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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.

Study links certain metabolites to stem cell function in the intestine

Molecules called ketone bodies may improve stem cells’ ability to regenerate new intestinal tissue.

Anne Trafton | MIT News Office
August 22, 2019

MIT biologists have discovered an unexpected effect of a ketogenic, or fat-rich, diet: They showed that high levels of ketone bodies, molecules produced by the breakdown of fat, help the intestine to maintain a large pool of adult stem cells, which are crucial for keeping the intestinal lining healthy.

The researchers also found that intestinal stem cells produce unusually high levels of ketone bodies even in the absence of a high-fat diet. These ketone bodies activate a well-known signaling pathway called Notch, which has previously been shown to help regulate stem cell differentiation.

“Ketone bodies are one of the first examples of how a metabolite instructs stem cell fate in the intestine,” says Omer Yilmaz, the Eisen and Chang Career Development Associate Professor of Biology and a member of MIT’s Koch Institute for Integrative Cancer Research. “These ketone bodies, which are normally thought to play a critical role in energy maintenance during times of nutritional stress, engage the Notch pathway to enhance stem cell function. Changes in ketone body levels in different nutritional states or diets enable stem cells to adapt to different physiologies.”

In a study of mice, the researchers found that a ketogenic diet gave intestinal stem cells a regenerative boost that made them better able to recover from damage to the intestinal lining, compared to the stem cells of mice on a regular diet.

Yilmaz is the senior author of the study, which appears in the Aug. 22 issue of Cell. MIT postdoc Chia-Wei Cheng is the paper’s lead author.

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Adult stem cells, which can differentiate into many different cell types, are found in tissues throughout the body. These stem cells are particularly important in the intestine because the intestinal lining is replaced every few days. Yilmaz’ lab has previously shown that fasting enhances stem cell function in aged mice, and that a high-fat diet can stimulate rapid growth of stem cell populations in the intestine.

In this study, the research team wanted to study the possible role of metabolism in the function of intestinal stem cells. By analyzing gene expression data, netbet online sports bettingCheng discovered that several enzymes involved in the production of ketone bodies are more abundant in intestinal stem cells than in other types of cells.

When a very high-fat diet is consumed, cells use these enzymes to break down fat into ketone bodies, which the body can use for fuel in the absence of carbohydrates. However, because these enzymes are so active in intestinal stem cells, these cells have unusually high ketone body levels even when a normal diet is consumed.

To their surprise, the researchers found that the ketones stimulate the Notch signaling pathway, which is known to be critical for regulating stem cell functions such as regenerating damaged tissue.

“Intestinal stem cells can generate ketone bodies by themselves, and use them to sustain their own stemness through fine-tuning a hardwired developmental pathway that controls cell lineage and fate,” Cheng says.

In mice, the researchers showed that a ketogenic diet enhanced this effect, and mice on such a diet were better able to regenerate new intestinal tissue. When the researchers fed the mice a high-sugar diet, they saw the opposite effect: Ketone production and stem cell function both declined.

Stem cell function

The study helps to answer some questions raised by Yilmaz’ previous work showing that both fasting and high-fat diets enhance intestinal stem cell function. The new findings suggest that stimulating ketogenesis through any kind of diet that limits carbohydrate intake helps promote stem cell proliferation.

“Ketone bodies become highly induced in the intestine during periods of food deprivation and play an important role in the process of preserving and enhancing stem cell activity,” Yilmaz says. “When food isn’t readily available, it might be that the intestine needs to preserve stem cell function so that when nutrients become replete, you have a pool of very active stem cells that can go on to repopulate the cells of the intestine.”

The findings suggest that a ketogenic diet, which would drive ketone body production in the intestine, might be helpful for repairing damage to the intestinal lining, which can occur in cancer patients receiving radiation or chemotherapy treatments, Yilmaz says.

The researchers now plan to study whether adult stem cells in other types of tissue use ketone bodies to regulate their function. Another key question is whether ketone-induced stem cell activity could be linked to cancer development, because there is evidence that some tumors in the intestines and other tissues arise from stem cells.

“If an intervention drives stem cell proliferation, a population of cells that serve as the origin of some tumors, could such an intervention possibly elevate cancer risk? That’s something we want to understand,” Yilmaz says. “What role do these ketone bodies play in the early steps of tumor formation, and can driving this pathway too much, either through diet or small molecule mimetics, impact cancer formation? We just don’t know the answer to those questions.”

The research was funded by the National Institutes of Health, a V Foundation V Scholar Award, a Sidney Kimmel Scholar Award, a Pew-Stewart Trust Scholar Award, the MIT Stem Cell Initiative, the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund, the Koch Institute Dana Farber/Harvard Cancer Center Bridge Project, and the American Federation of Aging Research.

Mentorship and scholarship keep summer biology research program strong

Support from Squire Booker PhD ’94 and the Bernard S. and Sophie G. Gould Fund helps MSRP-bio students excel.

Laura Carter | School of Science
August 20, 2019

When you get a call offering you the chance to get involved in research at MIT, says Squire Booker PhD ’94, as he did when he was a student back home in Beaumont, Texas, with no summer plans, you don’t say no. This is how he joined seven other students from around the United States as the first class in the MIT Student Research Program (MSRP), even though the start date was only days away. “I was given the opportunity to get out of Texas, the opportunity to go to a big cosmopolitan city, the opportunity to go to MIT. So, I got a plane ticket and flew up a few days later,” says Booker.

Thirty-three summers later, back on campus to deliver the doctoral graduation ceremony speech, where he had lunch with several current members and fellow alumni of the program, Booker insists that he has no regrets with his decision.

Booker was one of three from that inaugural class who remained at MIT to pursue a PhD to continue the research he started during the program. He was incredibly fortunate, he notes, to get a “perfect match” placement, working with former professors of biology Bill Johnson and Chris Walsh on a project that aligned with his interests of combining chemistry and biology. He didn’t have much more of an idea of his preferred area of study than that.

Prior to arriving at MIT, given the lack of exposure to science, he didn’t know what research entailed, or what scientists did every day. But he says he quickly fell in love with the subject and his research group, even joining their summer lab softball team.

Although Walsh left MIT the year Booker was accepted as a PhD student, he easily shifted into the lab of Novartis Professor of Chemistry Emeritus JoAnne Stubbe, a new faculty member at the time, who was also working on the interface of chemistry and biology and provided the amount of hands-on support he needed as a new graduate student. “Ever since leaving the lab, she’s been my number one supporter,” he says of Stubbe.

Stubbe and her research inspired the direction Booker’s education took. He continues to conduct research revolving around proteins and catalysis reactions as a professor at Penn State University and a principal investigator with the Howard Hughes Medical Institute. Now, he heads a large lab group himself.

From mentee to mentor

Booker oversees an average of 10 group members at any given time, not including undergraduate students. Like his mentor, he tries to be very hands-on, resorting to email when he’s traveling — which is often. He admitted with a chuckle that his students keep track of where he is at any given time by following his Twitter account. Always trying to find ways to include motivated students who approach him about contributing to his research, the only time Booker turns them away is for their benefit — if they have a full course load and additional time on research will overload their schedules. He even considers high school students.

The first high school student to join his lab was Martin McLaughlin ’15, who Booker describes fondly as “aggressively motivated” and “trembling with excitement to do research.” Within the first week, McLaughlin was taking the initiative to use his lunch breaks from school to bike to Booker’s lab. Martin’s results, which were published in Science in collaboration with Professor Cathy Drennan in the MIT Department of Biology, introduced Booker into a new niche: crystallography.

When McLaughlin asked to continue working on the discovery with Drennan as an undergraduate at MIT, he didn’t hesitate to agree. McLaughlin had moved into Drennan’s lab a week into his first semester.

Research for all

Not all students share this drive to delve into research. Like Booker himself, many aren’t even aware of possibilities to get involved in science and consider a career in research. It’s still hard, he says, even though “people are more serious about this diversity thing,” as he calls it, than when he was first starting his education.

Booker tries to reach out, especially to other minority students, through several programs, much like the MSRP, an invaluable program. While on campus this past spring, Booker met with current and past MSRP students.

One of those students was Jeandele Elliot, a chemical engineering student at Howard University from Saint Lucia in the Caribbean, who is working in the Jing-Ke Weng Lab in the Department of Biology this summer on a molecule that can protect pollen grains. For her, meeting Booker was another connection the program affords her. “The MSRP program has been beneficial to me in a special way since it has connected me with people I can really relate to,” she said.

The advice he gave to Elliot, and the others in the same position he was in once, was to prepare netbet sports bettingfor exciting careers. The program is not just a steppingstone into research, he proclaimed, but it places participants with the best mentors and being privy to the best frontiers. Booker was delighted that some of the 25 current and past participants then attended MIT for graduate school as he did.

Tsehai A.J. Grell PhD ’18, a current chemistry graduate student in Drennan’s research group and an alumnus of MSRP, calls Squire Booker a “labhold” name — a household name in the lab. “As an African-American professor of biochemistry, an alumnus of my department, and a leader in my field, he instantly became one of my role models,” Grell said. “This was further solidified when I found out that he was a part of the first cohort of MSRP students, the summer research program which is responsible for me enrolling in MIT’s graduate program.”

Grell reminisced on his experience and the spring luncheon with Booker. “Because MSRP was such a foundational experience in my career, I am always enthused to interact with the current MSRP cohort and to encourage them to make the most of this opportunity, as it can be a pivotal summer in their careers,” says Grell. In addition, he said, “the excitement of the students is palpable and contagious. It reenergizes me and gives me purpose.”

Elliott, Grell, and Booker are three of more than 800 students from institutions with limited research opportunities who have participated in the MSRP, which was divided into two subcategories in 2003: general and biology, the latter of which has hosted 450 students. Since 2003, the MRSP-Bio program has been administered by Mandana Sassanfar, a biology lecturer in charge of the Department of Biology’s diversity and outreach programs. Since then, nearly 70 MSRP alumni have, like Booker, continued their research as graduate students at MIT.

Going for Gould

Bernard “Bernie” Gould ’32, who received his BS from MIT, was a longstanding and beloved biochemistry professor in the Department of Biology, well known for being an incredibly dedicated mentor to biology and pre-med students at MIT for nearly 40 years. His wife, Sophia Gould CMP ’48, shared his passion for counseling students. To honor this investment in encouraging student learning, the Goulds’ son, Michael, and his wife, Sara Moss, founded the Bernard S. and Sophia G. Gould Fund in 2016. Gould is a philanthropist and the retired chairman and CEO of Bloomingdales. Moss is the vice chairman of Estée Lauder Companies. The Gould Fellow Fund sponsors students, such as Elliott, in MSRP-Bio. Each year, Gould and Moss return to the MIT campus to meet with students benefitting from their support.

Recently, the couple has designated a second fund, which will aid in extending the academic careers of students interested in the life sciences by providing support for MSRP-Bio alumni entering into the MIT biology graduate program.

Six of the 16 Gould Fellowship alumni who have graduated from college have already been admitted to MIT as graduate students. “This is an exceptionally high rate by any standards, which demonstrates the amazing success of this initiative,” says Sassanfar. “Gould Fellows are truly grateful for the generosity of Mike and Sara and are very eager to succeed and give back to their communities,” a goal that is always stressed by the founders.

With successful role models from previous MSRP cohorts, like Booker, combined with philanthropy from those like Gould and Moss, who believe strongly in supporting the education of our next generation of scientists, students are given the opportunity to thrive.

An emerging view of RNA transcription and splicing

Whitehead Institute scientists find chemical modification contributes to trafficking between non-membrane-bound compartments that control gene expression.

Nicole Davis | Whitehead Institute
August 9, 2019

Cells often create compartments to control important biological functions. The nucleus is a prime example; surrounded by a membrane, it houses the genome. Yet cells also harbor enclosures that are not membrane-bound and more transient, like oil droplets in water. Over the past two years, these droplets (called “condensates”) have become increasingly recognized as major players in controlling genes. Now, a team led by Whitehead Institute scientists helps expand this emerging picture with the discovery that condensates play a role in splicing, an essential activity that ensures the genetic code is prepared to be translated into protein. The researchers also reveal how a critical piece of cellular machinery moves between different condensates. The team’s findings appear in the Aug. 7 online issue of Nature.

“Condensates represent a real paradigm shift in the way molecular biologists think about gene control,” says senior author Richard Young, a member of the Whitehead Institute and professor of biology at MIT. “Now, we’ve added a critical new layer to this thinking that enhances our understanding of splicing as well as the major transcriptional apparatus RNA polymerase II.”

Young’s lab has been at the forefront of studying how and when condensates form as well as their functions in gene regulation. In the current study, Young and his colleagues, including first authors Eric Guo and John Manteiga, focused their efforts on a key transition that happens when genes undergo transcription — an early step in gene activation whereby an RNA copy is created from the genes’ DNA template. First, all of the molecular machinery needed to make RNA, including a large protein complex known as RNA polymerase II, assembles at a given gene. Then, specific chemical modifications to RNA polymerase II allow it to begin transcribing DNA into RNA. This shift from so-called transcription initiation to active transcription also involves another important molecular transition: As RNA molecules begin to grow, the splicing apparatus must also move in and carry out its job.

“We wanted to step back and ask, ‘Do condensates play an important role in this switch, and if so, what mechanism might be responsible?’” explains Young.

For roughly three decades, it has been recognized that the factors required for splicing are stored in compartments called speckles. Yet whether these speckles play an active role in splicing, or are simply storage vessels, has remained unclear.

Using confocal microscopy, the Whitehead team discovered condensates filled with components of the splicing machinery in the vicinity of highly active genes. Notably, these structures exhibited similar liquid-like characteristics to those condensates described in prior studies from Young’s lab that are involved in transcription initiation.

“These findings signaled to us that there are two types of condensates at work here: one involved in transcription initiation and the other in splicing and transcriptional elongation,” said Manteiga, a graduate student in Young’s lab.

With two different condensates at play, the researchers wondered: How does the critical transcriptional machinery, specifically RNA polymerase II, move from one condensate to the other?

Guo, Manteiga, and their colleagues found that chemical modification, specifically the addition of phosphate groups, serves as a kind of molecular switch that alters the protein complex’s affinity for a particular condensate. With fewer phosphate groups, it associates with the condensates for transcription initiation; when more phosphates are added, it enters the splicing condensates. Such phosphorylation occurs on one end of the protein complex, which contains a specialized region known as the C-terminal domain (CTD). Importantly, the CTD lacks a specific three-dimensional structure, and previous work has shown that such intrinsically disordered regions can influence how and when certain proteins are incorporated into condensates.

“It is well-documented that phosphorylation acts as a signal to help regulate the activity of RNA polymerase II,” says Guo, a postdoc in Young’s lab. “Now, we’ve shown that it also acts as a switch to alter netbet sports bettingthe protein’s preference for different condensates.”

In light of their discoveries, the researchers propose a new view of splicing compartments, where speckles serve primarily as warehouses, storing the thousands of molecules required to support the splicing apparatus when they are not needed. But when splicing is active, the phosphorylated CTD of RNA Pol II serves as an attractant, drawing the necessary splicing materials toward the gene where they are needed and into the splicing condensate.

According to Young, this new outlook on gene control has emerged in part through a multidisciplinary approach, bringing together perspectives from biology and physics to learn how properties of matter predict some of the molecular behaviors he and his team have observed experimentally. “Working at the interface of these two fields is incredibly exciting,” says Young. “It is giving us a whole new way of looking at the world of regulatory biology.”

Support for this work was provided by the U.S. National Institutes of Health, National Science Foundation, Cancer Research Institute, Damon Runyon Cancer Research Foundation, Hope Funds for Cancer Research, Swedish Research Council, and German Research Foundation DFG.

Study furthers radically new view of gene control

Along the genome, proteins form liquid-like droplets that appear to boost the expression of particular genes.

Anne Trafton | MIT News Office
August 8, 2019

In recent years, MIT scientists have developed a new model for how key genes are controlled that suggests the cellular machinery that transcribes DNA into RNA forms specialized droplets called condensates. These droplets occur only at certain sites on the genome, helping to determine which genes are expressed in different types of cells.

In a new study that supports that model, researchers at MIT and the Whitehead Institute for Biomedical Research have discovered physical interactions between proteins and with DNA that help explain why these droplets, which stimulate the transcription of nearby genes, tend to cluster along specific stretches of DNA known as super enhancers. These enhancer regions do not encode proteins but instead regulate other genes.

“This study provides a fundamentally important new approach to deciphering how the ‘dark matter’ in our genome functions in gene control,” says Richard Young, an MIT professor of biology and member of the Whitehead Institute.

Young is one of the senior authors of the paper, along with Phillip Sharp, an MIT Institute Professor and member of MIT’s Koch Institute for Integrative Cancer Research; and Arup K. Chakraborty, the Robert T. Haslam Professor in Chemical Engineering, a professor of physics and chemistry, and a member of MIT’s Institute for Medical Engineering and Science and the Ragon Institute of MGH, MIT, and Harvard.

Graduate student Krishna Shrinivas and postdoc Benjamin Sabari are the lead authors of the paper, which appears in Molecular Cell on Aug. 8.

“A biochemical factory”

Every cell in an organism has an identical genome, but cells such as neurons or heart cells express different subsets of those genes, allowing them to carry out their specialized functions. Previous research has shown that many of these genes are located near super enhancers, which bind to proteins called transcription factors that stimulate the copying of nearby genes into RNA.

About three years ago, Sharp, Young, and Chakraborty joined forces to try to model the interactions that occur at enhancers. In a 2017 Cell paper, based on computational studies, they hypothesized that in these regions, transcription factors form droplets called phase-separated condensates. Similar to droplets of oil suspended in salad dressing, these condensates are collections of molecules that form distinct cellular compartments but have no membrane separating them from the rest of the cell.

In a 2018 Science paper, the researchers showed that these dynamic droplets do form at super enhancer locations. Made of clusters of transcription factors and other molecules, these droplets attract enzymes such as RNA polymerases that are needed to copy DNA into messenger RNA, keeping gene transcription active at specific sites.

“We had demonstrated that the transcription machinery forms liquid-like droplets at certain regulatory regions on our genome, however we didn’t fully understand how or why these dewdrops of biological molecules only seemed to condense around specific points on our genome,” Shrinivas says.

As one possible explanation for that site specificity, the research team hypothesized that weak interactions between intrinsically disordered regions of transcription factors and other transcriptional molecules, along with specific interactions between transcription factors and particular DNA elements, might determine whether a condensate forms at a particular stretch of DNA. Biologists have traditionally focused on “lock-and-key” style interactions between rigidly structured protein segments to explain most cellular processes, but more recent evidence suggests that weak interactions between floppy protein regions also play an important role in cell activities.

In this study, computational modeling and experimentation revealed that the cumulative force of these weak interactions conspire together with transcription factor-DNA interactions to determine whether a condensate of transcription factors will form at a particular site on the genome. Different cell types produce different transcription factors, which bind to different enhancers. When many transcription factors cluster around the same enhancers, weak interactions between the proteins are more likely to occur. Once a critical threshold concentration is reached, condensates form.

“Creating these local high concentrations within the crowded environment of the cell enables the right material to be in the right place at the right time to carry out the multiple steps required to activate a gene,” Sabari says. “Our current study begins to tease apart how certain regions of the genome are capable of pulling off this trick.”

These droplets form on a timescale of seconds to minutes, and they blink in and out of existence depending on a cell’s needs.

“It’s an on-demand biochemical factory that cells can form and dissolve, as and when they need it,” Chakraborty says. “When certain signals happen at the right locus on a gene, the condensates form, which concentrates all of the transcription molecules. Transcription happens, and when the cells are done with that task, they get rid of them.”

A new view

Weak cooperative interactions between proteins may also play an important role in evolution, the researchers proposed in a 2018 Proceedings of the National Academy of Sciences paper. The sequences of intrinsically disordered regions of transcription factors need to change only a little to evolve new types of specific functionality. In contrast, evolving new specific functions via “lock-and-key” interactions requires much more significant changes.

“If you think about how biological systems have evolved, they have been able to respond to different conditions without creating new genes. We don’t have any more genes that a fruit fly, yet we’re much more complex in many of our functions,” Sharp says. “The incremental expanding and contracting of these intrinsically disordered domains could explain a large part of how that evolution happens.”

Similar condensates appear to play a variety of other roles in biological systems, offering a new way to look at how the interior of a cell is organized. Instead of floating through the cytoplasm and randomly bumping into other molecules, proteins involved in processes such as relaying molecular signals may transiently form droplets that help them interact with the right partners.

“This is a very exciting turn in the field of cell biology,” Sharp says. “It is a whole new way of looking at biological systems that is richer and more meaningful.”

Some of the MIT researchers, led by Young, have helped form a company called Dewpoint Therapeutics to develop potential treatments for a wide variety of diseases by exploiting cellular condensates. There is emerging evidence that NetBet live casinocancer cells use condensates to control sets of genes that promote cancer, and condensates have also been linked to neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease.

The research was funded by the National Science Foundation, the National Institutes of Health, and the Koch Institute Support (core) Grant from the National Cancer Institute.

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.

A new way to block unwanted genetic transfer

Researchers identify a strategy to prevent mobile genetic elements from breaching the bacterial cell wall.

Raleigh McElvery | Department of Biology
August 6, 2019

We receive half of our genes from each biological parent, so there’s no avoiding inheriting a blend of characteristics from both. Yet, for single-celled organisms like bacteria that reproduce by splitting into two identical cells, injecting variety into the gene pool isn’t so easy. Random mutations add some diversity, but there’s a much faster way for bacteria to reshuffle their genes and confer evolutionary advantages like antibiotic resistance or pathogenicity.

Known as horizontal gene transfer, this process permits bacteria to pass pieces of DNA to their peers, in some cases allowing those genes to be integrated into the recipient’s genome and passed down to the next generation.

The Grossman lab in the MIT Department of Biology studies one class of mobile DNA, known as integrative and conjugative elements (ICEs). While ICEs contain genes that can be beneficial to the recipient bacterium, there’s also a catch — receiving a duplicate copy of an ICE is wasteful, and possibly lethal. The biologists recently uncovered a new system by which one particular ICE, ICEBs1, blocks a donor bacterium from delivering a second, potentially deadly copy.

“Understanding how these elements function and how they’re regulated will allow us to determine what drives microbial evolution,” says Alan Grossman, department head and senior author on the netbet sports betting appstudy. “These findings not only provide insight into how bacteria block unwanted genetic transfer, but also how we might eventually engineer this system to our own advantage.”

Former graduate student Monika Avello PhD ’18 and current graduate student Kathleen Davis are co-first authors on the study, which appeared online in Molecular Microbiology on July 30.

Checks and balances

Although plasmids are perhaps the best-known mediators of horizontal transfer, ICEs not only outnumber plasmids in most bacterial species, they also come with their own tools to exit the donor, enter the recipient, and integrate themselves into the recipient’s chromosome. Once the donor bacterium makes contact with the recipient, the machinery encoded by the ICE can pump the ICE DNA from one cell to the other through a tiny channel.

For horizontal transfer to proceed, there are physical barriers to overcome, especially in so-called Gram-positive bacteria, which boast thicker cell walls than their Gram-negative counterparts, despite being less widely studied. According to Davis, the transfer machinery essentially has to “punch a hole” through the recipient cell. “It’s a rough ride and a waste of energy for the recipient if that cell already contains an ICE with a specific set of genes,” she says.

Sure, ICEs are “selfish bits of DNA” that persist by spreading themselves as widely as possible, but in order to do so they must not interfere with their host cell’s ability to survive. As Avello explains, ICEs can’t just disseminate their DNA “without certain checks and balances.”

“There comes a point where this transfer comes at a cost to the bacteria or doesn’t make sense for the element,” she says. “This study is beginning to get at the question of when, why, and how ICEs might want to block transfer.”

The Grossman lab works in the Gram-positive Bacillus subtilis, and had previously discovered two mechanisms by which ICEBs1 could prevent redundant transfer before it becomes lethal. The first, cell-cell signaling, involves the ICE in the recipient cell releasing a chemical cue that prohibits the donor’s transfer machinery from being assembled. The second, immunity, initiates if the duplicate copy is already inside the cell, and prevents the replicate from being integrated into the chromosome.

However, when the researchers tried eliminating both fail-safes simultaneously, rather than re-instating ICE transfer as they expected, the bacteria still managed to obstruct the duplicate copy. ICEBs1 seemed to have a third blocking strategy, but what might it be?

The third tactic

In this most recent study, they’ve identified the mysterious blocking mechanism as a type of “entry exclusion,” whereby the ICE in the recipient cell encodes molecular machinery that physically prevents the second copy from breaching the cell wall. Scientists had observed other mobile genetic elements capable of exclusion, but this was the first time anyone had witnessed this phenomenon for an ICE from Gram-positive bacteria, according to Avello.

The Grossman lab determined that this exclusion mechanism comes down to two key proteins. Avello identified the first protein, YddJ, expressed by the ICEBs1 in the recipient bacterium, forming a “protective coating” on the outside of the cell and blocking a second ICE from entering.

But the biologists still didn’t know which piece of transfer machinery YddJ was blocking, so Davis performed a screen and various genetic manipulations to pinpoint YddJ’s target. YddJ, it turned out, was obstructing another protein called ConG, which likely forms part of the transfer channel between the donor and recipient bacteria. Davis was surprised to find that, while Gram-negative ICEs encode a protein that’s quite similar to ConG, the Gram-negative YddJ equivalent is actually much different.

“This just goes to show that you can’t assume the transfer machinery in Gram-positive ICEs like ICEBs1 are the same as the well-studied Gram-negative ICEs,” she says.

The team concluded that ICEBs1 must have three different mechanisms to prevent duplicate transfer: the two they’d previously uncovered plus this new one, exclusion.

Cell-cell signaling allows a cell to spread the word to its neighbors that it already has a copy of ICEBs1, so there’s no need to bother assembling the transfer machinery. If this fails, exclusion kicks in to physically block the transfer machinery from penetrating the recipient cell. If that proves unsuccessful and the second copy enters the recipient, immunity will initiate and prevent the second copy from being integrated into the recipient’s chromosome.

“Each mechanism acts at a different step, because none of them alone are 100 percent effective,” Grossman says. “That’s why it’s helpful to have multiple mechanisms.”

They don’t know all the details of this transfer machinery just yet, he adds, but they do know that YddJ and ConG are key players.

“This initial description of the ICEBs1 exclusion system represents the first report that provides mechanistic insights into exclusion in Gram-positive bacteria, and one of only a few mechanistic studies of exclusion in any conjugation system,” says Gary Dunny, a professor of microbiology and immunology at the University of Minnesota who was not involved in the study. “This work is significant medically because ICEs can carry “cargo” genes such as those conferring antibiotic resistance, and also of importance to our basic understanding of horizontal gene transfer systems and how they evolve.”

As researchers continue to probe this blocking mechanism, it might be possible to leverage ICE exclusion to design bacteria with specific functions. For instance, they could engineer the gut microbiome and introduce beneficial genes to help with digestion. Or, one day, they could perhaps block horizontal gene transfer to combat antibiotic resistance.

“We had suspected that Gram-positive ICEs might be capable of exclusion, but we didn’t have proof before this,” Avello says. Now, researchers can start to speculate about how pathogenic Gram-positive species might control the movement of ICEs throughout a bacterial population, with possible ramifications for disease research.

This work was funded by research and predoctoral training grants from the National Institute of General Medical Sciences of the National Institutes of Health.

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 netbet sports betting appdelicate 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.