Research Area: Stem Cell and Developmental Biology

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.

Uncovering the “must-haves” of tissue regeneration
Nicole Davis | Whitehead Institute
November 27, 2018

Cambridge, MA.  – The ability to regrow missing or damaged body parts is one of the great marvels of modern biology. In an effort to lay bare the biological underpinnings of this phenomenon, scientists at Whitehead Institute have begun to define the core features that are required for regeneration in flatworms. Their research, which appears online November 27 in Cell Reports, reveals that a set of cellular and molecular responses — previously thought to be essential for regeneration following amputations and other major injuries — is in fact dispensable.

“This is a real surprise,” said senior author Peter Reddien, a Member of Whitehead Institute, professor of biology at Massachusetts Institute of Technology, and investigator with the Howard Hughes Medical Institute. “These responses are broad, prominent attributes of tissue regeneration and repair and, a reasonable bet was that they function to bring about regeneration.”

About eight years ago, Reddien and his team described a set of biological activities that are triggered by injuries that remove tissue. Whereas a cut or a scrape removes little if any tissue, more damaging injuries, like amputations, cause significant tissue loss. That missing tissue must be regenerated to ensure the organism retains its proper anatomical proportions.

A series of cellular and molecular activities — known collectively as the missing tissue response — were believed to enable this regeneration to occur. They include the sustained action of genes that respond to injury, a period of intense cell division in areas surrounding the wound, and a general increase in cell death throughout the body. “This happens prominently, not only in planarians but also in other organisms capable of regeneration, so we suspected that the missing tissue response must play a very fundamental role in regeneration,” recalled Reddien.

What types of injuries require the missing tissue response for repair, and what is the function of the missing tissue response in regeneration? Graduate student and first author Aneesha Tewari, Reddien and colleagues, including Sarah Stern and Isaac Oderberg, set out to uncover the answers. This work forms the basis of their latest Cell Reports study.

The researchers harnessed an earlier discovery that a gene known as follistatin is required for the missing tissue response in flatworms (known as planarians). By using molecular tools to inhibit this gene, they could block the missing tissue response and observe what happens under various wound conditions, ranging from minimal (the removal of an eye, for example) to moderate (the removal of the pharynx or part of the head) to significant tissue loss (the removal of a complete side of the body). Remarkably, in every case, the missing tissue was regenerated, albeit much more slowly than it would be otherwise.

“These results tell us that what the missing tissue response is really doing is simply pushing the foot down on the gas pedal — basically accelerating the process of regeneration,” explained Reddien. “If you can’t accelerate, you’ll still get there, it just takes longer.”

Tewari, Reddien, and their colleagues also cracked a thorny mystery surrounding the missing tissue response. Although their results show that it is not required across a wide range of injuries, there is one lingering instance in which regeneration failed to occur when they blocked the missing tissue response: head amputation.

“This was a big puzzle,” said Tewari. “It left us wondering whether or not we could generalize our findings to all types of wounds — is there something special about the head that makes it uniquely dependent on the missing tissue response?”

The answer, it turns out, is no. When follistatin is blocked, a key signaling protein, called Wnt1, kicks into overdrive. And when that happens, the tissue destined to form the head does not receive the positional cues it needs to properly regrow, which means regeneration fails to proceed. But, when both the missing tissue response and Wnt1 are blocked, the head does indeed regenerate, the team uncovered.

Taken together, the researchers’ findings begin to clarify what is essential for regeneration to take place and what is not. “Our study greatly simplifies the picture of what it takes to regenerate,” said Reddien. “And that’s an important step along the path towards dissecting the central elements of regeneration in animals that do regenerate well, like flatworms, and then applying that knowledge to understand what the limits might be in those animals that don’t regenerate as well, like humans.”

This research was supported by the NIH (R01GM080639), the National Science Foundation, the Eleanor Schwartz Charitable Foundation, and the Howard Hughes Medical Institute.

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Peter Reddien’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 Howard Hughes Medical Institute Investigator and a professor of biology at Massachusetts Institute of Technology.
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Full citation:
Cell Reports,  Vol. 25, Is. 9, P2577-2590.E3, November 27, 2018, DOI:https://doi.org/10.1016/j.celrep.2018.11.004
“Cellular and molecular responses unique to major injury are dispensable for planarian regeneration”
Aneesha G. Tewari (1,2), Sarah R. Stern (1,2), Isaac M. Oderberg (1,2,4), and Peter W. Reddien (1,2,3)
 1.Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
3. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
4. Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Plant characteristics shaped by parental conflict
Greta Friar | Whitehead Institute
November 19, 2018

CAMBRIDGE, Mass. – Different subpopulations of a plant species can have distinct traits, varying in size, seed count, coloration, and so on. The primary source of this variation is genes: different versions of a gene can lead to different traits. However, genes are not the only determinant of such traits, and researchers are learning more about another contributor: epigenetics. Epigenetic factors are things that regulate genes, altering their expression, and like genes they can be inherited from generation to generation, even though they are independent of the actual DNA sequences of the genes.

One epigenetic mechanism is DNA methylation, in which the addition of chemical tags called methyl groups can turn genes on or off. Genes that share the identical DNA sequence but have different patterns of methylation are called epialleles. Several studies have shown that epialleles, like different versions of genes, can cause differences in traits between plant subpopulations, or strains, but whether genetic factors are also at play can be difficult to determine.

The lab of Whitehead Member Mary Gehring, who is also an associate professor at Massachusetts Institute of Technology, has described evidence that epialleles alone can lead to different heritable traits in plants. In research published online November 5 in the journal PLoS Genetics, Gehring, along with co-first authors and former lab members Daniela Pignatta and Katherine Novitzky, showed that altering the methylation state of the gene HDG3 in different strains of the plant Arabidopsis thaliana was enough to cause changes in seed weight and in the timing of certain aspects of seed development.

In plants, methylation states of genes change most frequently during seed development, when genes are switched on or off to progress development of the organism. This period is also when a conflict of interest arises in the genome of each seed between the parts inherited from its mother and father. The mother plants produce seeds fertilized by different fathers at the same time. It’s in the mother’s interest to give an equal share of nutrients to each seed—to have many smaller seeds. But it’s in the father’s interest for its seed to get the most nutrients and grow larger. This conflict plays out through an epigenetic mechanism called imprinting, in which, through differential methylation between the father’s and mother’s copies of a gene, one parent’s copy is silenced in the offspring so that only the other parent’s version of the gene is expressed.

The gene HDG3 is imprinted in one strain of Arabidopsis so that only the father’s copy is expressed. Gehring and her team found that when the strain loses its paternal imprinting, the timing of seed development is affected and the plant ends up with smaller seeds. This is consistent with the theory of imprinting: When the father’s genes have the advantage, the seeds are larger than when both parents’ genes are equally expressed.

Other experiments tested the effect of either activating or silencing HDG3 by methylation in a variety of scenarios, both in a separate strain of Arabidopsis in which the gene starts off silenced, as well as in crosses between the two strains. The researchers found that altering the methylation state of the gene was sufficient to affect seed size and the timing of seed development. In the crosses, these traits depended on whether the paternal copy of the gene came from the strain in which HDG3 was normally silenced or the strain in which it was normally activated.

Altogether these experiments demonstrate a link between changes in methylation state and differences in seed development and size. This suggests that epialleles in natural populations function much like variations in genes, creating heritable traits that differ within the larger population.

This work was funded by the National Science Foundation (NSF grant 1453459).

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Mary Gehring’s primary affiliation is with Whitehead Institute for Biomedical Research, where her laboratory is located and all her research is conducted. She is also an associate professor of biology at Massachusetts Institute of Technology.
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Full citation:
“A variably imprinted epiallele impacts seed development”
PLoS Genetics, online November 5 2018, https://doi.org/10.1371/journal.pgen.1007469
Daniela Pignatta (1,3), Katherine Novitzky (1,3), P. R. V. Satyaki (1), Mary Gehring (1,2)
1. Whitehead Institute for Biomedical Research, Cambridge, MA, United States of America
2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, United States of America
3. These authors contributed equally to this work.
Only in Your Head
Greta Friar | Whitehead Institute
October 9, 2018

Cambridge, Mass. — Brain development is a delicately choreographed dance in which cell division and differentiation into mature cell types must be performed in the right balance for normal growth. In order to better understand factors affecting brain development, Whitehead Institute researchers investigated a genetic mutation that leads to a brain-specific developmental disorder in spite of the gene’s prevalent expression in other cell types.

Kinetochore null protein 1 (KNL1) acts throughout the body during cell division to help ensure the accurate segregation of chromosomes into each daughter cell. A mutation in the KNL1 gene caused by a single change in its DNA sequence leads to microcephaly, a condition in which the brain fails to properly develop, causing babies to be born with small heads, often accompanied by intellectual disabilities and other health problems. In an article published online October 9 in the journal Cell Reports, Whitehead Institute Founding Member Rudolf Jaenisch and colleagues investigated how this KNL1 mutation can lead to microcephaly without affecting other cell types, providing important insights into the underlying basis of microcephaly and the role that KNL1 normally plays in brain development.

“The key question we were interested in was why, if the gene is ubiquitously expressed, is there a brain-specific phenotype,” says Jaenisch, who is also a professor of biology at the Massachusetts Institute of Technology.

Jaenisch lab graduate student Attya Omer Javed, a co-first author on the paper along with past lab members Yun Li and Julien Muffat, used CRISPR-Cas9 to recreate the mutation—a point mutation, or one-letter change in the DNA sequence of the KNL1 gene—in several different cell types derived from human stem cells in the lab. Of the three cell types tested, they found that only the neural progenitor cells, early stage cells that become brain cells, appeared to be affected.

As the brain develops, each neural progenitor can either keep dividing to increase the overall number of cells in the brain, or it can mature into a differentiated brain cell, at which point it is no longer able to divide. For a healthy brain to develop, there needs to be a careful balance between these two processes of proliferation and differentiation. If the progenitors take too long to differentiate, the developing brain won’t have the specific cells it needs to assemble. But if all of the cells differentiate too quickly, before they can divide, there will be a shortage of cells and the brain will be too small.

“Neural progenitors are going through many cell cycles, dividing quickly during brain development. Even a small defect could accumulate to have a huge impact,” Omer Javed says.

The researchers discovered that neural progenitors with the KNL1 mutation differentiated prematurely at the cost of proliferation, resulting in the small brain size that characterizes microcephaly. The brain cells with the mutation also were at a greater risk of cell death, disruption of the cell cycle, ending up with the wrong number of chromosomes, and malfunctions during attempted cell division.

KNL1’s role is in the kinetochore, an assembly of proteins that operate during mitosis to attach chromosomes to the machinery that will pull them apart into the daughter cells. This is why the KNL1 mutation negatively affects cell division. Co-author and Whitehead Member Iain Cheesemanhelped identify KNL1’s role in the kinetochore as a postdoctoral researcher years ago, and his expertise provided an opportunity for collaboration between his lab and Jaenisch’s.

“I have always found it interesting that inherited mutations to the kinetochore seem to lead to microcephaly,” Cheeseman says. “Investigating KNL1 together was an exciting chance to combine our labs’ diverse scientific knowledge.”

In order for the researchers to study the cells in an environment that more closely mimicked a human brain, they used a 3D cell culture technique to grow organoids made up of neural progenitors. Omer Javed found that the neural progenitors were extremely sensitive, as the organoids with the mutation expressed the microcephaly phenotype after as little as two weeks of growth.

Omer Javed then looked for differences between neural progenitors and the other cell types that would explain the brain-specific effects of the mutation. Even with the mutation, the KNL1 gene appeared able to make a functioning protein, explaining its lack of effect on the other cell types. So Omer Javed turned her attention to factors involved in regulating gene expression. For many of our genes to be expressed, first sections called introns must be removed, or spliced out, in order for the correct DNA sequence to then be read into RNA and then translated into a functional protein.

Omer Javed found that the KNL1 mutation created a site for splicing inhibitors to bind and silence the KNL1 gene by preventing it from being read into RNA. She also found a disparity in the level of a protein involved in this process between the cell types: the inhibitory splicing protein hnRNPA1 was much more prevalent in neural progenitors than elsewhere. When hnRNPA1 came across the site caused by the mutation, it prevented the gene from being expressed. The high quantity of hnRNPA1 in neural progenitors appears to be the main factor mediating the brain-specific effects of the mutation.

The work complements and extends previous investigations by the researchers into how neural progenitor proliferation may have contributed to the evolution of large human brains, as well as studies investigating why neural progenitors are so vulnerable to the Zika virus, which has been associated with microcephaly. Given their work suggesting that KNL1 could be a regulator of brain size, Omer Javed hopes that future research will reveal its role in the evolution of the human brain.

 

This research was supported by Boehringer Ingelheim Fonds, the Simons Foundation, the International Rett Syndrome Foundation, Brain & Behavior Research Foundation, the European Leukodystrophy Association, the National Institutes of Health (NIH grants HD 045022, R37-CA084198 and 1U19AI131135-01). Jaenisch is a cofounder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics.

 

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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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Full citation:
“Microcephaly modeling of kinetochore mutation reveals a brain-specific phenotype”
Cell Reports, online October 9, 2018
Attya Omer (1,2,8), Yun Li (2,3,4,8), Julien Muffat (2,4,5,8), Kuan-Chung Su (2), Malkiel A. Cohen (2), Tenzin Lungjangwa (2), Patrick Aubourg (1,6), Iain M. Cheeseman (2,7), and Rudolf Jaenisch (2,7).
1. Université Paris-Saclay, ED 569, 5 Rue Jean-Baptiste Clément, 92290 Châtenay-Malabry, France
2.  Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA
3. Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M4G 0A4, Canada
4. Department of Molecular Genetics, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada
5. Program in Neurosciences and Mental Health, The Hospital for Sick Children, 686 Bay Street, Toronto, ON M5G 0A4, Canada
6. INSERM U1169, CHU Bicêtre Paris Sud, Le Kremlin-Bicêtre, France.
7. Department of Biology, MIT, 31 Ames Street, Cambridge, MA 02139, USA
8. These authors contributed equally
The Y chromosome: Holding steadfast in a sea of change
Nicole Davis
August 2, 2018

The human Y chromosome is, in many ways, a study in contrasts. For decades, scientists have struggled to dissect its evolution in part because it does not have a genetic partner (or homolog), as all of the other human chromosomes do. That solitary existence means the Y chromosome is subject to some unusual evolutionary pressures. For example, it does not swap genetic material with a homologous chromosome — a practice known as recombination that other chromosomes follow — along the lion’s share of its length. However, its lack of recombination presents a unique opportunity: Because so much of its own genetic material stays put, scientists can trace the history of individual human Y chromosomes much further back in time than other chromosomes — in fact, they can go as far back as the data will allow.

That is precisely the approach taken by a team of Whitehead Institute researchers, led by Whitehead Institute Director David Page, who is also a professor of biology at the Massachusetts Institute of Technology and a Howard Hughes Medical Institute investigator. Their work is published in the August 2nd online issue of the American Journal of Human Genetics. Page and his colleagues, including graduate student and first author Levi Teitz, set out to examine a series of regions on the Y chromosome called amplicons — vast stretches of DNA, from tens of thousands to millions of nucleotides in length, which are present in two or more copies per chromosome. While the DNA contained in amplicons is often highly repetitive, it also houses biologically important genes. Although the precise functions of many of these genes remains to be determined, some have been found to play important roles in the development of sperm cells and testicular cancer. However, the amplicons vary drastically among species, so scientists cannot look to other organisms such as mice or chimpanzees to help reconstruct their past.

Page’s team zeroed in on these amplicons. Specifically, they looked at how the number of amplicon copies varies from one person’s Y chromosome to another. The researchers developed sophisticated computational tools to analyze DNA sequencing data collected from more than 1,200 males as part of the 1000 Genomes Project. What they discovered was quite surprising. Although the amplicons are quite variable, they found that overall, the configuration of amplicon copies on the Y chromosome has been painstakingly maintained over the last 300,000 years of human evolution. That means that despite the high level of mutation the chromosome experiences, evolutionary forces work to counteract this change and preserve its ancestral structure.

More work is needed to determine which aspects of the amplicons’ structure are important for chromosome biology, and in turn proper male development and fertility. However, the efforts of Teitz, Page, and their colleagues shed new light on the unusual tricks the solo chromosome uses to maintain its genomic integrity.

This research is supported by the National Institutes of Health and the Howard Hughes Medical Institute.

 

Written by Nicole Davis

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David Page’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 Howard Hughes Medical Institute Investigator and a Professor of Biology at the Massachusetts Institute of Technology.

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Full citation:

“Selection Has Countered High Mutability to Preserve the Ancestral Copy Number of Y Chromosome Amplicons in Diverse Human Lineages”

American Journal of Human Genetics, online August 2, 2018.

Levi S. Teitz (1,2), Tatyana Pyntikova (1), Helen Skaletsky (1,3), and David C. Page (1,2,3).

1. Whitehead Institute, Cambridge, MA 02142, USA

2. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA

3. Howard Hughes Medical Institute, Whitehead Institute, Cambridge, MA 02142, USA

Fasting boosts stem cells’ regenerative capacity

A drug treatment that mimics fasting can also provide the same benefit, study finds.

Anne Trafton | MIT News Office
May 1, 2018

As people age, their intestinal stem cells begin to lose their ability to regenerate. These stem cells are the source for all new intestinal cells, so this decline can make it more difficult to recover from gastrointestinal infections or other conditions that affect the intestine.

This age-related loss of stem cell function can be reversed by a 24-hour fast, according to a new study from MIT biologists. The researchers found that fasting dramatically improves stem cells’ ability to regenerate, in both aged and young mice.

In fasting mice, cells begin breaking down fatty acids instead of glucose, a change that stimulates the stem cells to become more regenerative. The researchers found that they could also boost regeneration with a molecule that activates the same metabolic switch. Such an intervention could potentially help older people recovering from GI infections or cancer patients undergoing chemotherapy, the researchers say.

“Fasting has many effects in the intestine, which include boosting regeneration as well as potential uses in any type of ailment that impinges on the intestine, such as infections or cancers,” says Omer Yilmaz, an MIT assistant professor of biology, a member of the Koch Institute for Integrative Cancer Research, and one of the senior authors of the study. “Understanding how fasting improves overall health, including the role of adult stem cells in intestinal regeneration, in repair, and in aging, is a fundamental interest of my laboratory.”

David Sabatini, an MIT professor of biology and member of the Whitehead Institute for Biomedical Research and the Koch Institute, is also a senior author of the paper, which appears in the May 3 issue of Cell Stem Cell.

“This study provided evidence that fasting induces a metabolic switch in the intestinal stem cells, from utilizing carbohydrates to burning fat,” Sabatini says. “Interestingly, switching these cells to fatty acid oxidation enhanced their function significantly. Pharmacological targeting of this pathway may provide a therapeutic opportunity to improve tissue homeostasis in age-associated pathologies.”

The paper’s lead authors are Whitehead Institute postdoc Maria Mihaylova and Koch Institute postdoc Chia-Wei Cheng.

Boosting regeneration

For many decades, scientists have known that low caloric intake is linked with enhanced longevity in humans and other organisms. Yilmaz and his colleagues were interested in exploring how fasting exerts its effects at the molecular level, specifically in the intestine.

Intestinal stem cells are responsible for maintaining the lining of the intestine, which typically renews itself every five days. When an injury or infection occurs, stem cells are key to repairing any damage. As people age, the regenerative abilities of these intestinal stem cells decline, so it takes longer for the intestine to recover.

“Intestinal stem cells are the workhorses of the intestine that give rise to more stem cells and to all of the various differentiated cell types of the intestine. Notably, during aging, intestinal stem function declines, which impairs the ability of the intestine to repair itself after damage,” Yilmaz says. “In this line of investigation, we focused on understanding how a 24-hour fast enhances the function of young and old intestinal stem cells.”

After mice fasted for 24 hours, the researchers removed intestinal stem cells and grew them in a culture dish, allowing them to determine whether the cells can give rise to “mini-intestines” known as organoids.

The researchers found that stem cells from the fasting mice doubled their regenerative capacity.

“It was very obvious that fasting had this really immense effect on the ability of intestinal crypts to form more organoids, which is stem-cell-driven,” Mihaylova says. “This was something that we saw in both the young mice and the aged mice, and we really wanted to understand the molecular mechanisms driving this.”

Metabolic switch

Further studies, including sequencing the messenger RNA of stem cells from the mice that fasted, revealed that fasting induces cells to switch from their usual metabolism, which burns carbohydrates such as sugars, to metabolizing fatty acids. This switch occurs through the activation of transcription factors called PPARs, which turn on many genes that are involved in metabolizing fatty acids.

The researchers found that if they turned off this pathway, fasting could no longer boost regeneration. They now plan to study how this metabolic switch provokes stem cells to enhance their regenerative abilities.

They also found that they could reproduce the beneficial effects of fasting by treating mice with a molecule that mimics the effects of PPARs. “That was also very surprising,” Cheng says. “Just activating one metabolic pathway is sufficient to reverse certain age phenotypes.”

Jared Rutter, a professor of biochemistry at the University of Utah School of Medicine, described the findings as “interesting and important.”

“This paper shows that fasting causes a metabolic change in the stem cells that reside in this organ and thereby changes their behavior to promote more cell division. In a beautiful set of experiments, the authors subvert the system by causing those metabolic changes without fasting and see similar effects,” says Rutter, who was not involved in the research. “This work fits into a rapidly growing field that is demonstrating that nutrition and metabolism has profound effects on the behavior of cells and this can predispose for human disease.”

The findings suggest that drug treatment could stimulate regeneration without requiring patients to fast, which is difficult for most people. One group that could benefit from such treatment is cancer patients who are receiving chemotherapy, which often harms intestinal cells. It could also benefit older people who experience intestinal infections or other gastrointestinal disorders that can damage the lining of the intestine.

The researchers plan to explore the potential effectiveness of such treatments, and they also hope to study whether fasting affects regenerative abilities in stem cells in other types of tissue.

The research was funded by the National Institutes of Health, the V Foundation, a Sidney Kimmel Scholar Award, a Pew-Stewart Trust Scholar Award, the Kathy and Curt Marble Cancer Research Fund, the MIT Stem Cell Initiative through Fondation MIT, the Koch Institute Frontier Research Program through the Kathy and Curt Marble Cancer Research Fund, the American Federation of Aging Research, the Damon Runyon Cancer Research Foundation, the Robert Black Charitable Foundation, a Koch Institute Ludwig Postdoctoral Fellowship, a Glenn/AFAR Breakthroughs in Gerontology Award, and the Howard Hughes Medical Institute.

Computing changes in cell fate

Meena Chakraborty ’19 has spent two years in the lab of Nobel Prize winner Philip Sharp, combining computer science and wet lab techniques to study the impact of microRNAs on gene expression.

Raleigh McElvery
May 2, 2018

When Meena Chakraborty was eleven years old, her parents took her to South Africa to show her what life was like outside her hometown of Lexington, Massachusetts. The trip was first and foremost a family vacation, but what struck Chakraborty, now a junior at MIT, was neither the sights nor safaris, but their visit to a children’s hospital. Looking back, she identifies that experience as the catalyst that spurred her current career path, centered on three years of biology research with implications for human health.

“I remember being astounded that the patients there were my age,” she says. “I had all these things in my life to look forward to, while they were fighting HIV and might not survive. That’s when I started thinking that I could do something to counter disease, and studying biology seemed like the best way to do that.”

Up until that point, she’d intended to be a writer. So when it came time to choose a college, she initially shied away from MIT, fearing it would be too “tech-focused.”

“Even though I was primarily interested in biology, I still wanted diversity in terms of the academic subjects and the people around me,” she says. “But it became clear that MIT really encourages you to step outside your major. Every undergrad has to complete a Humanities, Arts, or Social Sciences concentration, and I chose philosophy. Those classes have become a staple of my undergrad experience, and allowed me to keep in touch with my love for writing while still focusing on my science.”

Given her propensity for math, she declared Course 6-7 (Computer Science and Molecular Biology), as a means to develop analytical tools to decipher large data sets and better understand biological systems. The summer after her freshman year, she had her first chance to marry these two skills in a real-world setting: she began working in the lab of Nobel Prize winner Philip Sharp, located in the Koch Institute for Integrative Cancer Research.

This was her first foray into computational biology, but it wasn’t her first time at the Koch — she’d shadowed two graduates students in the Irvine lab for a summer as a junior in high school. This time, though, as an undergraduate, she was assigned her own project, under the guidance of postdoctoral fellow Salil Garg. Together, they’ve studied a type of RNA known as microRNA (miRNA) for the past two years.

Messenger RNA (mRNA) — perhaps the most well-known of the RNAs — constitutes the intermediate step between DNA and the final product of gene expression: the protein. In contrast, miRNAs are never translated into proteins. Instead, they bind to complementary sequences in target mRNAs, preventing those mRNAs from being turned into proteins, and blocking gene expression.

This miRNA-directed silencing is widespread and complex. In some cases, miRNAs silence single genes. In others, multiple miRNAs coordinate to turn groups of genes on and off in concert, thereby controlling entire sets of genes that interact with one another.  For example, two years ago, Chakraborty’s mentor used computational methods to pinpoint a group of poorly expressed, understudied “nonclassical” miRNAs that appear to coordinate the expression of pluripotency genes. Pluripotency gene levels dictate the behavior and fate of embryonic stem cells — non-specialized cells awaiting instructions to “differentiate” and assume a particular cell type (skin cell, blood cell, neuron, and so on).

Chakraborty then used a technique known as fluorescence-activated cell sorting (FACS) to determine how nonclassical miRNAs affect gene expression in embryonic stem cells. She used a FACS assay to detect miRNA activity, engineering special DNA and inserting it into mouse embryonic stem cells. The DNA contained two genes: one encoding a red fluorescent protein with a place for miRNAs to bind, and another that makes a blue fluorescent protein and lacks this miRNA attachment site. When the miRNA binds to the gene expressing the red fluorescing protein, it is silenced, and the cell makes fewer red proteins compared to blue ones, whose production remains unhindered.

“We know when miRNAs are active, they will reduce the expression of the red florescent protein, but not the blue one,” she says. “And that’s precisely what we’ve seen with these nonclassical miRNAs, suggesting that they are active in the cell.”

Chakraborty is excited about what this finding could mean for cancer research. A growing number of studies have shown that some cancers arise when miRNAs fail to help embryonic stem cells interconvert between cell states.

Although she spends anywhere from four to 20 hours a week in lab, Chakraborty hasn’t lost sight of her extracurriculars. As co-president of the Biology Undergraduate Student Association, she serves as a liaison between biology students and faculty, coordinating events to connect the two. As the discussion chair for the Effective Altruism Club, she promotes dialogue between student club members regarding charities — how these organizations can maximize their donations, and how the public should decide which ones to support. As a volunteer for the non-profit Help at Your Door, she inputs grocery lists from senior citizens and disabled individuals into a computer program, and then coordinates with community members to deliver the specified order.

Last summer, she was accepted into the Johnson & Johnson UROP Scholars Program, joining approximately 20 fellow undergraduate women in STEM research at MIT during the summer term. Her cohort attended faculty presentations, workshops, and networking events geared towards post-graduate careers in the sciences.

“I really appreciated that program, because I think a lot of women are afraid of science due to societal norms,” she says. “I remember originally thinking I wouldn’t be good at computer science or math, and now here I am combining both skills with wet lab techniques in my research.”

Most recently, Chakraborty was a recipient of the 2017-2018 Barry Goldwater Scholarship Award, selected from a nationwide field of candidates nominated by university faculty. She will also remain on campus this coming summer to conduct faculty-mentored research as part of the MIT Amgen Scholars Program.

After she graduates in 2019, Chakraborty intends to pursue a PhD in a biology-related discipline, perhaps computational biology. After that, the options are endless — professor, consultant, research scientist. She’s still weighing the possibilities, and doesn’t seem too concerned about selecting one just yet.

“I know I’m going in the right direction, because it hits me every time I finish a challenging assignment or whenever I figure out a new approach in the lab,” she says. “When I complete a task like that with the help of friends and mentors, there’s this sense of pride and a feeling that I can’t believe how much I’ve learned in just once semester. The way my brain considers problems and finds solutions is just so different from the way it was three years ago when I first started out as a freshman.”

Photo credit: Raleigh McElvery
Single-cell database to propel biological studies

Whitehead team analyzes transcriptomes for roughly 70,000 cells in planarians, creates publicly available database to drive further research.

Nicole Davis | Whitehead Institute
April 20, 2018

A team at Whitehead Institute and MIT has harnessed single-cell technologies to analyze over 65,000 cells from the regenerative planarian flatworm, Schmidtea mediterranea, revealing the complete suite of actives genes (or “transcriptome”) for practically every type of cell in a complete organism. This transcriptome atlas represents a treasure trove of biological information on planarians, which is the subject of intense study in part because of its unique ability to regrow lost or damaged body parts. As described in the April 19 advance online issue of the journal Science, this new, publicly available resource has already fueled important discoveries, including the identification of novel planarian cell types, the characterization of key transition states as cells mature from one type to another, and the identity of new genes that could impart positional cues from muscles cells — a critical component of tissue regeneration.

“We’re really at the beginning of an amazing era,” says senior author Peter Reddien, a member of Whitehead Institute, professor of biology at MIT, and investigator with the Howard Hughes Medical Institute. “Just as genome sequences became indispensable resources for studying the biology of countless organisms, analyzing the transcriptomes of every cell type will become another fundamental tool — not just for planarians, but for many different organisms.”

The ability to systematically reveal which genes in the genome are active within an individual cell flows from a critical technology known as single-cell RNA sequencing. Recent advances in the technique have dramatically reduced the per-cell cost, making it feasible for a single laboratory to analyze a suitably large number of cells to capture the cell type diversity present in complex, multi-cellular organisms.

Reddien has maintained a careful eye on the technology from its earliest days because he believed it offered an ideal way to unravel planarian biology. “Planarians are relatively simple, so it would be theoretically possible for us to capture every cell type. Yet they still have a sufficiently large number of cells — including types we know little or even nothing about,” he explains. “And because of the unusual aspects of planarian biology — essentially, adults maintain developmental information and progenitor cells that in other organisms might be present transiently only in embryos — we could capture information about mature cells, progenitor cells, and information guiding cell decisions by sampling just one stage, the adult.”

Two and a half years ago, Reddien and his colleagues — led by first author Christopher Fincher, a graduate student in Reddien’s laboratory — set out to apply single-cell RNA sequencing systematically to planarians. The group isolated individual cells from five regions of the animal and gathered data from a total of 66,783 cells. The results include transcriptomes for rare cell types, such as those that comprise on the order of 10 cells out of an adult animal that consists of roughly 500,000 to 1 million cells.

In addition, the researchers uncovered some cell types that have yet to be described in planarians, as well cell types common to many organisms, making the atlas a valuable tool across the scientific community. “We identified many cells that were present widely throughout the animal, but had not been previously identified. This surprising finding highlights the great value of this approach in identifying new cells, a method that could be applied widely to many understudied organisms,” Fincher says.

“One main important aspect of our transcriptome atlas is its utility for the scientific community,” Reddien says. “Because many of the cell types present in planarians emerged long ago in evolution, similar cells still exist today in various organisms across the planet. That means these cell types and the genes active within them can be studied using this resource.”

The Whitehead team also conducted some preliminary analyses of their atlas, which they’ve dubbed “Planarian Digiworm.” For example, they were able to discern in the transcriptome data a variety of transition states that reflect the progression of stem cells into more specialized, differentiated cell types. Some of these cellular transition states have been previously analyzed in painstaking detail, thereby providing an important validation of the team’s approach.

In addition, Reddien and his colleagues knew from their own prior, extensive research that there is positional information encoded in adult planarian muscle — information that is required not only for the general maintenance of adult tissues but also for the regeneration of lost or damaged tissue. Based on the activity pattern of known genes, they could determine roughly which positions the cells had occupied in the intact animal, and then sort through those cells’ transcriptomes to identify new genes that are candidates for transmitting positional information.

“There are an unlimited number of directions that can now be taken with these data,” Reddien says. “We plan to extend our initial work, using further single-cell analyses, and also to mine the transcriptome atlas for addressing important questions in regenerative biology. We hope many other investigators find this to be a very valuable resource, too.”

This work was supported by the National Institutes of Health, the Howard Hughes Medical Institute, and the Eleanor Schwartz Charitable Foundation.

Study suggests method for boosting growth of blood vessels and muscle

Activating proteins linked to longevity may help to increase endurance and combat frailty in the elderly.

Anne Trafton | MIT News Office
March 22, 2018

As we get older, our endurance declines, in part because our blood vessels lose some of their capacity to deliver oxygen and nutrients to muscle tissue. An MIT-led research team has now found that it can reverse this age-related endurance loss in mice by treating them with a compound that promotes new blood vessel growth.

The study found that the compound, which re-activates longevity-linked proteins called sirtuins, promotes the growth of blood vessels and muscle, boosting the endurance of elderly mice by up to 80 percent.

If the findings translate to humans, this restoration of muscle mass could help to combat some of the effects of age-related frailty, which often lead to osteoporosis and other debilitating conditions.

“We’ll have to see if this plays out in people, but you may actually be able to rescue muscle mass in an aging population by this kind of intervention,” says Leonard Guarente, the Novartis Professor of Biology at MIT and one of the senior authors of the study. “There’s a lot of crosstalk between muscle and bone, so losing muscle mass ultimately can lead to loss of bone, osteoporosis, and frailty, which is a major problem in aging.”

The first author of the paper, which appears in Cell on March 22, is Abhirup Das, a former postdoc in Guarente’s lab who is now at the University of New South Wales in Australia. Other senior authors of the paper are David Sinclair, a professor at Harvard Medical School and the University of New South Wales, and Zolt Arany, a professor at the University of Pennsylvania.

Race against time

In the early 1990s, Guarente discovered that sirtuins, a class of proteins found in nearly all animals, protect against the effects of aging in yeast. Since then, similar effects have been seen in many other organisms.

In their latest study, Guarente and his colleagues decided to explore the role of sirtuins in endothelial cells, which line the inside of blood vessels. To do that, they deleted the gene for SIRT1, which encodes the major mammalian sirtuin, in endothelial cells of mice. They found that at 6 months of age, these mice had reduced capillary density and could run only half as far as normal 6-month-old mice.

The researchers then decided to see what would happen if they boosted sirtuin levels in normal mice as they aged. They treated the mice with a compound called NMN, which is a precursor to NAD, a coenzyme that activates SIRT1. NAD levels normally drop as animals age, which is believed to be caused by a combination of reduced NAD production and faster NAD degradation.

After 18-month-old mice were treated with NMN for two months, their capillary density was restored to levels typically seen in young mice, and they experienced a 56 to 80 percent improvement in endurance. Beneficial effects were also seen in mice up to 32 months of age (comparable to humans in their 80s).

“In normal aging, the number of blood vessels goes down, so you lose the capacity to deliver nutrients and oxygen to tissues like muscle, and that contributes to decline,” Guarente says. “The effect of the precursors that boost NAD is to counteract the decline that occurs with normal aging, to reactivate SIRT1, and to restore function in endothelial cells to give rise to more blood vessels.”

These effects were enhanced when the researchers treated the mice with both NMN and hydrogen sulfide, another sirtuin activator.

Vittorio Sartorelli, a principal investigator at the National Institute of Allergy and Infectious Diseases who was not involved in the research, described the experiments as “elegant and compelling.” He added that “it will be of interest and of clinical relevance to evaluate the effect of NMN and hydrogen sulfide on the vascularization of other organs such as the heart and brain, which are often damaged by acutely or chronically reduced blood flow.”

Benefits of exercise

The researchers also found that SIRT1 activity in endothelial cells is critical for the beneficial effects of exercise in young mice. In mice, exercise generally stimulates growth of new blood vessels and boosts muscle mass. However, when the researchers knocked out SIRT1 in endothelial cells of 10-month-old mice, then put them on a four-week treadmill running program, they found that the exercise did not produce the same gains seen in normal 10-month-old mice on the same training plan.

If validated in humans, the findings would suggest that boosting sirtuin levels may help older people retain their muscle mass with exercise, Guarente says. Studies in humans have shown that age-related muscle loss can be partially staved off with exercise, especially weight training.

“What this paper would suggest is that you may actually be able to rescue muscle mass in an aging population by this kind of intervention with an NAD precursor,” Guarente says.

In 2014, Guarente started a company called Elysium Health, which sells a dietary supplement containing a different precursor of NAD, known as NR, as well as a compound called pterostilbene, which is an activator of SIRT1.

The research was funded by the Glenn Foundation for Medical Research, the Sinclair Gift Fund, a gift from Edward Schulak, and the National Institutes of Health.

A blueprint for regeneration

Whitehead Institute researchers uncover framework for how stem cells determine where to form replacement structures.

Lisa Girard | Whitehead Institute
March 15, 2018

Researchers at Whitehead Institute have uncovered a framework for regeneration that may explain and predict how stem cells in adult, regenerating tissue determine where to form replacement structures.

In a paper that appeared online March 15 in the journal Science, the researchers describe a model for planarian (flatworm) eye regeneration that is governed by three principles acting in concert, which inform how progenitor cells behave in regeneration. The model invokes positional cues that create a scalable map; self-organization that attracts progenitors to existing structures; and progenitor cells that originate in a diffuse spatial zone, rather than a precise location, allowing flexibility in their path. These principles appear to dictate how progenitor cells decide where to go during regeneration to recreate form and function, and they bring us closer to a systems-level understanding of the process.

From previous work, the researchers knew that stem cells are likely reading out instructions from neighboring tissues to guide their path, and it became clear that the process faces some serious challenges in regeneration. “We realized that positional information has to move; it needs to change during regeneration in order to specify the new missing parts to be regenerated. This revised information can then guide progenitor cells that are choosing to make new structures to differentiate into the correct anatomy at the correct locations,” says the paper’s senior author Peter Reddien, a Whitehead Institute researcher, an MIT professor of biology, and a Howard Hughes Medical Institute (HHMI) investigator. “There is a puzzle that emerges, however. Since positional information shifts after injury during regeneration, there is a mismatch between the positional information pattern and the remaining anatomy pattern. Realizing this mismatch exists was a trigger for our study. We wanted to understand how stem cells making particular tissues decide where to go and differentiate. Is it based on anatomy, or is it based on positional information? And when those two things are not aligned, how do they decide?”

Reddien and his lab have spent over a decade unraveling the mysteries of regeneration using a small flatworm, called the planarian. If a planarian’s head is amputated, or its side is removed, each piece will regenerate an entire animal. In order to understand how progenitors decide where to go in the noisy environment of animal regeneration, the researchers used the planarian eye, a visible organ that is small enough to be removed without serious injury and has the added advantage of having defined progenitor cell molecular markers.

The researchers devised a simple experiment to resolve the question of how the progenitors decide where to go: amputate the animal’s head, then after three days remove one of the eyes from the head piece. What they found was that progenitor cells would nucleate a new eye in a position anterior (closer to the tip) to the remaining eye, rather than in the “correct” position specified by the anatomy, symmetrical with the current eye. However, if the same experiment is done, except with one of the eyes removed from the head piece earlier — the same day as the amputation, rather than three days later — there is a different result: The new eye is nucleated at a position symmetrical to the remaining eye, at the “correct” position according to the anatomy, suggesting that when the choices are conflicting, anatomical self-organizing dynamics win.

These simple rules guide the system to successful regeneration and also yield surprising outcomes when the system is pushed to its limits, producing alternative stable anatomical states with three-, four-, and five-eyed animals. Resecting both the side of an animal and amputating the head can place progenitors far enough from existing, self-organizing attractors that they miss them, allowing them to nucleate a new attractor — a third eye — in the head fragments. “If the migrating progenitors are too close to the attractor that is the existing eye, it will suck them in, just like a black hole; if they are far enough, they can escape,” says Kutay Deniz Atabay, first author on the paper and a graduate student in the Reddien lab.

When the researchers did the same surgery on a three-eyed animal, the progenitors miss the attraction of existing eyes and form a five-eyed animal. In each of these cases, all of the eyes are functionally integrated into the brain and exhibit the same light-avoidance response of the planarian eye. The researchers also observed that these alternative anatomical states endure, revealing rules for maintenance of existing and alternative anatomical structures. “This recipe helps provide an explanation for a fundamental problem of animal regeneration, which is how migratory progenitors make choices that lead the system to regenerate and maintain organs,” Atabay says.

“These studies contribute to a proposed framework for regeneration in which competing forces, self-organization, and extrinsic cues, are the guideposts impacting the choice of progenitor targeting in regeneration; and those two forces together determine the outcome,” Reddien says.

This work was supported by the National Institutes of Health (NIH) and the HHMI. Additional funding was provided by the MIT Presidential Fellowship Program and a National Defense Science and Engineering Graduate Fellowship.