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.

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

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.

“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