How a Molecular Matchmaker Is Furthering Alzheimer’s Research

A new drug helping natural enzymes target a dysregulated protein represents a step forward in Alzheimer’s research.

By Rebecca DeGiosio

There’s plenty of fish in the sea…and that’s especially true of our cells. Scientists estimate that a single cell contains over 42 million proteins. These proteins—biomolecules formed from chains of amino acids—are diverse in size, shape, and function. They range from enzymes that speed up chemical reactions to structural proteins that give our cells shape. Proteins can also modify each other; for instance, enzymes known as kinases can fuse phosphate groups to other proteins, “phosphorylating” their targets, while phosphatases remove these groups. 

Despite this complex ecosystem of molecules, the functions of these proteins are well-balanced…most of the time. In disease and disorder, there’s often an imbalance in the amount of different proteins. There can also be unusual interaction of proteins, or a lack of normal interactions. Now, researchers are developing new drugs to correct these interactions. This forms a large part of Alzheimer’s research, since specific proteins become dysregulated in Alzheimer’s disease (AD).

Tau: The Lynchpin of Alzheimer’s

AD is a type of condition called a tauopathy: a disorder caused by dysfunction of a protein called Tau. In AD, Tau is hyperphosphorylated: too many phosphates are added to it. This seems to result from unusual interactions between Tau and its kinases. This phosphorylation changes the structure of Tau, causing it to aggregate into filaments called neurofibrillary tangles (NFTs). NFTs are a hallmark of AD, and it’s long been thought that removing phosphates from Tau (or getting rid of some Tau altogether) could slow or stop AD progression. 

Luckily, there are phosphatases in our cells designed to do just that…but they target other proteins, too. That could cause a host of nasty side effects. So how do we get them to target Tau exclusively?

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Plan of a “TAC”

In 2001, researchers from UCLA published a groundbreaking experiment. They fused a drug called ovalicin, which binds a protein involved in cancer growth to another protein that helps break down dysfunctional or excess proteins. They found that this hybrid molecule was able to force the two molecules to interact, leading to degradation of the cancer-promoting protein. In doing so, they described the first PROTAC: PROtein TArgeting Chimera.

Since then, many scientists have explored similar ways to rig connections between specific proteins and molecules. Most of these experiments aim to cause degradation of a protein, like the original PROTAC. This is in part because protein aggregation is common in disorder. For example, in AD, another protein called amyloid beta also accumulates in neurons, forming clumps called plaques. In Parkinson’s disease, a protein called α-synuclein clumps into structures called Lewy bodies. Helping our cells break down these too-abundant proteins could improve brain function and clinical outcomes.

However, Jingfen Su and colleagues from Huazhong University of Science and Technology wondered if a similar strategy could instead be used to dephosphorylate Tau as an alternative treatment for AD. In 2021, their Alzheimer’s research led them to develop the first DEPTAC, or DEPhosphorylation TArgeting Chimera, promoting the interaction of Tau and a phosphatase called PP2A. Now, the same research group is expanding and refining their DEPTAC technology.

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DEPTAC: A Flexible Molecule

The DEPTAC is composed of three parts: 1) the part that recognizes and binds Tau protein (the “warhead”), 2) the part that recognizes and binds a phosphatase (the “anchor”), and 3) a “linker” element which joins the two. While keeping the warhead constant, Su and colleagues systematically tested different anchors for efficiency in dephosphorylating Tau. This could be measured using antibodies that specifically bind phosphorylated Tau. 

They found that sequences designed to bind different phosphatases were all capable of reducing Tau phosphorylation, though with differing efficacy. They also tested different linkers and warhead sequences. Intriguingly, they found that these two elements had synergistic effects on the efficacy of the DEPTAC; certain combinations seemed to work best. This suggests that if DEPTACs are developed for other proteins and diseases, it will take careful optimization of all components to yield an effective therapeutic.

Su and colleagues went on to further study one particularly effective DEPTAC they called D16. First, to confirm that Tau and the phosphatase (PP2A) were actually interacting, they used a method called fluorescence resonance energy transfer, or FRET. In this assay, two proteins are tagged with different fluorescent molecules. When these molecules are in very close proximity (like when Tau and PP2A are interacting), they change each other’s properties in such a way that the wavelength of light emitted is altered. This changes the color of the light. Using this technique, Su and colleagues found that the presence of D16 significantly increased interaction of Tau and PP2A in cells.

Next, the researchers wondered if dephosphorylation of Tau by D16 could help clear up Tau accumulations like NFTs. To create a cellular model of AD, they “seeded” rat neurons with small Tau aggregates. These aggregates go on to spur further Tau phosphorylation and aggregation, forming NFTs. However, treatment with D16 reversed these accumulations, suggesting it could have value in Alzheimer’s research.

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DEPTAC as a Therapeutic

However, that experiment was performed in neurons grown outside the body. Thus, to test whether D16 can improve AD-like features in animals, the researchers used a strain of mice called P301L mice. These mice have a mutation which makes Tau more prone to hyperphosphorylation and aggregation. After injecting D16 into these mice, Su and colleagues found that a single dose significantly reduced phosphorylated Tau as well as total Tau levels in the hippocampus, a brain region linked with learning and memory.

Finally, it was unclear whether reducing Tau phosphorylation this way would actually lead to improved cognitive function. To test this, they used another AD model, 3×Tg mice. These mice have mutations in three AD-linked genes, one of them being the gene that makes Tau. 3×Tg mice have significant impairments to several forms of learning and memory.

The researchers subjected mice treated with D16 to several tasks designed to test their memory. One such test was a novel-object recognition task. This relies on the tendency of mice to explore new objects. For this test, mice are acclimated to a space with two identical objects. The mice are then removed, and one of the objects is exchanged for a new, distinct object before the mice are returned. If the mice successfully remember the original, familiar object, they’ll be more likely to explore the new object. 3×Tg mice treated with DEPTAC explored the new object more than untreated 3×Tg mice, suggesting their memory improved.

Alzheimer’s research and new drug development

DEPTACs, like other protein-targeting chimeras, could revolutionize Alzheimer’s research as well as drug development for other diseases. They still have a way to go, however, before they’ll make it to your doctor’s office or pharmacy. DEPTACs have yet to be tested in humans for safety and efficacy. And while several PROTACs are currently being investigated in clinical trials, they still bear several drawbacks that may be shared by DEPTACs.

Firstly, PROTACs and DEPTACs are rather large, as therapeutics go. This limits their ability to travel throughout the body, and especially across the blood-brain barrier (BBB). The BBB is a sheet of cells protecting the brain from unapproved molecules in the bloodstream. While Su and colleagues demonstrate that D16 is able to get through the BBB and reach several brain areas in mice, it’s not clear if it can permeate enough of the (much larger) human brain to have any therapeutic effect. The size of these molecules also makes their production more difficult than smaller drugs, making for a more expensive medication.

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Also, these chimeras may target the wrong proteins to some extent. This so-called “off-targeting” could cause dangerous side effects in patients. Though Su and colleagues confirmed that D16 does not dephosphorylate MAP2, a protein similar in sequence to Tau, nor several other proteins usually targeted by PP2A, still other proteins could be affected. Further studies will need to robustly test the specificity of a Tau-targeted DEPTAC.

Still, DEPTACs could represent an important step forward in Alzheimer’s research, as well as research on other disorders caused by unusual protein phosphorylation. More broadly, similar chimeras could continue to manipulate proteins in new ways. We may one day direct proteins to organelles of our choosing, or better control their secretion and uptake by certain cell types. 

Perhaps then, human disease will have truly met its match.

This study was published in the peer-reviewed journal Science Bulletin.

References

Békés, M., Langley, D. R., & Crews, C. M. (2022). PROTAC targeted protein degraders: the past is prologue. Nature Reviews Drug Discovery, 21(3), 181–200. https://doi.org/10.1038/s41573-021-00371-6

Ho, B., Baryshnikova, A., & Brown, G. W. (2018). Unification of protein abundance datasets yields a quantitative Saccharomyces cerevisiae proteome. Cell Systems, 6(2), 192–205. https://doi.org/10.1016/j.cels.2017.12.004

Sakamoto, K. M., Kim, K. B., Kumagai, A., Mercurio, F., Crews, C. M., & Deshaies, R. J. (2001). Protacs: Chimeric molecules that target proteins to the Skp1–Cullin–F box complex for ubiquitination and degradation. Proceedings of the National Academy of Sciences, 98(15), 8554–8559. https://doi.org/10.1073/pnas.141230798

Su, J., Xiao, Y., Wei, L., Lei, H., Sun, F., Wang, W., … & Wang, J. Z. (2024). Generation of tau dephosphorylation-targeting chimeras for the treatment of Alzheimer’s disease and related tauopathies. Science Bulletin, 69(8), 1137–1152. https://doi.org/10.1016/j.scib.2024.01.019 

Zheng, J., Tian, N., Liu, F., Zhang, Y., Su, J., Gao, Y., … & Wang, J. Z. (2021). A novel dephosphorylation targeting chimera selectively promoting tau removal in tauopathies. Signal Transduction and Targeted Therapy, 6(1), 269. https://doi.org/10.1038/s41392-021-00669-2