By Deborah Borfitz
November 12, 2021 | Researchers in Germany are developing a lateral flow assay that aims to inform people if they are at heightened risk of developing Alzheimer’s disease (AD) and the robustness of their “cognitive reserve” to stave off symptoms of degenerative brain changes—all by measuring levels of three microRNAs (miRNAs) circulating in their blood. The idea is to identify the 10% of aging adults most in need of a visit to a memory clinic and more expensive, cumbersome diagnostics such as cerebrospinal fluid analysis and brain MRIs, says André Fischer, research group leader at the DZNE (German Center for Neurodegenerative Diseases) site in Göttingen and professor in the department of psychiatry and psychotherapy at University Medical Center Göttingen.
It could also give the other 90% peace of mind and perhaps spur them to take protective measures, including exercising their bodies and brains more and adopting a dementia-fighting diet, he adds. The test “could be very cheap, probably something you could buy yourself at CVS and retake every six months to look for changes.”
As chronicled in a recent edition of EMBO Molecular Medicine (DOI: 10.15252/emmm.202013659), the trio of miRNAs could provide early warning of impending dementia among people with mild cognitive impairment (MCI) several years before they convert to AD. The miRNAs may also be therapeutic targets for drugs that counteract their expression levels to possibly “reinstate memory function,” says Fischer.
MicroRNAs are of growing scientific interest for several reasons, he adds. For starters, only about 2,500 of the little molecules exist in RNA and they’re easy to measure and remarkably stable—resisting degradation even if they’re left at room temperature for five days. They also play key roles in the regulation of cellular processes and, much like the hormone system, mediation of signaling between organs.
That means MiRNAs could provide a lot of potentially useful information about synapse loss in the brain, inflammatory responses, metabolic changes, and cardiovascular pathologies. Multiple studies have already shown that miRNA plays a causative role in neurodegeneration, including AD.
It appears possible to use just a handful of miRNAs to better understand multiple biological pathways, says Fischer. With inflammation where cytokines are typically measured, for example, a single miRNA might instead provide information on the entire inflammatory process.
Building Evidence
In a reversal of the usual approach to biomarker research, Fischer explains, he and his colleagues did not use patients as the discovery cohort but instead curated a disease signature and then looked for it in people. Typical practice is to look for signatures based on observed differences between patient cohorts and healthy controls using bioinformatics, machine learning, and artificial intelligence.
“But this is always biased… because something else was used to make the diagnosis,” he says. “You will never be able to find anything really early [this way].”
Here, the researchers started with a variety of approaches in healthy individuals and otherwise healthy animals to identify the three critical miRNAs and then tried to find that signature in clinical cohorts. Importantly, altered expression of the signature reflects aberrant neuronal and immune processes that could potentially correspond to changes in the brain and thus serve as targets for RNA therapeutics, he says.
The miRNA signature was initially correlated with cognition in a cohort of 132 healthy and young humans who took cognitive tests and donated blood samples. Differences in cognition were seen, having nothing to do with sex or education level, although nobody was demented, continues Fischer.
A mouse model was then used as a proxy for a longitudinal study of age-associated memory decline in people, reducing the wait for results from 30 years to 16.5 months. This involved water maze training of the animals followed by a memory test and measuring miRNAs in their blood every 1.5 months. Differences in expression were seen starting at 12 months of age (equivalent to roughly 58 human years).
Out of 55 aging responsive miRNAs, seven linked to cognition in healthy humans in a recent genome-wide association study and the chosen three—miR-181a-5p, miR-148a-3p, and miR-146a-5p—could also be linked to neuronal function and inflammation.
Further evidence came from patients with MCI involved in a DZNE study that has been running for years in collaboration with university clinics throughout Germany. Among those in whom the blood marker was highly elevated, about 90% developed Alzheimer’s disease within two years.
“We therefore see an increased blood level of these three microRNAs as a harbinger of dementia,” Fischer says. “We estimate that in humans this biomarker indicates a development that is about two to five years in the future.”
To test the hypothesis that altered levels of the three-miRNA signature control cellular processes essential for cognitive function in the human brain, researchers employed brain organoids. Findings were in line with the data obtained in mouse cultures, with heightened levels of the miRNA inducing gene expression changes and related biological processes linked to cellular stress and decreases in genes representing synaptic function-related processes.
This suggests that analysis of circulating miRNA that inform about central nervous system mechanisms may be suitable to the development of corresponding RNA-based therapies, Fischer says.
Promising Therapeutics
The consensus on why most drugs developed for AD over the last decade have failed in clinical trials is because the wrong patients are being treated, says Fischer. Getting treated early in the disease process is also crucial because the therapeutic effect of causative therapies is likely to be minimal in patients with advanced neurodegeneration.
To the surprise of many, the U.S. Food and Drug Administration recently granted aducanumab (an amyloid-β–directed antibody marketed as Aduhelm) accelerated approval for the treatment of AD and it was not without proof, he says. Basis of the approval was the drug’s effect on a surrogate endpoint—reduction of amyloid beta plaque—and its intended users are patients with AD-related MCI or mild dementia.
During clinical trials, early identification of AD was accomplished by the traditional invasive battery of tests, he notes. But in the real world, people typically don’t get diagnosed that way. More often, they’re dragged by relatives into a memory clinic at a relatively advanced stage of the disease.
RNA therapeutics look particularly promising, Fischer says, and enthusiasm is building thanks in part to the Pfizer and Moderna mRNA vaccines. Although the underlying mechanisms of messenger RNA and microRNA are “slightly different” (a mRNA is regulated by several miRNAs), both are thought to be useful in treating neurodegenerative diseases.
Aside from not yet having a cure for AD, the other big challenge is not having an ideal disease marker—non-invasive, easily measured, inexpensive, and producing rapid results—enabling routine screening, as happens for cancer, Fischer says. The newly discovered miRNA signature could possibly fill that gap so people could get screened every other year, starting around age 60, and the 10% high-risk population would get the more rigorous diagnostic workup and early treatment with drugs such as aducanumab.
Clinical Testing
Creation of the miRNA-based screening assay is the most immediate goal, Fischer emphasizes. At the population level, many app-based cognitive tests are already in use among the worried well checking for early signs of AD. “It would be important to also have a molecular marker that can be easily measured.”
DZNE researchers are already working with the German biotech company Fassisi to develop the analysis of the three-miRNA signature into a lateral flow assay, says Fischer. He expects the final product to be affordable and as simple to administer as a home pregnancy or COVID-19 test with a fingerprick of blood.
Detection is currently being done with next-generation sequencing and reagents that cost roughly $100 each, he points out. The new assay, which is expected to be ready to deploy early next year, would initially be rolled out as a screening tool to a large longitudinal cohort of about 1,500 patients and healthy controls in Germany.
This is the same group of patients used in the proof-of-concept study, Fischer says, to enable a comparison of results from sequencing and the new assay. An additional group of 860 individuals in the U.S. would next join the study.
Researchers will also attempt to stratify patients into groups who receive treatment and see how they are responding to a particular drug, says Fischer. “If the cognition gets better, we also will see that the signature gets normalized.”
If all goes well, clinical trials testing whether the assay could be used as a medical product might be pursued as required by various regulatory bodies, he adds. The test could potentially be market-ready within two years.