17 Jul 2018

When tau strays into neuronal synapses, microglia see food. That is one possible interpretation from a handful of presentations at the joint Keystone symposia—Advances in Neurodegenerative Disease Research and Therapy / New Frontiers in Neuroinflammation—held June 17–21 in Keystone, Colorado. Researchers reported that neuronal hyperactivity drives tau from microtubules into presynaptic terminals, where it latches onto vesicles. Others saw tau congregate on both the pre- and postsynaptic sides, along with complement proteins that bait microglia. What’s worse, microglia not only engulf neurons afflicted by tau pathology, they may also help tau spread across the brain, claimed scientists. Collectively, the findings suggest that microglia gorge—for better or worse—on neurons harboring misplaced forms of tau.

Synaptic Tau: Which Side Are You On?
As a protein that stabilizes microtubules, tau primarily resides inside cells. However, because mounting evidence points to tau’s travels between neurons, researchers have started investigating how the cytosolic protein winds up beyond the boundaries of the plasma membrane. Previous studies have reported that neuronal activity stokes tau secretion (Feb 2014 news). At Keystone, meeting organizer Li Gan of the Gladstone Institute of Neurological Disease in San Francisco presented news from her investigation of how neuronal firing shifts tau’s locale inside the cell. Gan is in the process of moving her lab to Weill Cornell Medical College in New York.

Gan’s postdoc Tara Tracy investigated which proteins buddy up with tau in the cell, and whether these liaisons change when neurons fire. The researchers ran an APEX2 interaction screen in i3 neurons. Made to resemble cortical neurons, i3 neurons were generated from human induced pluripotent stem cells (iPSCs) using a protocol previously developed by Gan and colleagues (Wang et al., 2017; Fernanadopulle et al., 2018). In these proximity screens, proteins that touch or closely mingle with an APEX2-tagged protein, in this case tau, become biotinylated and can then be identified by mass spectrometry (for review of method, see Lam et al., 2015). 

Appetite for Tau. Cultured microglia (red: Iba1, green: tau fibrils] ingest tau fibrils (yellow inside cell). [Courtesy of Marcus Chin, Gan lab.]

Tracy reported that under basal conditions, tau mingled primarily with microtubule and cytoskeletal proteins, as expected. But 30 minutes after stimulating the cells with a high concentration of potassium chloride to depolarize the membrane, tau had ditched these microtubule partners in favor of synaptic proteins, including multiple SNAREs, which facilitate the fusion of synaptic vesicles with the synaptic membrane. Of the 207 tau interactors the scientists identified, 52 associated with tau in stimulated neurons only. By zeroing in on the exact residues that touched tau from each protein, the researchers further concluded that tau hooked up with vesicular proteins from the cytosolic side, as opposed to the vesicle lumen. They concluded that when neurons are triggered, tau rapidly relocates into presynaptic terminals, where it associates with synaptic vesicles but does not enter them. Gan proposed that tau’s attachment to outgoing synaptic cargo could help tau exit the cell.

The findings mesh with a recent study led by Patrik Verstreken of KU Leuven in Belgium, which found that tau mislocalized to the presynapses and latched onto vesicles via an interaction with synaptogyrin-3. This essentially clumped the vesicles and prevented their efficient release (Feb 2018 news). Synaptogyrin-3 was among the proteins identified in Gan’s screen. At Keystone, Joseph McInnes of Baylor College of Medicine in Houston, first author on Verstreken’s study, told Alzforum that Gan’s findings beautifully demonstrated tau’s dramatic change of locale in the face of stimulation. Furthermore, McInnes proposed that KCl stimulation, a relatively harsh treatment that activates numerous kinases, might trigger tau phosphorylation. Thus, KCl stimulation could serve as a model for tauopathy.

Bearing news from the other side of the synapse, Morgan Sheng of Genentech in South San Francisco reported that in a mouse model of tauopathy, tau is in cahoots with postsynaptic proteins. Numerous studies have reported that tau’s movement from axons into the dendritic compartment, which is speckled with postsynaptic proteins and structures, bodes poorly for neurons. To explore tau’s movement into the postsynapse, Sheng analyzed the postsynaptic density (PSD) proteome in nine-month-old P301S mice. At this age, the mice have no overt neurodegeneration, but their neurons contain plenty of phospho-tau and show inklings of synaptic deficits.

A mass spec analysis of the proteins in the PSD revealed a striking amount of phospho-tau there. Sheng noted that due to the imperfect purification process, a fair amount of presynaptic proteins also popped up in the proteomic analysis, making it difficult to say for certain which side of the synapse phospho-tau came from. Similarly, immuno-gold labeling of P301S neurons revealed clusters of phospho-tau on both sides of the synapse.

What’s more, Sheng told the audience that the PSD preps contained complement C1q, a key component of the complement cascade. Sheng claimed that all three C1q subunits could be found in the PSDs of nine-month-old P301S mice. He noted that neurons do not produce C1q, and suggested it came from glia. Using super-resolution microscopy and immune-electron microscopy, Sheng found that the C1q sat on the extracellular side of the neuronal membrane, immediately adjacent to, but not within, the synapse. C1q appeared sandwiched between pre- and postsynaptic markers. Previous work from Beth Stevens and Cynthia Lemere’s labs have reported that overzealous pruning of complement-tagged synapses leads to synapse loss in AD mouse models (Aug 2013 conference news; Nov 2015 conference news). Now, Sheng’s work implicates tau pathology as an “eat-me” signal as well.

Finally, Sheng investigated whether microglia truly engulf synapses in P301S mice. He found that by nine, but not six, months of age, microglia contained both the pre- and postsynaptic proteins synapsin and PSD95, respectively. The amount of synaptic proteins microglia contained correlated with levels of phospho-tau. Treating the mice with an anti-C1q antibody prevented this synaptic engulfment and blocked synapse loss. In all, the findings support the idea that microglia munching on tau-laden synapses promotes synaptic loss.

Marco Colonna of Washington University in St. Louis told Alzforum that he found Sheng’s proteomics approach convincing. He speculated that when tau accumulates in the synapse, it could trigger changes in the conformation of membranes there. This by itself could signal microglia to pump out complement. Whether TREM2, the lipid-sensing microglial receptor that Colonna discovered, plays any role in that process is an open question, he said.

McInnes, whose work supports the idea that tau exerts its toxic effects from the presynapse, said Sheng’s approach could not definitively nail down tau’s whereabouts, nor the source of its synaptoxicity, to either the pre- or postsynaptic side. McInnes proposed that, rather than in response to a shift in membrane structure, microglia might douse synapses with complement in response to waning synaptic activity brought about by tau’s corralling of vesicles.

In his talk, Naruhiko Sahara of the National Institute of Radiological Sciences in Chiba, Japan, reported that microglia engulfed tau-laden neurons in rTg4510 mouse models of tauopathy. Previously, Sahara had used tau PET and TSPO PET imaging in these mice to track neurofibrillary pathology and microglial activation, respectively. Both rose together, but the latter continued unabated even after tau pathology had plateaued (Ishikawa et al., 2018). At Keystone, Sahara described the results of two-photon confocal microscopy through cranial windows in rTg4510 mice. He injected adeno-associated viruses (AAVs) equipped with fluorescent tags driven by microglial or neuron-specific promoters. This enabled him to visualize microglia and neurons, respectively. He also injected PBB3, a fluorogenic dye that labels tau tangles and serves as a tau PET tracer. Daily tracking of the interactions between these cells in five- to seven-month-old mice revealed that microglia not only nibbled on PBB3-labeled neurons, but ultimately killed them. Microglia also killed neurons without tau pathology, yet seemed to have a preference for those with it.

Finally, Sahara reported a dramatic increase in complement proteins C1q and C3 in the brains of mice as microglia reached their peak of neuron consumption. Sahara’s findings meshed with Sheng’s, as both implicated a complement-driven microglial response in the demise of neurons burdened with tau pathology.

In the latter part of her talk, Gan investigated yet another potential consequence of microglia’s appetite for tau: The cells might serve as unwitting transporters of tau across the brain. Others have reported that in addition to trans-synaptic travels between connected neurons, tau may spread via microglia (Oct 2015 news). To investigate, Chao Wang from Gan’s lab used a model developed by Virginia Lee at the University of Pennsylvania in Philadelphia, in which injection of tau fibrils into aged PS19 tauopathy mice triggers the propagation of tau pathology throughout the brain (Iba et al., 2013). Wang found that depleting microglia, using the CSF1R agonist PLX3397, cut tau propagation by half, suggesting the cells play a role in spreading tau.

Wang went on to detail how cultured primary microglia that take up tau fibrils enlist the transcription factor NFκB to switch on inflammatory genes. The researchers generated mice in which NFκB was constitutively active, or repressed, only in microglia. While all microglia gobbled up tau (see image above), those with active NFκB retained less of it than their NFκB-inactive counterparts.

Gan wondered what would happen to the tau fibrils ingested by microglia. The researchers crossed the NFκB /microglia animals to PS19 mice. Those that repressed microglial NFκB had far less spread of tau inclusions and they performed better on spatial memory tests. Mice with constituitively active NFκB in their microglia had more tau spread. Incidentally, forcing NFκB activation in microglia triggered cognitive deficits irrespective of transgenic tau, suggesting that overzealous microglia might harm healthy neurons, perhaps by vigorous synaptic pruning, Wang told Alzforum at his poster.

Overall, Gan proposed that activated microglia act not merely as trash cans for tau, but rather process and release it again. On his poster, Wang also reported that microglia acetylate tau before releasing it, which could imply that tau emerges from microglia in a more toxic form than when it went in. He is investigating this. Wang told Alzforum that his findings do not contradict the idea that tau spreads between neurons independently of microglia, but instead implicate activated microglia as one possible route of tau processing and dissemination.—Jessica Shugart

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References

News Citations

1. Neurons Release Tau in Response to Excitation 27 Feb 2014
2. Tau Uses Synaptogyrin-3 to Clump Synaptic Vesicles 6 Feb 2018
3. Curbing Innate Immunity Boosts Synapses, Cognition 8 Aug 2013
4. Microglia Control Synapse Number in Multiple Disease States 3 Nov 2015
5. Deadly Delivery: Microglia May Traffic Tau Via Exosomes 9 Oct 2015

Research Models Citations

1. Tau P301S (Line PS19)

Paper Citations

1. Wang C, Ward ME, Chen R, Liu K, Tracy TE, Chen X, Xie M, Sohn PD, Ludwig C, Meyer-Franke A, Karch CM, Ding S, Gan L. Scalable Production of iPSC-Derived Human Neurons to Identify Tau-Lowering Compounds by High-Content Screening. Stem Cell Reports. 2017 Oct 10;9(4):1221-1233. Epub 2017 Sep 28 PubMed.
2. Fernandopulle MS, Prestil R, Grunseich C, Wang C, Gan L, Ward ME. Transcription Factor-Mediated Differentiation of Human iPSCs into Neurons. Curr Protoc Cell Biol. 2018 Jun;79(1):e51. Epub 2018 May 18 PubMed.
3. Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, Ting AY. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods. 2015 Jan;12(1):51-4. Epub 2014 Nov 24 PubMed.
4. Ishikawa A, Tokunaga M, Maeda J, Minamihisamatsu T, Shimojo M, Takuwa H, Ono M, Ni R, Hirano S, Kuwabara S, Ji B, Zhang MR, Aoki I, Suhara T, Higuchi M, Sahara N. In Vivo Visualization of Tau Accumulation, Microglial Activation, and Brain Atrophy in a Mouse Model of Tauopathy rTg4510. J Alzheimers Dis. 2018;61(3):1037-1052. PubMed.
5. Iba M, Guo JL, McBride JD, Zhang B, Trojanowski JQ, Lee VM. Synthetic tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of Alzheimer's-like tauopathy. J Neurosci. 2013 Jan 16;33(3):1024-37. PubMed. Correction.

Further Reading

No Available Further Reading