The presence of a third copy of chromosome 21 causes all the other chromosomes to squish inward, not unlike when people in a crowded elevator must narrow their stance when one more person squeezes in. The main effects of this are more genetic interactions within each chromosome and less interactions among them. These changes and differences in DNA conformation within the cell nucleus lead to changes in how genes are transcribed and therefore expressed, causing important differences in cell function that affect brain development.
In Down syndrome, the third copy of chromosome 21 causes a reorganization of the 3D configuration of the entire genome in a key cell type of the developing brain, a new study shows. The resulting disruption of gene transcription and cell function are so similar to those seen in cellular aging, or senescence, that the scientists leading the study found they could use anti-senescence drugs to correct them in cell cultures.
The study published in Cell Stem Cell therefore establishes senescence as a potentially targetable mechanism for future treatment of Down syndrome, said Hiruy Meharena, a new assistant professor at the University of California San Diego who led the work as a Senior Alana Fellow in the Alana Down Syndrome Center at MIT.
“There is a cell-type specific genome-wide disruption that is independent of the gene dosage response,” Meharena said. “It’s a very similar phenomenon to what’s observed in senescence. This suggests that excessive senescence in the developing brain induced by the third copy of chromosome 21 could be a key reason for the neurodevelopmental abnormalities seen in Down syndrome.”
The study’s finding that neural progenitor cells (NPCs), which develop into major cells in the brain including neurons, have a senescent character is remarkable and novel, said senior author Li-Huei Tsai, but it is substantiated by the team’s extensive work to elucidate the underlying mechanism of the effects of abnormal chromosome number, or aneupoloidy, within the nucleus of the cells.
“This study illustrates the importance of asking fundamental questions about the underlying mechanisms of neurological disorders,” said Tsai, Picower Professor of Neuroscience, director of the Alana Center, and of The Picower Institute for Learning and Memory at MIT. “We didn’t begin this work expecting to see senescence as a translationally relevant feature of Down syndrome, but the data emerged from asking how the presence of an extra chromosome affects the architecture of all of a cell’s chromosomes during development.”
Genomewide changes
Meharena and co-authors spent years measuring distinctions between human cell cultures that differed only by whether they had a third copy of chromosome 21. Stem cells derived from volunteers were cultured to turn into NPCs. In both the stem cells and the NPCs, the team examined 3D chromosome architecture, several metrics of DNA structure and interaction, gene accessibility and transcription, and gene expression. They also looked at the consequences of the gene expression differences on important functions of these developmental cells, such as how well they proliferated and migrated in 3D brain tissue cultures. Stem cells were not particularly different, but NPCs were substantially affected by the third copy of chromosome 21.
Overall, the picture that emerged in NPCs was that the presence of a third copy causes all the other chromosomes to squish inward, not unlike when people in a crowded elevator must narrow their stance when one more person squeezes in. The main effects of this “chromosomal introversion,” meticulously quantified in the study, are more genetic interactions within each chromosome and less interactions among them. These changes and differences in DNA conformation within the cell nucleus lead to changes in how genes are transcribed and therefore expressed, causing important differences in cell function that affect brain development.
Treated as senescence
For the first couple of years as these data emerged, Meharena said, the full significance of the genomic changes were not apparent, but then he read a paper showing very similar genomic rearrangement and transcriptional alterations in senescent cells.
After validating that the Down syndrome cells indeed bore such a similar signature of transcriptional differences, the team decided to test whether anti-senescence drugs could undo the effects. They tested a combination of two: dasatinib and quercetin. The medications improved not only gene accessibility and transcription, but also the migration and proliferation of cells.
That said, the drugs have very significant side effects—dasatinib is only given to cancer patients when other treatments have not done enough—so they are not appropriate for attempting to intervene in brain development amid Down syndrome, Meharena said. Instead an outcome of the study could be to inspire a search for medications that could have anti-senolytic effects with a safer profile.
Senescence is a stress response of cells. At the same time, years of research by former MIT biology professor Angelika Amon, who co-directed the Alana Center with Tsai, has shown that aneuploidy is a source of considerable stress for cells. A question raised by the new findings, therefore, is whether the senescence-like character of Down syndrome NPCs is indeed the result of an aneuploidy induced stress and if so, exactly what that stress is.
Another implication of the findings is how excessive senescence among brain cells might affect people with Down syndrome later in life. The risk of Alzheimer’s disease is much higher at a substantially earlier age in the Down syndrome population than among people in general. In large part this is believed to be because a key Alzheimer’s risk gene, APP, is on chromosome 21, but the newly identified inclination for senescence may also accelerate Alzheimer’s development.
In addition to Meharena and Tsai, the paper’s other authors are Asaf Marco, Vishnu Dileep, Elana Lockshin, Grace Akatsu, James Mullahoo, Ashley Watson, Tak Ko, Lindsey Guerin, Fatema Abdurrob, Shruti Rengarajan, Malvina Papanastasiou and Jacob Jaffe.
The Alana Foundation, the LuMind Foundation, Burroughs Wellcome Fund, UNCF-Merck and the National Institutes of Health funded the research.
A path for addressing Alzheimer’s blood-brain barrier impairment
Brain's "keep out" system is compromised in Alzheimer's disease
Detail from a painting by co-author Leyla Akay inspired by the paper.
By developing a lab-engineered model of the human blood-brain barrier (BBB), neuroscientists at MIT’s Alana Down Syndrome Center have discovered how the most common Alzheimer’s disease risk gene causes amyloid protein plaques to disrupt the brain’s vasculature and showed they could prevent the damage with medications already approved for human use.
About 25 percent of people have the APOE4 variant of the APOE gene, which puts them at substantially greater risk for Alzheimer’s disease. Almost everyone with Alzheimer’s, and even some elderly people without, suffer from cerebral amyloid angiopathy (CAA), a condition in which amyloid protein deposits on blood vessel walls impairs the ability of the BBB to properly transport nutrients, clear out waste and prevent the invasion of pathogens and unwanted substances.
In the new study, published June 8 in Nature Medicine, the researchers pinpointed the specific vascular cell type (pericytes) and molecular pathway (calcineurin/NFAT) through which the APOE4 variant promotes CAA pathology.
The research indicates that in people with the APOE4 variant, pericytes in their vessels churn out too much APOE protein, explained senior author Li-Huei Tsai, Picower Directory & Professor of Neuroscience and co-Director of the Alana Down Syndrome Center. APOE causes amyloid proteins, which are more abundant in Alzheimer’s disease, to clump together. Meanwhile, the diseased pericytes’ increased activation of the calcineurin/NFAT molecular pathway appears to encourage the elevated APOE expression.
There are already drugs that suppress the pathway. Currently they are used to subdue the immune system after a transplant. When the researchers administered some of those drugs, including cyclosporine A and FK506, to the lab-grown BBBs with the APOE4 variant, they accumulated much less amyloid than untreated ones did.
“We identify that there is a specific genetic pathway that is expressed differently in a population that is susceptible to Alzheimer’s disease,” said study lead author Joel Blanchard, a postdoc in Tsai’s lab. “By identifying this we could identify drugs that change this pathway back to a non-diseased state and correct this outcome that’s associated with Alzheimer’s.”
Building barriers
To investigate the connection between Alzheimer’s, the APOE4 variant and CAA, Blanchard, Tsai and co-authors coaxed human induced pluripotent stem cells to become the three types of cells that make up the BBB: brain endothelial cells, astrocytes and pericytes. Pericytes were modeled by mural cells that they tested extensively to ensure they exhibited pericyte-like properties and gene expression.
Grown for two weeks within a three-dimensional hydrogel scaffold, the BBB model cells assembled into vessels that exhibited natural BBB properties, including low permeability to molecules and expression of the same key genes, proteins and molecular pumps as natural BBBs. When immersed in culture media high in amyloid proteins, mimicking conditions in Alzheimer’s disease brains, the lab-grown BBB models exhibited the same kind of amyloid accumulation seen in human disease.
With a model BBB established, they then sought to test the difference APOE4 makes. They showed by several measures that APOE4-carrying BBB models accumulated more amyloid from culture media than those carrying APOE3, the more typical and healthy variant.
To pinpoint how APOE4 makes that difference, they engineered eight different versions covering all the possible combinations of the three cell types having either APOE3 or APOE4. When exposed these month-old models to amyloid-rich media, only versions with APOE4 pericyte-like mural cells showed excessive accumulation of amyloid proteins. Replacing APOE4 mural cells with APOE3-carrying ones reduced amyloid deposition. These results put blame for CAA-like pathology squarely on pericytes.
To further validate the clinical relevance of these findings, the team also looked at APOE expression in samples of human brain vasculature in the prefrontal cortex and the hippocampus, two regions crucially affected in Alzheimer’s disease. Consistent with the team’s lab BBB model, people with APOE4 showed higher expression of the gene in the vasculature, and specifically in pericytes, than people with APOE3.
“That is a salient point of this paper,” said Tsai. “It’s really cool because it stresses the cell-type specific function of APOE.”
A pathway toward treatment?
The next step was to determine how APOE4 becomes so overexpressed by pericytes. The team therefore identified hundreds of transcription factors – proteins that determine how genes are expressed – that were regulated differently between APOE3 and APOE4 pericyte-like mural cells. Then they scoured that list to see which factors specifically impact APOE expression. A set of factors that were upregulated in APOE4 cells stood out: ones that were part of the calcineurin/NFAT pathway. They observed similar upregulation of the pathway in pericytes from human hippocampus samples.
As part of their investigation of whether elevated signaling activity of this pathway caused increased amyloid deposition and CAA, they tested cyclosporine A and FK506 because they tamp pathway activity down. They found that the drugs reduced APOE expression in their pericyte-like mural cells and therefore APOE4-mediated amyloid deposits in the BBB models. They also tested the drugs in APOE4-carrying mice and saw that the medicines reduced APOE expression and amyloid buildup.
Blanchard and Tsai noted that the drugs can have significant side effects, so their findings might not suggest using exactly those drugs to address CAA in patients.
“Instead it points toward the value of understanding the mechanism,” Blanchard said. “It allows one to design a small molecule screen to find more potent drugs that have less off-target effects.”
In addition to Blanchard and Tsai, the paper’s other authors are Michael Bula, Jose Davila-Velderrain, Leyla Akay, Lena Zhu, Alexander Frank, Matheus Victor, Julia Maeve Bonner, Hansruedi Mathys, Yuan-Ta Lin, Tak Ko, David Bennett, Hugh Cam, and Manolis Kellis.
The Robert A. and Renee E. Belfer Family Foundation, the Cure Alzheimer’s Fund, The National Institutes of Health, the Glenn Foundation for Medical Research and the American Federation for Aging Research funded the research.
Written by David Orenstein, Picower Institute for Learning and Memory
Human Models for Neuroscience
Stem cell, genetic technologies enable sophisticated studies of human brain cells and brain "organoids"
An organoid, or "minibrain," labeled to show neurons and glial cells (credit: Kwanghun Chung lab)
Scientists cannot perform many experiments directly on people’s brains, but with new technologies, they can create human brain tissue models and ask important questions via experiments on those. Their findings could help them find new ways to improve brain health.
For decades, neuroscientists seeking to better understand human neurological conditions and develop new therapies have worked with the obvious limitation that a living patient’s brain is not open for investigation or experimentation at the genetic, molecular or cellular scale where many of the brain’s mysteries hide. Nonetheless, they’ve made extraordinary progress with studies in animal models and post-mortem human tissue.
But now neuroscientists are in a whole new era. Three technological breakthroughs over the past 12 years have given them a revolutionary way to study human brain disease: They can create cultures of brain cells derived from individual patients, and even engineer complex, three-dimensional “organoids” that mimic key aspects brain tissue. Scientists are only beginning to harness the potential of these new human cell and tissue models, and Picower Institute labs are helping lead the way. ADSC Director Li-Huei Tsai, and Professors Mriganka Sur and Kwanghun Chung are among a global vanguard that is making and analyzing these new patient-derived testbeds and applying them to study conditions such as Down syndrome, Alzheimer’s disease, Rett syndrome, and Zika virus infection.
While the whole field’s progress with these new capabilities has been rapid, so has been the recognition of their limits. That’s why rather than replacing animal models and other research methods, these new models are becoming integrated as powerful tools to complement broader research programs where findings from multiple methods often enhance each other’s value.
The new wave of breakthroughs began in 2007 when scientists showed how to take a cell from an individual’s body (often a skin cell) and to “reprogram” it to become an “induced pluripotent stem cell” (iPSC) that can then be biochemically guided to become any other cell, like a neuron or a supporting astrocyte or microglia. This development allowed scientists to make the brain cells that they of course would never directly extract from patients. By 2013, scientists began using iPSCs to grow 3D cultures of multiple brain cell types, or “organoids,” that can reproduce key aspects of brain development and intercellular interactions. That same year, scientists demonstrated that a technique called CRISPR/Cas9, could be used for precise genetic editing. Scientists quickly began using that to manipulate the genes in their iPSC-grown cultures and organoids, creating “isogenic pairs” where two otherwise identical stem cells contain a disease-causing or healthy version of a gene.
Tsai and lab members Jay Penney and William Ralvenius summarized and celebrated the significance of these breakthroughs for Alzheimer’s disease research in an August 2019 paper in Molecular Psychiatry.
“In little more than a decade since the advent of human iPSC technologies we have developed the ability to generate all the main brain cell types from pluripotent cells,” they wrote. “Increasingly complex 3D co-culture systems are also emerging that allow us to reconstitute many of the key interactions between brain cells. These technologies have already contributed greatly to our understanding of human development and human disease.”
Sur agreed, “It is perhaps the only way one can study a direct human model – it’s astonishing to be able to grow brain cells from a patient with a disease, derived from that person’s genetic material.”
Abundant applications
Picower labs have embarked on numerous studies using the models. In 2018, Tsai’s lab, led by Yuan-Ta Lin and Jinsoo Seo, published a paper in Neuron in which they used 2D single-cell-type iPSC cultures and 3D mixed-cell-type organoids to study the differences made by two versions of the leading risk gene for Alzheimer’s disease, APOE. People with the APOE4 variant are at much higher risk for the disease than APOE3 carriers but scientists haven’t been sure why. Tsai and Lin’s team used CRISPR/Cas9 to make isogenic pairs of neurons, astrocytes and microglia and spotted several key differences that likely help explain how APOE4 raises disease risk. For instance, APOE4 neurons secreted more potentially harmful amyloid proteins, APOE4 astrocytes showed dysregulated cholesterol metabolism and cleared less amyloid. APOE4 microglia, too, did a poorer job of getting rid of amyloid buildup. Using CRISPR to change APOE4 to APOE3, meanwhile, improved cell activity.
Elsewhere in the the Tsai lab, Joel Blanchard is using iPSCs to model the blood-brain barrier, which stringently filters what comes into and goes out of the brain through the blood stream, so he can see how APOE variants affect that. Researchers are also looking at how myelination – the process of wrapping neurons in a fatty sheath to improve their electrical conductivity – may differ in Alzheimer’s.
As part of the work in the Alana Down Syndrome Center, Tsai’s lab is also using iPSC-based cultures to study the difference that having a third copy of chromosome 21 makes in gene expression in a variety of brain cells and in the development of organoids. At October’s Society for Neuroscience (SfN) annual meeting, the team presented some initial results. Hiruy Meharena observed significant physical changes within chromosomes in various brain cell types with the syndrome’s three copies of chromosome 21 (trisomy) vs. when they have two copies (disomy). These changes, which appear especially prevalent in neural progenitor cells, occur genomewide and result in substantial differences in gene expression that are associated with brain development. Meanwhile, Lin showed how organoids grown from trisomy iPSCs vs. disomy ones have smaller diameters after 30 days of growth and show gene expression differences that may hinder development. Elana Lockshin focused on differences in glial cells, finding, for instance, that trisomy astrocytes don’t migrate during development as readily as in disomy ones.
Elana Lockshin, a member of the lab of Li-Huei Tsai, presents research on how neural support cells, or glia, are affected in trisomy 21.
Sur’s lab has been able to make important findings by using iPSCs as part of its studies of Rett syndrome, an autism-like disorder. In a study published in 2017 in Molecular Psychiatry, a team led by Nikolaos Mellios used isogenic 2D iPSC cultures to find that the disease-causing mutation in the gene MeCP2 led to misregulated forms of RNA that alter a key molecular pathway in early neural and brain development. They also used organoids to show that if they corrected regulation of those RNAs, they could restore healthier development. The study helped to show that Rett syndrome may begin to affect health even earlier than the onset of systems in toddlerhood.
Both the Tsai and Sur labs frequently collaborate with Kwanghun Chung, whose research group is dedicated to developing tools and technologies to help fellow scientists better visualize and quantitatively analyze tissues from the scale of whole human brains down to subcellular components like the synaptic connections between neurons. Chung has not only aided the Sur’ lab’s Rett syndrome studies but has also been working with MIT Health Sciences and Technology Professor Lee Gehrke in his lab’s efforts to quantify differences that may help explain what hinders the growth of organoids – and brains – with Zika infection.
“Organoids were pivotal in helping scientists discover how Zika causes microcephaly,” said Chung lab postdoc Alex Albanese. “We are taking a more in depth look at how Zika is actually modifying the structure and cell populations inside the organoids.”
Innovating past limits
A large part of the work in Chung’s lab, which is led by Albanese and Justin Swaney, aims to overcome some key fundamental limits of organoids. Though they are sometimes called “minibrains,” they really aren’t lab replicas of the real thing. A human brain, while extraordinarily complex, has a well-defined geography. Even though organoids are much simpler – with thousands of cells rather than about 100 billion – they are much more variable in how they turn out. While a real brain will develop highly distinct regions and exactly the right number of ventricles in the right places, organoids will recapitulate only an approximation of, say, cortical structure and may have enough ventricles to look more like Swiss cheese than a brain. Albanese calls this the “snowflake” problem, alluding to how organoids can differ so widely.
So how can they still be valuable models? One has to know how to assess them. Chung’s lab has developed advanced tissue processing technologies that can clarify, preserve, label and enlarge tissues, including organoids, so that properties like physiology and cellular function can be highlighted at all scales. Moreover, his lab has developed an imaging pipeline using light-sheet microscopy that is capable of capturing enormous amounts of data quickly (15 minutes per organoid), so that technicians can thoroughly image many organoids in a day.
Another limitation of organoids is that they aren’t actually that small. A millimeter or two of diameter may seem tiny, but that’s still big enough to present challenges. Traditional microscopes can’t image far enough into them to resolve what’s going on with deeper cells. Chung’s technologies overcome that problem both by clarifying tissue and labeling cells and proteins, but that requires chemically fixing the organoids. Yildirim’s “three photon” microscope technology doesn’t label cells as richly, but it can image all the way through organoids while they are alive and active. That’s how Sur’s lab was able to witness the erratic migration of new neurons. They’ve developed microfluidic multiple-well housings for organoids and devised methods for holding them perfectly steady for long-term imaging while still allowing nutrients and oxygen to circulate around them.
Mriganka Sur’s lab has developed a system for holding an organoid very stably while nutrients circulate around.
Indeed a significant problem associated with large size is that with no blood vessels to carry oxygen and nutrients in or to take waste out, the innermost cells can die. Keeping either the nutrients and oxygen, or the organoid, in motion helps keep them healthier than just growing them in dishes, research has shown. The Tsai lab recently began growing organoids by the thousands using a clever bioreactor device that keeps them spinning in the incubator. Postdoc Ping-Chieh Pao said the system saves space and uses less growth media, while yielding higher quality organoids with more mature neurons and less cell death.
Blood vessels are also integral to brain function. For instance, dysfunction in the blood-brain barrier plays a potentially pivotal role in Alzheimer’s. To build a human model of Alzheimer’s that explicitly accounts for vasculature in sickness and in health, Tsai and Blanchard recently secured a grant from the National Institutes of Health to build a “brain on a chip” that will engineer connections to unify their blood-brain barrier model with a co-culture of many brain cell types. The addition of vasculature and cell types like the oligodendrocytes that produce myelination will simulate a richer degree of Alzheimer’s complexity than human iPSC-based models have to date.
Yet another limitation of organoids has been that they don’t mimic multiple brain regions all that clearly, though they can be chemically coaxed to trend toward one or another. For that reason, organoids don’t provide much of a testbed for understanding the functional significance of inter-regional circuits. Delepine said the Sur lab is interested in experimenting with growing multiple organoids of different regional character on a chip and then nurturing connections between them to see if they can replicate such circuits.
Multiple models together
While Picower researchers and colleagues elsewhere work to make the most of human disease models, there are some limits that seem sure to endure, both for technical and ethical reasons. That means that traditional models, including animals, will remain integral to neuroscience research.
After all, organoids don’t think, behave or remember. They can’t gather any sensory input and do not exhibit consciousness. Lacking all these basic capabilities, they naturally can’t provide any data about how disease affects those vital functions of daily existence. In a recent study where Sur collaborated with MIT biologist Rudolf Jaenisch to test new drugs with the potential to treat Rett syndrome, Jaenisch’s lab screened the compounds on stem-cell derived human neurons, but Sur’s lab tested the most promising ones in mouse models, because that was the only way to see if they improved cognition and behavior.
“The question was, were they valid in a much more functional system,” Sur said.
Mellios’s study, too, drew upon and also utilized findings in Rett model mice.
“Our work in these two studies shows how mouse work can complement human work,” he said.
Especially when fighting disease, researchers will always draw upon whatever systems they can to help them find answers.
New method visualizes groups of neurons as they compute
Fluorescent probe could allow scientists to watch circuits within the brain and link their activity to specific behaviors.
Neurons in a mouse brain are labeled purple. In green, neurons are labeled with a fluorescent probe that reveals electrical activity.
Using a fluorescent probe that lights up when brain cells are electrically active, MIT and Boston University researchers have shown that they can image the activity of many neurons at once, in the brains of mice.
This technique, which can be performed using a simple light microscope, could allow neuroscientists to visualize the activity of circuits within the brain and link them to specific behaviors, says Edward Boyden, the Y. Eva Tan Professor in Neurotechnology and a professor of biological engineering and of brain and cognitive sciences at MIT.
“If you want to study a behavior, or a disease, you need to image the activity of populations of neurons because they work together in a network,” says Boyden, who is also a member of MIT’s McGovern Institute for Brain Research, Media Lab, and Koch Institute for Integrative Cancer Research, and is a member of the Alana Down Syndrome Center.
Using this voltage-sensing molecule, the researchers showed that they could record electrical activity from many more neurons than has been possible with any existing, fully genetically encoded, fluorescent voltage probe.
Boyden and Xue Han, an associate professor of biomedical engineering at Boston University, are the senior authors of the study, which appears in the Oct. 9 online edition of Nature. The lead authors of the paper are MIT postdoc Kiryl Piatkevich, BU graduate student Seth Bensussen, and BU research scientist Hua-an Tseng.
Seeing connections
Neurons compute using rapid electrical impulses, which underlie our thoughts, behavior, and perception of the world. Traditional methods for measuring this electrical activity require inserting an electrode into the brain, a process that is labor-intensive and usually allows researchers to record from only one neuron at a time. Multielectrode arrays allow the monitoring of electrical activity from many neurons at once, but they don’t sample densely enough to get all the neurons within a given volume. Calcium imaging does allow such dense sampling, but it measures calcium, an indirect and slow measure of neural electrical activity.
In 2018, Boyden’s team developed an alternative way to monitor electrical activity by labeling neurons with a fluorescent probe. Using a technique known as directed protein evolution, his group engineered a molecule called Archon1 that can be genetically inserted into neurons, where it becomes embedded in the cell membrane. When a neuron’s electrical activity increases, the molecule becomes brighter, and this fluorescence can be seen with a standard light microscope.
In the 2018 paper, Boyden and his colleagues showed that they could use the molecule to image electrical activity in the brains of transparent worms and zebrafish embryos, and also in mouse brain slices. In the new study, they wanted to try to use it in living, awake mice as they engaged in a specific behavior.
To do that, the researchers had to modify the probe so that it would go to a subregion of the neuron membrane. They found that when the molecule inserts itself throughout the entire cell membrane, the resulting images are blurry because the axons and dendrites that extend from neurons also fluoresce. To overcome that, the researchers attached a small peptide that guides the probe specifically to membranes of the cell bodies of neurons. They called this modified protein SomArchon.
MIT researchers developed a light-sensitive protein that can be embedded into neuron membranes, where it emits a fluorescent signal that indicates how much voltage a particular cell is experiencing. Microscope images of SomArchon-expressing neurons in cortex layer 2/3 (left), hippocampus (middle), and striatum (right)
“With SomArchon, you can see each cell as a distinct sphere,” Boyden says. “Rather than having one cell’s light blurring all its neighbors, each cell can speak by itself loudly and clearly, uncontaminated by its neighbors.”
The researchers used this probe to image activity in a part of the brain called the striatum, which is involved in planning movement, as mice ran on a ball. They were able to monitor activity in several neurons simultaneously and correlate each one’s activity with the mice’s movement. Some neurons’ activity went up when the mice were running, some went down, and others showed no significant change.
“Over the years, my lab has tried many different versions of voltage sensors, and none of them have worked in living mammalian brains until this one,” Han says.
Using this fluorescent probe, the researchers were able to obtain measurements similar to those recorded by an electrical probe, which can pick up activity on a very rapid timescale. This makes the measurements more informative than existing techniques such as imaging calcium, which neuroscientists often use as a proxy for electrical activity.
“We want to record electrical activity on a millisecond timescale,” Han says. “The timescale and activity patterns that we get from calcium imaging are very different. We really don’t know exactly how these calcium changes are related to electrical dynamics.”
With the new voltage sensor, it is also possible to measure very small fluctuations in activity that occur even when a neuron is not firing a spike. This could help neuroscientists study how small fluctuations impact a neuron’s overall behavior, which has previously been very difficult in living brains, Han says.
Mapping circuits
The researchers also showed that this imaging technique can be combined with optogenetics— a technique developed by the Boyden lab and collaborators that allows researchers to turn neurons on and off with light by engineering them to express light-sensitive proteins. In this case, the researchers activated certain neurons with light and then measured the resulting electrical activity in these neurons.
This imaging technology could also be combined with expansion microscopy, a technique that Boyden’s lab developed to expand brain tissue before imaging it, make it easier to see the anatomical connections between neurons in high resolution.
“One of my dream experiments is to image all the activity in a brain, and then use expansion microscopy to find the wiring between those neurons,” Boyden says. “Then can we predict how neural computations emerge from the wiring.”
Such wiring diagrams could allow researchers to pinpoint circuit abnormalities that underlie brain disorders, and may also help researchers to design artificial intelligence that more closely mimics the human brain, Boyden says.
The MIT portion of the research was funded by Edward and Kay Poitras, the National Institutes of Health, including a Director’s Pioneer Award, Charles Hieken, John Doerr, the National Science Foundation, the HHMI-Simons Faculty Scholars Program, the Human Frontier Science Program, and the U.S. Army Research Office.
By
Ann Trafton, MIT News Office
Study helps explain varying outcomes for cancer, Down Syndrome
Differences in chromosome number may underlie variation among genetically identical individuals
Colors represent variability of responses by cells with extra chromosomes
Aneuploidy is a condition in which cells contain an abnormal number of chromosomes, and is known to be the cause of many types of cancer and genetic disorders, including Down Syndrome. The condition is also the leading cause of miscarriage.
Disorders caused by aneuploidy are unusual in that the severity of their effects often varies widely from one individual to another.
For example, nearly 90 percent of fetuses with three copies of chromosome 21, the cause of Down Syndrome, will miscarry before birth. In other cases, people with the condition will live until they are over 60 years old.
Researchers have previously believed that this variation is the result of differences in the genetic makeup of those individuals with the condition.
But in a paper published today in the journal Cell, researchers at MIT reveal that aneuploidy alone can cause this significant variability in traits, in otherwise genetically identical cells.
The finding could have significant implications for cancer treatment, since it could explain why genetically identical cancer cells may respond differently to the same therapy.
An immediate impact
Aneuploidy originates during cell division, when the chromosomes do not separate properly or are not equally partitioned between the two daughter cells. This leads the cells, which in humans would normally have 46 chromosomes, to develop with either too many or too few chromosomes.
To study the effects of the condition, the researchers induced either chromosome loss or gain in genetically identical baker’s yeast cells. They chose baker’s yeast because the cells behave in a very similar way to human cells, according to Angelika Amon, the Kathleen and Curtis Marble Professor of Cancer Research, co-Director of the Alana Down Syndrome Center, and a member of the Koch Institute.
The induced changes had an immediate impact on the cells.
“We induced aneuploidy, and we found that the response was very variable from cell to cell,” Amon says. “Some cells slowed down their cycle completely, so that they could no longer divide, whereas others kept dividing quite normally and only experienced a small effect.”
The researchers carried out a systematic analysis, investigating the effect on the cells of gaining or losing a variety of different chromosomes. They found that in each case, even though individual cells had gained or lost the same chromosome, they behaved very differently from each other.
“So that really suggested that every single chromosome gained or lost had this effect, in that the responses (in each case) were quite variable,” Amon says.
Microscopy image of dividing cells, with chromosomes in green. The chromosome in the middle is lagging, which can lead to incorrect chromosome number.
Beyond cell division
Microscopy image of dividing cancer cells, with chromosomes in green. The chromosome in the middle is lagging, which can lead to incorrect chromosome number.
The researchers also investigated the impact of aneuploidy on other biological pathways, such as transcription, the first stage of gene expression in which a segment of DNA is copied into RNA.
They found that here too, the effects of aneuploidy were varied across otherwise identical cells.
The cells’ response to environmental changes also varied considerably, suggesting that aneuploidy has an impact on the robustness of many, if not all, biological processes.
To ensure the response is not an effect that is unique to baker’s yeast cells, the researchers then studied the impact of aneuploidy on mice, and found the same levels of variability, Amon says.
“This suggests that the aneuploidy state itself could create variability, and that could provide an additional explanation of why diseases that are caused by aneuploidy are so variable,” Amon says.
Tumors, for example, are known to contain different populations of cells, some of which are quite different to each other in their genetic makeup. These genetic differences have often been blamed when chemotherapy or other treatments have been unsuccessful, as it was believed that the therapy may not have targeted all of the cells within the tumor.
“Unfortunately our paper suggests that tumors don’t even need to be heterogeneous genetically, the very fact that they have aneuploidy could lead to very variable outcomes, and that represents a significant challenge for cancer therapy,” Amon says.
Understanding the consequences of aneuploidy on cellular phenotypes is a fundamental question that has important implications for the treatment of several diseases, such as cancer and Down Syndrome, according to Giulia Rancati of the Institute of Medical Biology at the Agency for Science, Technology and Research (A*STAR) in Singapore, who was not involved in the research.
“This new exciting work adds an additional layer of understanding of how aneuploidy causes phenotypic variation, by revealing an unexpectedly high cell-to-cell variability between cells harboring the same aneuploidy karyotype,” Rancati says. “It would be interesting to test if this property of the aneuploid state might positively contribute to the evolution of cancer cells, which are known to develop drug resistance at high frequency.”
The researchers are now hoping to carry out further studies to investigate the origins of the variability, Amon says.
The results suggest that subtle changes in gene dosage across many genes, caused by the change in chromosome numbers, can promote alternate behaviors.
“We’re now trying to track down which the key genes are, and which the key pathways are,” she says. “Once we can understand what the key pathways are that cause this variability, we can start to think about targeting those pathways, to combat alternate outcomes in cancer treatment, for example.”
Helen Knight | MIT News correspondent
Mapping the brain at high resolution
New 3-D imaging technique can reveal, much more quickly than other methods, how neurons connect throughout the brain
Neural structures imaged using a new high-resolution, nanoscale imaging system.
Researchers have developed a new way to image the brain with unprecedented resolution and speed. Using this approach, they can locate individual neurons, trace connections between them, and visualize organelles inside neurons, over large volumes of brain tissue.
The new technology combines a method for expanding brain tissue, making it possible to image at higher resolution, with a rapid 3-D microscopy technique known as lattice light-sheet microscopy. In a paper appearing in Science Jan. 17, the researchers showed that they could use these techniques to image the entire fruit fly brain, as well as large sections of the mouse brain, much faster than has previously been possible. The team includes researchers from MIT, the University of California at Berkeley, the Howard Hughes Medical Institute, and Harvard Medical School/Boston Children’s Hospital.
This technique allows researchers to map large-scale circuits within the brain while also offering unique insight into individual neurons’ functions, says Edward Boyden, the Y. Eva Tan Professor in Neurotechnology, an associate professor of biological engineering and of brain and cognitive sciences at MIT, and a member of the Alana Down Syndrome Center.
“A lot of problems in biology are multiscale,” Boyden says. “Using lattice light-sheet microscopy, along with the expansion microscopy process, we can now image at large scale without losing sight of the nanoscale configuration of biomolecules.”
Boyden is one of the study’s senior authors, along with Eric Betzig, a senior fellow at the Janelia Research Campus and a professor of physics and molecular and cell biology at UC Berkeley. The paper’s lead authors are MIT postdoc Ruixuan Gao, former MIT postdoc Shoh Asano, and Harvard Medical School Assistant Professor Srigokul Upadhyayula.
Large-scale imaging
In 2015, Boyden’s lab developed a way to generate very high-resolution images of brain tissue using an ordinary light microscope. Their technique relies on expanding tissue before imaging it, allowing them to image the tissue at a resolution of about 60 nanometers. Previously, this kind of imaging could be achieved only with very expensive high-resolution microscopes, known as super-resolution microscopes.
In the new study, Boyden teamed up with Betzig and his colleagues at HHMI’s Janelia Research Campus to combine expansion microscopy with lattice light-sheet microscopy. This technology, which Betzig developed several years ago, has some key traits that make it ideal to pair with expansion microscopy: It can image large samples rapidly, and it induces much less photodamage than other fluorescent microscopy techniques.
“The marrying of the lattice light-sheet microscope with expansion microscopy is essential to achieve the sensitivity, resolution, and scalability of the imaging that we’re doing,” Gao says.
Imaging expanded tissue samples generates huge amounts of data — up to tens of terabytes per sample — so the researchers also had to devise highly parallelized computational image-processing techniques that could break down the data into smaller chunks, analyze it, and stitch it back together into a coherent whole.
In the Science paper, the researchers demonstrated the power of their new technique by imaging layers of neurons in the somatosensory cortex of mice, after expanding the tissue volume fourfold. They focused on a type of neuron known as pyramidal cells, one of the most common excitatory neurons found in the nervous system. To locate synapses, or connections, between these neurons, they labeled proteins found in the presynaptic and postsynaptic regions of the cells. This also allowed them to compare the density of synapses in different parts of the cortex.
Mouse neurons in yellow, with cyan and magenta markers for synapses, imaged with the new technique
MIT researchers have developed a method to perform large-scale, 3D imaging of brain tissue. Here, they image the entire fruit fly brain.
Using this technique, it is possible to analyze millions of synapses in just a few days.
“We counted clusters of postsynaptic markers across the cortex, and we saw differences in synaptic density in different layers of the cortex,” Gao says. “Using electron microscopy, this would have taken years to complete.”
The researchers also studied patterns of axon myelination in different neurons. Myelin is a fatty substance that insulates axons and whose disruption is a hallmark of multiple sclerosis. The researchers were able to compute the thickness of the myelin coating in different segments of axons, and they measured the gaps between stretches of myelin, which are important because they help conduct electrical signals. Previously, this kind of myelin tracing would have required months to years for human annotators to perform.
This technology can also be used to image tiny organelles inside neurons. In the new paper, the researchers identified mitochondria and lysosomes, and they also measured variations in the shapes of these organelles.
Circuit analysis
The researchers demonstrated that this technique could be used to analyze brain tissue from other organisms as well; they used it to image the entire brain of the fruit fly, which is the size of a poppy seed and contains about 100,000 neurons. In one set of experiments, they traced an olfactory circuit that extends across several brain regions, imaged all dopaminergic neurons, and counted all synapses across the brain. By comparing multiple animals, they also found differences in the numbers and arrangements of synaptic boutons within each animal’s olfactory circuit.
In future work, Boyden envisions that this technique could be used to trace circuits that control memory formation and recall, to study how sensory input leads to a specific behavior, or to analyze how emotions are coupled to decision-making.
“These are all questions at a scale that you can’t answer with classical technologies,” he says.
The system could also have applications beyond neuroscience, Boyden says. His lab is planning to work with other researchers to study how HIV evades the immune system, and the technology could also be adapted to study how cancer cells interact with surrounding cells, including immune cells.
The research was funded by K. Lisa Yang and Y. Eva Tan, John Doerr, the Open Philanthropy Project, the National Institutes of Health, the Howard Hughes Medical Institute, the HHMI-Simons Faculty Scholars Program, the U.S. Army Research Laboratory and Army Research Office, the US-Israel Binational Science Foundation, Biogen, and Ionis Pharmaceuticals.
Anne Trafton, MIT News Office
Brain Wave Stimulation May Improve Alzheimer’s Symptoms
A combination of light and sound can improve hallmarks of Alzheimer's in mice
Microscope image of a light & sound treated mouse brain, cells labeled in blue, amyloid plaques in red.
By exposing mice to a unique combination of light and sound, MIT neuroscientists have shown that they can improve cognitive and memory impairments similar to those seen in Alzheimer’s patients. Individuals with Down syndrome have a high risk of developing Alzheimer’s Disease.
This noninvasive treatment, which works by inducing brain waves known as gamma oscillations, also greatly reduced the number of amyloid plaques found in the brains of these mice. Plaques were cleared in large swaths of the brain, including areas critical for cognitive functions such as learning and memory.
“When we combine visual and auditory stimulation for a week, we see the engagement of the prefrontal cortex and a very dramatic reduction of amyloid,” says Li-Huei Tsai, director of MIT’s Picower Institute for Learning and Memory and the Alana Down Syndrome Center, and the senior author of the study.
Further study will be needed, she says, to determine if this type of treatment will work in human patients. The researchers have already performed some preliminary safety tests of this type of stimulation in healthy human subjects.
MIT graduate student Anthony Martorell and Georgia Tech graduate student Abigail Paulson are the lead authors of the study, done in collaboration with Alana Investigator Ed Boyden’s lab, which appears in the March 14 issue of Cell.
Memory improvement
The brain’s neurons generate electrical signals that synchronize to form brain waves in several different frequency ranges. Previous studies have suggested that Alzheimer’s patients have impairments of their gamma-frequency oscillations, which range from 25 to 80 hertz (cycles per second) and are believed to contribute to brain functions such as attention, perception, and memory.
In 2016, Tsai and her colleagues first reported the beneficial effects of restoring gamma oscillations in the brains of mice that are genetically predisposed to develop Alzheimer’s symptoms. In that study, the researchers used light flickering at 40 hertz, delivered for one hour a day. They found that this treatment reduced levels of beta amyloid plaques and another Alzheimer’s-related pathogenic marker, phosphorylated tau protein. The treatment also stimulated the activity of debris-clearing immune cells known as microglia.
In that study, the improvements generated by flickering light were limited to the visual cortex. In their new study, the researchers set out to explore whether they could reach other brain regions, such as those needed for learning and memory, using sound stimuli. They found that exposure to one hour of 40-hertz tones per day, for seven days, dramatically reduced the amount of beta amyloid in the auditory cortex (which processes sound) as well as the hippocampus, a key memory site that is located near the auditory cortex.
“What we have demonstrated here is that we can use a totally different sensory modality to induce gamma oscillations in the brain. And secondly, this auditory-stimulation-induced gamma can reduce amyloid and Tau pathology in not just the sensory cortex but also in the hippocampus,” says Tsai, a founding member of MIT’s Aging Brain Initiative.
The researchers also tested the effect of auditory stimulation on the mice’s cognitive abilities. They found that after one week of treatment, the mice performed much better when navigating a maze requiring them to remember key landmarks. They were also better able to recognize objects they had previously encountered.
They also found that auditory treatment induced changes in not only microglia, but also the blood vessels, possibly facilitating the clearance of amyloid.
Dramatic effect
Brain cells called microglia, labeled green, change shape after light treatment
The researchers then decided to try combining the visual and auditory stimulation, and to their surprise, they found that this dual treatment had an even greater effect than either one alone. Amyloid plaques were reduced throughout a much greater portion of the brain, including the prefrontal cortex, where higher cognitive functions take place. The microglia response was also much stronger.
“These microglia just pile on top of one another around the plaques,” Tsai says. “It’s very dramatic.”
The researchers found that if they treated the mice for one week, then waited another week to perform the tests, many of the positive effects had faded, suggesting that the treatment would need to be given continually to maintain the benefits.
In an ongoing study, the researchers are now analyzing how gamma oscillations affect specific brain cell types, in hopes of discovering the molecular mechanisms behind the phenomena they have observed. Tsai says she also hopes to explore why the specific frequency they use, 40 hertz, has such a profound impact.
The combined visual and auditory treatment has already been tested in healthy volunteers, to assess its safety, and the researchers are now beginning to enroll patients with early-stage Alzheimer’s to study its possible effects on the disease.
“Though there are important differences among species, there is reason to be optimistic that these methods can provide useful interventions for humans,” says Nancy Kopell, a professor of mathematics and statistics at Boston University, who was not involved in the research. “This paper and related studies have the potential for huge clinical impact in Alzheimer’s disease and others involving brain inflammation.”
