DC: What’s That New Stuff in and Around Nuclei of AD Cells?

Series - Society for Neuroscience Annual Meeting 2011:

At this year’s annual meeting of the Society for Neuroscience, the number of mini- and nano-symposia dedicated to the molecular stalwarts of Alzheimer’s disease, Aβ and tau, was strikingly down. The trend may reflect both the field’s branching out toward other mechanisms and its shift toward more clinical and preclinical studies. In keeping with this shift, many of the Aβ presentations at the conference, held 12-16 November in Washington, DC, focused on treatment strategies or diagnostic potential. The former included a session on anti-Aβ strategies (see ARF related conference story), while the latter featured two posters from Charles Glabe’s lab that generated some hubbub and a novel tau correlate unveiled by Ben Wolozin’s group at Boston University.

Glabe, at the University of California, Irvine, has over the years generated a slew of antibodies to oligomeric forms of Aβ, notably the A11 and OC polyclonal antibodies that recognize a common structural conformation shared by different types of protein aggregates (see ARF related news story). At SfN, Glabe reported on a new monoclonal antibody whose properties raised eyebrows among other scientists. M78 recognizes a fibril-specific epitope in amino acids 6 to 12 of Aβ, but also binds large multimers and fibrils of α-synuclein and islet amyloid polypeptide. In that regard, it behaves similarly to A11, but in other aspects it seems unique. On his poster, Suhail Rasool in Glabe’s lab reported that M78 cross-reacts with some sort of inclusion in transgenic mice that is nuclear—not, as would be expected, extracellular, or even cytosolic. It also binds amyloid plaques. In another poster, Anna Pensalfini, in the same lab, reported that M78 similarly decorates cell nuclei and plaques in postmortem brain tissue taken from demented patients and cognitively normal people. The findings hint that the antibody may be detecting an early marker of pathology. Researchers with whom Alzforum at the meeting were intrigued while scratching their heads as to what this antibody might be picking up.

Probing the brains of six- to 14-month-old triple-transgenic (3xTg mice), Rasool found that M78 immunoreactivity appears in the nucleus at about 12 months of age. Scientists report variable pathology in these animals, but Glabe and colleagues find they accumulate intracellular Aβ (see ARF related Webinar) at about nine months and extracellular plaques at about 14 months. Rasool reported that M78 nuclear immunoreactivity coincides with cytosolic staining with the antibody 6E10, which recognizes the N-terminal of Aβ. Glabe told Alzforum that the new monoclonal seems to predominantly stain neurons, but also some oligodendrocytes and astrocytes. The researchers could not definitively say if it cross-reacts with any components in microglia. Most M78-positive nuclei lit up in the TUNEL test for DNA fragmentation, a marker of apoptotic cell death. M78 also recognized plaques that are not detected by 6E10 or by 4G8, another antibody that also binds the N-terminal of Aβ. It is unclear if M78-positive plaques are of a different kind. Rasool found thioflavin S-positive cores in only some of these plaques.

Pensalfini’s poster told a similar story about human tissue. She, too, found rampant nuclear M78 staining throughout the frontal cortex (Brodmann areas 4, 9, and 11) and in the hippocampus. Again, M78 correlated with 6E10 staining of the cytosol. In brain tissue from one non-demented person, Pensalfini used electron microscopy to confirm the M78 staining was indeed nuclear. The antibody also detected plaques in human tissue, though not necessarily in conjunction with the nuclear staining. In some tissue samples, M78 stained only nuclei; in others, it reacted only with plaques, and in yet others, it picked up both nuclei and plaques. Looking at tissue from 28 people with various levels of plaque deposition, Pensalfini found that M78-positive nuclei seem to appear before plaques, peak as plaque deposition ramps up, and then decline as plaques become widespread in the brain. Glabe told ARF that he thought this might indicate the antibody is detecting a marker of early- to mid-stage pathology, correlating with Braak plaque stages I and II.

As with the transgenic mice, M78-reactivity in human tissue seems to coincide with perturbations to DNA. Pensalfini found that DAPI staining for the latter goes down as M78 immunoreactivity goes up. Glabe suggested that these cells might be losing their nucleic acids because they are TUNEL positive, but other scientists were not so sure. For example, Dave Morgan, from the University of South Florida, Tampa, was intrigued by the posters, but thought DAPI might somehow be masked.

With what, exactly, does M78 react in transgenic mice and human brain? That’s the key question that Glabe said the lab is trying to answer. The researchers immunoprecipitated cell extracts with M78 and will employ mass spectrometry and other biophysical techniques to identify what the antibody pulled down. Morgan and some other scientists told Alzforum that M78 might actually bind some form of tau. Glabe is reserving judgment on that until more data come in, but he did tell ARF that of four transgenic AD models examined so far, M78 detects inclusions in nuclei of only 3xTg mice. These animals carry a tau mutation and develop tau pathology by six months of age and advanced neurofibrillary tangles by 20 months. The other three models have no tau pathology. Interestingly, Glabe also said that the most robust M78 staining seen so far in human tissue was from a patient with corticobasal degeneration, a tauopathy. In this initial sample, M78 did not bind brain tissue from people who had Parkinson’s disease or multiple system atrophy.

Ben Wolozin of Boston University was likewise intrigued by M78, but he had a different idea about what it binds. He suggested RNA-binding proteins. That might explain the antibody’s penchant for the nucleus, and it would fit with emerging work from Wolozin’s lab on stress granules. These cellular inclusions, which are laden with RNA-binding proteins, appear in response to “normal” stresses a cell might encounter and are reversible in this context; however, they also pop up in reaction to accumulating toxic proteins such as mutant huntingtin and TDP-43 (see ARF related Webinar). Wolozin has been looking to see if stress granules might be a common bellwether of neurodegeneration. At SfN, Tara Vanderweyde, a Ph.D. student in Wolozin’s lab, reported on her poster that stress granules coincide with tau pathology.

Vanderweyde used P301L tau mice (JPNL3) as a model system. She found that inclusions positive for the RNA-binding protein T cell internal antigen-1 (TIA-1), a common stress granule marker, accumulated in these animals as tau pathology progressed. The TIA-1 stress granules co-localized with tau and turned up predominantly in neurons, but some were also present in microglia early in the disease process, and in astrocytes at all stages. Interestingly, Vanderweyde reported that tristetraprolin (TTP), another stress granule marker, also accumulated in these animals but only associated with tau late in disease. Another stress granule marker that goes by the mouthful ras-GTPase-activating protein SH3-domain-binding protein (G3BP) identified granules that never associated with tau. The findings suggest that tauopathies could feature distinct forms of granules. In a related tau model (Tg4510), TIA-1, TTP, and G3BP accumulated with disease progression, and TIA-1 seemed to interact directly with tau, since the two co-immunoprecipitated.

Vanderweyde told Alzforum that she thinks G3BP could be an early marker of disease, accumulating in stress granules before tau pathology kicks in. “I think what is most striking is that people focus on tau, but doing that could miss an entire subset of neurons that may be sick,” she said. TTP, on the other hand, could mark late disease, which would be in keeping with its role in P-bodies, a type of granule that captures mRNAs for degradation.

Are these stress granules relevant to human conditions? As a first glimpse, Vanderweyde looked at tissue from six cases of Alzheimer’s and found the same scenario in human cells as in the mice. TIA-positive stress granules occurred predominantly in neurons and associated with tau in the perinuclear space. Wolozin told ARF that he is planning to look for stress granules in more samples of AD and other diseases. Vanderweyde said that they are also looking at Aβ mouse models. What, if anything, stress granules might mean in AD is something they plan to investigate. Whether Glabe’s M78 monoclonal recognizes tau, RNA-binding proteins, or something entirely different also remains to be seen.—Tom Fagan.

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References

News Citations

  1. DC: Anti-Aβ Treatments Old and New
  2. Amyloid Oligomer Antibody—One Size Fits All?

Webinar Citations

  1. Intraneuronal Aβ: Was It APP All Along?
  2. Stress Granules and Neurodegenerative Disease—What’s the Scoop?

Other Citations

  1. 3xTg mice

Further Reading

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