While this external digestion may initially shrink plaques, it could potentially backfire, Sole-Domenech reported. He found that when microglia were first allowed to gorge themselves on small Aβ fibrils before encountering large aggregates, the cells filled up their lysosomes with fibrils, then later spewed them out onto large Aβ aggregates via lysosomal synapses.

Akin to the way the process is regulated in LDL-digesting macrophages, Sole-Domenech and Maxfield found that signaling via MyD88, downstream of toll-like receptor 4 and CD14 receptors, instigated digestive exophagy in microglia.

How might signaling through TREM2, a microglial receptor that promotes phagocytosis of Aβ aggregates, influence this external digestion? Strikingly, Jacquet and Sole-Domenech found that microglia deficient in TREM2 doubled down on digestive exophagy, unleashing even more lysosomal contents onto Aβ plaques. He hypothesized that TREM2-deficient microglia might ramp up these external digestive mechanisms to compensate for their deficits in phagocytosis. In particular, TREM2-deficient microglia poorly polymerize actin, and phagocytosis requires more actin polymerization than does digestive exophagy, Sole-Domenech told Alzforum. “It is therefore ‘easier’ for the cells to exocytose lysosomal contents during digestive exophagy,” he added. In support of this idea, he said wild-type and TREM2-deficient microglia form lysosomal synapses with equal measure when they are treated with GM-CSF, which boosts actin polymerization.

In all, Sole-Domenech cast digestive exophagy as a double-edged sword: It allows microglia to cut Aβ plaques down to size without internalizing them, but if microglia also fill themselves with smaller Aβ aggregates via phagocytosis, they run the risk of regurgitating—and potentially spreading—undigested toxic amyloidogenic peptides.

This exact flavor of microglial indigestion was the topic of Anja Schneider’s talk at AD/PD. Schneider, of the German Center for Neurodegenerative Diseases in Bonn, used an isotope-labeling technique to track the evolution of aggregates within the mouse brain. When 5xFAD mice reached 6 months of age, she swapped out their regular chow with food containing ¹³C-labeled lysine. This heavy isotope can be distinguished from the lighter ¹²C variety using nanoSIMS, a form of mass spectrometry that can track isotopes with nanoscale resolution. Schneider was then able to distinguish between relatively younger and older proteins within the mouse brain.

Schneider found that roughly 30 percent of Aβ aggregates resided inside of cells. Microglia stomached most of that intracellular pool within their lysosomes. Isotope scanning of the lysosomal aggregates revealed that Aβ peptides in the core were substantially older than those in the outer shell, suggesting that fresh Aβ peptides were continually joining up with older aggregates that were sitting within the lysosome.

The results took Schneider by surprise. “We had assumed that Aβ aggregates are being degraded in the lysosome, not that they grow there.”

Schneider came to similar conclusions about tau. This protein accumulated within microglial lysosomes of P301L mice and, once again, younger tau proteins surrounded a comparatively ancient aggregate core. Schneider proposed that instead of clearing amyloid or tau aggregates, microglial phagocytosis might sometimes lead to the formation of amyloidogenic seeds. If the contents of these lysosomes were to be secreted, say, by a mechanism like digestive exophagy, this could lead to propagation rather than containment. Schneider and Sole-Domenech agreed that their findings complement each other, and are consistent with the hypothesis that buildup of aggregates within lysosomes could ultimately contribute to amyloid dissemination. In light of these findings, Schneider believes the field should approach therapies aiming to cautiously increase microglial phagocytosis.

The Ikezus’ data indicate that the microglial production of extracellular vesicles exacerbates tau pathology. Exactly how cutting off EV secretion might also counteract microglial activation needs further investigation, Ikezu said. Much as constipation reduces appetite, blocking the release of EVs apparently blocks microglial phagocytosis. Ikezu believes that phagocytosis of apoptotic neurons, damaged synapses, or aggregated proteins is an essential part of the microglial transition into the DAM state, so this could explain how TSG101 deletion ultimately douses microglial activation, she told Alzforum. It is also possible that the EVs contain inflammatory ligands, such as nucleic acids or mitochondrial proteins, that push microglia into the DAM state, she added.

In a startling example of microglia gone terribly wrong, Charles Arber, working in the lab of Selina Wray at University College London reported that the cells are the sole purveyors of the amyloidogenic peptide that causes familial British dementia. This rare genetic disorder is marked by amyloid plaques and tau tangles, but its plaques contain no Aβ. They are made of a peptide cleaved from the C-terminus of the transmembrane protein ITM2B. Mutations that cause FBD disrupt a stop codon in the ITM2B gene, resulting in production of the aggregation-prone, 34-amino acid “A-Bri” peptide.

Together with Sarah Wiethoff, Arber acquired fibroblasts from two people with FBD and two controls, and differentiated them into iPSCs. Try as she might, Emma Augustin from Arber’s lab found no trace of ITM2B, the A-Bri peptide, or any phenotype whatsoever, in iPSC-derived neurons from the two people with FBD. Taking a hint from other gene-expression datasets that suggested ITM2B was expressed in microglia, the scientists differentiated the iPSCs into microglia. Lo and behold, microglia from both control and patient samples expressed ITM2B, and A-Bri was found only in FBD. Microglia were also spotted manufacturing A-Bri in brain samples from a person with FBD, as well as from a person with familial Danish dementia, a related disorder caused by mutations in the same gene.

A dive into single-cell transcriptomic datasets revealed that ITM2B is a disease-associated microglia (DAM) signature gene, rising in step with TREM2, Tyrobp, and other DAM genes. What does this mean? Arber proposed that FBD could be sparked when an inflammatory event provokes microglia to shift into the DAM-like state. In support of this idea, Arber said that many people develop FBD symptoms after experiencing such events, such as a flu infection or a stroke. In other words, in people carrying a causative ITM2B mutation, an otherwise beneficial response to, say, a viral infection or a brain trauma, causes microglia, of all cells, to produce an amyloidogenic protein that leads to dementia.

David Holtzman of Washington University in St. Louis called the production of A-Bri by microglia “an amazingly cool finding. I don’t know of any other extracellular amyloid that is mostly produced by microglia.” Regarding the idea that the disorder could be sparked by neuroinflammation, Holtzman wondered if familial British dementia, like AD, starts with a long preclinical phase where plaques steadily grow. If so, how would one reconcile that long asymptomatic period with the idea that symptoms of disease surface soon after an inflammatory event? Arber said those remain open questions, given the dearth of preclinical samples or biomarkers for this disease. “Still, it is exciting to think that inflammation is part of the onset and progression,” Arber said.

This sampling of microglial behavior on display at AD/PD offers but a glimpse of the myriad ways the cells react to different stimuli. How do these reactions—and the transcriptional states that control them—influence neurodegenerative disease? In the next part of this series, read how scientists grapple with this question by marrying cutting edge omics and human microglial cell culture techniques.—Jessica Shugart