Research Area: Neurobiology

New approach enables a closer look at brain cell organelle

Microglia are involved in brain development, as well as neurodegeneration and brain cancer. A new approach from the Jaenisch Lab allows researchers to isolate and analyze microglia phagosomes.

Greta Friar | Whitehead Institute
August 14, 2024

Microglia are the immune system’s front-line enforcers in the brain. They are cells that patrol the brain and destroy anything harmful that they encounter, from invading bacteria to cellular debris. They also remove plaques and prune dysfunctional synapses between neurons. Microglia eliminate their targets by eating them: they envelope material and seal it in bubble-like organelles called phagosomes. A phagosome can then fuse with other organelles that break down its contents.

Microglial phagosomes play important roles in brain development, brain function and a plethora of brain diseases, including neurodegeneration and brain cancer. Therefore, understanding microglial phagosome biology could help to develop new therapies for currently untreatable brain diseases. However, microglia and their organelles have been difficult to study because existing stem cell and animal models insufficiently resemble microglia in the human brain, and because microglia, as vigilant immune patrollers, react to even subtle stimuli and so experimental conditions can trigger changes in the cells that confound analyses.

To overcome those issues, Whitehead Institute Founding Member Rudolf Jaenisch, also a professor of biology at the Massachusetts Institute of Technology; University of Freiburg Professor of Neuropathology Marco Prinz; and University of Freiburg neuropathologist Emile Wogram, who began this project as a postdoctoral researcher in Jaenisch’s lab, have developed a method to isolate and analyze microglia phagosomes in a rapid, gentle, and unbiased fashion.

In research shared in the journal Immunity on August 15, the researchers describe how they can isolate and profile phagosomes from stem cell-derived microglia and fresh human brain tissue. They also share new insights into phagosome biology in the human brain, regarding synaptic pruning and generation of NAD+, a broadly used molecule in the brain, by microglia.

The method that the researchers developed to isolate phagosomes from cells uses immunoprecipitation, in which antibodies latch on to a specific target protein on an organelle’s surface. When the antibodies are collected, they pull the organelles with them. This technique avoids many chemical perturbations that might alter the microglial profile. Sometimes researchers genetically engineer a target for the antibodies, but in order to isolate phagosomes from human brain tissue, Wogram had to find a naturally expressed target. Eventually, he and colleagues found one: the protein CD68.

The researchers first isolated phagosomes from stem cell-derived microglia. They co-cultured the microglia with other brain cell types to create a more brain-like environment, which led to a better match between brain and stem cell-derived microglia gene expression. They triggered some of the microglia to enter an inflammatory or disease-like state to see how that affected the phagosomes. Additionally, Wogram collaborated with the neurosurgery department at the University of Freiburg to get access to brain tissues immediately after their removal during surgery. He isolated phagosomes from brain tissue within a half hour of its removal, allowing him to profile the organelles before their contents could change much.

The profiles that the researchers built included what proteins and metabolites the phagosomes contained, and the whole-cell gene expression profile. The profiles differed significantly between sets of phagosomes, but the researchers identified a core of consistent proteins, including many known and also some unknown phagosome proteins. The results showed that phagosomes contain sensitive signaling molecules that allow them to react quickly to even subtle environmental stimuli.

Additionally, the protein contents of the co-cultured microglia provided strong evidence that when microglia prune synapses, they predominantly prune the side that sends a signal and not the side that receives one. This insight could be useful for understanding how microglia interact with synapses in health and disease.

The researchers also gained insights into a key metabolic pathway that occurs inside of microglia. In excess, the molecule quinolinic acid can be toxic to neurons; it is implicated as involved in many neurodegenerative diseases. However, cells can use quinolinic acid to make NAD+, a molecule broadly used to carry out essential cellular functions. Microglia are the only brain cells that generate NAD+. Wogram and colleagues found that key steps in this process occur in phagosomes. Phagosomes are therefore necessary both for removing excess quinolinic acid to prevent toxicity and for helping to generate NAD+ in the brain.

Finally, Wogram used brain tissues to compare phagosomes from within a tumor to those in the surrounding healthy tissue. The phagosomes in the tumor contained excess quinolinic acid. Although follow-up studies would be needed to confirm the results, these findings are consistent with research that suggests cancer cells use quinolinic acid to fuel their growth.

Collectively, these findings illuminate aspects of phagosome biology and the roles that phagosomes may play in normal brain development and maintenance, as well as in cancer and neurodegeneration. The researchers also anticipate that their method could prove useful for profiling other organelles, especially when the organelles need to be rapidly isolated from human tissue.

Unusual Labmates: Meet tardigrades, the crafters of nature’s ultimate survival kit

Whitehead Institute Member Siniša Hrvatin is studying tardigrades to decode the mechanisms enabling their survival in extreme environmental conditions. Learn about the biology of these microscopic “water bears” and what makes them a particularly fascinating model organism.

Shafaq Zia | Whitehead Institute
July 23, 2024

Tardigrades, also affectionately known as “water bears” or “moss piglets”, are remarkable microscopic organisms that have captured the imagination of scientists and nature enthusiasts alike.

With adults measuring anywhere from 0.2 to 1.2 millimeters in length — as big as a grain of salt — tardigrades possess the astounding ability to survive harsh environmental conditions. These resilient creatures have been found in habitats ranging from the depths of oceans and hot radioactive springs to the frigid expanses of Antarctica. It is their unparalleled adaptability that makes them invaluable as a model organism for researchers like Whitehead Institute Member Siniša Hrvatin, who’s studying physiological adaptation in animals with a focus on states that can slow down tissue damage, disease progression, and even aging.

Follow along to learn what’s behind tardigrades’ nearly indestructible nature, how researchers at Whitehead Institute — and beyond — are studying them, and what insights this work can offer into long-term organ preservation, space exploration, and more.

Big discovery of a tiny creature

In 1773, German naturalist Johann August Ephraim Goeze was analyzing moss samples under a microscope when he stumbled upon an unusual creature. Captivated by its peculiar appearance, he continued his observations and documented the discovery of Kleiner Wasserbär, translating to “little water bear”, in his publication. This work also featured the first-ever drawing of a tardigrade.

Since then, researchers’ understanding of this remarkable organism has evolved alongside advancements in imaging technology. Today, tardigrades are recognized as bilaterally symmetrical invertebrates with two eyes and eight chubby legs adorned with hook-like claws. Often described as a mix between nematodes and insects, these extremophiles are able to withstand freezing, intense radiation, vacuum of outer space, desiccation, chemical treatments, and possibly more.

And the best part? Despite their otherworldly appearance and surprising capabilities, tardigrades share plenty of similarities with larger, more complex organisms, including possessing a primordial brain, muscles, and even a digestive system.

The biology of an extremophile

Researchers trace the evolutionary origins of tardigrades back to panarthropods, a group that includes now-extinct worm-like organisms called lobopodians. To date, over a thousand species of tardigrades have been identified, with terrestrial species inhabiting environments like moss, leaf litter, and lichen, grassland, and deserts while aquatic ones are found in both fresh and saltwater.

Little is known about tardigrades’ diet but researchers are particularly drawn to herbivorous ones that like to munch on single-celled algae and thrive in water. There’s good reason for it: algae are inexpensive to grow in the lab with just light and basic nutrients. But it’s not just their diet that makes tardigrades an attractive model organism — they also have a short generation time (11 to 14 days), with eggs hatching within a four-day span. In fact, some species are able to reproduce without sexual reproduction through a process called parthenogenesis, during which the female egg undergoes cell division without fertilization by a male gamete.

Although genomic resources for studying tardigrades are limited to only a few species, researchers from Keio University and University of Edinburgh have successfully sequenced the genome of a moss-residing tardigrade commonly used in research called Hypsibius exemplaris. Its genome is less than half the size of a Drosophila melanogaster genome, consisting of 105 million base pairs that serve as the building blocks of DNA.

In spite of their small genome — and only a few thousand cells in the body — tardigrades have a well-defined miniaturized body plan, consisting of a head and four segments, that holds valuable insights for researchers looking to decode their adaptation prowess.

Inside tardigrade research at Whitehead Institute

In 2022, as Hrvatin was setting up his lab at Whitehead Institute, a question lingered in his mind. “I was trying to find animals that can survive being frozen for long periods of time and then continue living,” he says. “But there are not that many that fit the bill.”

Then, an undergraduate student at Massachusetts Institute of Technology (MIT) expressed her enthusiasm for astrobiology — the study of life across the universe — and highlighted tardigrades as a favorite among space researchers. Hrvatin was intrigued.

Up until this point, his research had centered upon two states of dormancy, or reduced metabolic activity, in animals: hibernation and a shorter, less intense torpor. But tardigrades possessed a survival mechanism unlike any other. When faced with harsh conditions like dehydration, they would expel water, retract their head and legs, and curl up in a small, dry ball, entering a state of suspended animation called crytobiosis or tun formation.

For decades, researchers hypothesized that the tun state might be responsible for tardigrades’ unparalleled ability to withstand a myriad of environmental assaults, including extremely low temperature. However, recent work has revealed that these animals utilize a separate and unique adaptation, distinct from the tun state, to survive being frozen for extended periods. In fact, preliminary evidence from a preprint by a team of scientists at UC Berkeley and UC San Francisco illustrates unique patterns of how tardigrades survive freezing while hydrated in water.

This phenomenon is markedly different from hibernation and its cousin torpor. “Unlike animals lowering their body temperature, we’re talking about putting tardigrades at minus 180 degrees Celsius, and then thawing them,” says Hrvatin. In fact, cryobiosis is so intense that tardigrades’ metabolic activity drops to undetectable levels, rendering them virtually, but not quite, dead. The organisms can then remain in this state from months to years, only to revive as healthy when conditions become favorable once again.

Frozen in time

In 2014, a group of Japanese researchers at Tokyo’s National Institute for Polar Research undertook an intriguing experiment. They began by thawing moss samples collected from East Antarctica in November 1983. Then, they carefully teased apart each sample using tweezers to retrieve tardigrades that might be nestled within. Among the tardigrades the researchers found, two stood out: Sleeping Beauty 1 and Sleeping Beauty 2 who were believed to be undergoing cold induced-dormancy. Turns out, the researchers were right — within the first day of being placed in the Petri dish with water, the tardigrades began exhibiting slow movements despite having been frozen for over 30 years.

The Swiss army knife in tardigrades’ toolbox

Yet, the remarkable resilience of tardigrades continues to baffle scientists. Recently, they’ve uncovered what could be another potential weapon in the creatures’ arsenal: intrinsically disordered proteins or IDPs. Picture them as putty — a group of proteins that do not have a well-defined three-dimensional structure and can interact with other molecules to produce a range of different outcomes. Some researchers have linked these tardigrade-specific IDPs to the animals extraordinary resilience: under extreme heat, these proteins remain stable. And when desiccated, they form protective glasses that shield cells and vital enzymes from dehydration.

If confirmed, the implications of this work would extend beyond tardigrades’ survival, potentially revolutionizing dry vaccine storage and the development of drought-resistant crops.

With programmable pixels, novel sensor improves imaging of neural activity

New camera chip design allows for optimizing each pixel’s timing to maximize signal to noise ratio when tracking real-time visual indicator of neural voltage, described in a new paper from a team in the Wilson Lab published in Nature Communications.

David Orenstein | The Picower Institute for Learning and Memory
June 7, 2024
Microscope system sharpens scientists’ view of neural circuit connections

To study plasticity in the brain, neuroscientists seek to track it at high resolution across whole cells, which is challenging in part because brain tissue is notorious for scattering light and making images fuzzy. A newly described technology described in a paper in Scientific Reports improves the clarity and speed of using two-photon microscopy to image synapses in the live brain. The paper was co-authored by Elly Nedivi, the William R. (1964) and Linda R. Young Professor of Neuroscience in the Picower Institute for Learning and Memory and the Department of Biology.

David Orenstein | The Picower Institute for Learning and Memory
June 4, 2024
New findings activate a better understanding of Rett syndrome’s causes

Rett syndrome is caused by mutations to the gene MECP2, which is highly expressed in the brain and appears to play important roles in maintaining healthy neurons. Researchers led by Rudolf Jaenisch have used cutting-edge techniques to create an epigenome map of MECP2, which may help guide future research on the disease.

Greta Friar | Whitehead Institute
April 25, 2024
Individual neurons mix multiple RNA edits of key synapse protein, study finds

Neurons stochastically generated up to eight different versions of a protein-regulating neurotransmitter release, which could vary how they communicate with other cells.

David Orenstein | The Picower Institute for Learning and Memory
September 25, 2023

Neurons are talkers. They each communicate with fellow neurons, muscles, or other cells by releasing neurotransmitter chemicals at “synapse” junctions, ultimately producing functions ranging from emotions to motions. But even neurons of the exact same type can vary in their conversational style. A new open-access study in Cell Reports by neurobiologists at The Picower Institute for Learning and Memory highlights a molecular mechanism that might help account for the nuanced diversity of neural discourse.

The scientists made their findings in neurons that control muscles in Drosophila fruit flies. These cells are models in neuroscience because they exhibit many fundamental properties common to neurons in people and other animals, including communication via the release of the neurotransmitter glutamate. In the lab of Troy Littleton, Menicon Professor in MIT’s departments of Biology and Brain and Cognitive Sciences, which studies how neurons regulate this critical process, researchers frequently see that individual neurons vary in their release patterns. Some “talk” more than others.

In more than a decade of studies, Littleton’s lab has shown that a protein called complexin has the job of restraining spontaneous glutamate chatter. It clamps down on fusion of glutamate-filled vesicles at the synaptic membrane to preserve a supply of the neurotransmitter for when the neuron needs it for a functional reason, for instance to simulate a muscle to move. The lab’s studies have identified two different kinds of complexin in flies (mammals have four) and showed that the clamping effectiveness of the rare but potent 7B splice form is regulated by a molecular process called phosphorylation. How the much more abundant 7A version is regulated was not known, but scientists had shown that the RNA transcribed from DNA that instructs the formation of the protein is sometimes edited in the cell by an enzyme called ADAR.

In the new study from Littleton’s team, led by Elizabeth Brija PhD ’23, the lab investigated whether RNA editing of complexin 7A affects how it regulates glutamate release. What she discovered was surprising. Not only does RNA editing of complexin 7A have a significant impact on how well the protein prevents glutamate release, but also this can vary widely among individual neurons because they can stochastically mix and match up to eight different editions of the protein. Some edits were much more common than others on average, but 96 percent of the 200 neurons the team examined had at least some editing, which affected the structure of an end of the protein called its C-terminus. Experiments to test some of the consequences of this structural variation showed that different complexin 7A edits can dramatically affect the level of electrical current measurable at different synapses. That varying level of activity can also affect the growth of the synapses the neurons make with muscle. RNA editing of the protein might therefore endow each neuron with fine degrees of communication control.

“What this offers the nervous system is that you can take the same transcriptome and by alternatively editing various RNA transcripts, these neurons will behave differently,” Littleton says.

Moreover, Littleton and Brija’s team found that other key proteins involved in synaptic glutamate release, such as synapsin and Syx1A, are also sometimes edited at quite different levels among the same population of neurons. This suggests that other aspects of synaptic communication might also be tunable.

“Such a mechanism would be a robust way to change multiple features of neuronal output,” Brija, Littleton, and colleagues wrote.

The team tracked the different editing levels by meticulously extracting and sequencing RNA from the nuclei and cell bodies of 200 motor neurons. The work yielded a rich enough dataset to show that any of three adenosine nucleotides encoding two amino acids in the C-terminus could be swapped for another, yielding eight different editions of the protein. A slim majority of complexin 7A went unedited in the average neuron, while the seven edited versions composed the rest with widely varying degrees of frequency.

To investigate the functional consequences of some of the different editions, the team knocked out complexin and then “rescued” flies by adding back in unedited or two different edited versions. The experiments showed a stark contrast between the two edited proteins. One, which occurs more commonly, proved to be a less effective clamp than unedited complexin, barely preventing spontaneous glutamate release and upticks in electrical current. The other turned out to be more effective at clamping than the unedited version, keeping a tight lid on glutamate release and synaptic output. And while both of the edited versions showed a tendency to drift away from synapses and into the neuron’s axon, the long branch that extends from the cell body, the edition that clamped well prevented any overgrowth of synapses while the one that clamped poorly provided only a meager curb.

Because multiple editions are often present in neurons, Brija and the team did one more set of experiments in which they “rescued” complexin-less flies with a combination of unedited complexin and the weak-clamping edition. The result was a blend of the two: reduced spontaneous glutamate release than with just the weakly clamping edition alone. The findings suggest that not only does each edition potentially fine-tune glutamate release, but that combinations among them can act in a combinatorial fashion.

In addition to Brija and Littleton the paper’s other authors are Zhuo Guan and Suresh Jetti.

The National Institutes of Health, The JPB Foundation, and The Picower Institute for Learning and Memory supported the research.

Study connects neural gene expression differences to functional distinctions

Researchers compared a pair of superficially similar motor neurons in fruit flies to examine how their differing use of the same genome produced distinctions in form and function

David Orenstein | The Picower Institute for Learning and Memory
August 23, 2023
Brady Weissbourd named Klingenstein-Simons Fellow

Three-year fellowship will support Weissbourd’s research on how the C. hemisphaerica jellyfish survives and thrives by constantly making new neurons.

David Orenstein | The Picower Institute for Learning and Memory
July 20, 2023

The Clytia hemisphaerica jellyfish is not only a hypnotically graceful swimmer, but also an amazing neuron-manufacturing machine with a remarkable ability to expand and regenerate its nervous system.

Now, thanks to a prestigious Klingenstein-Simons Fellowship Award in Neuroscience, MIT Assistant Professor Brady Weissbourd will study how the tiny, transparent animals use this ability to build, organize, and rebuild a stable, functional, and robust nervous system throughout their lives.

“As we look more broadly across the animal kingdom it is amazing to see how similar the basic biology is of animals that look completely different — even jellyfish have neurons similar to our own that generate their behavior,” says Weissbourd, a faculty member in MIT’s Department of Biology and The Picower Institute for Learning and Memory, whose work to engineer genetic access to C. hemisphaerica in 2021 established it as a new neuroscience model organism. “At the same time, it could be just as important to examine what is different across species, particularly when it comes to some of the incredible capabilities that have evolved.”

Weissbourd is just one of 13 researchers nationally to be recognized with this fellowship, which provides $300,000 over three years. It will enable Weissbourd’s lab to tackle several questions raised by the jellyfish’s prodigious production of neurons. Where does the constant stream of newborn neurons come from, and what guides them to their eventual places in the jellyfish’s mesh-like neural network? How does the jellyfish organize these ever-changing neural populations — for instance, into functional circuits — to enable its various behaviors?

Another question hails from the surprising results of an experiment in which Weissbourd ablated the entire class of the neurons that the jellyfish uses to fold up its umbrella-shaped body — about 10 percent of the 10,000 or so neurons that it has. He found that within a week enough new neurons had taken their place that the folding behavior was restored. Weissbourd’s studies will also seek to determine how the animal can so readily bounce back from the destruction of a whole major neural network and the behavior it produces.

“We were studying the neural control of a particular behavior and stumbled across this shocking observation that the subnetwork that controls this behavior is constantly changing size and can completely regenerate,” Weissbourd says. “We want to understand the mechanisms that allow this network to be so robust, including the ability to rebuild itself from scratch. I’m very grateful to the Klingenstein Fund and the Simons Foundation for supporting our work.”