Tau proteins come in two varieties in the brain, depending on how neurons splice the mRNA. The four-repeat (4R) form underlies several tauopathies, including frontotemporal dementia, but researchers have struggled to directly compare its toxicity with that of the three-repeat version. A study in the June 1 issue of Neuron reports an antisense oligonucleotide strategy for switching between 3R- and 4R-tau expression in mice, allowing scientists to evaluate the two isoforms in the same genetic background. The researchers, led by Timothy Miller of Washington University in St. Louis, found that increasing the proportion of human 4R tau ramped up tau phosphorylation and aggregation, made brain seizures more severe, and interrupted the animals’ nesting behavior, all while total levels of tau remained unchanged.

The scientists also reduced 4R tau in a mouse model of frontotemporal dementia using the same oligonucleotide strategy, suggesting the approach might have therapeutic potential. “We predict that [converting] 4R to 3R tau may treat some 4R mouse models of tauopathies, and patients with mutations in the tau gene,” Miller told Alzforum. “This is a novel approach to perhaps treat patients with particular tau mutations.”

Neurons produce tau with three or four repeated segments, depending on whether they splice out exon 10 from the mRNA. This exon harbors almost half of the 53 known tau mutations (Ghetti et al., 2015). Though results of in vitro experiments suggest that 4R tau aggregates more readily (Adams et al., 2010), but in vivo proof has been challenging, not least because humans and mice produce different ratios of the two isoforms.

Miller and colleagues approached the problem by developing antisense oligonucleotides (ASOs) that force translation of exon 10, converting 3R to 4R tau in neurons without changing the total level of tau protein in the cells.

4R Problem.

Oligonucleotides that shift tau splicing from 3R (left) to 4R (right) exacerbate phospho-tau immunoreactivity. [Courtesy of Timothy Miller and Neuron.]

The researchers infused the isoform-switching ASOs into the brain ventricles of three- to four-month-old htau transgenic mice, which express no mouse tau and mostly human 3R tau (Andorfer et al., 2003). One month later, these mice accumulated almost twice as much 4R MAPT mRNA as the control htau mice that received scrambled ASOs or saline. The proportion of 4R proteins also increased. When the researchers examined the brains of these animals at 12 months, they saw more high-molecular-weight tau, and greater levels of phosphorylation compared with control mice. Both phenomena are signs of pathology. 

Mice with elevated 4R tau developed neurological problems. Unlike the 3R htau mice, they did not build proper nests, a task that relies on a normally functioning hippocampus and cerebral cortex. The researchers also tested for hyperexcitability, since tau appears to regulate electrical activity at the synapse and has been linked to epileptiform activity in AD mouse models (Dec 2013 newsIttner et al., 2010). After receiving an injection of pentylenetetrazole, which ramps up electrical activity in the brain, the seizures endured by the 3R to 4R ASO-treated mice were more severe than those in controls.

Taken together, the results suggest that 4R tau is indeed toxic in the brain. “The data all seemed to line up together, suggesting that 4R tau was inducing pathological changes in the mice,” said co-first author Kathleen Schoch of Washington University. “While observations tell us that 4R tau is not the only actor in tauopathies, this paper elegantly shows that it can be a major one,” said Gil Rabinovici of the University of California, San Francisco.

However, as the authors pointed out, and others agreed, unresolved questions regarding the toxicity of the two isoforms remain. For example, the results cannot explain the neurodegeneration seen in Pick’s disease, a rare frontotemporal dementia whose tangles are predominately composed of 3R tau, and in Alzheimer’s disease, where aggregates have equal amounts of the two forms.

To see if they could drive the repeat tau ratio in the other direction, the researchers gave htau mice ASOs that caused spliceosomes to skip over exon 10. After treatment, 4R tau levels fell to less than half those of the control mice. Schoch saw a similar drop when she treated a second mouse model, the 4R tau-overexpressing mutant tau N279K (Dawson et al., 2007). 

The researchers have not yet examined the behavioral and pathologic effects of 4R to 3R conversion in the htau and N279K mice. Even so, they believe their experiments provide proof of concept that ASOs that skip exon 10 can shift the ratio of tau isoforms in mice toward 3R. If effective in people, ASOs could in the future have a therapeutic benefit for treating 4R tauopathies, they propose.

“Splicing is a great target for tauopathy. For some reason it has been understudied, despite the many tau mutations known to affect it,” said Chad Dickey of the University of South Florida in Tampa. “It can be very specifically targeted [to a particular gene] as well, as this study has shown.” Exon skipping to restore dystrophin levels has been tested in a small Phase 2 study of Duchenne’s muscular dystrophy, where it slightly improved muscle function in young boys (Voit et al., 2014). 

Miller and colleagues are working with Ionis Pharmaceuticals of Carlsbad, California, which provided the ASOs for the study, and are considering exon skipping to treat genetic and sporadic tauopathies. “That [the approach] may have benefit for sporadic neurodegenerative disease is very exciting,” said Rabinovici.—Patricia Waldron

Patricia Waldron is a science writer based in Dryden, New York.

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References

Research Models Citations

  1. htau

News Citations

  1. Is Dendritic Tau to Blame for AD-Related Hyperexcitability?

Paper Citations

  1. . Invited review: Frontotemporal dementia caused by microtubule-associated protein tau gene (MAPT) mutations: a chameleon for neuropathology and neuroimaging. Neuropathol Appl Neurobiol. 2015 Feb;41(1):24-46. PubMed.
  2. . Three repeat isoforms of tau inhibit assembly of four repeat tau filaments. PLoS One. 2010 May 25;5(5):e10810. PubMed.
  3. . Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms. J Neurochem. 2003 Aug;86(3):582-90. PubMed.
  4. . Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models. Cell. 2010 Aug 6;142(3):387-97. Epub 2010 Jul 22 PubMed.
  5. . Loss of tau elicits axonal degeneration in a mouse model of Alzheimer's disease. Neuroscience. 2010 Aug 11;169(1):516-31. Epub 2010 Apr 29 PubMed.
  6. . Safety and efficacy of drisapersen for the treatment of Duchenne muscular dystrophy (DEMAND II): an exploratory, randomised, placebo-controlled phase 2 study. Lancet Neurol. 2014 Oct;13(10):987-96. Epub 2014 Sep 7 PubMed.

Further Reading

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Primary Papers

  1. . Increased 4R-Tau Induces Pathological Changes in a Human-Tau Mouse Model. Neuron. 2016 Jun 1;90(5):941-7. Epub 2016 May 19 PubMed.