The brain’s resident immune cells have a taste for tau, and anti-tau antibodies act like the secret sauce. According to a June 9 study in Scientific Reports, microglia dine on pathogenic forms of tau released from brain tissue affected by Alzheimer’s disease. The cells internalized tau more readily when anti-tau antibodies were added to the mix, and the researchers found that the Fc (stem) portion of the antibody was required for this enhancement. While in vivo experiments are still lacking, the findings hint that microglia play a role in clearing toxic tau from the brain, and that the success of future tau immunotherapies may hinge upon improving the action of these brain sentinels.
“Our data strongly suggest that if tau exists in the extracellular space, it will be taken up and degraded by microglia, and antibodies could enhance the process,” said Steven Paul of Weill Cornell Medical College in New York, the paper’s senior author.
In healthy neurons, tau stabilizes the microtubules that ferry important proteins between the cell body and the axon’s outer reaches. When hyperphosphorylated, tau ditches microtubules and forms intracellular tangles instead. Researchers have found that in addition to intracellular aggregates, tau can exit neurons and enter neighboring ones, thus spreading pathology throughout the brain (see Mar 2009 conference news; Jun 2009 news).
Efforts to target pathogenic forms of tau using active and passive immunizations have exploded. A handful of therapies are in, or about to enter, Phase 1 trials, while a plethora of others are in preclinical development (see Pedersen and Sigurdsson, 2015). In preclinical studies, anti-tau antibodies reduced tauopathy in transgenic mice and improved cognitive function (see Boutajangout et al., 2011; Chai et al., 2011; Sep 2013 news).
However, the way these antibodies work their magic is still unknown. They could enter neurons and bind to intracellular tau, shuttling the protein into lysosomes and/or preventing it from spreading to other cells (see Congdon et al., 2013; Gu et al., 2013; Collin et al., 2014). Alternatively, the antibodies could latch onto extracellular tau. Using their Fc receptors, microglia would then internalize these tau-antibody complexes and digest them. Either or both mechanisms could occur in the AD brain, and may vary depending upon the antibody and the form of tau that it targets.
First author Wenjie Luo and colleagues tested whether microglia ingested tau, and if it did, whether antibodies could influence the process. They started by isolating sarkosyl-insoluble tau from postmortem AD brain samples that were riddled with tau tangles. This fraction of tau was highly enriched with paired helical filament (PHF)-tau, which is thought to represent a pathogenic species. Then the researchers incubated this insoluble tau with primary microglia derived from neonatal mouse brains. After two days, the researchers found that only 20 percent of total tau and 5 percent of hyperphosphorylated tau remained in the media, whereas most of the tau remained when microglia were absent. Using confocal microscopy, the researchers observed hyperphosphorylated tau within discrete puncta inside the microglia, as well as some large tau aggregates attached to their cell membranes. They saw a dramatic rise in intracellular tau within the first two hours of incubation, followed by a steep decline. They concluded that microglia internalized and then degraded tau derived from AD brain tissue.
The researchers next measured whether microglia would internalize tau released from brain slices. They added microglia to cultures of thawed brain slices from P301S transgenic mice, which harbor an abundance of tau tangles. The researchers found that the tau concentration in the slice culture medium initially rose, and then plummeted as microglia internalized the protein. In cultures without microglia, tau levels held steady after the initial bump. Tau levels also remained unchanged when researchers added media from microglial cultures to the slices, indicating that enzymes secreted by microglia did not degrade tau. The researchers observed a similar microglia-dependent reduction in tau derived from postmortem frontal cortex slice cultures of advanced-stage AD patients, and also found that microglia internalized Aβ40 and Aβ42 from those slices in addition to tau.
Interestingly, microglia also took a bite out of neurofibrillary tangles in the slice cultures, as measured by immunohistochemistry. How the microglia access the intracellular tangles is unclear, Paul said. It is important to note that because the slices measured only 10 microns thick, few cells survived slicing, and intracellular proteins (including tangles) could leak out into the medium. Paul would not speculate on whether microglia could somehow access the intracellular compartments. He said the most important aspect of the findings was that microglia readily internalize a variety of pathogenic tau species.
Next the researchers measured the effect of anti-tau antibodies on microglial internalization of tau. They mixed a fluorescently labeled AT8 antibody—which recognizes hyperphosphorylated tau—with P301S brain slices, washed away any unbound antibodies, and then added microglia. One hour later, they observed an abundant fluorescent signal inside the microglia, indicating that the cells had consumed the tau/antibody complexes. Next, the researchers mixed MC1—another anti-tau antibody that recognizes a pathological conformation of tau—with sarkosyl-insoluble tau isolated from AD brains. They found that microglia took up tau bound by MC1 more efficiently than tau alone, but that other IgG antibodies did not enhance uptake. This enhancement vanished when the researchers instead added a Fab fragment, which lacks the Fc effector portion of the antibody, to the tau prep.
“Their findings are as expected based on known functions of microglia, and further confirm the feasibility of tau immunotherapy,” wrote Einar Sigurdsson of New York University.
Paul proposed that using anti-tau antibodies could rev up flagging microglial clearance responses in the AD brain. Microglial functions, including phagocytosis, are known to decline with age, and variants in the AD risk genes TREM2 and CD33 exacerbate these deficits, Paul said (see July 2014 Webinar; Aug 2013 news; Griciuc et al., 2013). He proposed that defective microglia fail to clear both amyloid and tau in two parallel pathways that ultimately lead to AD. Paul proposed that antibodies that engage microglial Fc receptors could “supercharge” the cells’ cleanup capacities.
Takami Tomiyama of Osaka City University in Japan commented that the findings indicate that antibodies without Fc regions could have lower tau clearance rates in vivo, despite the fact that these smaller antibody fragments enter the brain more readily than their full-sized cousins. “Moreover, this notion would prompt us to select antibodies with a higher binding affinity to Fc receptors during the development of therapeutic antibodies,” he wrote.
Paul’s findings indicate that microglial internalization is one possible mechanism of antibody-mediated tau clearance, commented Yona Levites of the University of Florida in Gainesville. However, it is not yet possible to rule out the possibility that other non-microglial mechanisms, such as entrance of the antibodies into neurons, contribute to therapeutic effects in vivo. Levites added that emerging data from her lab and others suggests that Fab and single-chain variable fragment (scFv) antibodies clear tau in mouse models just as well as those with Fc portions attached.
Martin Citron of UCB Pharma in Brussels referenced similar reports, pointing out that researchers from Genentech/AC Immune recently presented findings at the AD/PD conference in Nice suggesting that antibodies with or without Fc regions cleared tau equally well. He added that it will be important to understand why, in the case of Paul’s antibody, the Fc portion was needed. “The paper does not address whether the Fab fragment is less active due to loss of effector function or loss of the avidity benefit of an antibody,” he wrote. “Clearly, more work is needed to fully understand the mechanisms of tau clearance and tau immunotherapy in cell systems and in vivo.”
Paul said his lab is currently investigating whether microglial function or mutations in AD risk genes such as TREM2 influence the efficacy of anti-tau antibodies in mouse models.—Jessica Shugart