Brain cells snap strands of DNA in many more places and cell types than researchers previously thought.
The urgency to remember a dangerous experience requires the brain to make a series of potentially dangerous moves: Neurons and other brain cells snap open their DNA in numerous locations — more than previously realized, according to a new study — to provide quick access to genetic instructions for the mechanisms of memory storage.
The extent of these DNA double-strand breaks (DSBs) in multiple key brain regions is surprising and concerning, says study senior author Li-Huei Tsai, Picower Professor of Neuroscience at MIT and director of The Picower Institute for Learning and Memory, because while the breaks are routinely repaired, that process may become more flawed and fragile with age. Tsai's lab has shown that lingering DSBs are associated with neurodegeneration and cognitive decline and that repair mechanisms can falter.
"We wanted to understand exactly how widespread and extensive this natural activity is in the brain upon memory formation because that can give us insight into how genomic instability could undermine brain health down the road," says Tsai, who is also a professor in the Department of Brain and Cognitive Sciences and a leader of MIT's Aging Brain Initiative. "Clearly, memory formation is an urgent priority for healthy brain function, but these new results showing that several types of brain cells break their DNA in so many places to quickly express genes is still striking."
In 2015, Tsai's lab provided the first demonstration that neuronal activity caused DSBs and that they induced rapid gene expression. But those findings, mostly made in lab preparations of neurons, did not capture the full extent of the activity in the context of memory formation in a behaving animal, and did not investigate what happened in cells other than neurons.
In the new study published July 1 in PLOS ONE, lead author and former graduate student Ryan Stott and co-author and former research technician Oleg Kritsky sought to investigate the full landscape of DSB activity in learning and memory. To do so, they gave mice little electrical zaps to the feet when they entered a box, to condition a fear memory of that context. They then used several methods to assess DSBs and gene expression in the brains of the mice over the next half-hour, particularly among a variety of cell types in the prefrontal cortex and hippocampus, two regions essential for the formation and storage of conditioned fear memories. They also made measurements in the brains of mice that did not experience the foot shock to establish a baseline of activity for comparison.
The creation of a fear memory doubled the number of DSBs among neurons in the hippocampus and the prefrontal cortex, affecting more than 300 genes in each region. Among 206 affected genes common to both regions, the researchers then looked at what those genes do. Many were associated with the function of the connections neurons make with each other, called synapses. This makes sense because learning arises when neurons change their connections (a phenomenon called "synaptic plasticity") and memories are formed when groups of neurons connect together into ensembles called engrams.
"Many genes essential for neuronal function and memory formation, and significantly more of them than expected based on previous observations in cultured neurons … are potentially hotspots of DSB formation," the authors wrote in the study.
In another analysis, the researchers confirmed through measurements of RNA that the increase in DSBs indeed correlated closely with increased transcription and expression of affected genes, including ones affecting synapse function, as quickly as 10-30 minutes after the foot shock exposure.
"Overall, we find transcriptional changes are more strongly associated with [DSBs] in the brain than anticipated," they wrote. "Previously we observed 20 gene-associated [DSB] loci following stimulation of cultured neurons, while in the hippocampus and prefrontal cortex we see more than 100-150 gene associated [DSB] loci that are transcriptionally induced."
Snapping with stress
In the analysis of gene expression, the neuroscientists looked at not only neurons but also non-neuronal brain cells, or glia, and found that they also showed changes in expression of hundreds of genes after fear conditioning. Glia called astrocytes are known to be involved in fear learning, for instance, and they showed significant DSB and gene expression changes after fear conditioning.
Among the most important functions of genes associated with fear conditioning-related DSBs in glia was the response to hormones. The researchers therefore looked to see which hormones might be particularly involved and discovered that it was glutocortocoids, which are secreted in response to stress. Sure enough, the study data showed that in glia, many of the DSBs that occurred following fear conditioning occurred at genomic sites related to glutocortocoid receptors. Further tests revealed that directly stimulating those hormone receptors could trigger the same DSBs that fear conditioning did and that blocking the receptors could prevent transcription of key genes after fear conditioning.
Tsai says the finding that glia are so deeply involved in establishing memories from fear conditioning is an important surprise of the new study.
"The ability of glia to mount a robust transcriptional response to glutocorticoids suggest that glia may have a much larger role to play in the response to stress and its impact on the brain during learning than previously appreciated," she and her co-authors wrote.
Damage and danger?
More research will have to be done to prove that the DSBs required for forming and storing fear memories are a threat to later brain health, but the new study only adds to evidence that it may be the case, the authors say.
"Overall we have identified sites of DSBs at genes important for neuronal and glial functions, suggesting that impaired DNA repair of these recurrent DNA breaks which are generated as part of brain activity could result in genomic instability that contribute to aging and disease in the brain," they wrote.
The National Institutes of Health, The Glenn Foundation for Medical Research, and the JPB Foundation provided funding for the research.
The Vertebrate Genomes Project may spell good news for the kakapo and the vaquita.
The world's heaviest parrot—representing one of the most ancestral branches of the parrot family tree—is nearly extinct, with barely 200 adults plodding the underbrush of four small islands. Whether the last of the kakapos have the genetic resilience to survive has long been unknown, and a question that only high-quality genomic analysis could answer.
But a high-quality genome assembly does not exist for the kakapo—nor for most of the 70,000 vertebrate species alive today. As a result, questions abound about how best to prevent the extinction of species like flightless kakapos and adorable vaquita dolphins.
Answers may come from the Vertebrate Genomes Project, which aims to generate high-quality reference genomes for every extant vertebrate species. In a flagship study in the journal Nature, the team presents methods and principles for sequencing and assembling high-quality reference genomes.
The team has applied this approach and principles to produce 16 high-quality reference genomes, one of which was the endangered kakapo, to help reveal if it is hardy enough to rebuild its population. The researchers found that extremely small populations of the endangered kakapo and vaquita have been able to survive their low numbers in the past since the last ice age over 10,000 years ago, by purging deleterious mutations that cause disease from inbreeding.
As long as humans do not kill off more of the last remaining animals, findings from the high-quality reference genomes give hope that these species could survive even with less than 100 individuals each.
"We call it the 'kitchen sink approach'—combining tools from several biotech companies to make this one high-quality genome assembly pipeline," says Rockefeller University's Erich D. Jarvis, chair of the Vertebrate Genomes Project. "Endangered species were the first to benefit from the new technology because, even though conservation is not my area of research, I felt it was a moral duty."
Genomes full of errors
High-quality reference genomes only exist for the celebrities of laboratory science—mice, fruit flies, zebrafish, and, of course, humans. For less popular species, there is often no reference genome or, perhaps worse, messy genomes stitched together from sequences obtained via quick and dirty methods. Compared to the new VGP genomes, up to 60% of the genes in such genomes have missing sequences, are entirely missing, or incorrectly assembled, the researchers found. It can take years to untangle the thousands of assembly errors per species.
Many false gene duplications were found, most caused by algorithms that do not properly separate out maternal and paternal chromosome sequences and instead interpret them as two separate sister genes. "We have thousands of genes in the literature that are false duplications. The genes are not actually there!" Jarvis says. "It is unconscionable to be working with some of these genomes."
The Vertebrate Genomes Project arose from the frustrations of hundreds of scientists working in its parent organization, the Genome 10K consortium, whose mission was to generate genome assemblies of 10,000 vertebrate species. The initial genome assemblies that the G10K and other groups generated were based on short 35 to 200 base pair reads, but these assemblies were highly incomplete. The VGP goal is to build a library of error-free reference genomes for all vertebrate species, which researchers and conservationists will be able to use readily, without dedicating months or years to fixing individual genes.
"We said, let's do some hard work on the front end, so that we can get high quality data on the back end," Jarvis says.
Vertebrate Genomes Project rollout
Many companies approached the Vertebrate Genomes Project, promising a single sequencing technology that would solve every problem with messy reference genomes. The Vertebrate Genomes Project assembly team tested each method on a single hummingbird, chosen both for its relatively small genome and because of Jarvis's research interests in vocal learning among bird species ("two birds with one stone," he quips). But every technology fell short. "None had all of the necessary components to make a high-quality assembly," Jarvis says. "So we combined many tools into one pipeline."
Their approach works. Organizations including the Earth Biogenome Project, the Darwin Tree of Life Project, and the New Zealand Genome Sequencing Project are already using the most advance version of the novel pipeline. Reference genomes that once took years to generate are now rolling out in weeks and months—all without the false duplications and other errors endemic to previous assemblies.
Scientists are already using the new data to study genes that render bats immune to COVID-19, and question long-standing conventions in basic science, such as whether there are meaningful differences among oxytocin and its receptors found in humans, birds, reptiles, and fish.
All told, 20 studies and 25 high-quality vertebrate genomes accompany the rollout of the novel pipeline. "The first high-quality genomes that we sequenced taught us so much about the technology and the biology that we decided to publish in these initial papers," Jarvis says. But plenty of work still lies ahead. "The next step is to sequence all 1,000 vertebrate genera, and then all 10,000 vertebrate families, and eventually every single vertebrate species."
Source: Rockefeller University
Original Study DOI: 10.1038/s41586-021-03451-0
"The question is which are okay, which are not okay."
- As the material that makes all living things what/who we are, DNA is the key to understanding and changing the world. British geneticist Bryan Sykes and Francis Collins (director of the Human Genome Project) explain how, through gene editing, scientists can better treat illnesses, eradicate diseases, and revolutionize personalized medicine.
- But existing and developing gene editing technologies are not without controversies. A major point of debate deals with the idea that gene editing is overstepping natural and ethical boundaries. Just because they can, does that mean that scientists should be edit DNA?
- Harvard professor Glenn Cohen introduces another subcategory of gene experiments: mixing human and animal DNA. "The question is which are okay, which are not okay, why can we generate some principles," Cohen says of human-animal chimeras and arguments concerning improving human life versus morality.
Find your dog's breed mix, personality and more with a simple cheek swab in this DNA kit.
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Forget being man’s best friend; dogs are everybody’s best friend. We treat our furry friends like royalty, because we recognize the value of having them around as long as possible. But while we like to view ourselves as knowing our dogs as well as our own family, there’s a lot that we remain unaware of. While humans regularly take DNA tests, due to the potential benefits of knowing one’s genetic makeup as well as family history, it is less common to know the genetics of your dog.
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What makes some people more likely to shiver than others?
Some people just aren't bothered by the cold, no matter how low the temperature dips. And the reason for this may be in a person's genes.
Our new research shows that a common genetic variant in the skeletal muscle gene, ACTN3, makes people more resilient to cold temperatures.
Around one in five people lack a muscle protein called alpha-actinin-3 due to a single genetic change in the ACTN3 gene. The absence of alpha-actinin-3 became more common as some modern humans migrated out of Africa and into the colder climates of Europe and Asia. The reasons for this increase have remained unknown until now.
Our recent study, conducted alongside researchers from Lithuania, Sweden and Australia, suggests that if you're alpha-actinin-3 deficient, then your body can maintain a higher core temperature and you shiver less when exposed to cold, compared with those who have alpha-actinin-3.
We looked at 42 men aged 18 to 40 years from Kaunas in southern Lithuania and exposed them to cold water (14℃) for a maximum of 120 minutes, or until their core body temperature reached 35.5℃. We broke their exposure up into 20-minute periods in the cold with ten-minute breaks at room temperature. We then separated participants into two groups based on their ACTN3 genotype (whether or not they had the alpha-actinin-3 protein).
While only 30% of participants with the alpha-actinin-3 protein reached the full 120 minutes of cold exposure, 69% of those that were alpha-actinin-3 deficient completed the full cold-water exposure time. We also assessed the amount of shivering during cold exposure periods, which told us that those without alpha-actinin-3 shiver less than those who have alpha-actinin-3.
Our study suggests that genetic changes caused by the loss of alpha-actinin-3 in our skeletal muscle affect how well we can tolerate cold temperatures, with those that are alpha-actinin-3 deficient better able to maintain their body temperature and conserve their energy by shivering less during cold exposure. However, future research will need to investigate whether similar results would be seen in women.
Skeletal muscles are made up of two types of muscle fibres: fast and slow. Alpha-actinin-3 is predominantly found in fast muscle fibres. These fibres are responsible for the rapid and forceful contractions used during sprinting, but typically fatigue quickly and are prone to injury. Slow muscle fibres on the other hand generate less force but are resistant to fatigue. These are primarily the muscle you'd use during endurance events, like marathon running.
Our previous work has shown that ACTN3 variants play an important role in our muscle's ability to generate strength. We showed that the loss of alpha-actinin-3 is detrimental to sprint performance in athletes and the general population, but may benefit muscle endurance.
This is because the loss of alpha-actinin-3 causes the muscle to behave more like a slower muscle fibre. This means that alpha-actinin-3 deficient muscles are weaker but recover more quickly from fatigue. But while this is detrimental to sprint performance, it may be beneficial during more endurance events. This improvement in endurance muscle capacity could also influence our response to cold.
While alpha-actinin-3 deficiency does not cause muscle disease, it does influence how our muscle functions. Our study shows that ACTN3 is more than just the "gene for speed", but that its loss improves our muscle's ability to generate heat and reduces the need to shiver when exposed to cold. This improvement in muscle function would conserve energy and ultimately increase survival in cold temperatures, which we think is a key reason why we see an increase in alpha-actinin-3 deficient people today, as this would have helped modern humans better tolerate cooler climates as they migrated out of Africa.
The goal of our research is to improve our understanding of how our genetics influence how our muscle works. This will allow us to develop better treatments for those who suffer from muscle diseases, like Duchenne muscular dystrophy, as well as more common conditions, such as obesity and type 2 diabetes. A better understanding of how variants in alpha-actinin-3 influences these conditions will give us better ways to treat and prevent these conditions in the future.
Victoria Wyckelsma, Postdoctoral Research Fellow, Muscle Physiology, Karolinska Institutet and Peter John Houweling, Senior Research Officer, Neuromuscular Research, Murdoch Children's Research Institute