Targeting extracellular Aβ with immunotherapy is one thing, but could antibodies even drive the clearance of tau, a predominantly intracellular protein? In the last few years, scientists have turned their attention to vaccinating against various forms of this potentially toxic protein, and the strategy seems to pay some dividends, at least in mice. Tau immunotherapy was well represented with new data at the 2nd International Conference on Neurodegenerative Disorders: Immunotherapy and Biomarkers, which took place 26-27 May 2011 at Uppsala University, Sweden. Even big pharmaceutical companies are now getting in on the act.

Martin Citron, Eli Lilly, Indianapolis, Indiana, is best known for his studies on the β-secretase that cleaves amyloid-β precursor protein (APP), but in Uppsala he confessed to being a bit of a closet tauist. He noted that the AD field has come to appreciate in the last five to 10 years that amyloid pathology is an early event in the disease, and that by the time patients become demented, interventions targeting Aβ may be too late. Two alternative strategies are to diagnose and target Aβ earlier, or to go for targets that lie downstream. Citron thought tau might fit the latter category because tau pathology continues to worsen in the symptomatic phase of the disease. The problem, he said, has been figuring out how to target it.

Citron reported on a passive immunotherapy strategy using antibodies (PHF1 and MC1) that recognize neurofibrillary tangles. He injected these into two different models: the JNPL3 mouse (Lewis et al., 2000), which expresses a human tau transgene containing the P301L mutation that causes frontotemporal dementia, and a mouse expressing human tau with the P301S mutation that has more aggressive pathology and also more consistent transgene expression (Allen et al., 2002). He treated two-month-old JNPL3 mice for four months with PHF1 and MC1 (15 mg/kg three times per week for two months, then 10 mg/kg twice per week). While neither antibody treatment altered total tau levels in the mice, they did lower the amount of insoluble tau stained by the AT8 antibody. Citron said the JNPL3 model varied greatly from animal to animal. For quantitative analysis, he developed an AT8-based enzyme-linked immunosorbent assay (ELISA) for insoluble tau. Citron believes that a 64 kDa, hyperphosphorylated tau fragment recognized by AT8 is the biochemical correlate of neurofibrillary tangles. He said the 64 kDa tau correlates with the extent of tangle pathology in both Alzheimer’s disease and transgenic mouse models. The AT8 ELISA detected no signal from brain tissue samples taken from human controls, but did detect tau in tissue from AD patients. He said neither the PHF1 nor MC1 interfere with the assay, indicating that it is suitable for monitoring the effects of these antibodies in vivo. With this ELISA, Citron detected significant reduction of AT8 tau reactivity in brain extracts from the treated JNPL3 mice compared to controls. Whether the ELISA could be used as a diagnostic test is unclear. That would require a test of biological fluid, such as CSF, and Citron said it is unlikely that much of the insoluble tau would make its way there.

Citron used the P301S model to measure behavioral effects, since the JNPL3 animals show no behavioral phenotype until they are quite old. In the P301S mice, tau pathology damages the spinal cord and the animals have severe motor problems by five months of age. Citron treated two-month-old animals for three months with twice-weekly doses (15 mg/kg) of both PHF1 and MC1. Treated mice performed better on the rotarod and lost less weight than did controls who got an equal amount of a generic immunoglobulin G. Similar to the JNPL3 animals, total tau stayed unchanged, but the ELISA revealed a decline in AT8-reactive insoluble tau. Tangle pathology is rampant in the P301S animals, but the immunotherapy attenuated it. Citron noted that the antibody treatment also appeared to improve neurodegeneration.

How does this passive immunotherapy work? Citron does not know, but said the simplest hypothesis would be that it neutralizes extracellular tau, which somehow is driving pathology. Tau is an intracellular protein, but evidence is accumulating that tau can be secreted from cells and taken up by others (see ARF related news story and ARF news story on Frost et al., 2009). Alternatively, the antibodies might be taken up into the cells and neutralize tau there, he said.

Einar Sigurdsson, New York University, also broached the subject of how tau immunotherapies work. He agreed that multiple mechanisms—extracellular and intracellular—are likely involved in tau immunotherapy. Sigurdsson’s group was the first to show that active immunization against tau can rescue phenotypes in mouse models and might be a viable strategy for tauopathies (see ARF related news story on Asuni et al., 2007 and ARF related news story on Boutajangout et al., 2010). His group also reported that passive tau immunotherapy clears tau pathology in mice and stems their functional decline (see Boutajangout et al., 2011). To address how these antibodies work, Sigurdsson has isolated immunoglobulin Gs (IgGs) from vaccinated mice, labeled them with a fluorescent tag (FITC), and then re-injected them into mice to see where they go. He showed that they not only enter the brain, but also seem to get inside neurons where they colocalize with tau. Interestingly, he said, the antibodies do not penetrate the brains of control mice—at least in quantities this method can detect. He believes a damaged blood-brain barrier explains why the antibodies gain access to the brains of the transgenic animals.

How do the antibodies get inside neurons? Sigurdsson said that work on ex-vivo slice cultures suggests cells take up the antibodies and that they colocalize with endosomal/lysosomal markers, such as LAMP2. Isolated lysosomal fractions from these cells contain the antibodies and tau. Sigurdsson’s group presented some of these data at the International Conference on Alzheimer’s Disease in Hawaii last July. He said that it is well known that several receptors on most cells, including neurons, bind antibodies. He speculated that antibodies enter via receptor-mediated endocytosis, traveling in endosomes that then fuse with autophagosomes containing tau aggregates. The antibodies would then drive disassembly of the aggregates, freeing tau up for lysosomal degradation. Alternatively, he suggested, antibodies might diffuse into cells through damaged membranes, bind to tau aggregates on the inner surface of the plasma membrane, and get processed by autophagosomes. Sigurdsson is also interested in a ubiquitin ligase system that binds antibodies attached to viruses and targets them for destruction. He thought this system might be involved in the tau antibody response as well.

Sigurdsson’s lab has newer monoclonals in development. One of them, 4E6G7, is a phospho-tau-specific antibody. It reduced tau pathology and also rescued cognition in the radial arm maze, a closed-field symmetrical maze, and in an object recognition test as well. Even though treatment began late, he saw improvements, said Sigurdsson. He thought this bodes well for translation to human studies.

Scientists are also looking to other tau immunotherapy strategies, including specifically targeting tau oligomers. Rakez Kayed, University of Texas Medical Branch, Galveston, reviewed his lab’s progress in this approach. He has made polyclonal (T22) and monoclonal (TOMA) antibodies to tau oligomers (see ARF related news story). In Uppsala, Kayed reported that his group isolated a specific tau oligomer directly from human AD brain by using the antibodies to immunoprecipitate tau from soluble fractions enriched with extracellular material. The oligomer is a dimer or possibly a trimer. Kayed said he is not sure which, because oligomer migration on electrophoresis gels does not always predict size. He said they also established a causative relationship between this putative tau dimer/trimer and memory deficits in mice. Specifically, injecting it into the brains of normal mice weakened their ability to remember novel objects.

TOMA injected into the brain reduced tau pathology in eight-month-old P301L and 14-month-old Tg2576 mice. Injected intravenously into the latter, the antibody rescued memory deficits as judged by a novel object recognition test. When given as a single intravenous injection (30 μg), TOMA reduced tau oligomers detected biochemically and immunohistochemically. Surprisingly, Kayed said, it did not reduce AT8-reactive tau species phosphorylated at serine 202/threonine 205, which is in contrast to other immunotherapies directed at tau, including those of Citron and Sigurdsson above. Kayed did not want to speculate why AT8-reactive tau was not reduced. He did claim that TOMA recognizes oligomers in human cerebrospinal fluid (CSF), suggesting that these oligomers, present at ng/ml levels, could become the basis for a new diagnostic test. In three separate experiments, two of them blinded, his group measured these oligomers in CSF taken from 25 AD patients and 25 normal controls, Kayed told the audience.—Tom Fagan.

This is Part 2 of a three-part series. See also Part 1 and Part 3. Read the entire series.

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References

News Citations

  1. Traveling Tau—A New Paradigm for Tau- and Other Proteinopathies?
  2. Double Paper Alert—Keystone Presentations Now in Press
  3. Tau Vaccine Detangles Mouse Brain
  4. San Diego: Tau Oligomer Antibodies Relieve Motor Deficits in Mice
  5. Uppsala: Conference Puts Uppsala on Immunotherapy Map
  6. Uppsala: Immunotherapy—Not Just for AD Anymore

Paper Citations

  1. . Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein. Nat Genet. 2000 Aug;25(4):402-5. PubMed.
  2. . Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein. J Neurosci. 2002 Nov 1;22(21):9340-51. PubMed.
  3. . Propagation of tau misfolding from the outside to the inside of a cell. J Biol Chem. 2009 May 8;284(19):12845-52. PubMed.
  4. . Immunotherapy targeting pathological tau conformers in a tangle mouse model reduces brain pathology with associated functional improvements. J Neurosci. 2007 Aug 22;27(34):9115-29. PubMed.
  5. . Immunotherapy targeting pathological tau prevents cognitive decline in a new tangle mouse model. J Neurosci. 2010 Dec 8;30(49):16559-66. PubMed.
  6. . Passive immunization targeting pathological phospho-tau protein in a mouse model reduces functional decline and clears tau aggregates from the brain. J Neurochem. 2011 Aug;118(4):658-67. PubMed.

Other Citations

  1. Read the entire series.

External Citations

  1. JNPL3 mouse

Further Reading