09 Mar 2022

The traditional view of tau is that of a rather dull microtubule-binding protein that occasionally goes rogue, wandering off into other cellular compartments where it stokes neurodegeneration. At Tau2022, a virtual meeting held February 22-23, scientists questioned this concept. Even in health, they said, tau leads a far more varied life. For one, it travels between neurons in a manner that commandeers the entire endolysosomal system. For another, cells require LRRK2 to internalize tau monomers, but not fibrils. Known for its association with Parkinson’s disease, LRRK2 was recently linked to primary tauopathies as well.

There’s more. In neurons from healthy human brain, tau was spotted within the nucleolus—the birthplace of ribosomes. It appeared right at home in this membraneless organelle, where it protects ribosomal DNA and keeps heterochromatin stable. Finally, tau appeared to play a role in ALS, as it shifted from the cytoplasm to synapses in people with this disease. There, tau cavorted with a protein called Drp1 to provoke mitochondrial fission.

Together, the findings suggest that tau plays a host of underappreciated and dynamic roles in health and in neurodegeneration, even in diseases not considered tauopathies.

Tau, A Frequent Traveler?
Mounting evidence suggests that in the context of neurodegenerative disease, aggregated forms of tau spread from cell to cell via templated misfolding. Scientists consider this propagation to be part of a pathological process, but Rick Livesey, who recently moved from University College London to Biogen, pointed out that tau has been spotted passing between healthy cells, as well. In previous studies, Livesey found that different forms of tau are actively secreted and taken up by iPSC-derived human excitatory neurons as part of a normal physiological process (Evans et al., 2018).

What cellular mechanism operates this shuffle? Lewis Evans in Livesey’s lab ran two genome-wide CRISPR screens in iPSC-derived neurons, asking which genes they needed to take up fluorescently labeled monomers and fibrils of tau. More than 200 popped out. Both screens identified genes throughout the entire endolysosomal trafficking system—everything from receptor-mediated endocytosis to endolysosomal fusion, autophagy, and even endoplasmic reticulum and Golgi trafficking.

Curiously, many of these genes also enable uptake of viruses, including influenza A, Zika, and SARS-CoV2. These three viruses start with receptor-mediated endocytosis and from there hijack the entire endosomal trafficking system to invade cells. A notable exception is Ebola, which uses a different mechanism—micropinocytosis—to barge into cells. In keeping with this distinction, Livesey reported virtually no overlap between genes involved in tau uptake and Ebola infection. Viruses have evolved to exploit the endolysosomal system to infect cells, but it is still unclear why tau uses the same machinery, or what process its cellular entry serves.

While the endolysosomal system seems necessary for both tau monomers and fibrils to get inside cells, Livesey identified differences in specific genes involved in the internalization of each. For example, LRP1 was required in the uptake of the former, but not the latter, confirming findings first reported at Tau2020 by researchers at Kenneth Kosik’s lab at the University of California, Santa Barbara (Mar 2020 conference news).

Secondly, Livesey reported that LRRK2—a multicatalytic protein infamous for its role in familial and sporadic Parkinson's disease—was essential for the entry of tau monomers, but not fibrils. This rang bells for Livesey, because Virginia Lee and colleagues at the University of Pennsylvania, Philadelphia, had found extensive AD-like tau tangle pathology in people with PD caused by LRRK2 mutations, and at Tau2020, Huw Morris and Edwin Jabbari at UCL had linked high LRRK2 expression to a genetic risk variant for the primary tauopathy progressive supranuclear palsy (Henderson et al., 2019; Mar 2020 conference news; Jabbari et al., 2021). A case-control study had also tied LRRK2 mutations to PSP and corticobasal degeneration (Sanchez-Contreras et al., 2017). 

Using iPSC-derived neurons without LRRK2 to probe further, Livesey found that the cells not only decline to ingest tau monomers, they also accumulated LRP1 on their surface. This suggested a link between LRRK2 and LRP1 in tau monomer internalization, but Livesey does not know how LRRK2 mediates internalization of LRP1.

When Livesey blocked LRRK2 kinase activity with an inhibitor rather than by knocking it out, cells stopped taking up both monomeric and fibrillar tau, despite retaining a functionally intact endolysosomal system. Why the inhibitor has a different effect than does deleting the gene remains to be seen. Complicating matters even further, neurons expressing the PD-causing G2019S LRRK2 mutation also ingested less of both forms of tau. This variant boosts LRRK2 kinase activity and compromises endolysosomal function.

Complex findings are par for the course for LRRK2, a large protein that has multiple functions in different cell types. While these studies were limited to neurons, Livesey believes that LRRK2 might facilitate passage of tau between glia as well.

Li Gan of Weill Cornell Medical College in New York was intrigued by the overlap between genes mediating uptake of tau and viruses. Once inside cells, viruses escape the confines of the endolysosomal system, and Gan wondered if tau makes it into the neuronal cytosol the same way. Livesey has not investigated this, but said others have proposed ideas ranging from tau buddying up with specific proteins that whisk it across the endolysosomal membrane, to tau seeds punching holes to escape into the cytosol (Jan 2021 news).

Is cellular release and uptake of tau part of its life cycle? Is this hand-off somehow beneficial, and if so, under what circumstances might it become pathological? Researchers posed different versions of this question during discussion. Hui Zheng of Baylor College of Medicine in Houston said previous studies from several labs have found that secreted monomers have been truncated by lysosomal processing, rendering them incompetent for seeding aggregation (Mar 2018 news; Xu et al., 2020). This suggests that cellular uptake and release serves as a beneficial clearance mechanism, Zheng said, adding that distinct pathways may be involved in proteopathic propagation of tau aggregates.

Maria Grazia Spillantini of the University of Cambridge, U.K., took a therapeutic view of these tau shenanigans. “If you were developing a LRRK2-based treatment for tauopathies, would you prefer that tau is taken into the cell, or left out?” she asked Livesey. Livesey has been mulling the question. “What it comes down to is, are there truly toxic extracellular species of tau, and if so, what is their target? I’m not sure we have answers to either of those questions,” he said.

Tau: Card-Carrying Member of the Nucleolus?
Tau’s reach does not end in the lysosomes, cytosol, or even the synapse. Mahmoud Maina, University of Sussex, Brighton, U.K., reported that tau also helps organize the heart of the cell—the nucleus and nucleolus. The protein had been spotted in these nucleic-acid-rich regions 30 years ago, but what it does there is a mystery (Loomis et al., 1990; Brady et al., 1995). Recently, tau has been linked to nuclear dalliances, for example disruption of the spliceosome, deformation of the nucleus, and a dangerous liaison with RNA (Apr 2021 news; Jan 2019 news; Jul 2017 news).

At Tau2022, Maina argued that tau’s presence in so many cellular compartments means it could have diverse physiological functions. “To truly understand tauopathies and find effective therapies, we need a deep understanding of tau’s function in these different locations,” Maina said.

Many Haunts. Tau (red) is found in myriad locations of the cell, including the nucleus and nucleolus. [Courtesy of Mahmoud Maina, University of Sussex.]

A membraneless organelle within the nucleus, the nucleolus is where ribosomes are born. There, ribosomal DNA is transcribed into rRNA, which is then readied to join the massive ribonucleoprotein conglomerate that translates RNA into protein. What business might tau have in this protein-making hub of the cell?

Maina addressed this first by asking whether tau resided within the nucleus or nucleolus in neurons. Using transmission electron microscopy and immunolabeling, he spotted tau within both compartments in human brain samples, SHSY5Y neurons, and iPSC-derived cortical neurons. Within the nucleus, tau occupied dense regions of heterochromatin surrounding the nucleolus, and also co-localized with fibrillarin, a nucleolar protein (Maina et al., 2018). Notably, Maina reported that nucleolar tau was not phosphorylated, suggesting its presence there is not a consequence of pathological modification.

Tau in the Nucleolus. Non-phosphorylated tau (green) and the nucleolar protein fibrillarin (arrowhead, red) co-mingle (yellow) within the nucleolus of human iPSC-derived neurons. [Courtesy of Mahmoud Maina, University of Sussex.]

Maina got a hint that tau might be a regular in the nucleolus when he noticed that it co-localized with the nucleolar remodeling complex (NoRC), which is an essential component of the nucleolar machinery responsible for silencing rDNAs. These repetitive sequences within the genome loop into the nucleolus, where they are transcribed in a highly regulated manner that moves to the bioenergetic beat of the cell. Because their repeats make them prone to recombination, rDNAs are protected by being tightly packaged into heterochromatin by NoRC until they are needed. Knocking down either of NoRC’s two main components—Snf2h or TIP5—relaxes the heterochromatin, ramping up rDNA transcription and jeopardizing its stability.

Maina was surprised when he realized that knocking down tau, which directly associated with TIP5, had a similar effect. In SHSY5Y cells, loss of tau led the heterochromatin to relax, rDNA transcription to rise, and more nucleoli to form per cell. Could tau be an integral part of the nucleolus, not merely a passerby? In line with this idea, Maina had previously reported that tau behaved like a classical nucleolar protein in response to stress, that is, it rapidly redistributed to the nucleoplasm and cytoplasm when cells were rattled by Aβ oligomers or glutamate (Maina et al., 2018). Maina believes tau shows some of the hallmark features of a nucleolar protein, and noted that dysfunction of the nucleolus has been implicated in multiple neurodegenerative diseases.

Curious about the unphosphorylated nature of nucleolar tau, Zheng asked Maina whether phosphorylation might be a barrier for tau’s entry into the nucleolus. Maina considers tau phosphorylation a stress response. He thinks that under conditions such as exposure to Aβ oligomers or synaptic toxicity, phosphorylated tau enters the nucleus, but is excluded from the nucleolus. He plans to study the dynamic interplay between stress, tau phosphorylation, and nucleolar function.

Last but not least, Ghazaleh Sadri-Vakili of Massachusetts General Hospital in Boston presented recently published findings implicating tau in mitochondrial dysfunction in amyotrophic lateral sclerosis (Petrozziello et al., 2022). Studies reported hyperphosphorylated tau in the primary motor cortices of people with ALS, and altered ratios of p-tau to total tau in their cerebrospinal fluid. Since tau is known to mislocalize to synapses in Alzheimer’s disease, Sadri-Vakili wondered if it does the same in ALS. Tiziana Petrozziello at Mass. General and colleagues in her lab investigated postmortem motor cortex samples from 55 people with ALS and 26 controls. Six ALS cases had a known disease mutation, including five with a C9ORF72 and one with an SOD1 mutation. Sadri-Vakili reported that, regardless of sex or mutation, neurons from cases showed tau to have shifted from the cytosol into synaptosomes, specifically isoforms phosphorylated on serines 396 and 404.

To find out what these isoforms might be doing there, Petrozziello isolated synaptoneurosomes from ALS brain samples, and added them to primary neuronal cultures. The ALS synaptosomes triggered release of reactive oxygen species, suggesting that something within these synaptic compartments, possibly tau that got taken up by the neurons, stressed out mitochondria. Sadri-Vakili and colleagues found profound mitochondrial dysfunction in their postmortem ALS samples, such as low levels of electron transport chain proteins and fewer, smaller mitochondria in people with ALS than controls.

Does this dysfunction come from tau? Previous studies have reported that phospho-tau binds to, and promotes activity of, the mitochondrial fission protein, DRP1 (Manczak and Reddy, 2012). In further experiments, Sadri-Vakili found both p-tau396 and DRP1 to be enriched within ALS synaptoneurosomes. When added to neuroblastoma cells in culture, these p-tau- and DRP1-laden packages triggered fission, resulting in smaller mitochondria. Finally, Sadri-Vakili reported that silencing DRP1, or reducing p-tau with the ubiquitin ligase linker QC-01-175, prevented mitochondrial destruction and release of reactive oxygen species (Silva et al., 2019). QC-01-175 stimulated tau degradation by the proteasome.

While mechanistic questions remain, the findings hint at an important connection between hyperphosphorylated tau and mitochondrial dysfunction in ALS, Sadri-Vakili said. They also dovetail with a recent study by Gan, which identified synaptic and mitochondrial proteins as key constituents of the tau interactome (Jan 2022 news).—Jessica Shugart

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References

News Citations

1. Tau Receptor Identified on Cell Surface 7 Mar 2020
2. Tau2020: Meeting for Tauopathies Debuts Genetic Variants 3 Mar 2020
3. Do Lysosomes Help Propagate Tau Seeds? 15 Jan 2021
4. Isotope Labeling Links Tau Production to Aβ Burden 28 Mar 2018
5. Tau, Speckle Wrecker, Disrupts the Nuclear Home 15 Apr 2021
6. Invasion of the Microtubules: Mutant Tau Deforms Neuronal Nuclei 19 Jan 2019
7. Tau Hooks Up with RNA to Form Droplets 19 Jul 2017
8. Survey of Tau Partners Highlights Synaptic, Mitochondrial Roles 27 Jan 2022

Paper Citations

1. Evans LD, Wassmer T, Fraser G, Smith J, Perkinton M, Billinton A, Livesey FJ. Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways. Cell Rep. 2018 Mar 27;22(13):3612-3624. PubMed.
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5. Xu Y, Du S, Marsh JA, Horie K, Sato C, Ballabio A, Karch CM, Holtzman DM, Zheng H. TFEB regulates lysosomal exocytosis of tau and its loss of function exacerbates tau pathology and spreading. Mol Psychiatry. 2020 May 4; PubMed.
6. Loomis PA, Howard TH, Castleberry RP, Binder LI. Identification of nuclear tau isoforms in human neuroblastoma cells. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8422-6. PubMed.
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9. Maina MB, Bailey LJ, Doherty AJ, Serpell LC. The Involvement of Aβ42 and Tau in Nucleolar and Protein Synthesis Machinery Dysfunction. Front Cell Neurosci. 2018;12:220. Epub 2018 Aug 3 PubMed.
10. Petrozziello T, Bordt EA, Mills AN, Kim SE, Sapp E, Devlin BA, Obeng-Marnu AA, Farhan SM, Amaral AC, Dujardin S, Dooley PM, Henstridge C, Oakley DH, Neueder A, Hyman BT, Spires-Jones TL, Bilbo SD, Vakili K, Cudkowicz ME, Berry JD, DiFiglia M, Silva MC, Haggarty SJ, Sadri-Vakili G. Targeting Tau Mitigates Mitochondrial Fragmentation and Oxidative Stress in Amyotrophic Lateral Sclerosis. Mol Neurobiol. 2022 Jan;59(1):683-702. Epub 2021 Nov 10 PubMed.
11. Manczak M, Reddy PH. Abnormal interaction between the mitochondrial fission protein Drp1 and hyperphosphorylated tau in Alzheimer's disease neurons: implications for mitochondrial dysfunction and neuronal damage. Hum Mol Genet. 2012 Jun 1;21(11):2538-47. PubMed.
12. Silva MC, Ferguson FM, Cai Q, Donovan KA, Nandi G, Patnaik D, Zhang T, Huang HT, Lucente DE, Dickerson BC, Mitchison TJ, Fischer ES, Gray NS, Haggarty SJ. Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models. Elife. 2019 Mar 25;8 PubMed.

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