bundles thereof, that comprise the pathological protein deposits that often characterize protein misfolding diseases ⁴. Protein quality control (proteostasis) mechanisms usually limit the accumulation of abnormally folded and aggregated proteins ⁵. However, with aging, pathogenic mutations in specific proteins, inoculation of preformed seeds, or perhaps other factors, protein quality control mechanisms can be overwhelmed, allowing protein aggregation to spiral out of control. The accumulation of protein aggregates may, or may not, have devastating consequences for the host ⁶.

Nervous tissue, with its lack of neuronal turnover, is particularly vulnerable to the effects of pathological protein aggregation. Indeed, the aggregation of specific proteins is known to feature prominently in the pathogenesis of many neurodegenerative diseases including Alzheimer’s, Parkinson’s and prion diseases ¹. In genetic forms of these diseases, mutated proteins appear to be more prone to aggregation, leading to the appearance of neurodegenerative disorders in earlier stages of life. Considering the relative inability of neurons to regenerate, accurate diagnosis in the early stages of these diseases should facilitate effective treatment while neuronal damage is not yet too extensive and/or irreversible. In this context, proteopathic seed amplification assays have provided means of detecting minute amounts [attograms to femtograms (ag-fg)] of self-propagating protein aggregates in diverse biological specimens by exploiting their inherent seeded polymerization growth mechanisms ^7–15.

The assembly of proteins into amyloid fibrils is akin to “one dimensional crystallization” ¹⁶. As with other crystallizations, the de novo, or spontaneous, formation of seeds in a solution of monomers usually takes much longer than the growth of preexisting seeds. In proteopathic seed amplification assays, a biospecimen is incubated in the presence of a vast stoichiometric excess of soluble protein monomers under appropriate conditions. If the specimen contains seeds, the assembly of monomers into amyloid fibrils occurs more rapidly than the de novo assembly that eventually occurs in the absence of seeds. Thus, seeds can be detected by comparing lag phases, or the reaction times required to detect amyloid that is newly formed from the monomeric “substrate” molecules. This use of “substrate” is analogous to the substrate in an enzymatic reaction rather than to a structure onto which something builds. As the substrate molecules are much more abundant than the seeds in a biospecimen, their conversion into amyloid fibrils can, in effect, provide billion-, or even trillion-fold amplifications of the seed ^17,18. Such amplification can enable ultrasensitive detection of even a few seed particles in a test sample. Here we highlight many of these types of assays, with an emphasis on their applications to human proteopathies.

PRION SEED AMPLIFICATION ASSAYS

The most highly developed proteopathic seed amplification assays are those for prions, with current assays allowing highly accurate molecular diagnoses of prion diseases in living patients ^15,19–21. The ability of infectious prions to induce the conversion of natively folded prion protein (PrP^C) into a prion-like protease-resistant form (PrP^res) in a cell-free system was first demonstrated in 1994 ²². This experimental system revealed prion strain- and sequence- specificities of this prion-seeded conversion reaction ^23–27, but did not support the continuous prion propagation that would be required for an amplification assay.

Protein misfolding cyclic amplification

Several years later Soto and coworkers developed a highly sensitive PrP^Sc (infectious scrapie prion protein) amplification method termed “protein misfolding cyclic amplification” (PMCA) ⁷. In this assay, attogram amounts of PrP^Sc present in a tissue sample can be amplified to detectable levels by its incubation with an excess of non-infected brain homogenate, which provides the PrP^C monomers needed for the polymerization process. By interleaving incubation and sonication steps, the newly synthesized misfolded aggregate is intermittently broken, providing more seeds, amplifying PrP^res and prion infectivity exponentially. The final product is then subjected to a proteinase K (PK)-treatment and proteinase-resistant fragments are revealed by Western blotting with an anti-PrP antibody. PrP^Sc can be amplified from a 10⁻¹² dilution of infected hamster brain homogenate containing ~26 PrP^Sc molecules ¹⁷. A key feature of PMCA is that it faithfully replicates the PrP^Sc structure such that the amplified products are fully infectious ^28,29. Thus, PMCA has been invaluable as an in vitro experimental system for studying prion propagation. On the other hand, infectious prion amplification can be a disadvantage for routine diagnostic applications when it would be preferable not to generate large amounts of infectivity. Notwithstanding that practical concern, PMCA has clear diagnostic potential as prions have been amplified from blood samples of experimentally infected animals at late ³⁰ and early ³¹ stages of the disease, as well as in urine samples from animals ³² and humans with variant Creutzfeldt-Jakob disease (vCJD) ³³.

To circumvent the need to use brain homogenates as a source of PrP^C substrate, Atarashi et al developed PMCA reaction conditions that allowed the use of bacterially expressed recombinant PrP^C as the reaction substrate ⁸. This assay was called rPrP PMCA and allowed for detection of ag-range amounts of PrP^Sc in 2–3 days compared to the 2–3 weeks required for conventional PMCA assays at that time. A subsequent rPrP PMCA permutation called quaking induced conversion (QuIC) allowed for the substitution of more reproducible shaking for sonication ¹².

Amyloid seeding assay

Meanwhile, Colby and colleagues also developed a highly sensitive assay for PrP^Sc using recombinant PrP^C as a substrate called the amyloid seeding assay (ASA) ⁹. The ASA, which was also shaken rather than sonicated, had the major practical advantages of having a multiwell plate-based assay format and a direct fluorescence readout. The basic principle was that PrP^Sc seeds in a sample could induce the formation of recombinant PrP amyloid fibrils which were then detected with the amyloid-sensitive dye thioflavin T (ThT). Frequent fluorescence measurements while the reactions progressed in a shaking fluorescence plate reader allowed convenient measurement of the relative lag phases of reactions seeded with prion-infected (e.g. sporadic CJD or sCJD) versus negative control brain homogenates. However, in contrast to most RT-QuIC assays, a phosphotungstate (PTA)-precipitation step was needed in order to purify the seeds prior to its use in the ASA.

RT-QuIC

By combining various aspects of the QuIC and ASA assays and further optimizations, Atarashi and colleagues developed the first real-time QuIC (RT-QuIC) assays which provided easier distinction of prion-infected and uninfected biospecimens such as cerebrospinal fluid (CSF) ^10,11, often without the need of doing a pre-clearing of the sample. Many laboratories have since contributed to the development of assays for most known prions of mammals, e.g.^34–36 and references therein. The analytical sensitivities of these assays often reach down in the low fg to ag range and typically meet or markedly exceed the sensitivities of animal bioassays for prions. RT-QuIC assays have been adapted to diverse tissues and biological fluids, including brain, skin, olfactory mucosa, blood, CSF, saliva, urine and feces ^{18,33,37–45}. Faster and more sensitive second-generation RT-QuIC assays for human prions allow for nearly 100% accurate antemortem diagnosis of sCJD when either CSF, nasal brushings, or both are tested ^21,41,46. Analysis of CSF specimens alone provide 92–96% overall diagnostic sensitivity (the percentage of sCJD cases giving positive assays) and nearly 100% specificity (the percentage of non-prion disease cases giving negative assays) in multiple independent studies by different research groups ^{21,40,41,46,47}. Prior to the availability of these assays, definite diagnoses of CJD required biochemical or immunohistological analysis of brain tissue, which is usually only available postmortem. According to the US Centers for Disease Control and Prevention (CDC), a positive RT-QuIC result in combination with neuropsychiatric symptoms provides a probable diagnosis of CJD, although immunodetection of PrP^res is still needed for a definite diagnosis (https://www.cdc.gov/prions/cjd/diagnostic-criteria.html). Similar diagnostic criteria are used by the UK National CJD Research and Surveillance Unit (https://www.cjd.ed.ac.uk/sites/default/files/criteria_0.pdf). Recent demonstrations of the detection of sCJD prions in the skin ⁴³ and multiple components of the eyes ⁴⁸ of all tested sCJD cases have raised the possibility that these tissues might also be useful diagnostically, as well as being potentially biohazardous sources of prion infectivity. Indeed, studies in prion-infected rodents have shown that prion seeding activity can be detected in skin far in advance of the onset of overt clinical signs of prion disease ³⁸. Detailed protocols for performing prion RT-QuIC assays have been reported elsewhere 20,49

Scrapie cell assay

Klohn and colleagues ⁵⁰ described a prototypic cellular seeding assay which exploits the ability of prions to infect cells and induce the de novo accumulation of PrP^Sc. Briefly, neuroblastoma-derived cell lines are incubated with dilutions of prion-infected brain homogenates from rodents, and the proportion of cells accumulating PrP^Sc (detected by an ELISPOT after three in vitro passages over ~ 2 weeks total) were found to correlate with the prion concentration in the original sample. The standard scrapie cell assay (SSCA) has been automated ^51,52 and is as sensitive as the mouse prion bioassay but is faster and less expensive, with the advantage of no use of animals. There have been many revealing applications of the scrapie cell assay, e.g. ^52–54, however, so far the assay has been limited to the measurement of rodent-adapted prion strains. Indeed, no cell line capable of supporting replication of human prion strains has been established and made available for research.

SEED AMPLIFICATION ASSAYS FOR SYNUCLEINOPATHIES

αSyn RT-QuIC and PMCA

To provide molecular diagnoses of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), several laboratories have now developed RT-QuIC-like assays for pathological forms of α-synuclein (αSyn^D) called αSyn RT-QuIC ^55–60 or αSyn-PMCA ⁶¹. These assays can have unprecedented sensitivity for αSyn^D down into the low fg range and detect up to 10⁸-fold dilutions of patients’ brain tissue. When applied to CSF specimens collected from living patients, the diagnostic sensitivities for PD and DLB have ranged from 88–96% and specificities from 82–100% ^{55,57,60–62}. Blinded comparisons of the performance of two of these assays on a large set of PD cases and healthy controls revealed a high degree of concordance in diagnostic performance ⁶². Whereas the earlier assays took 5–13 days, newer permutations take 1–2 d ⁵⁷ or 3 d ⁶⁰ with comparable diagnostic performance and the ability to detect αSyn^D seeds in CSF collected early in the clinical course of PD. Similarly rapid assays have been reported using postmortem submandibular gland tissue from PD and incidental Lewy body disease decedents, giving 100% sensitivity and 94% specificity for synucleinopathy ⁵⁹. αSyn^D seeding activity can often be detected in nasal brushings from synucleinopathy patients ⁶³. It is notable that studies have also reported evidence of strain-like differences between types of synucleinopathy-associated seeds 57,58.

Although the multiple initial analyses of αSyn RT-QuIC and related assays have been encouraging, more extensive studies will be required to fully understand their diagnostic and prognostic utilities and the extent to which αSyn^D seeding activities in accessible biospecimens might vary over time in individual patients. Such information will likely be important in understanding the extent to which the monitoring of αSyn^D seeding activities might be helpful in setting up and assessing the progress of clinical trials of treatments aimed at reducing αSyn^D burden in the brain.

HEK cell bioassay for MSA

Woerman and colleagues expressed yellow fluorescent protein-tagged αSyn in human embryonic kidney cells and found that phospshotungstic acid-precipitated extracts of brain homgenates from MSA decedents could be assayed on these cells 4 days after exposure by quantitating cells with fluorescent aggregates ^64,65. The assay had an analytical sensitivity of 70 pg/mL, and revealed differences in regional distribution of αSyn seeding activity between MSA patients. Interestingly, this assay was not sensitive to the αSyn seeds of PD cases ^64,65. The results provided evidence for the MSA and PD being distinct αSyn prion strains.

Hanabi assay

Recently, an ultrasonication-based assay was described which uses the HANdai Amyloid Burst Inducer (HANABI) system to amplify αSyn^{D 66}. Analogous to what is done in classical PMCA, the authors applied ultrasonication to break up αSyn^D oligomers in order to increase the nuclei from which the polymerization starts. The addition of αSyn pre-formed fibrils in artificial CSF enhanced the reaction speed in a dose-dependent fashion. Although ultrasonication provided faster reactions, it also increased the unseeded polymerization in negative control reactions. The analysis of human CSF from PD and control cases showed overlapping reaction kinetics, complicating the diagnostic sensitivity and specificity of this assay.

SEED AMPLIFICATION ASSAYS FOR TAUOPATHIES

Multiple neurodegenerative diseases involve the pathological accumulation of tau filaments, including AD, chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), frontotemporal dementia and Parkinsonism 17 MAPT (FTDP 17 MAPT), argyrophyllic grain disease (AGD), and Pick disease (PiD). In adult humans, the alternative splicing of the MAPT (microtubule-associated protein tau) gene gives rise to six tau isoforms. These isoforms differ from each other by the presence or absence of inserts near the N-terminus and by the number of microtubule binding domains near the C-terminus [i.e., three (3R) or four (4R) isoforms]. The tau pathologies of different diseases are characterized by the preferential deposition of 3R, 4R or both 3R and 4R tau isoforms. For example, AD and CTE cases accumulate roughly equivalent amounts of both 3R and 4R isoforms because all the isoforms contain the residues comprising the amyloid cores of the paired helical and straight tau filaments of these diseases ^67,68. PiD cases accumulate mainly 3R isoforms ^69,70,consistent with their compatibility with the distinct amyloid core of PiD filaments ⁷¹. In contrast, PSP, CBD, and FTDP 17 MAPT cases preferentially accumulate 4R isoforms, which, in the case of CBD, can again be rationalized by the core structure of the CBD-associated filaments ⁷².

Tau RT-QuIC assays

Initial studies by Margittai and colleagues showed that in sonicated, multiwell plate-based reactions synthetic fibrils formed with different 3R and 4R tau constructs had distinct seeding activities ⁷³ and also that AD brain homogenates could seed the fibrillization of tau constructs faster than control homogenates ⁷⁴. Since then, Saijo, Kraus, Metrick, and colleagues have developed ultrasensitive and selective tau RT-QuIC assays for disease-associated 3R, 4R, and 3R+4R tau aggregates, and some subclasses thereof ^6,75–78. Tau RT-QuIC performance can be improved, i.e., higher sensitivity can be achieved without compromising specificity, by systematic comparisons of conditions such as salts of the Hofmeister series ⁷⁷. These tau RT-QuIC assays can detect seeds in 10⁸-10¹⁰-fold dilutions of brain tissue, with orders of magnitude of selectivity for the types of tauopathy for which they were optimized. The optimization of each of these assays involved comparisons of various recombinant tau substrate constructs, cofactors and other reaction components and conditions. Use of a 3R tau fragment (K19CFh) as a substrate first allowed the development of the 3R tau RT-QuIC assay which specifically detects PiD seeds in brain tissue and postmortem CSF samples ⁷⁵. Then, to detect AD and CTE tau filaments, a substrate (τ306) spanning the entire amyloid core of the associated filaments was included in the reaction to allow the amplification of the seeds ⁶. Seeds of multiple diseases with 4R tauopathy can be detected with the recently developed 4R Tau RT-QuIC ⁷⁶. This is the first tau RT-QuIC assay to allow detection of tau seeds in antemortem, as well as, postmortem CSF specimens. Finally, we more recently used another tau substrate (K12CFh) to develop a single tau RT-QuIC assay for the detection and discrimination of tau seeds of AD and PiD ⁷⁸. This assay simplifies the testing for these types of tau seeds and indicates that they differ in conformational templating activity. As with the prion and αSyn RT-QuIC assays, the ability to detect tau seeds in assessible diagnostic specimens will be important for antemortem diagnostic utility of tau RT-QuIC assays. However, at present, further research is necessary to better establish any such utility.

Another valuable feature of the 4R RT-QuIC assay is its ability to differentiate of three classes of 4R tau seeds based on characteristics of the fibrillar reaction products, namely their relative enhancement of ThT fluorescence, and their β-sheet conformations as assessed by Fourier transform infrared (FTIR) spectroscopy: Class A is represented by FTDP-17 with P301L MAPT mutation; Class B includes the FTDP-17 with N279K mutation) and CBD; and Class C includes PSP ⁷⁶.

In applying tau RT-QuIC assays to biological specimens, it is important to bear in mind that various types of tau aggregates and seeding activities can be found at lower levels (usually by orders of magnitude) in brain tissue, at least, of individuals without primary tauopathies ^6,75–78. Thus, the diagnostic specificities of these ultrasensitive assays when applied to biospecimens may depend on quantitative, as well as qualitative, differences in tau seeding activities.

Biosensor cell assays

A highly sensitive and specific biosensor cell seeding assay for tau seeds has also been developed ^79,80 by exploiting the ability of tau aggregates to penetrate cells and induce the fibrillization of soluble endogenous tau ⁸¹. Briefly, when engineered HEK293T cell lines expressing 3R or 4R tau constructs fused to fluorescent proteins were exposed to exogenous proteopathic tau, these protein aggregates penetrated the cells and seeded the polymerization of intracellular tau ⁷⁹. The intracellular protein aggregation was detected by Fluorescence Resonance Energy Transfer (FRET), which was measured by fluorescence microscopy or flow cytometry. The exposure of the cells to increasing amounts of recombinant tau fibrils resulted in the gain of FRET signal, in a dose-dependent fashion. Moreover, the incubation of HEK293T cells expressing fluorescent 3R or 4R tau constructs, or both, with specific tauopathy brain lysates (e.g., from AD, CTE, PSP, CBD, AGD) brain lysates triggered the formation of tau inclusions, whereas no tau aggregation was induced by negative controls. This showed that the polymerization was promoted by aggregated tau and not other protein aggregates. Comparison of tau seeds from multiple sources were found to behave like different strains by inducing characteristic intracellular inclusions that could be faithfully propagated through many in vitro passages and then upon inoculation into transgenic mice ⁸². To compare the sensitivity of this technique to histology (the gold standard postmortem method to diagnose AD ⁸³), a time-course analysis in a tauopathy mouse model was performed. Tau seeding activity preceded histopathological detection by more than four weeks. Although these experimental systems have revealed much about the prion-like propagation and strain-dependent seeding capacity of tau aggregates, the practicality of these cellular models for routine clinical diagnostic purposes in clinical practice may be limited by the need for tissue cultures and either immunostaining or flow cytometry.

AMYLOID-β (Aβ) SEED AGGREGATION ASSAYS

Kinetic aggregation assay

In addition to the aforementioned neurofibrillary tangles of tau, amyloid plaques of the amyloid-β (Aβ) peptide are major lesions found in brains from Alzheimer’s disease patients ⁸⁴. To detect Aβ aggregates, Kelly and colleagues developed a seeded polymerization-based assay ⁸⁵. In this “kinetic aggregation assay”, Aβ aggregates from mammalian cell culture media, Caenorhabditis elegans lysates, and AD mouse brain homogenates seeded the fibrillization of monomeric Aβ_1–40 peptide, in a cell-free reaction containing ThT to monitor fibril formation over time. The amount of Aβ amyloid fibrils in the sample was proportional to the half time of the growth phase (t50). To prove the specificity of this assay, experiments with αSyn aggregates were also conducted and differences in the kinetic reactions were observed, with reactions seeded with 4.3 μg/mL Aβ_1–40 fibrils showing a t50 of ~5h while 14.5 μg/mL α-synuclein fibrils yielded a ~9-h t50. The authors stated that this selectivity is needed to allow the quantification of Aβ amyloid fibrils in tissues which can potentially contain other amyloid seeds; however, human biological specimens from patients with different neurodegenerative disorders were not assessed by this assay in order to prove its specificity.

Aβ-PMCA

Continuing along these lines, Soto and colleagues developed an assay called Aβ-PMCA that detects misfolded Aβ oligomers down to as little as 3 fmol in a multiwell plate-based assay with ThT readout as in RT-QuIC assays ⁸⁶. Application of Aβ-PMCA to CSF specimens from AD and non-AD cases provided discrimination between the two sets with 90% sensitivity and 92% specificity. As it is well known that cognitively normal or other non-AD cases can have some Aβ deposits in their brain tissue, the specificity of the Aβ-PMCA is presumably dependent upon disease-dependent differences in concentration of Aβ seeds in the CSF. Seed concentration is typically inversely correlated with lag time in seeded protein polymerization reactions, which likely explains the shorter lag phases that were observed with CSF samples from AD cases. This assay promises to provide a useful complement to the AD tau RT-QuIC assay described above in measuring the key pathological protein aggregates of AD.

HUNTINGTON DISEASE SEED AMPLIFICATION ASSAY

The applicability of an amyloid seeding assay of postmortem brain extracts from human Huntington diseases cases, and transgenic mice models thereof, was reported by Gupta and coworkers ⁸⁷. Misfolded huntingtin partially purified from the diseased brains induced amyloid formation of a largely polyglutamine substrate more rapidly than extracts from negative control brains. Although this assay is inhibited by components of crude brain tissue, hence the need for partial purification of the seeds from brain, it serves as a prototypic test for pathological forms of huntingtin.

CONCLUSIONS

Continuing work by many laboratories is yielding a growing panel of ultrasensitive assays for the various misfolded self-propagating protein aggregates that cause many neurodegenerative diseases. These assays provide important tools for fundamental research into the pathogenesis of these diseases. Moreover, the ability to detect miniscule amounts of such proteopathic aggregates as biomarkers in accessible specimens is allowing more accurate molecular diagnoses of diseases that can otherwise be difficult to discriminate, especially early in pathogenesis while appropriately targeted treatments are more likely to be successful. Importantly, given the frequency with which neurodegenerative disease patients have more than one type of aggregated protein in their CNS, the high sensitivity of these assays allows detection of both primary and secondary proteopathies. Such testing should also facilitate the development of new therapies by allowing clearer identification of cases and controls for clinical trials, as well as the measurement of specific protein seeds as key etiological biomarkers over the course of treatment.

Figure 1. Diagram of seeded polymerization in RT-QuIC reactions.

A. Reactions are set up in multiwell plates by diluting a biospecimen into reaction mixture containing a vast stoichiometric excess of an appropriate protein monomer (substrate) for the type of disease-associated seed being detected. The reaction also contains the amyloid-sensitive fluorescent dye, thioflavin T (ThT). When seeds are present in the biospecimen (left), they immediately start to grow (4) by recruiting new monomers. Elongated fibrils can promote secondary nucleation (5), i.e. the production of new seeding surfaces either by fragmentation or by providing lateral surfaces that can facilitate the ordering and conformational conversion of monomers into additional fibrils. Secondary nucleation contributes to the exponential growth of the new amyloid that enhances ThT fluorescence (dark blue trace). The lag phase in a seeded reaction represents the time it takes for the seeded amyloids to accumulate to levels that are detectable with ThT. Eventually, the reaction plateaus when all of the available monomer is converted to amyloid. In the absence of seeds in a negative control biospecimen (light blue trace), spontaneous nucleation (1–3) may occur, but only after a prolonged lag phase during which the kinetically unfavorable process of forming minimal stable nuclei (in this case a 6-mer) occurs ¹⁶. A key to developing an effective assay is finding substrates and assay conditions giving the greatest fold-separation between the lag phases of seeded versus unseeded reactions (e.g. see ⁷⁷). B. Representative primary AD tau RT-QuIC data comparing seeding by serial 10-fold dilutions of human familial AD (age 44) and cerebrovascular disease (CVD; age 53) brain tissue with reference to a 10⁰ dilution being solid brain tissue. Tau knock-out (KO) mouse brain served as a completely tau-free negative control. Traces from quadruplicate wells for each brain dilution are shown. AD brain could be diluted at least 10⁵-fold and 10³-fold further than the KO and CVD brains, respectively, and still seed positive reactions. Although the CVD brain was not recorded as having tau pathology by immunohistochemistry, the typically higher sensitivity of RT-QuIC assays likely allowed detection of tau aggregates that were below the detection limit of immunohistochemistry.

KEY POINTS

• Prion-like, self-propagating protein aggregates can cause, and serve as biomarkers, for multiple neurodegenerative diseases.

• The seeded polymerization growth mechanism of pathological protein aggregates has been exploited to develop ultrasensitive assays for many of these biomarkers.

• Some of these types of assays, e.g. prion and αSyn RT-QuIC assays, can provide highly sensitive and specific diagnoses of prion diseases and α-synucleinopathies when applied to patients’ cerebrospinal fluid or nasal brushings.

• Multiple ultrasensitive seed amplification assays have also been developed for many disease-associated types of tau and Aβ aggregates in biospecimens, but these assays are in earlier stages of diagnostic evaluation.

• The development of a broad panel of proteopathic seed amplification biomarker assays should facilitate the diagnosis of neurodegenerative diseases as well as the execution of clinical trials of potential therapeutics.

SYNOPSIS

To address the need for etiological biomarkers for neurodegenerative diseases involving protein aggregation, ultrasensitive cellular and cell-free assays have been developed based on the prion-like self-propagating (seeding) capacity of many such aggregates. The most practical and clinically validated of such assays so far are the prion RT-QuIC assays that allow nearly 100% accurate antemortem diagnosis of sporadic Creutzfeldt-Jakob disease using patients’ cerebrospinal fluid or nasal brushings. Analogous assays for synucleinopathies such as Parkinson disease and dementia with Lewy bodies are providing unprecedented diagnostic sensitivity using cerebrospinal fluid. Tau RT-QuIC assays can detect and discriminate different types of tau aggregates associated with Alzheimer disease and other tauopathies. Related assays have also been reported for disease-associated huntingtin and amyloid β aggregates, with the latter being applicable to Alzheimer’s cerebrospinal fluid. The development of a broad panel of seed amplification assays should improve diagnostics as well as the evaluation of potential therapeutics.