The reasons that drew Italian Francesca Peri to Germany were purely scientific. As a student at the University of Padua, she had the chance to work with Siegfried Roth one summer and decided to pursue her PhD in his laboratory at the University of Cologne to study signaling in Drosophila ovaries. As a postdoc, she joined the laboratory of Nobel laureate Christiane “Janni” Nüsslein-Volhard at the Max Planck Institute for Developmental Biology in Tübingen working with the transparent, tell-all zebrafish. With Nüsslein-Volhard’s encouragement, Peri followed where the cells took her, focusing on the macrophages that crawl into the developing brain to become the microglia that gobble up dying neurons during development and after injury (1).
As a group leader within the international halls of the European Molecular Biology Laboratory (EMBL) in Heidelberg, Peri continues to probe how these specialized cells develop, migrate, and perform their complicated clean-up routine. Along the way, her group has made some incredible cell biological findings, including how long-range calcium waves guide microglia to damage (2), how apoptotic neurons are engulfed and digested (3), and that microglia arise from a distinct sublineage of macrophages (4).
“Blocking digestion would be a good, generic way to stop [microglia] from killing neurons.”
She spoke with JCB about cellular indigestion and the advantages of having her nine-year-old tag along to a Gordon Conference.
During your postdoc, you switched from studying cell migration to microglia engulfment. How did that happen?
Macrophages are the ultimate moving cells. In the embryo they home to different organs to establish resident populations. I chose to focus on migration to the brain and then I started to wonder what do these cells do in the brain? They were reported to remove dying neurons and to respond to injury, which I found quite fascinating.
I remember the first time I actually saw them on the microscope. It was incredible. They move faster than any other cells in the brain, so I could watch it happening live. And they were full of neurons, because they actually eat, or engulf the neurons. “Oh wow,” I thought, “They are emptying out the brain.”
That opened up many different questions and experiments and because of Janni’s open and supportive attitude, I developed this microglia project. I’m still interested in their migration, but the main topic in my laboratory is what the microglia do and how they do it once they arrive in the brain.
Are microglia simply the clean-up crew?
Microglia play a role in all brain disorders. Sometimes they fail to remove sick neurons. At other times, they are hyperactive and become cytotoxic. If you remove microglia from adult mice they have cognitive impairments. During development, they also do synaptic pruning.
Our goal is to understand the ultimate purpose of these functions. Do microglia contribute to circuit formation, brain functionality, and development? How can microglia distinguish between a healthy and an unhealthy neuron or a synapse that needs to stay and one that needs to go?
How can a cell remove another cell or part of a cell without harming the rest of the tissue? This is highly relevant. It might teach us how to eventually trick these cells to remove neoplastic cells in the brain. Or, if they are being too active, we could modulate these responses.
You published the first real-time live analysis of microglial activity in zebrafish. What did you observe?
The microglia are highly motile migrating into the brain, but after a day and a half they “sit down” somewhere. There might be contact inhibition between different microglia in order to form a network that covers the whole brain volume. They are pretty fixed in one position, but they use long cellular extensions or branches. Some of those extensions will contact a dying neuron and a large phagosome will form at the tip of the extension. In the space of four minutes or so, this phagosome will quickly wrap around the neuronal cell body and close.
Then the branch rapidly retracts—moving around two microns per minute—and travels back to the cell body where the phagosome fuses with the lysosome and the neuronal material is digested.
When we knocked down the a1 subunit of the V0-ATPase proton pump, all of a sudden the cells had this phenotype of being full of vesicles and no longer eating neurons. Without this subunit, phagosomes and lysosomes accumulate, but they cannot fuse with one another.
You describe them as having “indigestion.” Could this be a therapeutic strategy for disorders with overactive microglia?
Absolutely. At the moment we are working on digestion because we have shown that digestion in the phagolysosomes is a very important step to allow the continued engulfment of neurons. If the cell cannot process this material, it will not keep eating. In many neurological disorders where these cells become aggressive phagocytes, blocking digestion would be a good, generic way to stop them from killing neurons.
RIDE THE WAVE
What signal is responsible for microglia crawling or reaching toward injured cells?
In both fish and mice, it’s ATP. Because ATP signaling often leads to calcium responses, we generated a transgenic line expressing a fluorescent calcium indicator in all brain cells to investigate ATP signaling dynamics. When we killed neurons with a laser, all the cells surrounding the injury experienced an increase in intracellular calcium signaling—with the signal high near the injury and low farther away.
It was very dramatic to see this wave of calcium traveling out from the site of injury. Next, we asked if the calcium gradient is necessary and sufficient to guide the microglia, using either calcium chelators to disrupt the wave or using photo-uncaging of calcium activators to elevate calcium without actually damaging neurons. This showed us that the calcium gradient is indeed part of the mechanism that guides the microglia. Surprisingly, however, we found that calcium elevation is not downstream of ATP signaling as initially expected, but it is upstream and responsible for ATP release.
What are the implications of that?
For me, that study was interesting from a pure biological point of view because we were asking, how do signals travel through tissues? How do you provide information to cells that have to respond to stimuli and find targets in these large areas? How would this information travel and be established across complex tissues such as the brain? How are these signals restricted? These are the next questions we would like to address.
How has light sheet microscopy changed your work?
Light sheet microscopy dramatically speeds up image acquisition. To give you an idea, the confocal microscope takes us roughly two minutes to image the whole brain but with the light sheet microscope, it takes two seconds. In this way we are able to image the whole brain, but also look at events that are really fast, for example vesicle fusion and neuronal activity.
“Calcium gradients are…part of the mechanism that guides microglia.”
In addition, the light sheet microscope has very low phototoxicity, which is extremely important when working with live specimens. The light sheet microscope has revolutionized looking at large samples in developmental biology.
What is special about working at EMBL?
We have a nine-year limited contract. It’s fantastic because there is a lot of turnover: every year you have some new colleagues. Also, you are very well funded and supported for nine years as a junior investigator. The idea is that then you will be ready for the next position. It is a formula that works well.
It’s also a highly interdisciplinary, very collaborative environment. You can take different, new approaches without making a huge investment.
Where would you like to head next?
I’m really open. I would consider an opportunity to be inspired by a different environment. For example, I could go to an institute that focuses on neurobiology, which would allow me to look at the role of microglia in brain development and functionality, or addressing more medical questions, like the role of microglia in regeneration.
At this stage of my career, moving will be a breath of fresh air. I read that you should change your job every seven years to keep excited.
Have you taken any good vacations lately?
We had an interesting experience this summer. My husband, who is also a biologist, and I both went to the Gordon Conference for Developmental Biology in Massachusetts and we brought our nine-year-old daughter with us.
She sat through the talks and at the beginning she was even taking notes! I will always keep these notes because they are so cute. She was drawing and writing about cancer and cell shapes. Then, she got bored and we had to activate the tablet so that she could watch Disney shows.
It was an enjoyable conference because I didn’t have that sense of guilt for being away. Actually, it was also a way to meet more people as they came over to interact with Giulietta.
Then we went to New York City for five days. We first went to the huge toy store FAO Schwarz because that was the bribe for Giulietta to sit through all those talks. Then we went to the Met to see a ballet. We went to the top of the Empire State Building. We did New York stuff. It was absolutely great.