Brain Disorder: ALS

CRISPR makes several Discovery of the Decade lists

by Sabbi Lall |

As we reach milestones in time, it’s common to look back and review what we learned. A number of media outlets, including National Geographic, NPR, The Hill, Popular Mechanics, Smithsonian Magazine, Nature, Mental Floss, CNBC, and others, recognized the profound impact of genome editing, adding CRISPR to their discovery of the decade lists.

“In 2013, [CRISPR] was used for genome editing in a eukaryotic cell, forever altering the course of biotechnology and, ultimately our relationship with our DNA.”
— Popular Mechanics

It’s rare for a molecular system to become a household name, but in less than a decade, CRISPR has done just that. McGovern Investigator Feng Zhang played a key role in leveraging CRISPR, an immune system found originally in prokaryotic – bacterial and archaeal – cells, into a broadly customizable toolbox for genomic manipulation in eukaryotic (animal and plant) cells. CRISPR allows scientists to easily and quickly make changes to genomes, has revolutionized the biomedical sciences, and has major implications for control of infectious disease, agriculture, and treatment of genetic disorders.

Scientists engineer new CRISPR platform for DNA targeting

by The Broad Institute |

A team that includes the scientist who first harnessed the revolutionary CRISPR-Cas9 and other systems for genome editing of eukaryotic organisms, including animals and plants, has engineered another CRISPR system, called Cas12b. The new system offers improved capabilities and options when compared to CRISPR-Cas9 systems.

In a study published today in Nature Communications, Feng Zhang and colleagues at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT, with co-author Eugene Koonin at the National Institutes of Health, demonstrate that the new enzyme can be engineered to target and precisely nick or edit the genomes of human cells. The high target specificity and small size of Cas12b from Bacillus hisashii (BhCas12b) as compared to Cas9 (SpCas9), makes this new system suitable for in vivo applications. The team is now making CRISPR-Cas12b widely available for research.

The team previously identified Cas12b (then known as C2c1) as one of three promising new CRISPR enzymes in 2015, but faced a hurdle: Because Cas12b comes from thermophilic bacteria — which live in hot environments such as geysers, hot springs, volcanoes, and deep sea hydrothermal vents — the enzyme naturally only works at temperatures higher than human body temperature.

“We searched for inspirations from nature,” Zhang said. “We wanted to create a version of Cas12b that could operate at lower temperatures, so we scanned thousands of bacterial genetic sequences, looking in bacteria that could thrive in the lower temperatures of mammalian environments.”

Through a combination of exploration of natural diversity and rational engineering of promising candidate enzymes, they generated a version of Cas12b capable of efficiently editing genomes in primary human T cells, an important initial step for therapeutics that target or leverage the immune system.

“This is further evidence that there are many useful CRISPR systems waiting to be discovered,” said Jonathan Strecker, a postdoctoral fellow in the Zhang Lab, a Human Frontiers Science program fellow, and the study’s first author.

The field is moving quickly: Since the Cas12b family of enzymes was first described in 2015 and demonstrated to be RNA-guided DNA endonucleases, several groups have have been exploring this family of enzymes. In 2017 a team from Jennifer Doudna’s lab at UC Berkeley reported that Cas12b from Alicyclobacillus acidoterrestris can mediate non-specific collateral cleavage of DNA in vitro. More recently, a team from the Chinese Academy of Sciences in Beijing reported that another Cas12b, from Alicyclobacillus acidiphilus, was used to edit mammalian cells.

The Broad Institute and MIT are sharing the Cas12b system widely. As with earlier genome editing tools, these groups will make the technology freely available for academic research via the Zhang lab’s page on the plasmid-sharing website Addgene, through which the Zhang lab has already shared reagents more than 52,000 times with researchers at nearly 2,400 labs in 62 countries to accelerate research.

Zhang is a core institute member of the Broad Institute of MIT and Harvard, as well as an investigator at the McGovern Institute for Brain Research at MIT, the James and Patricia Poitras Professor of Neuroscience at MIT, and an associate professor at MIT, with joint appointments in the departments of Brain and Cognitive Sciences and Biological Engineering.

Support for this study was provided by the Poitras Center for Psychiatric Disorders Research, the Hock E. Tan and K. Lisa Yang Center for Autism Research, the National Human Genome Research Institute, the National Institute of Mental Health, the National Heart, Lung, and Blood Institute, and other sources. Feng Zhang is an Investigator with the Howard Hughes Medical Institute.

References:

Strecker J, et al. Engineering of CRISPR-Cas12b for human genome editing. Nature Communications. Online January 22, 2019. DOI: 10.1038/s41467-018-08224-4.

H. Robert Horvitz

Learning from Worms

Bob Horvitz studies the nematode worm Caenorhabditis elegans. Only 1 mm long and containing fewer than 1000 cells, C. elegans has been key to discovering fundamental biological mechanisms that are conserved across species. Horvitz has focused on the genetic control of animal development and behavior, and on the mechanisms that underlie neurodegenerative disease. By identifying mutations that affect C. elegans behavior, Horvitz has revealed much about the genetic control of many aspects of nervous system development and of brain function, including how neural circuits control specific behaviors and how behavior is modulated by experience and by the environment.

 

What is CRISPR?

by The Brain |

CRISPR (which stands for Clustered Regularly Interspaced Short Palindromic Repeats) is not actually a single entity, but shorthand for a set of bacterial systems that are found with a hallmarked arrangement in the bacterial genome.

When CRISPR is mentioned, most people are likely thinking of CRISPR-Cas9, now widely known for its capacity to be re-deployed to target sequences of interest in eukaryotic cells, including human cells. Cas9 can be programmed to target specific stretches of DNA, but other enzymes have since been discovered that are able to edit DNA, including Cpf1 and Cas12b. Other CRISPR enzymes, Cas13 family members, can be programmed to target RNA and even edit and change its sequence.

The common theme that makes CRISPR enzymes so powerful, is that scientists can supply them with a guide RNA for a chosen sequence. Since the guide RNA can pair very specifically with DNA, or for Cas13 family members, RNA, researchers can basically provide a given CRISPR enzyme with a way of homing in on any sequence of interest. Once a CRISPR protein finds its target, it can be used to edit that sequence, perhaps removing a disease-associated mutation.

In addition, CRISPR proteins have been engineered to modulate gene expression and even signal the presence of particular sequences, as in the case of the Cas13-based diagnostic, SHERLOCK.

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Biologists discover function of gene linked to familial ALS

Anne Trafton |

MIT biologists have discovered a function of a gene that is believed to account for up to 40 percent of all familial cases of amyotrophic lateral sclerosis (ALS). Studies of ALS patients have shown that an abnormally expanded region of DNA in a specific region of this gene can cause the disease.

In a study of the microscopic worm Caenorhabditis elegans, the researchers found that the gene has a key role in helping cells to remove waste products via structures known as lysosomes. When the gene is mutated, these unwanted substances build up inside cells. The researchers believe that if this also happens in neurons of human ALS patients, it could account for some of those patients’ symptoms.

“Our studies indicate what happens when the activities of such a gene are inhibited — defects in lysosomal function. Certain features of ALS are consistent with their being caused by defects in lysosomal function, such as inflammation,” says H. Robert Horvitz, the David H. Koch Professor of Biology at MIT, a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, and the senior author of the study.

Mutations in this gene, known as C9orf72, have also been linked to another neurodegenerative brain disorder known as frontotemporal dementia (FTD), which is estimated to affect about 60,000 people in the United States.

“ALS and FTD are now thought to be aspects of the same disease, with different presentations. There are genes that when mutated cause only ALS, and others that cause only FTD, but there are a number of other genes in which mutations can cause either ALS or FTD or a mixture of the two,” says Anna Corrionero, an MIT postdoc and the lead author of the paper, which appears in the May 3 issue of the journal Current Biology.

Genetic link

Scientists have identified dozens of genes linked to familial ALS, which occurs when two or more family members suffer from the disease. Doctors believe that genetics may also be a factor in nonfamilial cases of the disease, which are much more common, accounting for 90 percent of cases.

Of all ALS-linked mutations identified so far, the C9orf72 mutation is the most prevalent, and it is also found in about 25 percent of frontotemporal dementia patients. The MIT team set out to study the gene’s function in C. elegans, which has an equivalent gene known as alfa-1.

In studies of worms that lack alfa-1, the researchers discovered that defects became apparent early in embryonic development. C. elegans embryos have a yolk that helps to sustain them before they hatch, and in embryos missing alfa-1, the researchers found “blobs” of yolk floating in the fluid surrounding the embryos.

This led the researchers to discover that the gene mutation was affecting the lysosomal degradation of yolk once it is absorbed into the cells. Lysosomes, which also remove cellular waste products, are cell structures which carry enzymes that can break down many kinds of molecules.

When lysosomes degrade their contents — such as yolk — they are reformed into tubular structures that split, after which they are able to degrade other materials. The MIT team found that in cells with the alfa-1 mutation and impaired lysosomal degradation, lysosomes were unable to reform and could not be used again, disrupting the cell’s waste removal process.

“It seems that lysosomes do not reform as they should, and material accumulates in the cells,” Corrionero says.

For C. elegans embryos, that meant that they could not properly absorb the nutrients found in yolk, which made it harder for them to survive under starvation conditions. The embryos that did survive appeared to be normal, the researchers say.

Robert Brown, chair of the Department of Neurology at the University of Massachusetts Medical School, describes the study as a major contribution to scientists’ understanding of the normal function of the C9orf72 gene.

“They used the power of worm genetics to dissect very fully the stages of vesicle maturation at which this gene seems to play a major role,” says Brown, who was not involved in the study.

Neuronal effects

The researchers were able to partially reverse the effects of alfa-1 loss in the C. elegans embryos by expressing the human protein encoded by the C9orf72 gene. “This suggests that the worm and human proteins are performing the same molecular function,” Corrionero says.

If loss of C9orf72 affects lysosome function in human neurons, it could lead to a slow, gradual buildup of waste products in those cells. ALS usually affects cells of the motor cortex, which controls movement, and motor neurons in the spinal cord, while frontotemporal dementia affects the frontal areas of the brain’s cortex.

“If you cannot degrade things properly in cells that live for very long periods of time, like neurons, that might well affect the survival of the cells and lead to disease,” Corrionero says.

Many pharmaceutical companies are now researching drugs that would block the expression of the mutant C9orf72. The new study suggests certain possible side effects to watch for in studies of such drugs.

“If you generate drugs that decrease C9orf72 expression, you might cause problems in lysosomal homeostasis,” Corrionero says. “In developing any drug, you have to be careful to watch for possible side effects. Our observations suggest some things to look for in studying drugs that inhibit C9orf72 in ALS/FTD patients.”

The research was funded by an EMBO postdoctoral fellowship, an ALS Therapy Alliance grant, a gift from Rose and Douglas Barnard ’79 to the McGovern Institute, and a gift from the Halis Family Foundation to the MIT Aging Brain Initiative.

From cancer to brain research: learning from worms

by Elizabeth Dougherty |

In Bob Horvitz’s lab, students watch tiny worms as they wriggle under the microscope. Their tracks twist and turn in every direction, and to a casual observer the movements appear random. There is a pattern, however, and the animals’ movements change depending on their environment and recent experiences.

“A hungry worm is different from a well-fed worm,” says Horvitz, David H. Koch Professor of Biology and a McGovern Investigator. “If you consider worm psychology, it seems that the thing in life worms care most about is food.”

Horvitz’s work with the nematode worm Caenorhabditis elegans extends back to the mid-1970s. He was among the first to recognize the value of this microscopic organism as a model species for asking fundamental questions about biology and human disease.

The leap from worm to human might seem great and perilous, but in fact they share many fundamental biological mechanisms, one of which is programmed cell death, also known as apoptosis. Horvitz shared the Nobel Prize in Physiology or Medicine in 2002 for his studies of cell death, which is central to a wide variety of human diseases, including cancer and neurodegenerative disorders. He has continued to study the worm ever since, contributing to many areas of biology but with a particular emphasis on the nervous system and the control of behavior.

In a recently published study, the Horvitz lab has found another fundamental mechanism that likely is shared with mice and humans. The discovery began with an observation by former graduate student Beth Sawin as she watched worms searching for food. When a hungry worm detects a food source, it slows almost to a standstill, allowing it to remain close to the food.
Postdoctoral scientist Nick Paquin analyzed how a mutation in a gene called vps-50, causes worms to slow similarly even when they are well fed. It seemed that these mutant worms were failing to transition normally between the hungry and the well-fed state.

Paquin decided to study the gene further, in worms and also in mouse neurons, the latter in collaboration with Yasunobu Murata, a former research scientist in Martha Constantine-Paton’s lab at the McGovern Institute. The team, later joined by postdoctoral fellow Fernando Bustos in the Constantine-Paton lab, found that the VPS-50 protein controls the activity of synapses, the junctions between nerve cells. VPS-50 is involved in a process that acidifies synaptic vesicles, microscopic bubbles filled with neurotransmitters that are released from nerve terminals, sending signals to other nearby neurons.

If VPS-50 is missing, the vesicles do not mature properly and the signaling from neurons is abnormal. VPS-50 has remained relatively unchanged during evolution, and the mouse version can
substitute for the missing worm gene, indicating the worm and mouse proteins are similar not only in sequence but also in function. This might seem surprising given the wide gap between the tiny nervous system of the worm and the complex brains of mammals. But it is not surprising to Horvitz, who has committed about half of his lab resources to studying the worm’s nervous system and behavior.

“Our finding underscores something that I think is crucially important,” he says. “A lot of biology is conserved among organisms that appear superficially very different, which means that the
understanding and treatment of human diseases can be advanced by studies of simple organisms like worms.”

Human connections

In addition to its significance for normal synaptic function, the vps-50 gene might be important in autism spectrum disorder. Several autism patients have been described with deletions that include vps-50, and other lines of evidence also suggest a link to autism. “We think this is going to be a very important molecule in mammals,” says Constantine-Paton. “We’re now in a position to look into the function of vps-50 more deeply.”

Horvitz and Constantine-Paton are married, and they had chatted about vps-50 long before her lab began to study it. When it became clear that the mutation was affecting worm neurons in a novel way, it was a natural decision to collaborate and study the gene in mice. They are currently working to understand the role of VPS-50 in mammalian brain function, and to explore further the possible link to autism.

The day the worm turned

A latecomer to biology, Horvitz studied mathematics and economics as an undergraduate at MIT in the mid-1960s. During his last year, he took a few biology classes and then went on to earn
a doctoral degree in the field at Harvard University, working in the lab of James Watson (of double helix fame) and Walter Gilbert. In 1974, Horvitz moved to Cambridge, England, where he worked with Sydney Brenner and began his studies of the worm.

“Remarkably, all of my advisors, even my undergraduate advisor in economics here at MIT, Bob Solow, now have Nobel Prizes,” he notes.

The comment is matter-of-fact, and Horvitz is anything but pretentious. He thinks about both big questions and small experimental details and is always on the lookout for links between the
worm and human health.

“When someone in the lab finds something new, Bob is quick to ask if it relates to human disease,” says former graduate student Nikhil Bhatla. “We’re not thinking about that. We’re deep in
the nitty-gritty, but he’s directing us to potential collaborators who might help us make that link.”

This kind of mentoring, says Horvitz, has been his primary role since he joined the MIT faculty in 1978. He has trained many of the current leaders in the worm field, including Gary Ruvkun
and Victor Ambros, who shared the 2008 Lasker Award, Michael Hengartner, now President of the University of Zurich, and Cori Bargmann, who recently won the McGovern’s 2016 Scolnick Prize in Neuroscience.

“If the science we’ve done has been successful, it’s because I’ve been lucky to have outstanding young researchers as colleagues,” Horvitz says.

Before becoming a mentor, Horvitz had to become a scientist himself. At Harvard, he studied bacterial viruses and learned that even the simplest organisms could provide valuable insights about fundamental biological processes.

The move to Brenner’s lab in Cambridge was a natural step. A pioneer in the field of molecular biology, Brenner was also the driving force behind the adoption of C. elegans as a genetic model organism, which he advocated for its simplicity (adults have fewer than 1000 cells, and only 302 neurons) and short generation time (only three days). Working in Brenner’s lab, Horvitz
and his collaborator John Sulston traced the lineage of every body cell from fertilization to adulthood, showing that the sequence of cell divisions was the same in each individual animal. Their landmark study provided a foundation for the entire field. “They know all the cells in the worm. Every single one,” says Constantine-Paton. “So when they make a mutation and something is weird, they can determine precisely which cell or set of cells are affected. We can only dream of having such an understanding of a mammal.”

It is now known that the worm has about 20,000 genes, many of which are conserved in mammals including humans. In fact, in many cases, a cloned human gene can stand in for a missing
worm gene, as is the case for vps-50. As a result, the worm has been a powerful discovery machine for human biology. In the early years, though, many doubted whether worms would be relevant. Horvitz persisted undeterred, and in 1992 his conviction paid off, with the discovery of ced-9, a worm gene that regulates programmed cell death. A graduate student in Horvitz’ lab cloned ced-9 and saw that it resembled a human cancer gene called Bcl-2. They also showed that human Bcl-2 could substitute for a mutant ced-9 gene in the worm and concluded that the two genes have similar functions: ced-9 in worms protects healthy cells from death, and Bcl-2 in cancer patients protects cancerous cells from death, allowing them to multiply. “This was the moment we knew that the studies we’d been doing with C. elegans were going to be relevant to understanding human biology and disease,” says Horvitz.

Ten years later, in 2002, he was in the French Alps with Constantine-Paton and their daughter Alex attending a wedding, when they heard the news on the radio: He’d won a Nobel Prize, along with Brenner and Sulston. On the return trip, Alex, then 9 years old but never shy, asked for first-class upgrades at the airport; the agent compromised and gave them all upgrades to business class instead.

Discovery machine at work

Since the Nobel Prize, Horvitz has studied the nervous system using the same strategy that had been so successful in deciphering the mechanism of programmed cell death. His approach, he says, begins with traditional genetics. Researchers expose worms to mutagens and observe their behavior. When they see an interesting change, they identify the mutation and try to link the gene to the nervous system to understand how it affects behavior.

“We make no assumptions,” he says. “We let the animal tell us the answer.”

While Horvitz continues to demonstrate that basic research using simple organisms produces invaluable insights about human biology and health, there are other forces at work in his lab. Horvitz maintains a sense of wonder about life and is undaunted by big questions.

For instance, when Bhatla came to him wanting to look for evidence of consciousness in worms, Horvitz blinked but didn’t say no. The science Bhatla proposed was novel, and the question
was intriguing. Bhatla pursued it. But, he says, “It didn’t work.”

So Bhatla went back to the drawing board. During his earlier experiments, he had observed that worms would avoid light, a previously known behavior. But he also noticed that they immediately stopped feeding. The animals had provided a clue. Bhatla went on to discover that worms respond to light by producing hydrogen peroxide, which activates a taste receptor.

In a sense, worms taste light, a wonder of biology no one could have predicted.

Some years ago, the Horvitz lab made t-shirts displaying a quote from the philosopher Friedrich Nietzsche: “You have made your way from worm to man, and much within you is still worm.”
The words have become an informal lab motto, “truer than Nietzsche could everhave imagined,” says Horvitz. “There’s still so much mystery, particularly about the brain, and we are still learning from the worm.”

Genome Editing with CRISPR – Cas9

Anne Trafton |

This animation depicts the CRISPR-Cas9 method for genome editing – a powerful new technology with many applications in biomedical research, including the potential to treat human genetic disease. Feng Zhang, a leader in the development of this technology, is a faculty member at MIT, an investigator at the McGovern Institute for Brain Research, and a core member of the Broad Institute.

 

Researchers find new actions of neurochemicals

by Catherine Delude |

Although the tiny roundworm Caenorhabditis elegans has only 302 neurons in its entire nervous system, studies of this simple animal have significantly advanced our understanding of human brain function because it shares many genes and neurochemical signaling molecules with humans. Now MIT researchers have found novel C. elegans neurochemical receptors, the discovery of which could lead to new therapeutic targets for psychiatric disorders if similar receptors are found in humans.

Dopamine and serotonin are members of a class of neurochemicals called biogenic amines, which function in neuronal circuitry throughout the brain. Many drugs used to treat psychiatric disorders, including depression and schizophrenia, target these signaling systems, as do cocaine and other drugs of abuse. Scientists have long known of a class of biogenic-amine receptors that are G protein-coupled receptors (GPCRs) and that, when activated, trigger a slow but long-lasting cascade of intracellular events that modulate nervous system activity.

A study in the July 3 issue of Science has found that in C. elegans these chemicals also act on receptors of a fundamentally different type. These receptors are chloride channels that open and close quickly in response to the binding of a neurochemical messenger. By allowing the passage of negatively charged chloride ions across the cell membrane, chloride channels can rapidly inactivate nerve cells.

“These results underscore the importance of determining whether, as in the C. elegans nervous system, a diversity of biogenic amine-gated chloride channels function in the human brain,” said H. Robert Horvitz of the McGovern Institute for Brain Research at MIT and senior author of the study. “If so, such channels might define novel therapeutic targets for neuropsychiatric disorders, such as depression and schizophrenia.”

In 2000, Horvitz’s group discovered that serotonin activates a chloride channel they called MOD-1, which inhibits neuronal activity in C. elegans.

In the current study, Niels Ringstad and Namiko Abe, a postdoctoral researcher and an undergraduate in Horvitz’s laboratory, respectively, looked for other ion channels that could be receptors for biogenic amines. Using both in vitro and in vivo methods, they surveyed the functions of 26 ion channels similar to MOD-1 and found three additional ion channels with an affinity for biogenic amines: dopamine activates one, serotonin another, and tyramine (the role of which in the human brain is unknown) a third. All three were chloride channels, like MOD-1.

“We now have four members of a family of chloride channels that can act as receptors for biogenic amines in the worm,” Ringstad said. “That these neurochemicals activate both GPCRs and ion channels means that they can have very complex actions in the nervous system, both as slow-acting neuromodulators and as fast-acting inhibitory neurotransmitters.”

It is unknown as yet whether an equivalent to this new class of worm receptor exists in the human brain, but Horvitz points out that worms have proved remarkably informative for providing insights into human biology. In 2002, Horvitz shared the Nobel Prize in Physiology or Medicine for the discovery based on studies of C. elegans of the mechanism of programmed cell death, a central feature of some neurodegenerative diseases and many other disorders in humans.

“Historically, studies of C. elegans have delineated mechanisms of neurotransmission that subsequently proved to be conserved in humans,” says Horvitz, the David H. Koch Professor of Biology at MIT and a Howard Hughes Medical Institute Investigator. “The next step is to look for chloride channels controlled by biogenic amines in mammalian neurons.”

This study was supported by the NIH, the Howard Hughes Medical Institute, the Life Sciences Research Foundation, and The Medical Foundation.