07 Sep 2018

Phosphorylated tau interferes with the finely tuned process of nuclear transport, according to a paper in the September 5 Neuron. Scientists co-led by Bradley Hyman, Massachusetts General Hospital, Charlestown, and Jeffrey Rothstein, Johns Hopkins University, Baltimore, found that when p-tau builds up in the neuronal cell body, it binds a particular protein in the nuclear pore called Nup98. P-tau then drags Nup98 out of the pore, rendering the pore leaky. Once in the cytoplasm, Nup98 causes p-tau to aggregate. All in all, the results point to a defect in nucleocytoplasmic transport in tauopathies, particularly AD, and suggest a new mechanism for tau aggregation.

Similar irregularities in nuclear transport have been reported both for amyotrophic lateral sclerosis (ALS), and Huntington’s disease. Alzforum first reported these findings when they were presented at last year’s Society for Neuroscience annual meeting (Dec 2017 conference news). 

“This group has uncovered a new aspect to the pathophysiology of tauopathy,” said Benjamin Wolozin, Boston University School of Medicine, who was not involved in the study. The study adds to a growing body of data that tauopathies have features in common with ALS and other diseases related to RNA-binding proteins. “It suggests a shared pathophysiology,” Wolozin said.

Leaky Pores. Phosphorylated tau comes off microtubules and builds up in the cell body (left). There, its interaction with the nuclear pore protein Nup98 snarls transport in and out of the opening (right). [Image courtesy of Eftekharzadeh et al., 2018.]

In AD, tau becomes phosphorylated, pops off microtubules, and builds up in dendrites and in the neuronal cell body. It is still unclear how the protein becomes toxic. Co-first authors Bahareh Eftekharzadeh at MGH and J. Gavin Daigle at Johns Hopkins searched for clues by asking if proteins in the perinuclear space interact with p-tau. Being an intrinsically disordered, floppy protein, tau undergoes phase separation, meaning individual proteins can band together to form liquid droplets within the cytoplasm (May 2017 conference news). Because disordered domains tend to interact with similarly disordered regions on other proteins, the scientists wondered if tau binds nucleoporins that sit in the nuclear pore complex. These sentinels help pass protein and RNA between the nucleus and cytoplasm; some contain a disordered domain at their N-terminal tail.

To find out, the researchers examined postmortem tissue from AD and control brains. They noticed that unlike the smooth, rounded neuronal nuclei of controls, those in AD brains appeared wrinkled and folded (see image at left). Some nucleoporins with disordered domains had seeped out into the cytoplasm instead of staying in the pores.

The group decided to focus on Nup98, a particularly abundant and highly disordered nucleoporin. High-resolution optical microscopy revealed that p-tau co-localized with Nup98 at the nuclear surface in hippocampal neurons from AD patients. In p–tau containing nuclei, Nup98 left the nuclear membrane and co-localized with more than 90 percent of tau tangles sitting in the cytoplasm. Other Nups stayed put in the nuclear membrane, suggesting the bulk of the complex remained, just in an altered form.

Did the two proteins interact directly? In surface plasmon resonance experiments, p-tau bound the N-terminus of Nup98. The C-terminus of this nucleoporin contains many negatively charged amino acids, a protein feature that tends to trigger tau aggregation. In aggregation assays, the C-terminus of Nup98 caused both wild-type tau and mutated P301L tau to aggregate, appearing alongside them in fibrils. This suggests that p-tau binds the exposed N-terminal region of Nup98 at the nuclear pore complex and tears it out. Once free of the complex, the newly exposed C-terminus of Nup98 helps tau clump up in the cytoplasm. “Maybe one mechanism for tau aggregation is this interaction with Nup98,” Rothstein told Alzforum.

What did this tau-Nup98 interaction do to the nuclear pore? While hippocampal nuclei from normal older adults admitted only dextran dyes that measured less than 40kDa, those from Braak stage III and VI patients were leakier, granting passage to 70- and 500-kDa molecules. Active import and export also seemed to be affected, because cytoplasmic proteins were found in the nucleus, and nuclear proteins turned up in the cytoplasm in neurons containing p-tau. This held up in vivo: Nuclei from rTg4510 mice admitted large dextran dyes that should have been excluded, whereas the normally nuclear Ran protein faded from the nucleus and appeared in the cytoplasm.

The researchers used doxycycline to turn off tau expression in rTg4510 mice when they were six months old, at which point they had abundant p-tau in their neurons but had only just begun to accumulate tangles. By 12 months, Ran protein returned to control levels in the nucleus and Nup98 settled back into the nuclear membrane, even though some stuck around in the tangles that had formed previously. The results suggest soluble forms of p-tau were responsible for the mis-sorting of Nup98 and problems with nucleo-cytoplasmic transport. Further, knocking down Nup98 with short hairpin RNAs restored Ran distribution to control levels.

The findings fit with previous studies reporting that TDP-43, mutant huntingtin, and repeat expansions in C9ORF72 all interfere with traffic in and out of the nucleus (Jan 2018 news; Freibaum et al., 2015; Grima et al., 2017). “Disrupted nucleocytoplasmic transport might be a general event in multiple neurodegenerative diseases,” said Eftekharzadeh. “Perhaps a small molecule or antisense oligonucleotide that could prevent the defect could be helpful in ALS, Huntington’s, and AD.”

Brian Freibaum, St. Jude’s Research Hospital, Memphis, Tennessee, agreed that this study suggests a common pathway between these diseases. “It clearly shows that one of the two proteins involved in Alzheimer’s disease is directly interacting with nuclear pore components,” he told Alzforum. “It almost certainly contributes to neurodegeneration, however, it remains unclear whether deregulation of the nuclear pore is an early or late event in disease progression.” Wolozin further pointed out that this study doesn’t address whether defective nucleo-cytoplasmic transport is toxic to neurons.

“This is a very interesting and important contribution to our understanding of tau’s effect on the nucleus,” said Bess Frost, University of Texas Health, San Antonio. The abnormal nucleocytoplasmic transport reported in this paper reminds her of studies that have reported deterioration in the nuclear pore complex proteins with aging, Frost said (D’Angelo et al., 2009). “The nucleus is where all the action happens in terms of gene expression and controlling the cell’s activity,” she told Alzforum. “Without proper sequestration of nuclear and cytoplasmic proteins, basic cellular controls don’t function properly.”

Rothstein said the next goal is to find a way to block p-tau from interacting with Nup98, and to see if that lessens the neuron’s dysfunction of slows disease progression. The scientists also want to decipher the mechanism behind Nup98 leaving the nuclear membrane.—Gwyneth Dickey Zakaib

Comments

1. • Diego Mastroeni
     
   • Posted: 07 Sep 2018

   Nucleocytoplasmic transport of molecules is essential for normal cell function. It is, in my opinion, the most basic and fundamental processes that initiate a cascade of events that leads to cellular aging and cell death. The mislocalization of molecules has been described in every neurological condition, including cancer, but to date, most of the work has been descriptive. The work done by Rothstein and Hyman nails down a direct mechanism associated with the nucleocytoplasmic dysfunction in Alzheimer’s disease. This is a critical step in developing targeted therapy, either tau-directed or the interaction between Nup98 and tau. Many nucleoporin therapies have already been developed in cancer, and just maybe, we can repurpose cancer drugs targeted to the nuclear transport machinery to restore nucleocytoplasmic homeostasis in Alzheimer’s disease.

   View all comments by Diego Mastroeni
2. • Jürgen Götz
     The University of Queensland
   • Posted: 12 Sep 2018

   I really liked this study, which elegantly combines work in human and transgenic tissue, in vitro and in primary cultures. The human brain and doxycycline rescue experiments in rTg4510 mice are especially compelling. I think the question (admittedly a difficult one) that remains for all pathological mechanisms that have been identified for tau is, what are their relative contributions to disease initiation and progression? In follow-up work it would be interesting to address whether and how murine and human tau differ in disrupting Nup98 and nucleocytoplamsic transport, and whether there is a role for specific post-translational modifications. Another interesting question is whether there are fundamental differences between primary and secondary tauopathies. Tau remains anything but a dull protein!

   View all comments by Jürgen Götz

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References

News Citations

1. Is There No End to Tau’s Toxic Tricks? 28 Dec 2017
2. Protein Liquid-Liquid Phase Transitions: The Science Is About to Gel 31 May 2017
3. TDP-43 Snarls Nuclear Traffic 17 Jan 2018

Research Models Citations

1. rTg(tauP301L)4510

Paper Citations

1. Freibaum BD, Lu Y, Lopez-Gonzalez R, Kim NC, Almeida S, Lee KH, Badders N, Valentine M, Miller BL, Wong PC, Petrucelli L, Kim HJ, Gao FB, Taylor JP. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. 2015 Sep 3;525(7567):129-33. Epub 2015 Aug 26 PubMed.
2. Grima JC, Daigle JG, Arbez N, Cunningham KC, Zhang K, Ochaba J, Geater C, Morozko E, Stocksdale J, Glatzer JC, Pham JT, Ahmed I, Peng Q, Wadhwa H, Pletnikova O, Troncoso JC, Duan W, Snyder SH, Ranum LP, Thompson LM, Lloyd TE, Ross CA, Rothstein JD. Mutant Huntingtin Disrupts the Nuclear Pore Complex. Neuron. 2017 Apr 5;94(1):93-107.e6. PubMed.
3. D'Angelo MA, Raices M, Panowski SH, Hetzer MW. Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells. Cell. 2009 Jan 23;136(2):284-95. PubMed.

Further Reading

News

1. Barring Export of Expanded Repeat RNAs Tames Their Toxicity 19 Jul 2017
2. Protein Liquid-Liquid Phase Transitions: The Science Is About to Gel 31 May 2017
3. Too Much of a Bad Thing: Protein Aggregates Snarl Nuclear Traffic 9 Dec 2015