13 Jul 2024

Tau in the blood can identify people in the earliest stages of Alzheimer’s disease, but plasma markers for other tauopathies, not to mention TDP-43 proteinopathies, have been hard to come by. Now, suddenly it seems, the field may have both. In the June 18 Nature Medicine, scientists led by Anja Schneider, German Center for Neurodegenerative Diseases, Bonn, report that extracellular vesicles floating in the blood may hold the key.

The ratio of three-repeat (3R) and four-repeat tau in these blebs of cytoplasm distinguished people with behavioral variant frontotemporal dementia (bvFTD) from people with progressive supranuclear palsy (PSP). These two primary tauopathies can be difficult to tell apart. Levels of TDP-43 in blood extracellular vesicles also identified people who have ALS or a form of FTD that causes this nuclear protein to accumulate in the cytoplasm.

The work impressed Robert Rissman, University of California, San Diego. “A lot of validation needs to be done, but if we can identify these diseases, then we can select whom to include in clinical trials. That would be a real benefit,” he said.

Henrik Zetterberg, University of Gothenburg, Sweden, told Alzforum the work was exciting. “By themselves, these markers are meaningless in plasma, but when Schneider isolates them from vesicles, the signal is strong, meaningful, and could become relevant to memory clinics,” he said. “We should all try to reproduce it.” Zetterberg has already begun to do so.

Frontotemporal dementias and ALS are part of a clinical spectrum that includes progressive supranuclear palsy and corticobasal degeneration. Many of the diseases on this spectrum are difficult to distinguish clinically, hence postmortem neuropathology still stands as the gold standard for diagnosis. In their search for a diagnostic test during a patient’s lifetime, preferably very early in their disease, scientists have scrutinized isoforms of tau, phosphorylated tau, and aggregates and modified forms of TDP43 for potential biomarkers, but results thus far have been equivocal or hampered by insufficient target protein in extracellular fluids, such as the plasma and the CSF. Schneider, who studies the role of EVs in cell-to-cell communication and in the spread of toxic proteins, wondered if these exvaginations of the intracellular milieu might offer a better chance of success.

In a pilot study, first authors Madhurima Chatterjee and Selcuk Özdemir isolated EVs from the plasma and CSF of 141 people in the Describe cohort, aka the DZNE Clinical Register Study of Neurodegenerative Disorders. They found that the ratio of 3R to 4R tau was much higher, around 2.6, in vesicles from 42 people clinically diagnosed with bvFTD than in those from 15 healthy controls, 23 people with AD, and 17 with semantic variant PPA, whose ratios all hovered around 1.0. Three-R and 4R ratios in 44 people with PSP were the lowest, at around 0.18. In PSP, aggregates of 4R tau predominate, whereas in FTD, 3R isoforms do. In AD, neurofibrillary tangles comprise a mix of 3R and 4R tau. In their pilot study, the scientists had found that the 3R/4R ratio nicely reflected the pathologies of each disease (image below).

Truth in Vesicles? The 3R/4R tau ratio (left) in plasma extracellular vesicles distinguishes bvFTD and PSP from controls and other dementias. EV TDP-43 does the same for ALS and bvFTD (right). [Courtesy of Chatterjee et al., 2024.]

A similar, though less robust, separation emerged in a larger Describe sub-cohort comprising 56 controls, 165 people with ALS, 179 with bvFTD, and 163 with PSP. Still, in specificity versus sensitivity plots, the tau ratio distinguished people with bvFTD from healthy controls and PSP with area-under-the-curve values of 0.91 and 0.89, respectively. AUCs for PSP versus controls, ALS, and bvFTD were in 0.96 to 0.98 range.

The ratios also correlated with disease severity. People with the lowest 3R/4R ratios in the PSP group posted the worst scores across a range of neurological, clinical, and functional measures. For bvFTD, higher ratios equated to worse scores. All told, this ratio looks promising as a marker for differential diagnosis.

What of TDP-43? The scientists found ample amounts of this protein in plasma EVs as well. Vesicles from people with ALS contained about 45 pg/ml, compared to about 9 pg/ml for healthy control and PSP EVs. EV TDP-43 distinguished ALS from controls/PSP with AUCs of 0.99, and separated ALS from bvFTD with slightly less accuracy, 0.91. Again, the more TDP-43 a person’s vesicles bore, the more severe their disease.

Since bvFTD is tricky to diagnose clinically, the scientists next tested these candidate markers in genetic and pathologically confirmed cases. In EVs from 28 people who carried mutations in C9ORF72, progranulin, valosin-containing protein, or TANK-binding kinase—all of which are linked to TDP-43 pathology—the 3R/4R ratio was around 1.0. In EVs from three people with mutations in the MAPT gene for tau, the 3R/4R ratio was three to four times higher, a robust difference. Consistent with this, 3R/4R tau ratios tracked low in 22 people with TDP-43 neuropathology and four people with confirmed 4R pathology.

As for TDP-43, EVs from people with neuropathologically confirmed pathology contained much more of the protein than did vesicles from tau pathology or MAPT mutation cases.

The ability to separate clinical FTD into FTD-tau and FTD-TDP-43 emerged as a key finding. That alone could be helpful as together, these two diseases account for about 90 percent of bvFTD cases. Chatterjee and colleagues found that when EV TDP-43 was high, the 3R/4R tau ratio was low, and vice versa. This enabled them to split the bvFTD group into its two major forms (image below). 

Which FTD Is It? High EV TDP-43 and low 3R/4R tau ratio identifies people with FTD-TDP43 in the Describe (left) and Sant Pau cohorts (right). The inverse indicates FTD-tau. Black lines depict low and high cut-off levels for each marker. [Courtesy of Chatterjee et al., 2024.]

To validate these findings, the researchers looked beyond Describe and measured the EV markers in a patient cohort from the Hospital de la Santa Creu i Sant Pau in Barcelona. This included 65 people with ALS, 58 with ALS-FTD, 50 with bvFTD, 41 with PSP, 50 healthy controls, and 23 people who had FTD mutations. By and large the Sant Pau and Describe data matched well. Notably, cut-off values calculated to reflect low and high tau ratios or TDP-43 were almost identical between the two cohorts (see image above).

Why would extracellular vesicles distinguish these various types of dementia where other tests, including of the proteins themselves in plasma, have failed? Plasma TDP-43, for example, did not differ among the diagnostic groups. Schneider told Alzforum that the vesicles may carry pathological forms, which might not show up in extracellular fluids.

In support of this, most of the EV TDP-43 and tau came from vesicles that tested positive for L1CAM. Some scientists think this cell adhesion molecule marks EVs that come from neurons in the brain—a controversial concept (Jun 2021 news). Zetterberg said it’s unclear where the vesicles come from, but he suspects it is the brain. For her part, Schneider does not think the origin of the EVs matters for diagnostic purposes, but might for monitoring in clinical trials. “If you want to measure target engagement, then it would be nice to measure a marker that comes from the brain,” she said. “We need better ways to detect brain-derived extracellular vesicles.”

This work could have implications beyond ALS and FTD. Aggregates of TDP-43 are found in people with AD and cause limbic-predominant, age-related TDP-43 encephalopathy, aka LATE (May 2019 news). “I think their EV TDP-43 assay is potentially a good one for LATE, and it should be tested,” Peter Nelson, University of Kentucky, Lexington, told Alzforum. He and Rissman had previously found that TDP-43 ticks up in astrocyte-derived EVs in LATE (Winston et al., 2022). “Whether it helps predict and stratify LATE might be a much bigger question than whether it can diagnose ALS, since LATE is much more common,” Nelson said. Schneider and colleagues noted that TDP-43 co-pathology in AD means faster clinical decline and that plasma EV TDP-43 might help stratify those cases, a point that Nelson raised as well.

Schneider wants to test asymptomatic carriers of FTD/ALS mutations to see how soon these markers can be detected in the course of disease. “It would also be interesting to test longitudinally in a clinical trial to see if we can get a target response,” she said.—Tom Fagan

News Citations

1. Neuronal Exosomes Embroiled in Controversy 10 Jun 2021
2. Introducing LATE—A Common TDP-43 Proteinopathy that Strikes After 80 1 May 2019

Paper Citations

1. Winston CN, Sukreet S, Lynch H, Lee VM, Wilcock DM, Nelson PT, Rissman RA.
   Evaluation of blood-based, extracellular vesicles as biomarkers for aging-related TDP-43 pathology.
   Alzheimers Dement (Amst). 2022;14(1):e12365. Epub 2022 Dec 15
   PubMed.

No Available Further Reading