Conventional wisdom holds that microglia are the main phagocytes of the brain, mopping up dead cells and debris. The reality is more complicated, according to scientists led by Jaime Grutzendler at Yale School of Medicine in New Haven, Connecticut. In today’s Science Advances, they show how microglia and astrocytes work as a team to clear dying neurons. The authors triggered apoptosis in single cortical neurons in the brains of living mice, then watched what happened to these cells over the next several days. Nearby microglia moved in to engulf the cell body and its proximal dendrites. Meanwhile, nearby astrocytes gobbled up the degenerating dendritic arbor. The two types of phagocyte each stayed in their lane, with sharp boundaries between their territories. Importantly, the efficiency of this cleanup crew waned as mice aged, with disposal of dead neurons taking twice as long in elderly animals.
- 2Phatal method lets scientists trigger apoptosis in a single neuron, then watch it die.
- One microglia devours its soma while astrocytes absorb the dendritic arbor.
- Both types of phagocyte need the receptor Mertk for this.
“This elegant study reveals a coordinated and finely orchestrated action of both microglia and astrocytes during cell death,” Marco Colonna and Simone Brioschi at Washington University in St. Louis wrote to Alzforum (full comment below). “It will be interesting to see how this technique will be exploited to study microglia- versus astrocyte-mediated phagocytosis in pathological settings.”
Although microglia have been regarded as the main phagocytic cells in the brain, astrocytes have been reported to gobble up apoptotic cells in developing brain and after ischemic damage, and to eat Aβ in vitro (Mar 2003 news; Iram et al., 2016; Morizawa et al., 2017). It was unknown under what conditions each cell type assumed cleanup duties, and whether they acted independently.
To investigate this, Grutzendler and colleagues deployed the technique two-photon apoptotic targeted ablation (2Phatal), which they had previously devised to observe the clearance of single cells over time in situ. In this procedure, the researchers apply a DNA-binding dye to the cortices of live mice through a cranial window. The dye penetrates up to 300 microns deep, labeling all nuclei. The scientists then train a two-photon laser on a single neuron and photobleach the dye with a pulse of light. This generates free radicals that damage DNA and trigger apoptosis in the targeted cell. The crime unfolds within a couple of hours, and causes no collateral damage to surrounding cells (Hill et al., 2017).
The 2Phatal technique enables researchers to induce apoptosis in the living brain with spatial and temporal precision, and follow the process dynamically, Grutzendler noted. “I’m not aware of any other technique that can look at the kinetics of cell death and engulfment,” he told Alzforum.
Winner Take All. Three nearby microglia (green) initially contact (left) a dying neuron (yellow). The middle one prevails and moves in (middle), while the others withdraw (right). [Courtesy of Damisah et al., Science Advances.]
Joint first authors Eyiyemisi Damisah and Robert Hill used 2Phatal in transgenic mice with fluorescently labeled microglia and astrocytes. The authors targeted a given neuron with 2Phatal, then watched with time-lapse two-photon microscopy. Within two to three hours, several microglia and astrocytes extended processes toward the dying cell. Initially, these processes intermingled, but a single microglia quickly “won out,” migrating toward the dying cell and swallowing it (see image above). Meanwhile, the other phagocytes drew back. The victorious microglia also covered and digested nearby dendrites (see video below).
Over and Done. Time-lapse of a microglia (green) moving in and engulfing a dying neuron (yellow). In reality, this took 24 hours. [Courtesy of Damisah et al., Science Advances.]
The dendritic arbor was another matter. As this fine neuropil degenerated into small apoptotic bodies, delicate astrocytic processes crept out to encapsulate and digest them (see image below). Unlike microglia, the astrocyte cell bodies did not move.
Because dendritic arbors can be quite large, extending long distances from the cell body, and are themselves surrounded by astrocytes, it makes sense that these cells would be tasked with tidying them up, Grutzendler noted. He believes the findings belie the idea that astrocytes are “nonprofessional phagocytes.” “They’re not just a backup phagocyte. We think they’re actively involved in the phagocytic process every time. They have a specialized function,” he told Alzforum.
Specialized Cleanup. Microglia (green) engulf the soma of a dying neuron (arrow, left), while astrocyte processes (red) swallow degenerating neurites (arrowhead, right). Dying cell is white, nuclei blue. [Courtesy of Damisah et al., Science Advances.]
Is this coordinated cleanup response specific to 2Phatal? The authors examined two other types of cell death, developmental and induced by viruses. In postmortem sections from developing mouse brain, microglia likewise surrounded apoptotic cell bodies and nearby processes, while astrocytes mopped up distant debris, with a sharp boundary between the two (see image at top of story). The same thing occurred in the cortices of mice infected by a virus.
Next, the authors searched for the mechanism behind this phagocytic response. Two tyrosine kinase receptors, Axl and Mertk, reside in the cell membrane of both astrocytes and microglia. These receptors mediate phagocytosis by sensing signals on the surface of infected or dying cells (Chung et al., 2013; Fourgeaud et al., 2016; Tufail et al., 2017). To probe their role, the authors induced 2Phatal in mice lacking them. In Axl knockouts, they saw no change in clearance efficiency. In Mertk knockouts, however, the time it took to clear the doomed neuron doubled from 48 to 96 hours. The lag occurred because microglia took longer to respond to dying cells; once they had contacted a shrunken neuron, the phagocytes devoured it at normal speed.
Does Mertk also control the interplay between microglia and astrocytes? The two cell types appear to communicate, because the faster microglia target a dying neuron, the less likely astrocytes are to extend their own processes toward it. Indeed, when the authors knocked Mertk out only in microglia, inhibiting their response, astrocytes attempted to compensate. Astrocytes recruited lysosomes into their processes and extended them to touch the dying neuron and digest it piecemeal. Similarly, when the authors completely ablated microglia from mouse brain with a toxin, the astrocytes around a dying neuron worked together to remove it. Their processes intermingled to wall off the neuronal corpse and nibble away at it (see image above). In both cases, the process was slower than microglial cleanup, taking twice as long.
“In the absence of microglia, astrocytes can take up the burden of cell body removal,” Damisah noted. Nonetheless, astrocyte cleanup appears to be less efficient than microglial. Like microglia, astrocytes also depend on Mertk to recognize dying cells, since they fail to respond when they lack this receptor. Intriguingly, Mertk mutations have been implicated in neurodegeneration (Gal et al., 2000).
Finally, the authors studied what happens with aging. In 26- to 28-month-old mice subjected to 2Phatal, removal of dead cells was slow, increasing from a maximum of about 32 hours in young mice to 64 hours. However, because the authors could not age reporter mice that long, they were unable to perform live imaging of glia to determine which part of the process was delayed: recognition, phagocytosis, or coordination between phagocytes.
Delayed removal of dead neurons in aging brains could potentially harm surrounding tissue, perhaps triggering an inflammatory process, Damisah speculated. “If there’s too much debris in the brain, does the whole system get clogged up?” she asked. In future work, she will investigate how these lingering corpses affect nearby cells and synapses. In fly brains, the failure to remove apoptotic neurons triggers neurodegeneration with age (Etchegaray et al., 2016).
Senescent cells are known to build up in aged brain, and likewise have been linked to neurodegenerative disease (Sep 2018 news). James Kirkland at the Mayo Clinic in Rochester, Minnesota, studies senescence. He believes the slowdown in phagocytic clearance with age could be relevant to that. “Potentially, all of these changes could be linked in some way to the accumulation of senescent microglia and astrocytes,” he wrote to Alzforum.—Madolyn Bowman Rogers
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