NEW YORK (360Dx) – Researchers from the National Institute of Allergy and Infectious Diseases have developed an assay for identifying tau aggregates that could aid in the diagnosis of Alzheimer's disease.
Detailed in a study published last month in Acta Neuropathologica, the assay uses the propensity of the tau protein filaments characteristic of Alzheimer's disease to form aggregates to detect the presence of these filaments at very low levels, which could enable more accurate and earlier detection of the condition.
According to Allison Kraus, a research fellow at the NIAID and first author on the study, the researchers are now working to apply the technique, which was developed using patient brain tissue, to more accessible samples like blood or cerebrospinal fluid. She noted that the assay has drawn significant interest from companies and research groups interested in using it for variouspurposes including selection of patients for drug trials and tracking response to therapy.
Alzheimer's is characterized by the accumulation of tau protein in the brain, and both tau and phosphorylated tau, along with amyloid beta 42, are among the most commonly used protein biomarkers for the disease.
The NIAID assay, called AD real-time quaking-induced conversion, or AD RT-QuIC, uses the self-aggregating nature of the tau filaments found in the brain of Alzheimer's patients to amplify the molecule, which could potentially allow clinicians to detect the presence of abnormal tau filaments at extremely low levels.
In the Acta Neuropathologica study, the researchers took Alzheimer's brain tissue and incubated it with recombinant tau protein that is induced by the abnormal tau filaments to form larger aggregates, the production of which can be tracked via fluorescent dyes.
"You can take these [tau] aggregates, and add recombinant protein, and the aggregates will corrupt that recombinant protein to give amplification of these assemblies," Kraus said. "And that cycle just keeps going and going exponentially, and we can measure that with the dye."
The technique was first developed for the detection of prion diseases, which similarly involve aggregates of misfolded proteins, Kraus said.
"It has been very successfully applied toward that end," she said. "We can detect prions in brain tissue and in CSF and in nasal brushings and even skin. So based on that premise we developed the Alzheimer's assay where we're able to amplify those tau [filaments] for very sensitive and quantitative detection."
Using the assay, the researchers were able to detect tau aggregates in Alzheimer's brain samples at dilutions as high as 1010-fold and could detect synthetic tau filaments in amounts as small as 16 femtograms.
In addition to being highly sensitive, the assay is also amenable to high throughput, Kraus said, noting that it is done in a 384-well plate format and that results are available within a day.
Alzheimer's biomarkers have been a major area of focus for proteomics research with groups exploring a variety of proteins in hopes of identifying markers that could allow for more convenient testing and earlier detection of the disease.
Interest in such markers is particularly strong within the pharmaceutical industry, where firms are looking for better approaches to selecting patients for clinical trials and tracking response to their therapies.
The industry has had little success, to date, in Alzheimer's therapeutics, and one line of thinking holds that these struggles are due in part to treating patients too late in the disease process. This has made development of tests for detecting the disease in its earliest stages a priority.
Kraus said that she and her colleagues are currently exploring how early in Alzheimer's development the assay could detect the condition. She noted that they had found it was able to detect tau aggregates in patients with subclinical disease who appeared cognitively normal, which suggests that the assay is "very sensitive and would work early on in disease."
She said, however, that this finding is based only on a limited number of patients and more extensive testing is needed.
The researchers will also need to demonstrate that the assay can work in more "diagnostically relevant specimens" like CSF or blood, Kraus said. She said she and her colleagues were currently working with collaborators to get access to the samples they need to adapt the assay to these specimens. She added that the history of the method in other diseases including prion conditions and Parkinson's, where it has been successfully applied to CSF, suggests that it can be adapted to such samples in the case of Alzheimer's, as well.
The case of prion diseases also demonstrates the potential clinical usefulness of the assay, Kraus said, noting that it is currently used by the National Prion Disease Pathology Surveillance Center to diagnose prion diseases.
The study authors noted that a similar amplification-based assay exists for Aβ oligomers, which are also characteristic of Alzheimer's patients. Previous work has shown that when used in patient CSF, that assay, called Aβ-PMCA, was able to diagnose the disease with a sensitivity of 90 percent and specificity of 92 percent, a level of performance that might be improved by the addition of the AD RT-QuIC tau assay.
The researchers are also exploring whether the tau assay can be used in combination with quaking-induced conversion assays for other aggregates to improve the diagnosis of other neurodegenerative diseases, Kraus said.
"These [tau] aggregates often occur as sort of comorbidities in other diseases," she said. "You can have Alzheimer's-like aggregates in synucleinopathies (like Parkinson's), and that can really confound some of these diagnoses. So we're looking to see if we can apply these assays to such specimens to understand the degree to which both types of aggregates may occur."