23 Dec 2016

Two techniques that detect prion protein oligomers in body fluids have made valuable improvements, say researchers. Each exploits the ability of certain toxic protein conformers to seed the misfolding and polymerization of the healthy, monomeric variety. Scientists led by Gianluigi Zanusso, University of Verona, Italy, report in the December 12 JAMA Neurology that real-time quaking-induced conversion (RT-QuIC) can now ferret out sporadic Creutzfeldt-Jakob disease (sCJD) prions in the CSF with near-perfect sensitivity and specificity. In the December 21 Science Translational Medicine, Claudio Soto of the University of Texas Medical School at Houston and colleagues report that they can use protein misfolding cyclic amplification (PMCA) to detect variant CJD (vCJD) in the blood. In a separate December 5 JAMA Neurology paper, Soto describes the potential application of the technology to detect α-synuclein in patients with Parkinson’s disease (PD). Scientists agree that these techniques will help better diagnose this group of devastating neurodegenerative disorders.

“These methods will have a major impact in the field,” wrote Inga Zerr, German Center for Neurodegenerative Diseases (DZNE), Göttingen, who was not involved in the studies. Jiri Safar of Case Western Reserve University in Cleveland agreed, saying the technology has widespread support amongst the small community of prion researchers. “It’s definitely a paradigm-changer, perhaps equivalent to the discovery of PCR amplification.” Safar directs the National Prion Disease Pathology Surveillance Center, where scientists monitor human cases of prion diseases for vCJD, the type that comes from eating beef contaminated with the prion that causes bovine spongiform encephalopathy (BSE).

Prion Hunters
Both PMCA and RT-QuIC hinge on the idea that protein monomers are slow to aggregate on their own, but if oligomers are present, fibrils come together quickly. PMCA was developed in Soto’s lab, but has been slow to catch on (Saborio et al., 2001). It mixes sample test fluid in a tube with normal protein monomers purified from healthy animal brain homogenates. If the sample contains no oligomers, the monomers stay monomers. In the presence of oligomers, monomers misfold, eventually incorporating into fibrils. After fibrils are allowed to grow for a while, mechanical forces break them into smaller oligomers, creating more seeds for another round of templated misfolding, and so on. This amplifies the oligomer signal so that the technique can detect as little as a single oligomer (Saá et al., 2006). Fibrils are then visualized by western blot with an anti-PrP antibody.

PMCA inspired RT-QuIC, which was developed by Byron Caughey at the National Institutes of Health in Bethesda, Maryland. The concept is similar, except the reaction takes place in a 96-well plate, the protein monomers are recombinant, and samples are shaken to hasten protein interactions and shorten incubation times. Amyloid fibrils are then detected by thioflavin T (ThT), a fluorescent dye, allowing the method to be completely automated. Since this technique is cheaper, easier, and faster than PMCA, it may be more widely adopted for routine lab use, said Safar. However, PMCA may complement RT-QuIC by detecting different types of prions and in the sorts of questions it can answer, he added.

RT-QuIC
Efforts are underway to optimize RT-QuIC technology even further. By shortening the recombinant protein and raising the reaction temperature, RT-QuIC has become faster and more accurate (Orrú et al., 2015). Zanusso and colleagues tested this new and improved assay retrospectively in CSF and olfactory mucosa samples from 23 people who were neurologically healthy, 81 people with non-prion neurological disorders, and 86 who were suspected sCJD patients. The Italian CJD surveillance system had initially diagnosed the last group with probable, possible, or suspected CJD. The patients were monitored until they either died or got another diagnosis. It turned out that 61 had a final diagnosis of sCJD, six a genetic form of CJD, and 17 a different neurological disease.

First authors Matilde Bongianni, Christina Orrú, and Bradley Groveman detected prions in the CSF and/or OM of all of the 61 confirmed sCJD patients, suggesting the RT-QuIC detection rate is 100 percent. None of the controls tested positive. The assay only detected prions in four of the six genetic CJD cases, suggesting the technique doesn’t work as well for prion strains that cause familial disease.

“The procedure should soon become the standard laboratory examination for the diagnosis of sporadic CJD,” wrote Paul Brown, formerly of the National Institutes of Health, in an accompanying editorial. “At last, we have a truly practical procedure for the diagnosis of sporadic CJD.” He added that it is still unclear whether it will detect prions in people who are asymptomatic.

RT-QuIC could be used to detect prions on hospital equipment or in transplanted tissue and organs, and monitor for the passage of prion diseases from animals to humans, said Safar. He and colleagues have run thousands of human CSF samples using RT-QuIC (Foutz et al., 2016). “It’s already changing our view of prion pathogenesis,” he said. Being able to detect minute quantities of prions has allowed researchers to detect the proteins in tissues where they hadn’t been seen before, he said.

A recent multicenter study validated RT-QuIC against neuropathologically confirmed cases of sporadic and genetic CJD (Cramm et al., 2016). It found that the assay had a sensitivity of 85 percent and a specificity of 99 percent. What’s more, it was almost perfectly reproducible between centers. Multiple labs are using it now, said Safar.

PMCA Struts Its Stuff in the Blood
Scientists are unsure why, but RT-QuIC poorly detects the vCJD strain of prion. In the United Kingdom, researchers estimate 30,000 people were exposed to this strain by eating BSE-tainted beef and could be asymptomatic carriers of the prion. Silent carriers are thought to have donated blood that caused infections in other people. A blood test for vCJD could prevent that. PMCA detects vCJD in blood from primates, and in human blood spiked with vCJD prions, but whether it detects the prion in samples from people who have CJD has been an open question (Jun 2014 news; Edgeworth et al., 2011). 

Now, Soto and colleagues have used PMCA to test blood samples from 14 human vCJD patients. As control samples, first author Luis Concha-Marambio used blood from 153 people who were either cognitively healthy or had sCJD, or another neurodegenerative or neurological disorder. PMCA detected prion in all 14 samples with vCJD but in none of the controls. The test worked in only a few microliters of blood. Though double-blind studies with larger numbers of patients will be required before researchers can draw firm conclusions about sensitivity and specificity, this suggests PMCA can detect vCJD in symptomatic patients. “Detection of abnormal PrP in blood of variant CJD cases bears huge potential for early and potentially presymptomatic testing for individuals at risk,” wrote Zerr. Whether it will help screen donated blood remains to be seen.

Can PMCA Work for Non-Prions?
Soto has already used PMCA to detect Aβ oligomers in the CSF of AD patients (Mar 2014 news). Though this assay has not been widely used for that purpose, he is now examining whether it can pick up oligomers of α-synuclein in the CSF as well. If α-synuclein works as a biomarker in PD, it could aid in clinical and differential diagnosis, he said.

First author Mohammad Shahnawaz tested the CSF of 76 patients clinically diagnosed with PD. He also tested 10 patients each with dementia with Lewy bodies (DLB) and multiple system atrophy (MSA)—two other α-synuclein-related disorders. He compared those samples to CSF collected from 97 controls who had other neurologic or neurodegenerative diseases, including AD, or who were cognitively healthy. PMCA detected α-synuclein oligomers in 67 of the 76 PD patients, all of the DLB patients, and in eight people with MSA. Because clinical diagnosis of PD is not completely accurate, some of the cases may have been misdiagnosed, the authors suggested. Twelve controls tested positive as well. Shahnawaz and colleagues wrote that five were AD cases, which often have α-synuclein aggregates as a co-pathology, and two of the positive controls went on to develop PD a few years later, hinting that the test could detect oligomers before symptoms appear. Notwithstanding, an overall specificity of 96.0 percent and sensitivity of 88.5 percent suggests the test could be helpful in diagnosing disease.

“This provides proof of concept that the technology can be adapted to work in Parkinson’s disease, at a very high level of sensitivity and specificity,” Soto told Alzforum. Only further studies can say whether this test will be useful for monitoring progression or detecting prodromal disease, the authors wrote.

Henrik Zetterberg, University of Gothenburg, Sweden, found the results very interesting. “I would almost call them groundbreaking,” he told Alzforum. Since there are currently no reliable PD biomarkers, this stands a chance of becoming widely adopted, he suggested. However, he pointed out that the assay is technically challenging, with a long incubation time, which might limit its routine use. RT-QuIC, with its shorter lag time, might be better adapted for PD, he said. Researchers recently detected α-synuclein aggregates in the CSF of people using that assay, though they used only a small number of PD cases (Fairfoul et al., 2016). 

“I’m excited to see many different labs using independent assays to confirm the potential use of α-synuclein oligomers as a marker for disease,” said Omar El-Agnaf, Hamad Bin Khalifa University, Doha, Qatar. He cautioned that the PMCA assay is not quantitative, and that the authors cannot rule out the possibility of cross-seeding from oligomers of other proteins, such as tau and Aβ, though they tested for this cross-seeding and did not observe it. He also noted that the reproducibility of the assay is questionable, so it may not be ideal for clinical use yet. “We have to work harder to find simpler, more sensitive, and specific assays that will help with diagnosis and prognosis,” said El-Agnaf.

Soto ultimately wants to use PMCA to test the blood of PD patients. He has founded a company, Amprion Inc., to commercialize the test.—Gwyneth Dickey Zakaib

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References

News Citations

1. A Blood Test for Preclinical Prion Disease, But Will It Be Used? 13 Jun 2014
2. Test Uses 'Seeding' to Detect Aβ Oligomers in Cerebrospinal Fluid 21 Mar 2014

Paper Citations

1. Saborio GP, Permanne B, Soto C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature. 2001 Jun 14;411(6839):810-3. PubMed.
2. Saá P, Castilla J, Soto C. Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J Biol Chem. 2006 Nov 17;281(46):35245-52. PubMed.
3. Orrú CD, Groveman BR, Hughson AG, Zanusso G, Coulthart MB, Caughey B. Rapid and sensitive RT-QuIC detection of human Creutzfeldt-Jakob disease using cerebrospinal fluid. MBio. 2015 Jan 20;6(1) PubMed.
4. Foutz A, Appleby BS, Hamlin C, Liu X, Yang S, Cohen Y, Chen W, Blevins J, Fausett C, Wang H, Gambetti P, Zhang S, Hughson A, Tatsuoka C, Schonberger LB, Cohen ML, Caughey B, Safar JG. Diagnostic and Prognostic Value of Human Prion Detection in Cerebrospinal Fluid. Ann Neurol. 2016 Nov 28; PubMed.
5. Cramm M, Schmitz M, Karch A, Mitrova E, Kuhn F, Schroeder B, Raeber A, Varges D, Kim YS, Satoh K, Collins S, Zerr I. Stability and Reproducibility Underscore Utility of RT-QuIC for Diagnosis of Creutzfeldt-Jakob Disease. Mol Neurobiol. 2016 Apr;53(3):1896-904. Epub 2015 Apr 1 PubMed.
6. Edgeworth JA, Farmer M, Sicilia A, Tavares P, Beck J, Campbell T, Lowe J, Mead S, Rudge P, Collinge J, Jackson GS. Detection of prion infection in variant Creutzfeldt-Jakob disease: a blood-based assay. Lancet. 2011 Feb 5;377(9764):487-93. PubMed.
7. Fairfoul G, McGuire LI, Pal S, Ironside JW, Neumann J, Christie S, Joachim C, Esiri M, Evetts SG, Rolinski M, Baig F, Ruffmann C, Wade-Martins R, Hu MT, Parkkinen L, Green AJ. Alpha-synuclein RT-QuIC in the CSF of patients with alpha-synucleinopathies. Ann Clin Transl Neurol. 2016 Oct;3(10):812-818. Epub 2016 Aug 28 PubMed.

Further Reading

Papers

1. Steinacker P, Blennow K, Halbgebauer S, Shi S, Ruf V, Oeckl P, Giese A, Kuhle J, Slivarichova D, Zetterberg H, Otto M. Neurofilaments in blood and CSF for diagnosis and prediction of onset in Creutzfeldt-Jakob disease. Sci Rep. 2016 Dec 8;6:38737. PubMed.
2. Abu Rumeileh S, Lattanzio F, Stanzani Maserati M, Rizzi R, Capellari S, Parchi P. Diagnostic Accuracy of a Combined Analysis of Cerebrospinal Fluid t-PrP, t-tau, p-tau, and Aβ42 in the Differential Diagnosis of Creutzfeldt-Jakob Disease from Alzheimer's Disease with Emphasis on Atypical Disease Variants. J Alzheimers Dis. 2017;55(4):1471-1480. PubMed.
3. Llorens F, Kruse N, Schmitz M, Gotzmann N, Golanska E, Thüne K, Zejneli O, Kanata E, Knipper T, Cramm M, Lange P, Zafar S, Sikorska B, Liberski PP, Mitrova E, Varges D, Schmidt C, Sklaviadis T, Mollenhauer B, Zerr I. Evaluation of α-synuclein as a novel cerebrospinal fluid biomarker in different forms of prion diseases. Alzheimers Dement. 2016 Nov 18; PubMed.
4. Foutz A, Appleby BS, Hamlin C, Liu X, Yang S, Cohen Y, Chen W, Blevins J, Fausett C, Wang H, Gambetti P, Zhang S, Hughson A, Tatsuoka C, Schonberger LB, Cohen ML, Caughey B, Safar JG. Diagnostic and Prognostic Value of Human Prion Detection in Cerebrospinal Fluid. Ann Neurol. 2016 Nov 28; PubMed.
5. Schmitz M, Cramm M, Llorens F, Müller-Cramm D, Collins S, Atarashi R, Satoh K, Orrù CD, Groveman BR, Zafar S, Schulz-Schaeffer WJ, Caughey B, Zerr I. The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases. Nat Protoc. 2016 Nov;11(11):2233-2242. Epub 2016 Oct 13 PubMed.

News

1. Protein Marker Distinguishes Prion Disease from Rapid Alzheimer’s 9 Jan 2015
2. News Brief: More Evidence for Aβ Spread Between People 29 Jan 2016