27 August 2013. A creepy idea is taking hold in Alzheimer’s research. Like a computer worm that travels through networks corrupting perfectly good files, toxic forms of tau bore from neuron to neuron turning good protein into bad along the way. Scientists at the Alzheimer’s Association International Conference (AAIC), held July 13-18 in Boston, discussed how aggregated tau makes this jump from neuron to neuron, how that affects the invaded cells, and how medicines might stop the process in its tracks. The upshot was that although researchers have come to associate this cell-to-cell transmission with misfolded tau, normal tau might travel the same way. Moreover, new data suggest that misfolded tau does not kill neurons directly or cut them off from their neural networks. Despite many gaps in current understanding, the transport of misfolded tau undoubtedly promotes dementia, leading many companies to pursue new therapeutics in the form of antibodies that grab onto the bad tau before it spreads.
Tau neurofibrillary tangle pathology as visualized at autopsy follows a distinct progression pathway in the AD brain. Starting in the locus coeruleus, it moves toward the transentorhinal cortex, hippocampus, and beyond. Importantly, it does not merely diffuse out radially from its point of origin; rather, it travels along neural circuits (Braak and Del Tredici, 2011). A similar spread occurs in animal models. For example, in mice that express mutant tau only in the entorhinal cortex, tau pathology travels to connected regions in the brain as the animals age (see Alzforum related news story). Researchers have come to believe that tau spreads like a prion, with pathological protein converting native protein to a misfolded, pathogenic form (see Alzforum related news story).
Cell-to-Cell Transmission—Not Just For Toxic Tau
Some researchers at AAIC turned their attention to how ordinary, non-mutant tau might move throughout the brain. Amy Pooler of King's College London, U.K., presented evidence that transport of wild-type tau among cells is a normal phenomenon. She also claimed it is induced by neuronal activity. In a paper that took the Alzheimer’s field by surprise, Pooler had reported previously that pathogenic tau hijacks this physiological transport process to move from cell to cell (see Braak et al., 2011). Specifically, Pooler found that cultured neurons stimulated with potassium chloride or glutamate released tau into the cell medium. The agonist (S)-AMPA, which stimulates AMPA glutamate receptors, had a similar effect. On the other hand, quenching neuronal activity with tetrodotoxin, or blocking synaptic vesicle release with tetanus toxin, reduced the release of tau in response to (S)-AMPA.
Pooler’s work convincingly shows that neuronal activity drives tau out of cultured neurons, commented David Holtzman of the Washington University School of Medicine in St. Louis, Missouri, in an email to Alzforum. Still to be determined, he added, are whether the same occurs in vivo and what the mechanism of tau release might be. Notably, active neurons also release amyloid-beta (see Alzforum related news story).
“Tau release may be a normal process in the healthy brain,” concluded Pooler in an email to Alzforum. Other evidence supports this idea, she added. Tau flows in the interstitial fluid of healthy mouse brains (see Alzforum related news story) and there is evidence that extracellular tau stimulates neurotransmitter receptors (Gómez-Ramos et al., 2008). The process of tau transfer could offer new potential drug targets, commented Benjamin Wolozin of Boston University.
Marc Diamond, also from Washington University, agreed. His group recently reported that once tau escapes into the extracellular space it can bind to heparan sulfate proteoglycans (HSPGs) on the surface of neighboring cells (see Holmes et al., 2013). Blocking HSPGs prevented tau transmission to other cells. HSPGs appear to act like a receptor for tau and also for α-synuclein, or by knocking out Ext1, an enzyme essential for HSPG synthesis. A suite of enzymes are known that modify HSPGs and turn them into tau receptors, noted Diamond. "These are all enzymes with active sites—potentially druggable," he told Alzforum. "This suggests an entirely new class of drug targets."
Modeling Sporadic Disease
Luc Buée of Inserm in Lille, France, has for years studied normal tau, in his case the wild-type protein that aggregates in the brains of people with sporadic tauopathies. Buée noted that a classic model for tau transmission, in which injected tau seeds spread from one part of the brain to others, relies on the delivery of mutant tau into mice primed to spread that pathology by their overexpression of human, wild-type tau. Classic Abeta seeding models work similarly (see Alzforum related news story; also Clavaguera et al., 2013). At AAIC in Boston, Buée described a similar model, with one crucial difference—it relies on only wild-type tau (see Alzforum related news story). Buée and colleagues used a lentivirus to deliver the wild-type human tau gene into the hippocampus of normal rats. Neurodegeneration and tangle-like structures spread from the site of injection along neuroanatomical pathways that connect networked parts of the brain. “Using wild-type tau, we were able to induce a type of pathology,” Buée concluded in an interview with Alzforum. This model could be a closer match for sporadic human disease, in which only wild-type tau is present.
To better understand the spread of tau from the viral injection site, Buée and colleagues added to the lentiviral vector a piece of DNA coding for V5. This peptide is widely used to tag and study the fate of proteins in the cell. That way, the researchers could distinguish the tau encoded by the injected gene from the endogenous version. To the researchers’ surprise, they observed that this V5-tagged tau spread all the way from the hippocampus to the olfactory bulb, traveling anterogradely along projection pathways. Buée told Alzforum that he believes tau undergoes some sort of active transfer from neuron to neuron. Like Pooler, he thinks tau transport is a normal process, and that when additional stressors turn tau toxic, its pathogenic kin follows the same trails.
Counterintuitively, the pathogenic tau may travel at a more leisurely pace. When the researchers expressed proline-301-leucine tau mutant, which causes neurodegeneration, they saw that deposits spread more slowly than the wild type. The researchers speculated that mutant tau does not spread so fast or so far because it rapidly aggregates, getting stuck in cells near the injection site. “This finding is particularly interesting. It suggests that it is the soluble form of tau that is propagated and that tangle formation might prevent the release of tau into extracellular space,” agreed Pooler. This does not preclude the role of mutations in aggressive tauopathies, since in that case all neurons have the mutant tau and it can cause damage without spreading, Buée noted. This new rat model of progressive tauopathy may be useful to investigate how tangles form, and also to test potential therapeutic compounds that may slow tau aggregation, Pooler added.
Full of Tangles, But Ready to Work
One transported, toxic tau forms intracellular tangles. Are these toxic waste dumps contributing to disease, or relatively safe places to store undesirable types of tau? Work presented by Susanne Wegmann, of Bradley Hyman’s group at Massachusetts General Hospital in Charlestown, suggests the latter. At AAIC, Wegman reported that tangled tau barely disrupts neural function or brain networks. The work was also reported by Wegmann’s colleague Kishore Kuchibhotla at the International Conference on Alzheimer’s and Parkinson’s Diseases in March (see Alzforum related news story).
Wegmann set out to directly compare tangle-laden neurons to neighboring, tangle-free nerve cells. She studied transgenic mice that overexpress human P301L tau and develop neurofibrillary tangles in their cortex. To assess individual neuron function, she used moving gray bars as visual stimuli to activate neurons in the visual cortex. As output, she recorded calcium flares in those neurons by in-vivo confocal microscopy and a calcium sensor called Yellow-Cameleon 3.6. Following the stimulation experiments, Wegmann sacrificed the mice and used immunostaining to identify neurons that contained, or lacked, tangles. She observed no difference in the responses of tangle-loaded neurons and their tangle-free neighbors.
“Even though these mice carry a high tangle load, they have no major neuronal network dysfunction in the visual cortex,” Wegmann concluded. She suggested that tangles themselves may not be directly neurotoxic. “These data…might even lend support to the idea that sequestration of tau into these tangles might be a protective mechanism,” added Pooler.
These findings have potentially good and bad implications, Buée told Alzforum. On the upside, it suggests that neurons with tangles in them remain functional and wired into the brain’s networks. If a treatment could clear away the toxic forms of tau, these neurons could be saved. However, since tangle-filled neurons remain connected, they likely could transmit their pathology to others in the network, further spreading the disease, said Buée. Other scientists interpreted Wegman’s finding as raising the notion that tangle-busting drugs could unleash more toxic forms of tau from a relatively less toxic storage compartment – a discussion that echoes from years past when plaque-busting drugs were being debated.
Modeling and Targeting Tau Transmission
If toxic tau travels, then a roadblock could halt disease, many researchers have reasoned. Nearly a dozen companies have turned their attention to immunotherapy using antibodies that bind tau and prevent its trans-synaptic spread, said Michael Hutton of Eli Lilly and Company in Windlesham, UK. Lily is among the companies pursuing this approach.
Those companies need ways to test their therapies. Zeshan Ahmed of Lilly’s Surrey, U.K. site was looking for a system that uses tau injection but is faster and more suitable for preclinical study of drug candidates. As with earlier models, Ahmed used brain extracts from old proline-301-serine-tau mice as the source of tau. But instead of targeting mice that over-express wild-type human tau, he injected it into young P301S mice. Already expressing mutant tau, these young mice were primed for tau transmission. Indeed, they demonstrated a “startling induction of tau pathology” within just 2 months, Ahmed said. Researchers have performed similar experiments injecting extracts from the brains of people with taopathies into P301S mice (see Alzforum related news story).
Mild tau pathology appeared just two weeks after injecting the brain extracts into the hippocampus. By one month, the damage had spread to the opposite side of the brain from the injection, and this contralateral pathology was more severe by two months. Again, the tau moved along neuroanatomical pathways instead of merely diffusing to neighboring regions. “It is a clear demonstration that the pathology spreads to regions that are synaptically connected,” said Hutton. “You have the tau pathology jumping across the synapses into a new brain area.” Despite this pathology, neurons remained present and alive.
“I like the speed and ease of this model,” said Wolozin. “The current models take a long time to see a bit of tau transfer.” The model will be useful for testing drugs as well as to understand tau transport biology, he suggested. The P301S strain could be crossed with knockouts for any number of genes suspected of involvement in tau transfer, and checked for effects on the post-injection pathology. The high level of tau aggregates also makes the mice suitable for biochemical studies, Wolozin said.
Hutton said the tau horizon is looking brighter than in earlier days, when efforts to affect tau phosphorylation and aggregation fizzled in human trials. Antibodies to block transfer are “currently the hottest area in tau therapeutics,” Hutton said. Lilly has already had some success reducing tauopathy in mice (Chai et al., 2011). Hutton predicts several of these therapies will move into the clinic over the next couple of years.—Amber Dance.
Caillierez R, Bégard S, Lécolle K, Deramecourt V, Zommer N, Dujardin S, Loyens A, Dufour N, Aurégan G, Winderickx J, Hantraye P, Déglon N, Buée L, Colin M. Lentiviral delivery of the human wild-type tay protein mediates a slow and progressive neurodegenerative tau pathology in the rat brain. Mol Ther. 2013 Jul;21(7):1358-68. Abstract.
Pooler AM, Phillips EC, Lau DH, Noble W, Hanger DP. Physiological release of endogenous tau is stimulated by neuronal activity. EMBO Rep. 2013 Apr;14(4):389-94. Abstract.