COMMENTARY Breakthroughs in antemortem diagnosis of neurodegenerative diseases COMMENTARY Glenn C. Tellinga,1 The World Health Organization forecasts that within 2 Abnormal cytoplasmic accumulation of a normally sol- decades neurodegenerative disorders will eclipse can- uble and unfolded protein called α-synuclein is the cer to become the foremost cause of death in the de- hallmark of diseases referred to as synucleinopathies. veloped world after cardiovascular disease. Accurate Neuronal deposition of α-synuclein aggregates in Lewy detection of pathological processes goes hand in hand bodies occurs in Parkinson’s disease (PD) and dementia with the goals of treatment and prevention and, in light with Lewy bodies (DLB). Yet another protein—the prion of their protracted but worsening clinical progression, protein (PrP)—is central to a group of interrelated dis- the earlier a diagnosis can be made the better. How- orders commonly referred to as prion diseases. ever, the challenge underlying accurate detection of Since the concept underlying the diagnostic ap- neurodegenerative diseases during their clinical phase proach taken by Metrick et al. (1) derives from studies is that specific biomarkers are not present at high of prions, it is worth reviewing what we have learned enough concentrations for routine detection in acces- about PrP and the applicability of these findings to sible specimens. Consequently, it has only been pos- other proteopathic diseases. The prion disorders are sible to definitively diagnose these conditions by transmissible neurodegenerative diseases affecting examination of brain pathology after death. The paper animals and humans. The most common human form by Metrick et al. (1) in PNAS addresses the issue of is Creutzfeldt–Jakob disease (CJD) which occurs most improved antemortem biomarker detection for a frequently as a sporadic, rapidly progressive condition spectrum of neurological disorders, using assays of older individuals. The discovery that CJD and which capitalize on prionic amplification of misfolded, kuru—an epidemic spread by cannibalism among tribal pathogenic proteins. peoples in Papua New Guinea—were transmissible and that they shared neuropathological features with Protein Misfolding and Prions in scrapie, a similarly infectious disorder of sheep and Neurodegenerative Diseases goats, led to their grouping in a category called trans- During neurodegenerative diseases biochemical missible spongiform encephalopathies (TSEs). Additional changes in particular proteins lead to their misfolding TSE epidemics were subsequently identified, in- and accumulation in specific neuronal populations. cluding bovine spongiform encephalopathy (BSE), Alzheimer’s disease (AD) is characterized by accumu- also known as “mad cow disease,” and chronic lation of extracellular plaques enriched with amyloid wasting disease (CWD), a contagious and rapidly beta (Aβ) peptide derived from the amyloid precursor spreading disease of deer, moose, elk, and other protein and intracellular neurofibrillary tangles com- cervids. Transmission of BSE to humans as variant posed of hyperphosphorylated microtubule-associated CJD (2), most likely by consumption of prion- proteintau.Ofthe6tauversions,3have3microtubule- contaminated foodstuffs, epitomizes the potential binding domains (3R isoforms) while the others con- for interspecies prion transmission. Pioneering studies tain 4 (4R isoforms). The involvement of tau in fronto- by Prusiner et al. (3) demonstrated that prions, the temporal dementia with parkinsonism-17, Pick disease causative agents of TSEs, were infectious proteins (PiD), progressive supranuclear palsy, argyrophilic devoid of DNA or RNA. According to the prion grain disease, and corticobasal degeneration results in hypothesis the infectious particle is composed of the blanket term of tauopathies to describe these dis- PrPSc, a conformationally altered pathogenic version orders. While tau deposits in PiD are composed pre- of a host-encoded protein, PrPC. Agent replication dominantly of 3R isoforms, roughly equivalent amounts occurs by a process in which PrPSc interacts with and of 3R and 4R isoforms are deposited in AD. Other templates its abnormal β-sheet conformation on PrPC tauopathies accumulate predominantly 4R isoforms. which is largely α-helical (4). The ensuing PrPSc is then aPrion Research Center (PRC), Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 Author contributions: G.C.T. wrote the paper. The author declares no competing interest. Published under the PNAS license. See companion article 10.1073/pnas.1909322116. 1Email: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1917054116 PNAS Latest Articles | 1of3 Downloaded by guest on September 26, 2021 capable of transforming additional PrPC, the result being expo- fidelity, speed, and specificity of protein aggregation depended nential prion accumulation. on whether the RT-QuIC assays were designed to amplify tau, α-synuclein, or PrP disease-associated seeds. Hofmeister salts Prionic Amplification of Proteopathic Seeds Facilitates were found to have pronounced and distinct effects on the ability Neurodegenerative Disease Diagnosis to differentiate between rapid seeded propagation of amyloids AlthoughearlyworkbyCaugheyandcoworkers(5)provided and the spontaneous aggregation of substrate protein in RT-QuIC important proof of concept that PrP conversion could be repli- assays for tau, α-synuclein, or PrP. While weakly hydrated salts cated in cell-free systems, the excessive amounts of PrPSc required to convert PrPC after prolonged periods of incubation precluded The paper by Metrick et al. in PNAS addresses the use of this approach as a diagnostic tool. Using a technique the issue of improved antemortem biomarker referred to as protein misfolding cyclic amplification (PMCA), Soto detection for a spectrum of neurological disorders, and coworkers (6) discovered that much smaller amounts of PrPSc were capable of converting PrPC derived from normal brains in a using assays which capitalize on prionic cell-free system. In PMCA, disruption of growing PrPSc oligomers amplification of misfolded, pathogenic proteins. by sonication generates multiple smaller templates for continued recruitment and conversion of PrPC. Using a simplified approach showed optimal effects on kinetics of fibril formation in AD- and referred to as quaking-induced conversion (QuIC), Caughey and prion-seeded reactions, seeding of a different form of tau by PiD coworkers (7) used bacterially expressed recombinant PrP (rPrP) as brain homogenate was fastest with more strongly hydrated an- conversion substrate and replaced sonication with alternating ions, and the α-synuclein RT-QuIC assay using seeds from patients cycles of incubation and shaking to facilitate fibril fragmentation with DLB was affected by yet other Hofmeister salts. Metrick et al. and reseeding. In this way minute starting amounts of prion seeds (1) also detected pronounced Hofmeister effects on the sensitivity could be amplified to a detectable level. In a further develop- of RT-QuIC assays. During the process of testing 4R tauopathy ment, the amyloid seeding assay (ASA) used the fluorescent dye brains they observed gains in sensitivity of up to a million-fold thioflavin T (ThT) to detect seeded polymerization of rPrP (8). By using sodium citrate compared to sodium chloride which had incorporating aspects of the ASA and QuIC, Caughey and co- been used in previous assay conditions. In contrast, no gains in workers (9) developed real-time QuIC (RT-QuIC) in which the seeding activity were seen for seeding assays from 3R or 3R/4R ability of prions to seed conversion of the rPrP template could be tauopathy cases. monitored in real time by incorporation of ThT into growing am- Metrick et al. (1) also found that Hofmeister effects impacted yloid fibrils. Since biospecimens containing as little as attogram amplification and detection in the context of diagnostically amounts of prion seeds can be detected after amplification by available specimens. While seed detection in brain homogenate many orders of magnitude, RT-QuIC has become a preferred using the AD tau RT-QuIC assay was enhanced by weakly hy- method for prion diagnosis in a variety of veterinary and medical drated anions, the trend was reversed in human blood plasma settings (10). Several RT-QuIC assays have been developed for where the effects of inhibitors were overcome by strongly hy- prion detection in accessible patient specimens including ce- drated salts. Sodium iodide increased the analytical sensitivity of rebral spinal fluid, nasal brushings, and skin (11–14). CJD prion seed detection by 6- to 32-fold in nasal brushings as Whereas the process of interactive conformational templating well as in brain homogenate and improved detection of prions in of normally folded host protein by a pathological counterpart was homogenates of ear biopsies from deer with CWD. This promising originally considered an exclusive property of PrP, increasing development raises the possibility that accessible ear punch bi- evidence linked this canonical mechanism to proteins involved in opsies might be used for antemortem monitoring of this un- the pathogenesis of additional neurodegenerative diseases (15). controlled prion disease of wild animals. Evidence of AD transmission to marmosets (16) was confirmed In summary, a rapidly emerging picture supports the self- and extended by investigators employing transgenic mouse