Article In Brief
Investigators developed an assay, 4R RT-QuIC, that can detect miniscule amounts of disease-associated tau seeding activity. Importantly, it is the first to demonstrate tau seeding activity in CSF collected from living patients.
A new assay shows promise for identifying specific tau aggregates associated with progressive supranuclear palsy (PSP), corticobasal degeneration, and other uncommon tauopathies.
Developed by scientists at the National Institute of Allergy and Infectious Diseases (NIAID), the assay— called real-time quaking-induced conversion (RT-QuIC)—amplifies small samples of misfolded protein, in this case 4-repeat tau, collected from cerebrospinal fluid (CSF) in living patients and autopsied tissue from pathologically-confirmed cases.
“It has become apparent that tau filaments have different conformations that can be faithfully propagated to yield various phenotypes and pathologies,” said Byron Caughey, PhD, a senior investigator at NIAID's Rocky Mountain Laboratories, who helped develop the assay. “We need a way to correctly identify these diseases.”
This is the first cell-free assay to demonstrate tau seeding activity in the CSF collected from living patients, he said.
Six tau isoforms are expressed in the human adult brain, three of which contain three microtubule binding repeats and three with four repeats, Dr. Caughey explained. Ultimately, treatments may have to target these different types of tau aggregates.
These biomarker assays could also be used to identify the right patients for clinical trials, and then test the study subject's response to an experimental drug over time, the researchers said.
Experts who were not involved with the current study said the assay is an advance that may help clinicians quickly diagnose specific tauopathies, recruit these patients into clinical trials, and ultimately, enable them to begin early and appropriate treatment. The findings from the study were published in the October 16 online edition of Acta Neuropathologica.
The Making of the Assay
Dr. Caughey and scientists at the NIAID's Rocky Mountain Laboratories previously helped develop an RT-QuIC assay to diagnose Creutzfeldt-Jakob disease (CJD). That was several years ago, and it is now used clinically for an accurate diagnosis of CJD.
“We have moved ahead to other neurodegenerative diseases after the success of the RT-QuIC for the diagnosis of CJD,” said Dr. Caughey.
The team has also developed an RT-QuIC assay for alpha-synuclein aggregates to help in the diagnosis of Parkinson's disease and Lewy body dementia. And they have developed assays for the 3-repeat tau aggregates of Pick disease and the mixed 3- and 4-repeat tau aggregates of Alzheimer's disease and chronic traumatic encephalopathy (CTE).
In this most recent set of experiments, they tested a 4-repeat tau RT-QuIC assay on different types of tauopathies that can be difficult to diagnose and discriminate clinically from other neurodegenerative diseases. [For more about how the assay works, see “The Science Explained: The RT-QuIC Assay.”]
Testing the Assay
To begin their testing, the scientists collected frozen postmortem frontal cortex samples from patients who had pathologically-confirmed cerebrovascular disease, diffuse Lewy body disease, multiple system atrophy, amyotrophic lateral sclerosis, primary age-related tauopathy, Pick disease, PSP, corticobasal degeneration, Alzheimer's disease, and frontotemporal lobar degeneration with several different MAPT mutations, including those on chromosome 17 (FTDP-17). They collected additional frozen brain samples of the superior frontal gyrus from patients with PSP, corticobasal degeneration, and FTDP-17 and neuropathologically normal control cases, provided by the Mayo Clinic in Jacksonville, FL. And they obtained data from previously collected samples of patients with CTE.
They tested CSF from postmortem PSP, corticobasal degeneration, and unaffected controls. They also had samples from patients who donated CSF for diagnosis and research, and postmortem CSF samples from patients who had Parkinson's disease, Alzheimer's disease, primary progressive aphasia, as well as patients who did not have neurological disorders.
They diluted PSP and corticobasal degeneration CSF-seeded synthetic fibrils tenfold serially into healthy pooled CSF for determination of an analytical sensitivity. They reported that the assay detected seeds in 106-109-fold dilutions of 4-repeat tauopathy brain tissue and was much less responsive to brains with other tauopathies.
They also found that specific diseases had four-repeat tau aggregates with different apparent conformations, which may help in teasing apart distinct tauopathies. For now, the assay worked best on the pathological brain tissue and ventricular CSF specimens compared to the CSF samples from living patients.
The seeding from the CSF collected from living patients did not seed as quickly as the pathological tissue from the post-mortem samples. Dr. Caughey said that the average assay responses from PSP and corticobasal degeneration cases were significantly faster than those samples obtained from individuals who were not diagnosed clinically with a 4-repeat tauopathy.
He said that the anomalous results from some cases and controls could be a result of a clinical misdiagnosis, but one cannot be sure in the absence of corresponding postmortem neuropathological data. The assay fluorescent signal was positive in 137 of 199 reactions seeded with samples from PSP cases and 24 of 48 reactions seeded with in samples from corticobasal syndrome or CBD patients.
The autopsy tissue samples collected from patients with mixed 3-repeat/4-repeat aggregates had undetected or significantly lower levels of 4-repeat seeding activity, he said.
Interestingly, they picked up a signal from some samples collected from autopsy tissue from patients with Pick disease, which characteristically involves 3-repeat tau deposition, but may occasionally have 4-repeat deposition as well.
Control cases had little or no tau seeding activity. The autopsied CSF samples also yielded some differences: The seven samples from PSP patients had shorter lag times than patients with corticobasal degeneration (and controls) and a higher peak fluorescence.
Dr. Caughey said that there is a lot more work to be done to develop a reliable antemortem diagnostic assay for the 4-R tauopathies.
“The assays that best achieve these goals are not natural, nor do they fully recapitulate the pathological processes that occur in vivo,” he and his colleagues wrote in the paper. “Highly influential compositional differences between these assays include the recombinant tau substrates, salts, and polyanionic cofactors.”
Dr. Caughey said that it would be important to confirm the findings in living patients using brain tissue collected at autopsy. Then, he added: “We can then figure out more clearly the diagnostic value of these biomarker assays.”
“It is very important to have an accurate diagnosis for treatment,” said Claudio Soto, PhD, Huffington distinguished university chair and professor of neurology and director of the Mitchell Center for Alzheimer's disease and related Brain Disorders at the University of Texas McGovern Medical School at Houston. Dr. Soto has also developed protein amplification technology for the diagnosis of CJD and for PD, and started a bioassay company called Amprion.
“This is good work,” he said of the new study. “It will have a big impact on diagnosis and ultimately treatment.”
“Obtaining an accurate diagnosis is essential for stratification into clinical trials,” added Kurt Giles, PhD, associate professor and director of the transgenic mouse core at the Institute for Neurodegenerative Diseases at the University of California, San Francisco.
“This paper builds on other papers where they developed and tested assays for other tau isoforms. This is the last piece of the puzzle. A more molecular diagnosis rather than a clinical one is a great step forward. This assay is not there yet but it is promising.”
“They have developed a sensitive seeding assay, but a question is whether this replicates what may be happening to tau inside the cells or are the conditions a little artificial, giving rise to a higher background of aggregation,” said Douglas Galasko, MD, professor of neuroscience at the University of California, San Diego. “The data from postmortem tissue, where 3R,R and 3/4R tauopathies show selective differences, is a useful starting point. These RT-QuIC assays for tau may be able to be refined and hopefully they will have one that could be used clinically.”
“The method looks quite solid but I have some reservations about the variability in the sample collection methods that could have altered the results,” said Nigel J. Cairns, PhD, professor of neuropathology at the Medical School of the University of Exeter.
“They collected specimens from a wide number of centers with varying collection methods. We now know that tubes that are used to collect cerebrospinal fluid vary and that proteins can bind differently to some of the plastics used.”
Also, he added, it would have been interesting to compare the results to a simple ELISA. They could have also compared it to imaging data. “There are a number of new technologies being tested to identify misfolded protein in blood.
Drs. Caughey, Giles, and Cairns had no competing interests. Dr. Soto is inventor on several patents related to the PMCA/Rt-QuIC technology and is currently founder, chief scientific officer, and member of the board of directors of Amprion Inc, a biotech company focusing on the commercial utilization of PMCA and RT-QuIC for high sensitive detection of misfolded protein aggregates implicated in a variety of neurodegenerative diseases. The University of Texas Health Science Center at Houston owns some patent applications related to the PMCA technology that have been licensed to Amprion Inc.
The Science Explained: The RT-QuIC Assay
WHAT IT IS: The assay exploits the seeded conversion of normal prion protein to the abnormal form and therefore detects disease associated prion protein in the CSF.
HOW IT WORKS: The assay amplifies small samples of misfolded protein collected from CSF in living patients and autopsied tissue from pathologically-confirmed cases. RT-QuIC usually starts with a small body fluid sample, usually CSF, collected from patients. Nasal brushings, urine, skin and eye components have been used for the assay.
In 4-repeat tau RT-QuIC assays, a small drop of diagnostic specimen that contains the tau aggregate or “seed” is mixed with a vast excess of monomers of a recombinant 4-repeat tau fragment (that is, the substrate). The aggregates incorporate the substrate and begin growing into recombinant fibrils. A fluorescent dye is used to detect the fibrils as they accumulate.
HOW IT IS APPLIED: The assay has been used to detect abnormal aggregates of prion protein and tau fragments in CJD, tauopathies, Lewy body disease, and other neurodegenerative disorders.