Article In Brief
Researchers identified post-translational modifications of toxic tau protein in the autopsy tissue of patients diagnosed and confirmed postmortem with Alzheimer's disease by using novel high-resolution proteomics mapping technology.
Using novel high-resolution proteomics mapping technology, scientists at Harvard Medical School identified post-translational modifications (PTMs) of toxic tau protein in the autopsy tissue of patients diagnosed and confirmed postmortem with Alzheimer's disease (AD).
The technique allowed them to identify PTMs at different stages of the disease, they reported in the December issue of Cell. These PTM maps could potentially help identify critical targets at the earliest time points in the disease process, setting the stage for disease staging and classification. Moreover, information from the maps could be useful for the design and testing of new therapies, the investigators suggested.
The senior author of the study Judith Steen, PhD, associate in neurology at Boston Children's Hospital and associate professor of neurology at Harvard Medical School, said growing evidence suggests that PTMs are common occurrences and regulate many functions of abnormal tau, including its aggregation and prion-like spread to other neurons. Dr. Steen, who trained in mass spectrometry-based proteomics, has continued to develop proteomics technologies to study human disease, first in the cancer world and now in neurology.
The maps enabled the research team to identify chemical changes in the tau protein as it becomes prion-like and spreads through the brain. This approach allowed them to identify key PTMs such as phosphorylation, acetylation, and ubiquitination.
“We began looking at autopsied tissue and using unbiased data analysis approaches, which allows data to direct our hypotheses,” said Dr. Steen.
“Then, using our workflow, we were able to see a lot more modifications in tau than anyone has seen before. When we analyzed the data, the tissue from Alzheimer's patients separated out from the controls. While everyone has their own lifestyle and general risk factors, there was a common endpoint. These PTMs were similar across all patients, and in some of the controls who may have been in the pre-clinical stages of disease.”
The tau PTMs showed patient heterogeneity at different stages of AD.
Study Design, Findings
To understand the functional significance of protein modification, the scientists set out to map possible PTMs—from the N- to the C-terminus. The scientists generated a high-resolution quantitative proteomics map using postmortem tissue (the frontal gyrus and angular gyrus) from 49 Alzheimer's patients and 42 sex-matched controls to identify PTMs. They identified 95 PTMs in several isoforms of tau.
The maps showed heterogeneity across the subjects, but a subset of the PTMs were far more common in occupancy and in frequency in AD patients compared to the controls.
The PTMs occurred in an ordered manner that ultimately culminated in aggregation. The patients with the worst pathology at death had more PTMs, and greater occupancy in the tissue studied. The ON and 4R tau isoforms were most prone to aggregation, according to findings from the study.
The scientists were then able to look at the modifications as they changed over time in the AD patients and in controls. In the early-to- moderate clinical stages, there were modifications linked to tau phosphorylation in the proline-rich region and in the C-terminus, the end of the protein chain. Then, there was additional phosphorylation in the C-terminus and proline-rich region, which was accompanied by cleavage of the tau protein.
Dr. Steen thinks that these proteomic responses set off a chain of events culminating in the acetylation and ubiquitination in the tau microtubule-binding domain, which correlates with progressively worsening clinical symptoms.
The team, which included scientists from medical centers around the world, also defined a minimum set of modifications—the PTMs that result in a tau prion-like molecule, which is thought to seed and propagate tau aggregation in nearby cells in the brain that leads to neuronal damage and atrophy becoming widespread during disease progression.
“We have identified a tau protein species critical for disease intervention at different stages of the disease,” said Dr. Steen.
If the findings hold up, she said that “we may be able to identify therapeutics that target PTMs and then test the potential therapeutics to see whether they can block the development of PTMs at earlier stages and prevent or slow the disease process.”
“The pictures from these maps tell us a story of a lifetime of stressors on the brain,” Dr. Steen said. “We are using a very different set of tools to see how this disease spreads. It is possible that therapeutics that prevent these modifications or create more beneficial chemical modifications could be used to treat Alzheimer's.”
Tissue samples in the study were collected from brain banks at the University of California, San Francisco, University of California Los Angeles, the Harvard Brain Tissue Resource Center, the University of Maryland Brain and Tissue Bank, and the National Institutes of Health NeuroBioBank. The study was funded by the Tau Consortium, a collaborative research program managed by the Rainwater Charitable Foundation in partnership with other funders.
“In recent years, it has become clear that AD is a heterogeneous disorder with diverse clinical presentations, age of onset, and mixed pathology,” said Eliezer Masliah, MD, director of the division of neuroscience at the National Institute on Aging (NIA), part of the National Institutes of Health.
“The mechanisms explaining this heterogeneity are not totally clear but are important to understand in terms of developing targeted therapeutics for the subtypes of AD.”
“This study is important and timely and provides an explanation for the heterogeneity of AD based on defined and very specific molecular changes or PTMs in tau microtube binding domains [MTBs]. In contrast, other studies utilizing novel high-resolution technologies, such as cryoEM, support the view that structural differences in tau fibrils might explain heterogeneity. The roles of PTMs on MTB in relationship with tau aggregation are mixed. Nevertheless, this paper seems to provide strong evidence supporting a role PTM has on tau-related pathologies in AD heterogeneity.”
He said that the “correlation between PTMs and tau's seeding activities is solid. Now the key is to test whether one can link this biochemical observation to drive the change of biology and pathology.”
The NIA is supporting a broad range of complementary grants using various biological and biophysical methods to validate this study as well, Dr. Masliah added.
“Together with some of other phospho-tau CSF and plasma biomarker studies published by Alzheimer's Disease Neuroimaging Initiative and Randall Bateman's group at Washington University School of Medicine, this paper also provides a strong justification that those p-tau biomarkers work,” he said. “In short, this paper has a potential for developing disease staging and classification. There are enough PTMs to permit this type of classification by artificial intelligence [AI], and it will potentially have a better resolution than most omics-related AI classification.”
“This is a comprehensive evaluation of the post-translational modifications of tau and how they relate to aggregation properties and disease in Alzheimer's,” said Alison M. Goate, DPhil, the Willard T.C. Johnson research professor of neurogenetics, and professor of genetics and genomic sciences, neuroscience, and neurology at Icahn School of Medicine at Mount Sinai.
“It gives us a clear picture of how modifications change at different stages of the disease in both soluble and insoluble tau. This work will help the field refine the choice of biomarkers used when looking for changes in cerebrospinal fluid and blood in AD patients at different stages of disease.”
Dr. Goate explained that the data are consistent with prior observations that phosphorylation of tau at residues serine 217 and threonine 181 are early events in disease pathogenesis, and demonstrate that acetylation changes occur somewhat later in the disease progression.
She added that it would be helpful to conduct a similar study using samples of blood and cerebrospinal fluid in AD patients and controls at different stages of disease, as well as studying other atypical forms of dementia. She agreed that the technique could be useful in identifying new treatment targets at different stages of the disease and stage-specific biomarkers.