Research Area: Neurobiology

With these neurons, extinguishing fear is its own reward
Picower Institute
January 16, 2020

When you expect a really bad experience to happen and then it doesn’t, it’s a distinctly positive feeling. A new study of fear extinction training in mice may suggest why: The findings not only identify the exact population of brain cells that are key for learning not to feel afraid anymore, but also show these neurons are the same ones that help encode feelings of reward.

The study, published Jan. 14 in Neuron by scientists at MIT’s Picower Institute for Learning and Memory, specifically shows that fear extinction memories and feelings of reward alike are stored by neurons that express the gene Ppp1r1b in the posterior of the basolateral amygdala (pBLA), a region known to assign associations of aversive or rewarding feelings, or “valence,” with memories. The study was conducted by Xiangyu Zhang, a graduate student, Joshua Kim, a former graduate student, and Susumu Tonegawa, Professor of Biology and Neuroscience at RIKEN-MIT Laboratory of Neural Circuit Genetics at the Picower Institute for Learning and Memory at MIT and Howard Hughes Medical Institute.

“We constantly live at the balance of positive and negative emotion,” Tonegawa said. “We need to have very strong memories of dangerous circumstances in order to avoid similar circumstances to recur. But if we are constantly feeling threatened we can become depressed. You need a way to bring your emotional state back to something more positive.”

Overriding fear with reward

In a prior study, Kim showed that Ppp1r1b-expressing neurons encode rewarding valence and compete with distinct Rspo2-expressing neurons in the BLA that encode negative valence. In the new study, Zhang, Kim and Tonegawa set out to determine whether this competitive balance also underlies fear and its extinction.

In fear extinction, an original fearful memory is thought to be essentially overwritten by a new memory that is not fearful. In the study, for instance, mice were exposed to little shocks in a chamber, making them freeze due to the formation of fearful memory. But the next day, when the mice were returned to the same chamber for a longer period of time without any further little shocks, freezing gradually dissipated and hence this treatment is called fear extinction training. The fundamental question then is whether the fearful memory is lost or just suppressed by the formation of a new memory during the fear extinction training.

While the mice underwent fear extinction training the scientists watched the activity of the different neural populations in the BLA. They saw that Ppp1r1b cells were more active and Rspo2 cells were less active in mice that experienced fear extinction. They also saw that while Rspo2 cells were mostly activated by the shocks and were inhibited during fear extinction, Ppp1r1b cells were mostly active during extinction memory training and retrieval, but were inhibited during the shocks.

These and other experiments suggested to the authors that the hypothetical fear extinction memory may be formed in the Ppp1r1b neuronal population and the team went on to demonstrate this vigorously. For this, they employed the technique previously pioneered in their lab for the identification and manipulation of the neuronal population that holds specific memory information, memory “engram” cells.  Zhang labeled Ppp1r1b neurons that were activated during retrieval of fear extinction memory with the light-sensitive protein channelrhodopsin. When these neurons were activated by blue laser light during a second round of fear extinction training it enhanced and accelerated the extinction. Moreover, when the engram cells were inhibited by another optogenetic technique, fear extinction was impaired because the Ppp1r1b engram neurons could no longer suppress the Rspo2 fear neurons. That allowed the fear memory to regain primacy.

These data met the fundamental criteria for the existence of engram cells for fear extinction memory within the pBLA Ppp1r1b cell population: activation and reactivation by recall and enduring and off-line maintenance of the acquired extinction memory.

Because Kim had previously shown Ppp1r1b neurons are activated by rewards and drive appetitive behavior and memory, the team sequentially tracked Ppp1r1b cell activity in mice that eagerly received water reward followed by food reward followed by fear extinction training and fear extinction memory retrieval. The overlap of Ppp1r1b neurons activated by fear extinction vs. water reward was as high as the overlap of neurons activated by water vs. food reward. And finally, artificial optogenetic activation of Ppp1r1b extinction memory engram cells was as effective as optogenetic activation of Ppp1r1b water reward-activated neurons in driving appetitive behaviors. Reciprocally, artificial optogenetic activation of water-responding Ppp1r1b neurons enhanced fear extinction training as efficiently as optogenetic activation of fear extinction memory engram cells. These results demonstrate that fear extinction is equivalent to bona fide rewards and therefore provide the neuroscientific basis for the widely held experience in daily life: omission of expected punishment is a reward.

What next?

By establishing this intimate connection between fear extinction and reward and by identifying a genetically defined neuronal population (Ppp1r1b) that plays a crucial role in fear extinction this study provides potential therapeutic targets for treating fear disorders like PTSD and anxiety, Zhang said.

From the basic scientific point of view, Tonegawa said, how fear extinction training specifically activates Ppp1r1b neurons would be an important question to address. More imaginatively, results showing how Ppp1r1b neurons override Rspo2 neurons in fear extinction raises an intriguing question about whether a reciprocal dynamic might also occur in the brain and behavior. Investigating “joy extinction” via these mechanisms might be an interesting research topic.

The research was supported by the RIKEN Brain Science Institute, the Howard Hughes Medical Institute and the JPB Foundation funded the research.

Whitehead Institute team develops new method to study human brain cells
Nicole Davis | Whitehead
November 25, 2019

A groundswell of evidence connects defects in the function of microglia, the brain’s resident immune cells, to neurodegenerative diseases, yet the tools for studying these cells in the laboratory have been limited. Now, a team of Whitehead Institute scientists has developed a new experimental platform for generating microglia from human stem cells that includes transplantation into newborn mice. As described online November 26 in the Proceedings of the National Academy of Sciences (PNAS), this new method yields microglial cells that resemble those in the human brain more closely than previous approaches, which could help enable future studies aimed at unravelling the role of microglia in neurodegeneration and other brain disorders.

“The dysfunction of microglia is implicated in a wide variety of brain conditions, and yet our knowledge of them, especially in humans, is really quite limited,” says senior author Rudolph Jaenisch, a Founding Member of the Whitehead Institute and professor of biology at the Massachusetts Institute of Technology. “This new approach will help us lift the hood on these important yet enigmatic brain cells.”

Microglia are increasingly recognized as key players in brain health and disease, but the majority of what is known about them comes from studies of mice, not humans. Yet human and mouse microglia are quite distinct — in humans, the cells are much larger, and have a more branched appearance, suggesting significant differences in their biology.

To address this gap in knowledge, multiple research teams have recently devised methods to generate microglia using human stem cells and grow them under laboratory conditions that mimic their natural environment. However, this approach has a fundamental drawback: the cultured cells do not look like microglia nor do they behave much like them, even though they display the appropriate molecular hallmarks.

“That really suggests to us that this is not the optimal approach to study how microglia are behaving in healthy and diseased brains,” says first author Devon Svoboda, a postdoctoral fellow in the Jaenisch lab. “We set out to create a new method in which the stem-cell derived microglial cells can reside in the brains of mice — one of the best models of the human brain that we have.”

Transplanting human cells into mice — creating “chimeras” — is a well-established technique. However, Svoboda and her colleagues discovered they needed to use special strains of mice that carry human genes for certain growth factors, called cytokines, which are required for microglial development and survival. The researchers utilized mice that carry human genes for four crucial cytokines: CSF1, IL3, SCF, and GM-CSF.

“What is special about these chimeras is really the mice we are using,” says Svoboda. “They express the human alleles of these cytokines which is key because the mouse versions are not able to communicate with receptors on human microglia, so the cells die.”

After transplanting the stem-cell derived microglia into these mice, the research team examined the cells’ morphology and their molecular characteristics. They found that the transplanted cells closely resembled those found in the human brain.

Further analyses revealed some striking differences between the team’s “chimera-grown” microglia and those grown in the laboratory using conventional cell culture methods. Surprisingly, Svoboda and her colleagues found that the cultured microglia showed strong similarities to the diseased microglia from patients with multiple sclerosis, another brain condition in which the cells are implicated.

“If you want to learn more about the role of microglia in disease, then studying them in culture is probably not the best way,” says Svoboda. “The chimeras and the in vitro methods really complement each other, and we think there is a place for both systems in microglia research going forward.”

The Whitehead-led team plans to extend their initial studies in several ways. One is to identify which cytokines and other growth factors are most crucial to microglial development. That knowledge could help improve existing cell culture methods and enable them to more closely mirror the cells’ natural environment. Another key direction is to use the new chimera-based system to create models of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease, to understand how microglia respond to diseased neurons and, in turn, how diseased microglia can impair neuron function.

“Our chimera-based method will give us a good handle to begin to stringently test the role of microglia in brain health and disease,” says Jaenisch. “This is an important step forward for the field.”

Support for this work was provided by the Cure Alzheimer’s Foundation, MassCATS, and NIH Grants R01 AG058002-01, R01 MH104610, R37 CA084198, and U19 AI131135 (to R.J.). L.D.S. is supported by NIH Grants R24 OD26440, AI32963, and CA034196. J.S. is supported by the National Institute of Child Health and Human Development (K99HD096049).

Written by Nicole Davis

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Rudolf Jaenisch’s primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.

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Paper cited:

Human iPSC-derived microglia assumer a primary microglia-like state after transplantation into the neonatal mouse brain.

PNAS, online November 26, 2019. DOI: 10.1073/pnas.1913541116

Devon S. Svoboda (1), M. Inmaculada Barrasa (1), Jian Shu (1,3), Rosalie Rietjens (1), Shupei Zhang (1), Maya Mitalipova (1), Peter Berube (3), Dongdong Fu (1), Leonard D. Shultz (4), George W. Bell (1), and Rudolf Jaenisch (1,2)

 

1. Whitehead Institute, Cambridge, MA 02142, USA

2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

3. Broad Institute of MIT and Harvard, Cambridge, MA 02142

4. The Jackson Laboratory Cancer Center, The Jackson Laboratory, Bar Harbor, ME 04609

MicroRNAs work together to tune gene expression in the brain
Raleigh McElvery
November 4, 2019

A new study from the MIT Department of Biology suggests we may need to re-think how certain RNAs operate to impact development and disease.

According to the “central dogma” of biology, DNA is converted into messenger RNA (mRNA) before being expressed as a protein. However, not all RNAs are destined to become proteins. MicroRNAs (miRNAs) are small, non-coding RNAs, which regulate a variety of cellular processes by binding to mRNAs and destabilizing them to reduce their expression.

A single miRNA can target hundreds of different mRNAs. And yet, on its own, an individual miRNA only represses the expression of each mRNA target by about 10-20%. Given that the effects of a single miRNA are so mild, researchers couldn’t understand how they could exert such powerful control over so many processes. One theory is that, rather than acting alone, perhaps multiple miRNAs bind to the same target mRNA in concert to exert enhanced repression. However, few studies have explored this idea in-depth, or identified examples of such co-regulation.

In a new study published in Genome Research on October 24, MIT biologists were able to pinpoint specific miRNAs that collaborate with one another to repress mRNA expression in the brain — adding credence to the notion that miRNAs often collaborate with one another.

“The idea that miRNAs may work by co-targeting sets of transcripts together has been around for a while,” says Jennifer Cherone, the study’s lead author. “But it’s only recently that certain key advances — like better annotations of where transcripts end and more accurate predictions of miRNA target sites — have allowed us to uncover these relationships and rigorously test them in the lab.”

Using powerful computational analyses to compare target sets of different miRNAs, Cherone was able to identify hundreds of distinct miRNAs, which — despite their sequence differences — bound many of the same mRNAs. Of all the tissues she examined, the brain appeared to have the most co-targeting. So she narrowed her focus to explore the overlapping functions of just two miRNAs that worked together there: miR-138 and miR-137.

“That was a really interesting observation and a functional demonstration of the overlap between these two miRNAs,” she says. “One miRNA can rescue the loss of a completely different miRNA if they share targets.”If she deleted miR-138 from her cells, they could no longer differentiate and become neurons. However, when she added miR-138’s co-targeting partner, miR-137, the cells were once again able to differentiate.

Cherone went on to identify an entire group of miRNAs within the brain, nine in total, that also shared similar targets. She selected several genes targeted by three or more of these miRNAs, and mutated every possible combination of the miRNA sites to determine their individual contributions. She ultimately established that subsets of the miRNAs could repress gene expression between five- and tenfold if they were expressed at the same time and bound close together.

According to Cherone, “seeing a tenfold repression by miRNAs is unheard of.” Such strong repression can have serious phenotypic consequences. She attributes this finding to the lab’s advanced computational strategies, which allowed them to systematically and unbiasedly identify the miRNAs that work together and their gene targets.

Why might a single gene be regulated by so many different miRNAs? There are more evolutionary paths to acquire sites for many different miRNAs than paths to acquire sites for the same miRNA. And, the authors explain, this arrangement may allow more precise control of cell type-specific expression.

Given that their miRNAs of interest primarily worked in the brain, the researchers wondered why this tissue might require so much co-targeting. One idea is that mRNAs in the brain tend to have longer regions where more miRNAs can bind to exert their effects. Another possibility is that mRNA expression in the brain must be especially fine-tuned, because too much or too little expression could have severe ramifications for neuronal function and development. For instance, fragile X-associated tremor/ataxia syndrome (FXTAS) can result from fairly subtle changes in proteins levels.

“Co-targeting appears to be widespread in many tissues, not just the brain,” says senior author Christopher Burge, a professor of biology at MIT. “This means that strategies to modulate the activity of a miRNA in a genetic or therapeutic context will be most effective when they take into account the levels of the other miRNAs that frequently partner with the miRNA of interest.”

“It’s time to start thinking of miRNAs as working together in networks, rather than functioning as individual units,” Cherone says. “If you want to know the function of a given miRNA, you have to understand the group it’s collaborating with, and explore its function within that group.”

Top image: Graphical illustration of co-targeting by miRNAs. Credit: Jennifer Cherone.

Citation:
“Cotargeting among microRNAs in the brain.”
Genome Research, online October 24, 2019, DOI: 10.1101/gr.249201.119
Jennifer M. Cherone, Vjola Jorgji, and Christopher B. Burge.

Study finds hub linking movement and motivation in the brain

Detailed observations in the lateral septum indicate region processes movement and reward information to help direct behavior.

David Orenstein | Picower Institute for Learning and Memory
September 19, 2019

Our everyday lives rely on planned movement through the environment to achieve goals. A new study by MIT neuroscientists at the Picower Institute for Learning and Memory at MIT identifies a well-connected brain region as a crucial link between circuits guiding goal-directed movement and motivated behavior.

Published Sept. 19 in Current Biology, the research shows that the lateral septum (LS), a region considered integral to modulating behavior and implicated in many psychiatric disorders, directly encodes information about the speed and acceleration of an animal as it navigates and learns how to obtain a reward in an environment.

“Completing a simple task, such as acquiring food for dinner, requires the participation and coordination of a large number of regions of the brain, and the weighing of a number of factors: for example, how much effort is it to get food from the fridge versus a restaurant,” says Hannah Wirtshafter PhD ’19, the study’s lead author. “We have discovered that the LS may be aiding you in making some of those decisions. That the LS represents place, movement, and motivational information may enable the LS to help you integrate or optimize performance across considerations of place, speed, and other environmental signals.”

Previous research has attributed important behavioral functions to the LS, such as modulating anxiety, aggression, and affect. It is also believed to be involved in addiction, psychosis, depression, and anxiety. Neuroscientists have traced its connections to the hippocampus, a crucial center for encoding spatial memories and associating them with context, and to the ventral tegmental area (VTA), a region that mediates goal-directed behaviors via the neurotransmitter dopamine. But until now, no one had shown that the LS directly tracks movement or communicated with the hippocampus, for instance by synchronizing to certain neural rhythms, about movement and the spatial context of reward.

“The hippocampus is one of the most studied regions of the brain due to its involvement in memory, spatial navigation, and a large number of illnesses such as Alzheimer’s disease,” says Wirtshafter, who recently earned her PhD working on the research as a graduate student in the lab of senior author Matthew Wilson, Sherman Fairchild Professor of Neurobiology. “Comparatively little is known about the lateral septum, even though it receives a large amount of information from the hippocampus and is connected to multiple areas involved in motivation and movement.”

Wilson says the study helps to illuminate the importance of the LS as a crossroads of movement and motivation information between regions such as the hippocampus and the VTA.

“The discovery that activity in the LS is controlled by movement points to a link between movement and dopaminergic control through the LS that that could be relevant to memory, cognition, and disease,” he says.

Tracking thoughts

Wirtshafter was able to directly observe the interactions between the LS and the hippocampus by simultaneously recording the electrical spiking activity of hundreds of neurons in each region in rats both as they sought a reward in a T-shaped maze, and as they became conditioned to associate light and sound cues with a reward in an open box environment.

In that data, she and Wilson observed a speed and acceleration spiking code in the dorsal area of the LS, and saw clear signs that an overlapping population of neurons were processing information based on signals from the hippocampus, including spiking activity locked to hippocampal brain rhythms, location-dependent firing in the T-maze, and cue and reward responses during the conditioning task. Those observations suggested to the researchers that the septum may serve as a point of convergence of information about movement and spatial context.

Wirtshafter’s measurements also showed that coordination of LS spiking with the hippocampal theta rhythm is selectively enhanced during choice behavior that relies on spatial working memory, suggesting that the LS may be a key relay of information about choice outcome during navigation.

Putting movement in context

Overall, the findings suggest that movement-related signaling in the LS, combined with the input that it receives from the hippocampus, may allow the LS to contribute to an animal’s awareness of its own position in space, as well as its ability to evaluate task-relevant changes in context arising from the animal’s movement, such as when it has reached a choice point, Wilson and Wirtshafter said.

This also suggests that the reported ability of the LS to modulate affect and behavior may result from its ability to evaluate how internal states change during movement, and the consequences and outcomes of these changes. For instance, the LS may contribute to directing movement toward or away from the location of a positive or negative stimulus.

The new study therefore offers new perspectives on the role of the lateral septum in directed behavior, the researchers added, and given the known associations of the LS with some disorders, it may also offer new implications for broader understanding of the mechanisms relating mood, motivation, and movement, and the neuropsychiatric basis of mental illnesses.

“Understanding how the LS functions in movement and motivation will aid us in understanding how the brain makes basic decisions, and how disruption in these processed might lead to different disorders,” Wirtshafter says.

A National Defense Science and Engineering Graduate Fellowship and the JPB Foundation funded the research.

BRAIN grant will fund new tools to study astrocytes
Picower Institute
August 27, 2019

In the brain, cells called astrocytes are at least as abundant as neurons, but are much less understood. While neuroscientists have begun to appreciate how closely astrocytes support the development and function of the neural circuit connections, or synapses, we depend on for all mental function – they haven’t been able to investigate many of the cells’ specific contributions because they have lacked the tools to manipulate them. With a new $1.5 million, three year grant from the U.S. government’s BRAIN Initiative, MIT neuroscientist Mriganka Sur will lead a three-lab partnership to develop new tools to study astrocytes.

“A great many genes of brain disorders involve astrocytes,” said Sur, Newton Professor of Neuroscience in The Picower Institute for Learning and Memory at MIT. “If you remove astrocytes from a culture in a dish, neurons will not make synapses. So it’s of great importance to be able to study and manipulate astrocytes in order to understand their role in the brain in modulating neurons.”

Sur, together with postdoc Grayson Oren Sipe and their collaborators, propose to create three tools both for their lab’s research and to share with the broader neuroscience community. Each tool is meant to give scientists the ability to experimentally manipulate key properties of astrocytes in living animals wherever in the brain they want (spatial control) and with the timing of their choosing (temporal control).

The first tool, to be developed in collaboration with Professor Rudolf Jaenisch of the Whitehead Institute and MIT Department of Biology, will give scientists a new way to manipulate astrocytes genetically. Sipe and Jaenisch lab postdoc Xin Tang will use the CRISPR/Cas9 gene editing technique to knock out with spatial and temporal precision two genes that allow astrocytes to receive the neuromodulator noradrenaline. Noradrenaline is crucial for stimulating arousal circuits in the brain, so the new manipulation will allow scientists to see what happens to neurons when astrocytes are selectively taken out of the arousal process.

Sipe notes that while the Sur and Jaenisch labs will target genes related to arousal, other neuroscientists could use the technique to target genes expressed in astrocytes that are related to other functions.

To create two other tool, Sur’s lab will work with both Jaenisch’s lab and the lab of Ed Boyden, Y. Eva Tan Professor of Neurotechnology. Boyden is affiliated with the MIT Media Lab, the Departments of Bioengineering and Brain and Cognitive Sciences, and the McGovern Institute for Brain Research. The tools will adapt for astrocytes a technology Boyden co-invented called optogenetics. Optogenetics changes cells to become responsive to flashes of visible light. Widely applied in neurons, optogenetics alters genes that regulate whether neurons electrically spike, giving scientists control over the cells’ electrical activity. In astrocytes, the team will target different genes related to calcium biochemistry.

One tool will give them temporal control over receptors that the cells use to regulate calcium levels around neurons, for instance to intentionally increase intercellular calcium levels. The other tool will allow scientists to experimentally disrupt the astrocytes’ uptake of the neurotransmitter glutamate by creating an ion channel called ChromeQ, that alters sodium ion currents within astrocytes. Manipulating ChromeQ to alter those currents will allow them to reduce glutamate uptake, which in turn will reduce intercellular calcium levels. By doing this specifically in the motor cortex, Sur’s team will be able to investigate the influence astrocytes have on neurons at the synaptic level in the process of motor learning in lab mice.

The grant started Aug. 15 and will run through July 2022.

How brain cells pick which connections to keep

Novel study shows protein CPG15 acts as a molecular proxy of experience to mark synapses for stabilization.

David Orenstein | Picower Institute for Learning and Memory
August 7, 2019

Brain cells, or neurons, constantly tinker with their circuit connections, a crucial feature that allows the brain to store and process information. While neurons frequently test out new potential partners through transient contacts, only a fraction of fledging junctions, called synapses, are selected to become permanent.

The major criterion for excitatory synapse selection is based on how well they engage in response to experience-driven neural activity, but how such selection is implemented at the molecular level has been unclear. In a new study, MIT neuroscientists have identified the gene and protein, CPG15, that allows experience to tap a synapse as a keeper.

In a series of novel experiments described in Cell Reports, the team at MIT’s Picower Institute for Learning and Memory used multi-spectral, high-resolution two-photon microscopy to literally watch potential synapses come and go in the visual cortex of mice — both in the light, or normal visual experience, and in the darkness, where there is no visual input. By comparing observations made in normal mice and ones engineered to lack CPG15, they were able to show that the protein is required in order for visual experience to facilitate the transition of nascent excitatory synapses to permanence.

Mice engineered to lack CPG15 only exhibit one behavioral deficiency: They learn much more slowly than normal mice, says senior author Elly Nedivi, the William R. (1964) and Linda R. Young Professor of Neuroscience in the Picower Institute and a professor of brain and cognitive sciences at MIT. They need more trials and repetitions to learn associations that other mice can learn quickly. The new study suggests that’s because without CPG15, they must rely on circuits where synapses simply happened to take hold, rather than on a circuit architecture that has been refined by experience for optimal efficiency.

“Learning and memory are really specific manifestations of our brain’s ability in general to constantly adapt and change in response to our environment,” Nedivi says. “It’s not that the circuits aren’t there in mice lacking CPG15, they just don’t have that feature — which is really important — of being optimized through use.”

Watching in light and darkness

The first experiment reported in the paper, led by former MIT postdoc Jaichandar Subramanian, who is now an assistant professor at the University of Kansas, is a contribution to neuroscience in and of itself, Nedivi says. The novel labeling and imaging technologies implemented in the study, she says, allowed tracking key events in synapse formation with unprecedented spatial and temporal resolution. The study resolved the emergence of “dendritic spines,” which are the structural protrusions on which excitatory synapses are formed, and the recruitment of the synaptic scaffold, PSD95, that signals that a synapse is there to stay.

The team tracked specially labeled neurons in the visual cortex of mice after normal visual experience, and after two weeks in darkness. To their surprise, they saw that spines would routinely arise and then typically disappear again at the same rate regardless of whether the mice were in light or darkness. This careful scrutiny of spines confirmed that experience doesn’t matter for spine formation, Nedivi said. That upends a common assumption in the field, which held that experience was necessary for spines to even emerge.

By keeping track of the presence of PSD95 they could confirm that the synapses that became stabilized during normal visual experience were the ones that had accumulated that protein. But the question remained: How does experience drive PSD95 to the synapse? The team hypothesized that CPG15, which is activity dependent and associated with synapse stabilization, does that job.

CPG15 represents experience

To investigate that, they repeated the same light-versus-dark experiences, but this time in mice engineered to lack CPG15. In the normal mice, there was much more PSD95 recruitment during the light phase than during the dark, but in the mice without CPG15, the experience of seeing in the light never made a difference. It was as if CPG15-less mice in the light were like normal mice in the dark.

Later they tried another experiment testing whether the low PSD95 recruitment seen when normal mice were in the dark could be rescued by exogenous expression of CPG15. Indeed, PSD95 recruitment shot up, as if the animals were exposed to visual experience. This showed that CPG15 not only carries the message of experience in the light, it can actually substitute for it in the dark, essentially “tricking” PSD95 into acting as if experience had called upon it.

“This is a very exciting result, because it shows that CPG15 is not just required for experience-dependent synapse selection, but it’s also sufficient,” says Nedivi, “That’s unique in relation to all other molecules that are involved in synaptic plasticity.”

A new model and method

In all, the paper’s data allowed Nedivi to propose a new model of experience-dependent synapse stabilization: Regardless of neural activity or experience, spines emerge with fledgling excitatory synapses and the receptors needed for further development. If activity and experience send CPG15 their way, that draws in PSD95 and the synapse stabilizes. If experience doesn’t involve the synapse, it gets no CPG15, very likely no PSD95, and the spine withers away.

The paper potentially has significance beyond the findings about experience-dependent synapse stabilization, Nedivi says. The method it describes of closely monitoring the growth or withering of spines and synapses amid a manipulation (like knocking out or modifying a gene) allows for a whole raft of studies in which examining how a gene, or a drug, or other factors affect synapses.

“You can apply this to any disease model and use this very sensitive tool for seeing what might be wrong at the synapse,” she says.

In addition to Nedivi and Subramanian, the paper’s other authors are Katrin Michel and Marc Benoit.

The National Institutes of Health and the JPB Foundation provided support for the research.

Speeding up drug discovery for brain diseases

Whitehead Institute team finds drugs that activate a key brain gene; initial tests in cells and mice show promise for rare, untreatable neurodevelopmental disorder.

Nicole Davis
August 2, 2019

A research team led by Whitehead Institute scientists has identified 30 distinct chemical compounds — 20 of which are drugs undergoing clinical trial or have already been approved by the FDA — that boost the protein production activity of a critical gene in the brain and improve symptoms of Rett syndrome, a rare neurodevelopmental condition that often provokes autism-like behaviors in patients. The new study, conducted in human cells and mice, helps illuminate the biology of an important gene, called KCC2, which is implicated in a variety of brain diseases, including autism, epilepsy, schizophrenia, and depression. The researchers’ findings, published in the July 31 online issue of Science Translational Medicine, could help spur the development of new treatments for a host of devastating brain disorders.

“There’s increasing evidence that KCC2 plays important roles in several different disorders of the brain, suggesting that it may act as a common driver of neurological dysfunction,” says senior author Rudolf Jaenisch, a founding member of Whitehead Institute and professor of biology at MIT. “These drugs we’ve identified may help speed up the development of much-needed treatments.”

KCC2 works exclusively in the brain and spinal cord, carrying ions in and out of specialized cells known as neurons. This shuttling of electrically charged molecules helps maintain the cells’ electrochemical makeup, enabling neurons to fire when they need to and to remain idle when they don’t. If this delicate balance is upset, brain function and development go awry.

Disruptions in KCC2 function have been linked to several human brain disorders, including Rett syndrome (RTT), a progressive and often debilitating disorder that typically emerges early in life in girls and can involve disordered movement, seizures, and communication difficulties. Currently, there is no effective treatment for RTT.

Jaenisch and his colleagues, led by first author Xin Tang, devised a high-throughput screen assay to uncover drugs that increase KCC2 gene activity. Using CRISPR/Cas9 genome editing and stem cell technologies, they engineered human neurons to provide rapid readouts of the amount of KCC2 protein produced. The researchers created these so-called reporter cells from both healthy human neurons as well as RTT neurons that carry disease-causing mutations in the MECP2 gene. These reporter neurons were then fed into a drug-screening pipeline to find chemical compounds that can enhance KCC2 gene activity.

Tang and his colleagues screened over 900 chemical compounds, focusing on those that have been FDA-approved for use in other conditions, such as cancer, or have undergone at least some level of clinical testing. “The beauty of this approach is that many of these drugs have been studied in the context of non-brain diseases, so the mechanisms of action are known,” says Tang. “Such molecular insights enable us to learn how the KCC2 gene is regulated in neurons, while also identifying compounds with potential therapeutic value.”

The Whitehead Institute team identified a total of 30 drugs with KCC2-enhancing activity. These compounds, referred to as KEECs (short for KCC2 expression-enhancing compounds), work in a variety of ways. Some block a molecular pathway, called FLT3, which is found to be overactive in some forms of leukemia. Others inhibit the GSK3b pathway that has been implicated in several brain diseases. Another KEEC acts on SIRT1, which plays a key role in a variety of biological processes, including aging.

In followup experiments, the researchers exposed RTT neurons and mouse models to KEEC treatment and found that some compounds can reverse certain defects associated with the disease, including abnormalities in neuronal signaling, breathing, and movement. These efforts were made possible by a collaboration with Mriganka Sur’s group at the Picower Institute for Learning and Memory, in which Keji Li and colleagues led the behavioral experiments in mice that were essential for revealing the drugs’ potency.

“Our findings illustrate the power of an unbiased approach for discovering drugs that could significantly improve the treatment of neurological disease,” says Jaenisch. “And because we are starting with known drugs, the path to clinical translation is likely to be much shorter.”

In addition to speeding up drug development for Rett syndrome, the researchers’ unique drug-screening strategy, which harnesses an engineered gene-specific reporter to unearth promising drugs, can also be applied to other important disease-related genes in the brain. “Many seemingly distinct brain diseases share common root causes of abnormal gene expression or disrupted signaling pathways,” says Tang. “We believe our method has broad applicability and could help catalyze therapeutic discovery for a wide range of neurological conditions.”

Support for this work was provided by the National Institutes of Health, the Simons Foundation Autism Research Initiative, the Simons Center for the Social Brain at MIT, the Rett Syndrome Research Trust, the International Rett Syndrome Foundation, the Damon Runyon Cancer Foundation, and the National Cancer Institute.

Study reveals how glial cells may play key epilepsy role

Mutation in disease model flies undermines maintenance of key ion balance.

David Orenstein | Picower Institute
May 2, 2019

A new study provides potential new targets for treating epilepsy and new fundamental insights into the relationship between neurons and their glial “helper” cells. In eLife, scientists at MIT’s Picower Institute for Learning and Memory report finding a key sequence of molecular events in which the genetic mutation in a fruit fly model of epilepsy leaves neurons vulnerable to becoming hyperactivated by stress, leading to seizures.

About 60 million people worldwide have epilepsy, a neurological condition characterized by seizures resulting from excessive neural activity. The “zydeco” model flies in the study experience seizures in a similar fashion. Since discovering zydeco, the lab of MIT neurobiologist Troy Littleton, the Menicon Professor in Neuroscience, has been investigating why the flies’ zydeco mutation makes it a powerful model of epilepsy.

Heading into the study, the team led by postdoc Shirley Weiss knew that the zydeco mutation was specifically expressed by cortex glial cells and that the protein it makes helps to pump calcium ions out of the cells. But that didn’t explain much about why a glial cell’s difficulty maintaining a natural ebb and flow of calcium ions would lead adjacent neurons to become too active under seizure-inducing stresses, such as fever-grade temperatures or the fly being jostled around.

The activity of neurons rises and falls based on the flow of ions — for a neuron to “fire,” for instance, it takes in sodium ions, and then to calm back down it releases potassium ions. But the ability of neurons to do that depends on there being a conducive balance of ions outside the cell. For instance, too much potassium outside makes it harder to get rid of potassium and calm down.

The need for an ion balance — and the way it is upset by the zydeco mutation — turned out to be the key to the new study. In a four-year series of experiments, Weiss, Littleton, and their co-authors found that excess calcium in cortex glia cells causes them to hyper-activate a molecular pathway that leads them to withdraw many of the potassium channels that they typically deploy to remove potassium from around neurons. With too much potassium left around, neurons can’t calm down when they are excited, and seizures ensue.

“No one has really shown how calcium signaling in glia could directly communicate with this more classical role of glial cells in potassium buffering,” Littleton says. “So this is a really important discovery linking an observation that’s been found in glia for a long time — these calcium oscillations that no one really understood — to a real biological function in glial cells, where it’s contributing to their ability to regulate ionic balance around neurons.”

Weiss’s work lays out a detailed sequence of events, implicating several specific molecular players and processes. That richly built knowledge meant that along the way, she and the team found multiple steps in which they could intervene to prevent seizures.

She started working the problem from the calcium end. With too much calcium afoot, she asked, what genes might be in a related pathway such that, if their expression was prevented, seizures would not occur? She interfered with expression in 847 potentially related genes and found that about 50 affected seizures. Among those, one stood out both for being closely linked to calcium regulation and also for being expressed in the key cortex glia cells of interest: calcineurin. Inhibiting calcineurin activity, for instance with the immunosuppressant medications cyclosprorine A or FK506, blocked seizures in zydeco mutant flies.

Weiss then looked at the genes affected by the calcineurin pathway and found several. One day at a conference where she was presenting a poster of her work, an onlooker mentioned that glial potassium channels could be involved. Sure enough, she found a particular one called “sandman” that, when knocked down, led to seizures in the flies. Further research showed that hyperactivation of calcineurin in zydeco glia led to an increase in a cellular process called endocytosis, in which the cell was bringing too much sandman back into the cell body. Without sandman staying on the cell membrane, the glia couldn’t effectively remove potassium from the outside.

When Weiss and her co-authors interfered to suppress endocytosis in zydeco flies, they also were able to reduce seizures, because that allowed more sandman to persist where it could reduce potassium. Sandman, notably, is equivalent to a protein in mammals called TRESK.

“Pharmacologically targeting glial pathways might be a promising avenue for future drug development in the field,” the authors wrote in eLife.

In addition to that clinical lead, the study also offers some new insights for more fundamental neuroscience, Littleton and Weiss said. While zydeco flies are good models of epilepsy, Drosophila’s cortex glia do have a property not found in mammals: They contact only the cell body of neurons, not the synaptic connections on their axon and dendrite branches. That makes them an unusually useful test bed to learn how glia interact with neurons via their cell body versus their synapses. The new study, for instance, shows a key mechanism for maintaining ionic balance for the neurons.

In addition to Weiss and Littleton, the paper’s other authors are Jan Melom, who helped lead the discovery of zydeco, postdoc Kiel Ormerod, and former postdoc Yao Zhang.

The National Institutes of Health and the JPB Foundation funded the research.

Study shows how specific gene variants may raise bipolar disorder risk

Findings could help inform new therapies, improve diagnosis.

David Orenstein | Picower Institute for Learning and Memory
January 18, 2019

A new study by researchers at the Picower Institute for Learning and Memory at MIT finds that the protein CPG2 is significantly less abundant in the brains of people with bipolar disorder (BD) and shows how specific mutations in the SYNE1 gene that encodes the protein undermine its expression and its function in neurons.

Led by Elly Nedivi, professor in MIT’s departments of Biology and Brain and Cognitive Sciences, and former postdoc Mette Rathje, the study goes beyond merely reporting associations between genetic variations and psychiatric disease. Instead, the team’s analysis and experiments show how a set of genetic differences in patients with bipolar disorder can lead to specific physiological dysfunction for neural circuit connections, or synapses, in the brain.

The mechanistic detail and specificity of the findings provide new and potentially important information for developing novel treatment strategies and for improving diagnostics, Nedivi says.

“It’s a rare situation where people have been able to link mutations genetically associated with increased risk of a mental health disorder to the underlying cellular dysfunction,” says Nedivi, senior author of the study online in Molecular Psychiatry. “For bipolar disorder this might be the one and only.”

The researchers are not suggesting that the CPG2-related variations in SYNE1 are “the cause” of bipolar disorder, but rather that they likely contribute significantly to susceptibility to the disease. Notably, they found that sometimes combinations of the variants, rather than single genetic differences, were required for significant dysfunction to become apparent in laboratory models.

“Our data fit a genetic architecture of BD, likely involving clusters of both regulatory and protein-coding variants, whose combined contribution to phenotype is an important piece of a puzzle containing other risk and protective factors influencing BD susceptibility,” the authors wrote.

CPG2 in the bipolar brain

During years of fundamental studies of synapses, Nedivi discovered CPG2, a protein expressed in response to neural activity, that helps regulate the number of receptors for the neurotransmitter glutamate at excitatory synapses. Regulation of glutamate receptor numbers is a key mechanism for modulating the strength of connections in brain circuits. When genetic studies identified SYNE1 as a risk gene specific to bipolar disorder, Nedivi’s team recognized the opportunity to shed light into the cellular mechanisms of this devastating neuropsychiatric disorder typified by recurring episodes of mania and depression.

For the new study, Rathje led the charge to investigate how CPG2 may be different in people with the disease. To do that, she collected samples of postmortem brain tissue from six brain banks. The samples included tissue from people who had been diagnosed with bipolar disorder, people who had neuropsychiatric disorders with comorbid symptoms such as depression or schizophrenia, and people who did not have any of those illnesses. Only in samples from people with bipolar disorder was CPG2 significantly lower. Other key synaptic proteins were not uniquely lower in bipolar patients.

“Our findings show a specific correlation between low CPG2 levels and incidence of BD that is not shared with schizophrenia or major depression patients,” the authors wrote.

From there they used deep-sequencing techniques on the same brain samples to look for genetic variations in the SYNE1 regions of BD patients with reduced CPG2 levels. They specifically looked at ones located in regions of the gene that could regulate expression of CPG2 and therefore its abundance.

Meanwhile, they also combed through genomic databases to identify genetic variants in regions of the gene that code CPG2. Those mutations could adversely affect how the protein is built and functions.

Examining effects

The researchers then conducted a series of experiments to test the physiological consequences of both the regulatory and protein coding variants found in BD patients.

To test effects of non-coding variants on CPG2 expression, they cloned the CPG2 promoter regions from the human SYNE1 gene and attached them to a “reporter” that would measure how effective they were in directing protein expression in cultured neurons. They then compared these to the same regions cloned from BD patients that contained specific variants individually or in combination. Some did not affect the neurons’ ability to express CPG2 but some did profoundly. In two cases, pairs of variants (but neither of them individually), also reduced CPG2 expression.

Previously Nedivi’s lab showed that human CPG2 can be used to replace rat CPG2 in culture neurons, and that it works the same way to regulate glutamate receptor levels. Using this assay they tested which of the coding variants might cause problems with CPG2’s cellular function. They found specific culprits that either reduced the ability of CPG2 to locate in the “spines” that house excitatory synapses or that decreased the proper cycling of glutamate receptors within synapses.

The findings show how genetic variations associated with BD disrupt the levels and function of a protein crucial to synaptic activity and therefore the health of neural connections. It remains to be shown how these cellular deficits manifest as biopolar disorder.

Nedivi’s lab plans further studies including assessing behavioral implications of difference-making variants in lab animals. Another is to take a deeper look at how variants affect glutamate receptor cycling and whether there are ways to fix it. Finally, she said, she wants to continue investigating human samples to gain a more comprehensive view of how specific combinations of CPG2-affecting variants relate to disease risk and manifestation.

In addition to Rathje and Nedivi, the paper’s other authors are Hannah Waxman, Marc Benoit, Prasad Tammineni, Costin Leu, and Sven Loebrich.

The JPB Foundation, the Gail Steel Fund, the Carlsberg Foundation, the Lundbeck Foundation and the Danish Council for Independent Research funded the study.