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Proteome Sciences Investigating Variety of Phospho-Tau Forms as Alzheimer's Markers


NEW YORK (GenomeWeb) – Proteome Sciences and its collaborators have identified a number of phosphorylated forms of tau protein that could serve as biomarkers for Alzheimer's disease.

In a study published last month in the Journal of Alzheimer's Disease, the researchers identified 47 different tau phosphopeptides covering 31 different phosphorylation sites and 11 tau phosphopeptides that were upregulated by 40 percent or more in Alzheimer's patients compared to controls. 

The findings suggest that these forms of phosphorylated tau could help with early detection of Alzheimer's, including with predicting patients with mild cognitive impairment who are likely to develop the disease, Ian Pike, Proteome Sciences' chief operating officer, told GenomeWeb.

The study also serves as a showcase for the company's TMTcalibrator technology, a workflow that enables more reliable measurement of lower abundance proteins, Pike said.

Phosphorylated tau is one of the most well-established protein biomarkers for Alzheimer's and, along with the CSF biomarkers amyloid-β and total tau, is widely used as an aid for diagnosing the disease, especially in clinical trial work. Like many proteins, tau can be phosphorylated at a number of different sites. However, Pike noted, to date Alzheimer's research has focused mainly on one form of the molecule, tau phosphorylated at the threonine residue 181 (pThr181).

In large part, this was a matter of happenstance, Pike suggested.

"It was a site to which a couple of groups were able to produce really good antibodies to, and so people were able to develop a diagnostic test using it," he said, adding that because this marker has performed relatively well it has remained the focal point of phospho-tau research in Alzheimer's.

But while the combination of tau, phospho-tau, and amyloid-β has, along with other clinical measures, proved effective at diagnosing Alzheimer's in patients in the later stages of the disease, the field still lacks good biomarkers for early detection. Alzheimer's drug development efforts have struggled, and some have suggested that treatments could be more effective if they are started earlier in the disease's progression. This makes early detection key both to treating patients and selecting clinical trial cohorts.

The JAD study did not address subjects with mild cognitive impairment but rather compared cerebrospinal fluid samples from three Alzheimer's patients to three controls.

"Obviously, at the moment these are no more than candidate markers that require further validation," Pike said. He noted, though, that several of the phospho-tau forms they measured in the experiment appeared to be more strongly regulated in Alzheimer's patients than the pThr181 form that is most commonly studied.

The hope, Pike said, is that other forms of phospho-tau may be involved earlier in the disease process than pThr181, making them useful for early detection of the disease. Looking at multiple forms of phospho-tau could provide the additional specificity and sensitivity required for early diagnosis, he added.

While the JAD study focused on phospho-tau, the analysis profiled the entire CSF proteome and identified around 100 proteins in addition to the tau phospho-forms that differed between Alzheimer's patients and controls, Pike said.

The researchers used Proteome Sciences TMTcalibrator technology for the work, which Pike said allowed them to detect low abundance molecules like various phospho-tau species while retaining mass spec's ability to generate comprehensive, unbiased proteomic profiles.

TMTcalibrator works by combining isobarically labeled peptides from both the fluid of interest (CSF, in this case) and the tissue in which the target proteins are produced (brain, in this case). By including the tissue digest at a higher concentration than the fluid digest (roughly two to one in the JAD study), researchers can ensure that even analytes present at only low abundance in the fluid digest are present at high abundance in the overall sample, making them more likely to be fragmented and detected by the mass spec.

Use of TMTcalibrator allowed the researchers to avoid using antibody enrichment, which, Pike said, could bias the findings towards one particular form of phospho-tau or require the use of multiple pulldowns using different antibodies.

"There is a risk if you use tau immunoprecipitation that you bias the population of tau that you pull out," he said. "So depending on which antibody you use you may get different populations of tau, and in that case you would need to do multiple pulldowns. So you would have lots of different comparisons, and the different amounts of tau you would get [in the different pulldowns] would make the whole calibration process relatively more difficult."

Likewise, while targeted mass spec might also have provided the required sensitivity, developing assays to each of the different tau phospho-forms would have been a difficult undertaking, Pike said.

"To cover all of the possible sites in tau with all of the possible combinations you would need over 100 different [assays], so it is somewhat impractical to do it by [selected-reaction monitoring]," he said.

Now that the researchers have identified a smaller set of candidate markers, an SRM-based approach is more practical, Pike said, adding that they now plan to develop SRM assays to some of the phospho-tau forms identified in the study.

There is not much evidence currently that these alternative phospho-tau forms could enable earlier detection of Alzheimer's than the commonly used pThr181 marker, but Pike suggested that this was in part because they have received relatively little attention.

"We haven't really had good ways of measuring them in CSF up till now," he said. "But clinically, in terms of understanding the pathology of neuronal damage from the initiation in the hippocampus and then all the way through the cortex and the rest of the brain, it is now well understood that you need phosphorylation of tau at a couple of sites that promotes its formation into small oligomers which are still soluble, and they can cross from the synapse and go from one neuron to the next."

The ability to measure a large variety of phospho-tau forms suggests that researchers "might be able to detect the earliest signs of these propagating forms of tau in CSF samples," Pike added. "There is certainly a lot of validation work to be done, but now that we know which peptides are there and can be detected, we have a much better chance of developing assays with the required sensitivity to help us do that."