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Bacterial immune system may be utilized as a tool harboring an impressive recording capacity, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair
Bacterial immune system may be utilized as a tool harboring an impressive recording capacity
Curator: Larry H. Bernstein, MD, FCAP
LPBI
Creating a DNA Record with CRISPR
Researchers repurpose a bacterial immune system to be a molecular recording device.
Utilizing the bacterial CRISPR/Cas adaptive immune system, researchers at Harvard have developed a method for permanently recording molecular events in living cells, according to a report published inSciencetoday (June 9). The system integrates specific synthetic DNA elements into the bacterial genomes in temporally-ordered arrays, which, once sequenced, can provide a readout of the bacteria’s timeline of DNA events.
“The importance of the work is in providing a proof of principle: that a fascinating bacterial immune system may be utilized as a tool harboring an impressive recording capacity,” said microbiologist Udi Qimron of Tel Aviv University who was not involved in the work.
The CRISPR/Cas system works by snipping short DNA elements from the genomes of infecting viruses, integrating those elements into the bacterium’s genome (at the CRISPR locus), and using the RNAs produced from the integrated elements to direct destruction of the corresponding virus. In essence, the bacterium keeps a DNA account of its viral foes, and uses it against them.
Integration of these viral DNA elements—or oligomers—into the CRISPR locus is nonrandom: the most recent viral elements are consistently integrated ahead of older viral elements in the array. Harvard’sGeorge Church and colleagues considered that this temporal ordering of integration could form the basis of a molecular recording device. If defined synthetic DNA oligomers could be integrated into CRISPR loci just as viral elements are, then sequencing the cells’ CRISPR loci would provide a log of which oligomers the cells had been exposed to and when, the researchers reasoned.
To test this idea, the team used an E. coli strain that contained a CRISPR DNA locus and a stripped-down version of the Cas protein machinery. The minimal machinery consisted of inducible versions of Cas1 and Cas2—enzymes required for integrating the DNA oligomers—but lacked all the Cas machinery required for virus destruction. The researchers found that, by introducing specific synthetic DNA sequences into these cells in a timed manner (different oligomers on different days, for example), the resulting sequences of the CRISPR loci did indeed accurately reflect the order in which the oligomers had been introduced.
“It’s the first demonstration of the ordered acquisition of intentionally introduced DNA sequences,” said bioengineer Adam Arkin of the University of California, Berkeley, who did not participate in the work.
Using directed evolution, the team went on to create new versions of Cas1 and Cas2 that could integrate oligomers in a subtly different and discernable way (though still temporally ordered) to that of wildtype Cas1 and 2. Putting these modified Cas enzymes under the control of a different inducer allowed the team to record DNA events in two different modes—depending on which versions of Cas1 and 2 were operational.
“Essentially, we’re measuring concentrations of nucleic acids,” said Church. “Ideally it would be messenger RNAs but in this case it is DNA. . . . This is a proof of concept on the way to other things,” he added.Church suggested, for example, that if a CRISPR/Cas system were to be combined with a reverse transcriptase—an enzyme that converts RNA to DNA—in cells or animals, it could be used to provide a record of which messenger RNAs are expressed, when.
Another possibility, suggested Arkin, is to use CRISPR/Cas-engineered bacteria to provide information about the other microorganisms present in an environment—be that the soil, the human gut, or wherever.
“[The bacteria] could kill a few neighboring [bugs], secrete an enzyme that cleaved their DNA, and express a competence system to take that DNA in,” Arkin said. “That sounds insane, but there are bacteria who do that naturally,” he added. The foreign microbial DNA could then be incorporated and logged at the bacteria’s CRISPR locus, he explained.
Such applications are as-yet distant possibilities, but the new paper, said Arkin, “conceptually sets the flag in the ground and says, ‘Here’s how we should move forward.’”
S.L. Shipman et al., “Molecular recordings by directed CRISPR spacer acquisition,” Science,doi:10.1126/science.aaf1175, 2016.
Molecular recordings by directed CRISPR spacer acquisition
Seth L. Shipman1,2,3,*, Jeff Nivala1,3,*, Jeffrey D. Macklis2, George M. Church1,3,†
The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR-Cas system of E. coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device.
An open mind and collaborative spirit have taken Hans Clevers on a journey from medicine to developmental biology, gastroenterology, cancer, and stem cells.
Ihave had to talk a lot about my science recently and it’s made me think about how science works,” says Hans Clevers. “Scientists are trained to think science is driven by hypotheses, but for [my lab], hypothesis-driven research has never worked. Instead, it has been about trying to be as open-minded as possible—which is not natural for our brains,” adds the Utrecht University molecular genetics professor. “The human mind is such that it tries to prove it’s right, so pursuing a hypothesis can result in disaster. My advice to my own team and others is to not preformulate an answer to a scientific question, but just observe and never be afraid of the unknown. What has worked well for us is to keep an open mind and do the experiments. And find a collaborator if it is outside our niche.”
“One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory.”
Clevers entered medical school at Utrecht University in The Netherlands in 1978 while simultaneously pursuing a master’s degree in biology. Drawn to working with people in the clinic, Clevers had a training position in pediatrics lined up after medical school, but then mentors persuaded him to spend an additional year converting the master’s degree to a PhD in immunology. “At the end of that year, looking back, I got more satisfaction from the research than from seeing patients.” Clevers also had an aptitude for benchwork, publishing four papers from his PhD year. “They were all projects I had made up myself. The department didn’t do the kind of research I was doing,” he says. “Now that I look back, it’s surprising that an inexperienced PhD student could come up with a project and publish independently.”
Clevers studied T- and B-cell signaling; he set up assays to visualize calcium ion flux and demonstrated that the ions act as messengers to activate human B cells, signaling through antibodies on the cell surface. “As soon as the experiment worked, I got T cells from the lab next door and did the same experiment. That was my strategy: as soon as something worked, I would apply it elsewhere and didn’t stop just because I was a B-cell biologist and not a T-cell biologist. What I learned then, that I have continued to benefit from, is that a lot of scientists tend to adhere to a niche. They cling to these niches and are not that flexible. You think scientists are, but really most are not.”
Here, Clevers talks about promoting a collaborative spirit in research, the art of doing a pilot experiment, and growing miniature organs in a dish.
Clevers Creates
Re-search? Clevers was born in Eindhoven, in the south of The Netherlands. The town was headquarters to Philips Electronics, where his father worked as a businessman, and his mother took care of Clevers and his three brothers. Clevers did well in school but his passion was sports, especially tennis and field hockey, “a big thing in Holland.” Then in 1975, at age 18, he moved to Utrecht University, where he entered an intensive, biology-focused program. “I knew I wanted to be a biology researcher since I was young. In Dutch, the word for research is ‘onderzoek’ and I knew the English word ‘research’ and had wondered why there was the ‘re’ in the word, because I wanted to search but I didn’t want to do re-search—to find what someone else had already found.”
Opportunity to travel. “I was very disappointed in my biology studies, which were old-fashioned and descriptive,” says Clevers. He thought medicine might be more interesting and enrolled in medical school while still pursuing a master’s degree in biology at Utrecht. For the master’s, Clevers had to do three rotations. He spent a year at the International Laboratory for Research on Animal Diseases (ILRAD) in Nairobi, Kenya, and six months in Bethesda, Maryland, at the National Institutes of Health. “Holland is really small, so everyone travels.” Clevers saw those two rotations more as travel explorations. In Nairobi, he went on safaris and explored the country in Land Rovers borrowed from the institute. While in Maryland in 1980, Clevers—with the consent of his advisor, who thought it was a good idea for him to get a feel for the U.S.—flew to Portland, Oregon, and drove back to Boston with a musician friend along the Canadian border. He met the fiancé of political activist and academic Angela Davis in New York City and even stayed in their empty apartment there.
Life and lab lessons. Back in Holland, Clevers joined Rudolf Eugène Ballieux’s lab at Utrecht University to pursue his PhD, for which he studied immune cell signaling. “I didn’t learn much science from him, but I learned that you always have to create trust and to trust people around you. This became a major theme in my own lab. We don’t distrust journals or reviewers or collaborators. We trust everyone and we share. There will be people who take advantage, but there have only been a few of those. So I learned from Ballieux to give everyone maximum trust and then change this strategy only if they fail that trust. We collaborate easily because we give out everything and we also easily get reagents and tools that we may need. It’s been valuable to me in my career. And it is fun!”
Clevers Concentrates
On a mission. “Once I decided to become a scientist, I knew I needed to train seriously. Up to that point, I was totally self-trained.” From an extensive reading of the immunology literature, Clevers became interested in how T cells recognize antigens, and headed off to spend a postdoc studying the problem in Cox Terhorst’s lab at Dana-Farber Cancer Institute in Boston. “Immunology was young, but it was very exciting and there was a lot to discover. I became a professional scientist there and experienced how tough science is.” In 1988, Clevers cloned and characterized the gene for a component of the T-cell receptor (TCR) called CD3-epsilon, which binds antigen and activates intracellular signaling pathways.
On the fast track in Holland. Clevers returned to Utrecht University in 1989 as a professor of immunology. Within one month of setting up his lab, he had two graduate students and a technician, and the lab had cloned the first T cell–specific transcription factor, which they called TCF-1, in human T cells. When his former thesis advisor retired, Clevers was asked, at age 33, to become head of the immunology department. While the appointment was high-risk for him and for the department, Clevers says, he was chosen because he was good at multitasking and because he got along well with everyone.
Problem-solving strategy. “My strategy in research has always been opportunistic. One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory. I think there is an art to doing pilot experiments. So we have always just set up systems in which something happens and then you try and try things until a pattern appears and maybe you formulate a small hypothesis. But as soon as it turns out not to be exactly right, you abandon it. It’s a very open-minded type of research where you question whether what you are seeing is a real phenomenon without spending a year on doing all of the proper controls.”
Trial and error. Clevers’s lab found that while TCF-1 bound to DNA, it did not alter gene expression, despite the researchers’ tinkering with promoter and enhancer assays. “For about five years this was a problem. My first PhD students were leaving and they thought the whole TCF project was a failure,” says Clevers. His lab meanwhile cloned TCF homologs from several model organisms and made many reagents including antibodies against these homologs. To try to figure out the function of TCF-1, the lab performed a two-hybrid screen and identified components of the Wnt signaling pathway as binding partners of TCF-1. “We started to read about Wnt and realized that you study Wnt not in T cells but in frogs and flies, so we rapidly transformed into a developmental biology lab. We showed that we held the key for a major issue in developmental biology, the final protein in the Wnt cascade: TCF-1 binds b-catenin when b-catenin becomes available and activates transcription.” In 1996, Clevers published the mechanism of how the TCF-1 homolog in Xenopus embryos, called XTcf-3, is integrated into the Wnt signaling pathway.
Clevers Catapults
COURTESY OF HANS CLEVERS AND JEROEN HUIJBEN, NYMUS
3DCrypt building and colon cancer.
Clevers next collaborated with Bert Vogelstein’s lab at Johns Hopkins, linking TCF to Wnt signaling in colon cancer. In colon cancer cell lines with mutated forms of the tumor suppressor gene APC, the APC protein can’t rein in b-catenin, which accumulates in the cytoplasm, forms a complex with TCF-4 (later renamed TCF7L2) in the nucleus, and caninitiate colon cancer by changing gene expression. Then, the lab showed that Wnt signaling is necessary for self-renewal of adult stem cells, as mice missing TCF-4 do not have intestinal crypts, the site in the gut where stem cells reside. “This was the first time Wnt was shown to play a role in adults, not just during development, and to be crucial for adult stem cell maintenance,” says Clevers. “Then, when I started thinking about studying the gut, I realized it was by far the best way to study stem cells. And I also realized that almost no one in the world was studying the healthy gut. Almost everyone who researched the gut was studying a disease.” The main advantages of the murine model are rapid cell turnover and the presence of millions of stereotypic crypts throughout the entire intestine.
Against the grain. In 2007, Nick Barker, a senior scientist in the Clevers lab, identified the Wnt target gene Lgr5 as a unique marker of adult stem cells in several epithelial organs, including the intestine, hair follicle, and stomach. In the intestine, the gene codes for a plasma membrane protein on crypt stem cells that enable the intestinal epithelium to self-renew, but can also give rise to adenomas of the gut. Upon making mice with adult stem cell populations tagged with a fluorescent Lgr5-binding marker, the lab helped to overturn assumptions that “stem cells are rare, impossible to find, quiescent, and divide asymmetrically.”
On to organoids. Once the lab could identify adult stem cells within the crypts of the gut, postdoc Toshiro Sato discovered that a single stem cell, in the presence of Matrigel and just three growth factors, could generate a miniature crypt structure—what is now called an organoid. “Toshi is very Japanese and doesn’t always talk much,” says Clevers. “One day I had asked him, while he was at the microscope, if the gut stem cells were growing, and he said, ‘Yes.’ Then I looked under the microscope and saw the beautiful structures and said, ‘Why didn’t you tell me?’ and he said, ‘You didn’t ask.’ For three months he had been growing them!” The lab has since also grown mini-pancreases, -livers, -stomachs, and many other mini-organs.
Tumor Organoids. Clevers showed that organoids can be grown from diseased patients’ samples, a technique that could be used in the future to screen drugs. The lab is also building biobanks of organoidsderived from tumor samples and adjacent normal tissue, which could be especially useful for monitoring responses to chemotherapies. “It’s a similar approach to getting a bacterium cultured to identify which antibiotic to take. The most basic goal is not to give a toxic chemotherapy to a patient who will not respond anyway,” says Clevers. “Tumor organoids grow slower than healthy organoids, which seems counterintuitive, but with cancer cells, often they try to divide and often things go wrong because they don’t have normal numbers of chromosomes and [have] lots of mutations. So, I am not yet convinced that this approach will work for every patient. Sometimes, the tumor organoids may just grow too slowly.”
Selective memory. “When I received the Breakthrough Prize in 2013, I invited everyone who has ever worked with me to Amsterdam, about 100 people, and the lab organized a symposium where many of the researchers gave an account of what they had done in the lab,” says Clevers. “In my experience, my lab has been a straight line from cloning TCF-1 to where we are now. But when you hear them talk it was ‘Hans told me to try this and stop this’ and ‘Half of our knockout mice were never published,’ and I realized that the lab is an endless list of failures,” Clevers recalls. “The one thing we did well is that we would start something and, as soon as it didn’t look very good, we would stop it and try something else. And the few times when we seemed to hit gold, I would regroup my entire lab. We just tried a lot of things, and the 10 percent of what worked, those are the things I remember.”
Greatest Hits
Cloned the first T cell–specific transcription factor, TCF-1, and identified homologous genes in model organisms including the fruit fly, frog, and worm
Found that transcriptional activation by the abundant β-catenin/TCF-4 [TCF7L2] complex drives cancer initiation in colon cells missing the tumor suppressor protein APC
First to extend the role of Wnt signaling from developmental biology to adult stem cells by showing that the two Wnt pathway transcription factors, TCF-1 and TCF-4, are necessary for maintaining the stem cell compartments in the thymus and in the crypt structures of the small intestine, respectively
Identified Lgr5 as an adult stem cell marker of many epithelial stem cells including those of the colon, small intestine, hair follicle, and stomach, and found that Lgr5-expressing crypt cells in the small intestine divide constantly and symmetrically, disproving the common belief that stem cell division is asymmetrical and uncommon
Established a three-dimensional, stable model, the “organoid,” grown from adult stem cells, to study diseased patients’ tissues from the gut, stomach, liver, and prostate
Regenerative Medicine Comes of Age
“Anti-Aging Medicine” Sounds Vaguely Disreputable, So Serious Scientists Prefer to Speak of “Regenerative Medicine”
Induced pluripotent stem cells (iPSCs) and genome-editing techniques have facilitated manipulation of living organisms in innumerable ways at the cellular and genetic levels, respectively, and will underpin many aspects of regenerative medicine as it continues to evolve.
An attitudinal change is also occurring. Experts in regenerative medicine have increasingly begun to embrace the view that comprehensively repairing the damage of aging is a practical and feasible goal.
A notable proponent of this view is Aubrey de Grey, Ph.D., a biomedical gerontologist who has pioneered an regenerative medicine approach called Strategies for Engineered Negligible Senescence (SENS). He works to “develop, promote, and ensure widespread access to regenerative medicine solutions to the disabilities and diseases of aging” as CSO and co-founder of the SENS Research Foundation. He is also the editor-in-chief of Rejuvenation Research, published by Mary Ann Liebert.
Dr. de Grey points out that stem cell treatments for age-related conditions such as Parkinson’s are already in clinical trials, and immune therapies to remove molecular waste products in the extracellular space, such as amyloid in Alzheimer’s, have succeeded in such trials. Recently, there has been progress in animal models in removing toxic cells that the body is failing to kill. The most encouraging work is in cancer immunotherapy, which is rapidly advancing after decades in the doldrums.
Many damage-repair strategies are at an early stage of research. Although these strategies look promising, they are handicapped by a lack of funding. If that does not change soon, the scientific community is at risk of failing to capitalize on the relevant technological advances.
Regenerative medicine has moved beyond boutique applications. In degenerative disease, cells lose their function or suffer elimination because they harbor genetic defects. iPSC therapies have the potential to be curative, replacing the defective cells and eliminating symptoms in their entirety. One of the biggest hurdles to commercialization of iPSC therapies is manufacturing.
Building Stem Cell Factories
Cellular Dynamics International (CDI) has been developing clinically compatible induced pluripotent stem cells (iPSCs) and iPSC-derived human retinal pigment epithelial (RPE) cells. CDI’s MyCell Retinal Pigment Epithelial Cells are part of a possible therapy for macular degeneration. They can be grown on bioengineered, nanofibrous scaffolds, and then the RPE cell–enriched scaffolds can be transplanted into patients’ eyes. In this pseudo-colored image, RPE cells are shown growing over the nanofibers. Each cell has thousands of “tongue” and “rod” protrusions that could naturally support rod and cone cells in the eye.
“Now that an infrastructure is being developed to make unlimited cells for the tools business, new opportunities are being created. These cells can be employed in a therapeutic context, and they can be used to understand the efficacy and safety of drugs,” asserts Chris Parker, executive vice president and CBO, Cellular Dynamics International (CDI). “CDI has the capability to make a lot of cells from a single iPSC line that represents one person (a capability termed scale-up) as well as the capability to do it in parallel for multiple individuals (a capability termed scale-out).”
Minimally manipulated adult stem cells have progressed relatively quickly to the clinic. In this scenario, cells are taken out of the body, expanded unchanged, then reintroduced. More preclinical rigor applies to potential iPSC therapy. In this case, hematopoietic blood cells are used to make stem cells, which are manufactured into the cell type of interest before reintroduction. Preclinical tests must demonstrate that iPSC-derived cells perform as intended, are safe, and possess little or no off-target activity.
For example, CDI developed a Parkinsonian model in which iPSC-derived dopaminergic neurons were introduced to primates. The model showed engraftment and enervation, and it appeared to be free of proliferative stem cells.
“You will see iPSCs first used in clinical trials as a surrogate to understand efficacy and safety,” notes Mr. Parker. “In an ongoing drug-repurposing trial with GlaxoSmithKline and Harvard University, iPSC-derived motor neurons will be produced from patients with amyotrophic lateral sclerosis and tested in parallel with the drug.” CDI has three cell-therapy programs in their commercialization pipeline focusing on macular degeneration, Parkinson’s disease, and postmyocardial infarction.
Keeping an Eye on Aging Eyes
The California Project to Cure Blindness is evaluating a stem cell–based treatment strategy for age-related macular degeneration. The strategy involves growing retinal pigment epithelium (RPE) cells on a biostable, synthetic scaffold, then implanting the RPE cell–enriched scaffold to replace RPE cells that are dying or dysfunctional. One of the project’s directors, Dennis Clegg, Ph.D., a researcher at the University of California, Santa Barbara, provided this image, which shows stem cell–derived RPE cells. Cell borders are green, and nuclei are red.
The eye has multiple advantages over other organ systems for regenerative medicine. Advanced surgical methods can access the back of the eye, noninvasive imaging methods can follow the transplanted cells, good outcome parameters exist, and relatively few cells are needed.
These advantages have attracted many groups to tackle ocular disease, in particular age-related macular degeneration, the leading cause of blindness in the elderly in the United States. Most cases of age-related macular degeneration are thought to be due to the death or dysfunction of cells in the retinal pigment epithelium (RPE). RPE cells are crucial support cells for the rods, cones, and photoreceptors. When RPE cells stop working or die, the photoreceptors die and a vision deficit results.
A regenerated and restored RPE might prevent the irreversible loss of photoreceptors, possibly via the the transplantation of functionally polarized RPE monolayers derived from human embryonic stem cells. This approach is being explored by the California Project to Cure Blindness, a collaborative effort involving the University of Southern California (USC), the University of California, Santa Barbara (UCSB), the California Institute of Technology, City of Hope, and Regenerative Patch Technologies.
The project, which is funded by the California Institute of Regenerative Medicine (CIRM), started in 2010, and an IND was filed early 2015. Clinical trial recruitment has begun.
One of the project’s leaders is Dennis Clegg, Ph.D., Wilcox Family Chair in BioMedicine, UCSB. His laboratory developed the protocol to turn undifferentiated H9 embryonic stem cells into a homogenous population of RPE cells.
“These are not easy experiments,” remarks Dr. Clegg. “Figuring out the biology and how to make the cell of interest is a challenge that everyone in regenerative medicine faces. About 100,000 RPE cells will be grown as a sheet on a 3 × 5 mm biostable, synthetic scaffold, and then implanted in the patients to replace the cells that are dying or dysfunctional. The idea is to preserve the photoreceptors and to halt disease progression.”
Moving therapies such as this RPE treatment from concept to clinic is a huge team effort and requires various kinds of expertise. Besides benefitting from Dr. Clegg’s contribution, the RPE project incorporates the work of Mark Humayun, M.D., Ph.D., co-director of the USC Eye Institute and director of the USC Institute for Biomedical Therapeutics and recipient of the National Medal of Technology and Innovation, and David Hinton, Ph.D., a researcher at USC who has studied how actvated RPE cells can alter the local retinal microenvironment.
Researchers from Harvard Medical School and Massachusetts General Hospital have completed the first stage of an important collaboration aimed at understanding the intricate variables of neuropsychiatric disease—something that currently eludes clinicians and scientists.
The research team, led by Isaac Kohane at HMS and Roy Perlis at Mass General, has created a neuropsychiatric cellular biobank—one of the largest in the world.
It contains induced pluripotent stem cells, or iPSCs, derived from skin cells taken from 100 people with neuropsychiatric diseases such as schizophrenia, bipolar disorder and major depression, and from 50 people without neuropsychiatric illness.
In addition, a detailed profile of each patient, obtained from hours of in-person assessment as well as from electronic medical records, is matched to each cell sample.
As a result, the scientific community can now for the first time access cells representing a broad swath of neuropsychiatric illness. This enables researchers to correlate molecular data with clinical information in areas such as variability of drug reactions between patients. The ultimate goal is to help treat, with greater precision, conditions that often elude effective management.
The cell collection and generation was led by investigators at Mass General, who in collaboration with Kohane and his team are working to characterize the cell lines at a molecular level. The cell repository, funded by the National Institutes of Health, is housed at Rutgers University.
“This biobank, in its current form, is only the beginning,” said Perlis, director of the MGH Psychiatry Center for Experimental Drugs and Diagnostics and HMS associate professor of psychiatry. “By next year we’ll have cells from a total of four hundred patients, with additional clinical detail and additional cell types that we will share with investigators.”
A current major limitation to understanding brain diseases is the inability to access brain biopsies on living patients. As a result, researchers typically study blood cells from patients or examine post-mortem tissue. This is in stark contrast with diseases such as cancer, for which there are many existing repositories of highly characterized cells from patients.
The new biobank offers a way to push beyond this limitation.
A Big Step Forward
While the biobank is already a boon to the scientific community, researchers at MGH and the HMS Department of Biomedical Informatics will be adding additional layers of molecular data to all of the cell samples. This information will include whole genome sequencing and transcriptomic and epigenetic profiling of brain cells made from the stem cell lines.
Collaborators in the HMS Department of Neurobiology, led by Michael Greenberg, department chair and Nathan Marsh Pusey Professor of Neurobiology, will also work to examine characteristics of other types of neurons derived from these stem cells.
“This can potentially alter the entire way we look at and diagnose many neuropsychiatric conditions,” said Perlis.
One example may be to understand how the cellular responses to medication correspond to the patient’s documented responses, comparing in vitro with in vivo. “This would be a big step forward in bringing precision medicine to psychiatry,” Perlis said.
“It’s important to recall that in the field of genomics, we didn’t find interesting connections to disease until we had large enough samples to really investigate these complex conditions,” said Kohane, chair of the HMS Department of Biomedical Informatics.
“Our hypothesis is that here we will require far fewer patients,” he said. “By measuring the molecular functioning of the cells of each patient rather than only their genetic risk, and combining that all that’s known of these people in terms of treatment response and cognitive function, we will discover a great deal of valuable information about these conditions.”
Added Perlis, “In the early days of genetics, there were frequent false positives because we were studying so few people. We’re hoping to avoid the same problem in making cellular models, by ensuring that we have a sufficient number of cell lines to be confident in reporting differences between patient groups.”
The generation of stem cell lines and characterization of patients and brain cell lines is funded jointly by the the National Institute of Mental Health, the National Human Genome Research Institute and a grant from the Centers of Excellence in Genomic Science program.
On C.T.E. and Athletes, Science Remains in Its Infancy
Alzheimer’s disease is the most common form of dementia, characterized by two pathological hallmarks: amyloid-β plaques and neurofibrillary tangles1. The amyloid hypothesis of Alzheimer’s disease posits that the excessive accumulation of amyloid-β peptide leads to neurofibrillary tangles composed of aggregated hyperphosphorylated tau2, 3. However, to date, no single disease model has serially linked these two pathological events using human neuronal cells. Mouse models with familial Alzheimer’s disease (FAD) mutations exhibit amyloid-β-induced synaptic and memory deficits but they do not fully recapitulate other key pathological events of Alzheimer’s disease, including distinct neurofibrillary tangle pathology4, 5. Human neurons derived from Alzheimer’s disease patients have shown elevated levels of toxic amyloid-β species and phosphorylated tau but did not demonstrate amyloid-β plaques or neurofibrillary tangles6, 7, 8, 9, 10, 11. Here we report that FAD mutations in β-amyloid precursor protein and presenilin 1 are able to induce robust extracellular deposition of amyloid-β, including amyloid-β plaques, in a human neural stem-cell-derived three-dimensional (3D) culture system. More importantly, the 3D-differentiated neuronal cells expressing FAD mutations exhibited high levels of detergent-resistant, silver-positive aggregates of phosphorylated tau in the soma and neurites, as well as filamentous tau, as detected by immunoelectron microscopy. Inhibition of amyloid-β generation with β- or γ-secretase inhibitors not only decreased amyloid-β pathology, but also attenuated tauopathy. We also found that glycogen synthase kinase 3 (GSK3) regulated amyloid-β-mediated tau phosphorylation. We have successfully recapitulated amyloid-β and tau pathology in a single 3D human neural cell culture system. Our unique strategy for recapitulating Alzheimer’s disease pathology in a 3D neural cell culture model should also serve to facilitate the development of more precise human neural cell models of other neurodegenerative disorders.
Figure 2: Robust increases of extracellular amyloid-β deposits in 3D-differentiated hNPCs with FAD mutations.close
a, Thin-layer 3D culture protocol. HC, histochemistry; IF, immunofluorescence; IHC, immunohistochemistry. b, Amyloid-β deposits in 6-week differentiated control and FAD ReN cells in 3D Matrigel (green, GFP; blue, 3D6; scale bar, …
In the March 28th, 2016 issue of the journal Nature Medicine, Mark Tuszynski and his colleagues from the University of California, San Diego, in collaboration with colleagues from Japan and Wisconsin, report that they were able to successfully coax stem cell-derived neurons to regenerate damaged corticospinal tracts in rats. Furthermore, this regeneration produced observable, functional benefits.
What is the “corticospinal tract” you ask? The corticospinal tracts are part of the “pyramidal tracts” that include the corticospinal and corticobulbar tracts. The pyramidal tracts are the main controllers of voluntary movement and connect their nerve fibers eventually to cells that serve voluntary muscles and allow them to contract. We call such nerves “motor nerves,” and the corticospinal nerve tracts are among the most important of the motor nerve tracts.
These neural tracts are collectively called “pyramidal tracts” because they pass through a small area of the brain stem known as the pyramids, which lie on the ventral side of the medulla oblongata. Both pyramidal tracts originate in the forebrain; specifically from the so-called “motor cortex” of the forebrain. The motor cortex lies just in front of the central sulcus of the forebrain. In the motor cortex, lies thousands of “upper motor neurons” that extend their axons down to the brain stem and spinal cord.
In the brain stem, the majority of these corticospinal tracts crossover (or decussate) to the other side of the brain stem and travel down the opposite side of the spinal cord. The corticospinal axons extend all the way down the spinal cord, until they make a connection (synapse) with a “lower motor neuron” that extends its axon to the skeletal muscles that it will direct to contract. The corticobulbar tract contains nerves that conduct nerve impulses from cranial nerves and these help the muscles of the face and neck contract, and are involved in facial expressions, swallowing, chewing, and so on.
Damage to the upper motor neurons as a result of a stroke can rob a person of the ability to move, since the muscles that are attached to the upper motor neurons cannot receive any signals to contract. Likewise, damage to the axonal tracts (also known as nerve fibers) can paralyze a patient and rob them of their ability to move.
“We humans use corticospinal axons for voluntary movement,” said Tuszynski. “In the absence of regeneration of this system in previous studies, I was doubtful that most therapies taken to humans would improve function. Now that we can regenerate the most important motor system for humans, I think that the potential for translation is more promising.”
Novel use of EPR spectroscopy to study in vivo protein structure
α-synuclein is a protein found abundantly throughout the brain. It is present mainly at the neuron ends where it is thought to play a role in ensuring the supply of synaptic vesicles in presynaptic terminals, which are required for the release of neurotransmitters to relay signals between neurons. It is critical for normal brain function.
However, α-synuclein is also the primary protein component of the cerebral amyloid deposits characteristic of Parkinson’s disease and its precursor is found in the amyloid plaques of Alzheimer’s disease. Although α-synuclein is present in all areas of the brain, these disease-state amyloid plaques only arise in distinct areas.
Imaging of isolated samples of α-synuclein in vitro indicate that it does not have the precise 3D folded structure usually associated with proteins. It is therefore classed as an intrinsically disordered protein. However, it was not known whether the protein also lacked a precise structure in vivo.
There have been reports that it can form helical tetramers. Since the 3D structure of a biological protein is usually precisely matched to the specific function it performs, knowing the structure of α-synuclein within a living cell will help elucidate its role and may also improve understanding of the disease states with which it is associated.
If α-synuclein remains disordered in vivo, it may be possible for the protein to achieve different structures, and have different properties, depending on its surroundings.
Techniques for determining protein structure
It has long been known that elucidating the structure of a protein at an atomic level is fundamental for understanding its normal function and behavior. Furthermore, such knowledge can also facilitate the development of targeted drug treatments. Unfortunately, observing the atomic structure of a protein in vivo is not straightforward.
X-ray diffraction is the technique usually adopted for visualizing structures at atomic resolution, but this requires crystals of the molecule to be produced and this cannot be done without separating the molecules of interest from their natural environment. Such processes can modify the protein from its usual state and, particularly with complex structures, such effects are difficult to predict.
The development of nuclear magnetic resonance (NMR) spectroscopy improved the situation by making it possible for molecules to be analyzed under in vivo conditions, i.e. same pH, temperature and ionic concentration.
More recently, increases in the sensitivity of NMR and the use of isotope labelling have enabled determinations of the atomic level structure and dynamics of proteins to be determined within living cells1. NMR has been used to determine the structure of a bacterial protein within living cells2 but it is difficult to achieve sufficient quantities of the required protein within mammalian cells and to keep the cells alive for NMR imaging to be conducted.
Electron paramagnetic resonance (EPR) spectroscopy for determining protein structure
Recently, researchers have managed to overcome these obstacles by using in-cell NMR and electron paramagnetic resonance (EPR) spectroscopy. EPR spectroscopy is a technique that is similar to NMR spectroscopy in that it is based on the measurement and interpretation of the energy differences between excited and relaxed molecular states.
In EPR spectroscopy it is electrons that are excited, whereas in NMR signals are created through the spinning of atomic nuclei. EPR was developed to measure radicals and metal complexes, but has also been utilized to study the dynamic organization of lipids in biological membranes3.
EPR has now been used for the first time in protein structure investigations and has provided atomic-resolution information on the structure of α-synuclein in living mammalians4,5.
Bacterial forms of the α-synuclein protein labelled with 15N isotopes were introduced into five types of mammalian cell using electroporation. Concentrations of α-synuclein close to those found in vivo were achieved and the 15N isotopes allowed the protein to be clearly defined from other cellular components by NMR. The conformation of the protein was then determined using electron paramagnetic resonance (EPR).
The results showed that within living mammalian cells α-synuclein remains as a disordered and highly dynamic monomer. Different intracellular environments did not induce major conformational changes.
Summary
The novel use of EPR spectroscopy has resolved the mystery surrounding the in vivo conformation of α-synuclein. It showed that α-synuclein maintains its disordered monomeric form under physiological cell conditions. It has been demonstrated for the first time that even in crowded intracellular environments α-synuclein does not form oligomers, showing that intrinsic structural disorder can be sustained within mammalian cells.
References
Freedberg DI and Selenko P. Live cell NMR Annu. Rev. Biophys. 2014;43:171–192.
Sakakibara D, et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 2009;458:102–105.
Yashroy RC. Magnetic resonance studies of dynamic organisation of lipids in chloroplast membranes. Journal of Biosciences 1990;15(4):281.
Alderson TA and Bax AD. Parkinson’s Disease. Disorder in the court. Nature 2016; doi:10.1038/nature16871.
Theillet FX, et al. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 2016; doi:10.1038/nature16531.
The corticospinal tract (CST) is the most important motor system in humans, yet robust regeneration of this projection after spinal cord injury (SCI) has not been accomplished. In murine models of SCI, we report robust corticospinal axon regeneration, functional synapse formation and improved skilled forelimb function after grafting multipotent neural progenitor cells into sites of SCI. Corticospinal regeneration requires grafts to be driven toward caudalized (spinal cord), rather than rostralized, fates. Fully mature caudalized neural grafts also support corticospinal regeneration. Moreover, corticospinal axons can emerge from neural grafts and regenerate beyond the lesion, a process that is potentially related to the attenuation of the glial scar. Rat corticospinal axons also regenerate into human donor grafts of caudal spinal cord identity. Collectively, these findings indicate that spinal cord ‘replacement’ with homologous neural stem cells enables robust regeneration of the corticospinal projection within and beyond spinal cord lesion sites, achieving a major unmet goal of SCI research and offering new possibilities for clinical translation.
Scientists build a living cellular organism with a genome smaller than any known in nature.
By Ruth Williams | March 24, 2016
By stripping down the genome of a mycoplasma bacterium to the minimal genes required for life,Craig Venter and colleagues have created a new organism with the smallest genome of any known cellular life form. The work, published in Sciencetoday (March 24), is the closest scientists have come to creating a cell in which every gene and protein is fully understood—but they are not quite there yet.
“In biology, as we’ve been trying to do genetic and biological engineering, we’re frustrated by the fact that . . . evolution has given us a real mess—it’s really just bubble gum and sticks, piecing together whatever works,” said biomedical engineer Chris Voigt of MIT who was not involved in the study. “This [work] is one of the first attempts at a grand scale to go in and try to clean up some of the mess . . . so that we can better understand the genetics.”
The quest to synthesize a minimal genome with only the essential genes for life is one researchers at the J. Craig Venter Institute (JCVI) in San Diego have been doggedly pursuing for the better part of two decades. Clyde Hutchison, an investigator at JCVI and lead author of the new study, explained the motivation: “We want to understand at a mechanistic level how a living cell grows and divides,” he told The Scientist, and yet, “there is no cell that exists where the function of every gene is known.” Possession of such fundamental knowledge, he added, would also put researchers “in a better position to engineer cells to make specific products,” like pharmaceuticals, Hutchinson said.
The team’s starting point was the bacterium Mycoplasma genitalium, which has the smallest known genome of any living cell with just 525 genes. However, it also has a very slow growth rate, making it difficult to work with. To practice synthesizing genomes and building new organisms, the team therefore turned to M. genitalium’s cousins, M. mycoides and M. capricolum, which have bigger genomes and faster growth rates. In 2010, Venter’s team successfully synthesized a version of the M. mycoides genome (JCVI-syn1.0) and placed it into the cell of a M. capricolum that had had its own genome removed. This was the first cell to contain a fully synthetic genome capable of supporting replicative life.
With the genome synthesis and transfer skills mastered, the next step was to make the genome smaller, explained Hutchison. One approach would be to delete the genes one by one and see which the cells could live without. But “we thought we knew enough, that it would be that much faster to design the genome, build it, and install it in a cell,” said Hutchison. The problem was, “we weren’t completely right about that,” he said. “It took quite a bit longer than we thought.”
Using JCVI-syn1.0 as their starting material, the researchers initially designed a minimal genome based on information from the literature and from mutagenesis studies that suggested which genes were likely essential. They divided this genome into eight overlapping segments and tested each one in combination with the complementary seven-eighths of the standard JCVI-syn1.0 genome. All but one of the designed segments failed to sustain viable cells.
Going back to the drawing board, the team decided to perform mutagenesis experiments on JCVI-syn1.0 to determine, categorically, which genes were required for life. Their experiments revealed that the genes fell into three groups: essential, nonessential, and quasiessential—those that aren’t strictly required, but without which growth is severely impaired. The failure to include these quasiessential genes in the initial design explained in large part why it had failed, explained Hutchison. “The concept of a minimal genome seems simple, but when you get into it, it’s a little more complicated,” he said. “There’s a trade-off between genome size and growth rate.”
Equipped with this knowledge, the team redesigned, synthesized, and tested new genome segments retaining the quasiessential genes. Three iterative cycles of testing later, the team had a genome that successfully supported life.
“This is a really pioneering next step in the use of synthetic biology,” said Leroy Hood, president of the Institute for Systems Biology in Seattle who also did not participate in the research.
Ultimately the team removed 428 genes from the JCVI-syn1.0 genome to create JCVI-syn3.0 with 473 genes (438 protein-coding genes and 35 RNA genes)—considerably fewer than the 525 genes of M. genitalium. Interestingly, the functions of around one-third of the genes (149) remain unknown. “I was surprised it was that high,” said Hood, “but I also think we kid ourselves about how much we know about the genomes of organisms. There’s still an enormous amount of dark matter.”
Some of these genes of unknown function appear to be conserved in higher eukaryotes, said Hutchison. “Those, in a way, are the most exciting,” he said, “because they might represent some new undescribed function that has spread through other life forms.”
C.A. Hutchison III et al., “Design and synthesis of a minimal bacterial genome,” Science, 351: 1414, 2016.
Design and synthesis of a minimal bacterial genome
A goal in biology is to understand the molecular and biological function of every gene in a cell. One way to approach this is to build a minimal genome that includes only the genes essential for life. In 2010, a 1079-kb genome based on the genome of Mycoplasma mycoides (JCV-syn1.0) was chemically synthesized and supported cell growth when transplanted into cytoplasm. Hutchison IIIet al. used a design, build, and test cycle to reduce this genome to 531 kb (473 genes). The resulting JCV-syn3.0 retains genes involved in key processes such as transcription and translation, but also contains 149 genes of unknown function.
INTRODUCTION In 1984, the simplest cells capable of autonomous growth, the mycoplasmas, were proposed as models for understanding the basic principles of life. In 1995, we reported the first complete cellular genome sequences (Haemophilus influenza, 1815 genes, and Mycoplasma genitalium, 525 genes). Comparison of these sequences revealed a conserved core of about 250 essential genes, much smaller than either genome. In 1999, we introduced the method of global transposon mutagenesis and experimentally demonstrated that M. genitalium contains many genes that are nonessential for growth in the laboratory, even though it has the smallest genome known for an autonomously replicating cell found in nature. This implied that it should be possible to produce a minimal cell that is simpler than any natural one. Whole genomes can now be built from chemically synthesized oligonucleotides and brought to life by installation into a receptive cellular environment. We have applied whole-genome design and synthesis to the problem of minimizing a cellular genome. RATIONALE Since the first genome sequences, there has been much work in many bacterial models to identify nonessential genes and define core sets of conserved genetic functions, using the methods of comparative genomics. Often, more than one gene product can perform a particular essential function. In such cases, neither gene will be essential, and neither will necessarily be conserved. Consequently, these approaches cannot, by themselves, identify a set of genes that is sufficient to constitute a viable genome. We set out to define a minimal cellular genome experimentally by designing and building one, then testing it for viability. Our goal is a cell so simple that we can determine the molecular and biological function of every gene.
RESULTS Whole-genome design and synthesis were used to minimize the 1079–kilobase pair (kbp) synthetic genome of M. mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology in combination with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three more cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kbp, 473 genes). Its genome is smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 has a doubling time of ~180 min, produces colonies that are morphologically similar to those of JCVI-syn1.0, and appears to be polymorphic when examined microscopically. CONCLUSION The minimal cell concept appears simple at first glance but becomes more complex upon close inspection. In addition to essential and nonessential genes, there are many quasi-essential genes, which are not absolutely critical for viability but are nevertheless required for robust growth. Consequently, during the process of genome minimization, there is a trade-off between genome size and growth rate. JCVI-syn3.0 is a working approximation of a minimal cellular genome, a compromise between small genome size and a workable growth rate for an experimental organism. It retains almost all the genes that are involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions, suggesting the presence of undiscovered functions that are essential for life. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.
Four design-build-test cycles produced JCVI-syn3.0.
(A) The cycle for genome design, building by means of synthesis and cloning in yeast, and testing for viability by means of genome transplantation. After each cycle, gene essentiality is reevaluated by global transposon mutagenesis. (B) Comparison of JCVI-syn1.0 (outer blue circle) with JCVI-syn3.0 (inner red circle), showing the division of each into eight segments. The red bars inside the outer circle indicate regions that are retained in JCVI-syn3.0. (C) A cluster of JCVI-syn3.0 cells, showing spherical structures of varying sizes (scale bar, 200 nm).
Abstract
We used whole-genome design and complete chemical synthesis to minimize the 1079–kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design.
A MATTER OF TIME: Optogenetics methods, which work on the millisecond timescale, allow for the finest level of temporal control over neuron excitation and inhibition. The chemogenetic tools, DREADDs and PSAMs-PSEMs, are ideal for the study of longer-lasting behaviors such as appetite, thirst, or anxiety because they work over a scale of the minutes-to-hours. The receptors are incorporated into specific neurons or cells using viruses. Ligands—CNO or salvinorin B (for DREADD receptors) or PSEMs—are administered via injection or drinking water. Both receptors and ligands are orthogonal, meaning they do not bind to anything else in the body. REDRAWN WITH PERMISSION OF SCOTT STERNSON FROM SCIENCE, 333:1292, 2011; NEUROENDOCRINOLOGY, 100:98, 2014
In a pharmacology lab at the University of North Carolina at Chapel Hill, doctoral student Reid Olsen, working with brain tissue harvested from a mouse just a few hours earlier, readies half a dozen dime-size slices for live calcium imaging. This mouse’s brain contains a genetically engineered receptor that Olsen has targeted to cells thought to control the making of new neurons in adult mice. He is about to use a synthetic drug to activate this receptor in the tissue. When it indeed works—just as he has predicted—he turns his attention to attempting to stimulate neurogenesis in a freely moving mouse that has the same engineered receptors in its brain.
Less than a decade ago, such precise control over neuronal activity in a dish, let alone in a living brain, was impossible. The drugs available to repress neurons or encourage them to fire would produce off-target effects or eliminate cell populations indiscriminately.
Working in the lab of Juan Song, Olsen is using a “designer receptor exclusively activated by a designer drug,” or DREADD. These modified G protein–coupled receptors (GPCRs) are usually either virally administered or bred into animals, then activated by a specific ligand that’s either injected or taken orally. Both the receptor and the ligand are designed to be orthogonal, effectively meaning they bind to each other but to nothing else.
Along with DREADDs, recently developed orthogonal ligand-gated ion channels called “pharmacologically selective actuator molecules” and “pharmacologically selective effector molecules” (PSAMs-PSEMs), are allowing researchers to dial up or dial down neuronal activity in living animals, with the goal of clarifying the brain wiring that controls appetite, thirst, anxiety, and many other behaviors.
Along with DREADDs, recently developed orthogonal ligand-gated ion channels called “pharmacologically selective actuator molecules” and “pharmacologically selective effector molecules” (PSAMs-PSEMs), are allowing researchers to dial up or dial down neuronal activity in living animals, with the goal of clarifying the brain wiring that controls appetite, thirst, anxiety, and many other behaviors.
“[DREADDs and PSAMs–PSEMs] are completely complementary methods, and they can in principle be used together in the same animal.—Scott Sternson, HHMI Janelia Research Campus”
These are not the only so-called chemical genetic, or “chemogenetic,” tools for controlling cells. Orthogonal kinases have been used in the brain to deduce the mechanisms underlying epilepsy, memory, and neuronal development. And inducible genetic systems, now in wide use for two decades—for example, tetracycline-dependent transcriptional promoters—are incredibly powerful for expressing specific genes at a particular point in an animal’s development, says Bruce Conklin, senior investigator at the University of California, San Francisco–affiliated Gladstone Institute of
Cardiovascular Disease, whose group pioneered the development of engineered GPCRs. At the other extreme of temporal control from inducible genetic systems, optogenetics—a set of methods that use light to activate genetically encoded opsins—is widely used for controlling brain cells on the millisecond time scale in vivo.
As tools, DREADDs and PSAMs-PSEMs allow control of neuronal activity over a middle ground—from minutes to hours. “These are the time scales that are most useful, in my opinion, for neurobiology experiments,” says Scott Sternson of the Howard Hughes Medical Institute’s Janelia Research Campus in Ashburn, Virginia, who has developed PSAMs-PSEMs but who also regularly uses DREADDs and optogenetics. (For a review, see Ann Rev Neurosci, 37:387-407, 2014.)
The Scientist talked to developers about the basics behind DREADDs and PSAMs-PSEMs. Here’s what they said.
DREADDs and PSAMs-PSEMs: A history
In 1991, scientists showed that engineering orthogonal GPCRs was possible, and first iterations of such tools, dubbed “receptor activated solely by a synthetic ligand” or RASSLs, came onto the scene in 1998.
Bryan Roth, of the UNC School of Medicine, made the second generation of RASSLs, which he called DREADDs, using an engineered muscarinic GPCR and, importantly, a ligand that was chemically inert (PNAS, 104:5163-68, 2007). Since the publication of that first paper on DREADDs, hundreds of labs have administered them in vivo, Roth says. This chemical genetic technique has the advantage of being easier to implement and less invasive than optogenetics, he adds.
Ligand-gated ion channel–based chemical genetic tools have their own history, but were not used in vivo in animals until 2011, when Sternson developed PSAMs-PSEMs. The researchers mutated ligand-binding domains and mixed and matched them to different ion-pore domains. But they altered the receptors and their ligands further so that they don’t interact with anything in the body. “As I was thinking about that system, I imagined I would want it to have easily optimizable, nontoxic ligands and the ability to tune the ion[-pore] or ligand-binding domain easily,” Sternson says.
DREADDs and PSAM-PSEM combinations in action
There are five different classes of DREADDs available, each designed for a different purpose:
hM3Dq raises calcium levels in a cell, causing burst firing;
hM4Di lowers cAMP and the activation of a particular potassium channel, causing neuronal silencing; also inhibits presynaptic neurotransmitter release;
GsD enhances cAMP, causing modulation signaling;
Rq(R165L) enhances arrestin signaling, a specific pathway that has been linked to the mechanisms of psychoactive drugs;
κ-opioid receptor DREADD or KORD quiets neurons and also inhibits presynaptic neurotransmitter release.
The synthetic ligand for each of the first four DREADDs is clozapine-N-oxide (CNO), whereas KORDs are activated by salvinorin B. That means combining DREADDs is now possible: Roth’s group showed recently that they could insert the hM3Dq and KORD to be able to activate and silence the same neurons (Neuron, 86:936-46, 2015).
Roth’s lab has also made light-activated (photocaged) CNO, which allows for more-precise control over the timing of DREADD receptor activation. He has not published any papers using this yet, but will provide the caged ligand to interested researchers upon request. To make use of photocaged CNO, however, you will need to surgically implant an optic cable to provide light to the brain region of interest. If you’re going to go to the trouble, you might consider optogenetics, Roth adds.
Scientists have paired different PSAMs with various ion channels and PSEMs in order to control neurons. Among the most popular:
PSAML141F, Y115F– 5HT3 HC is activated by the ligand PSEM89S, allowing cations to flow into the cell and boost excitability;
PSAML141F, Y115F – GlyR is activated by the ligand PSEM89S, silencing neurons;
PSAMQ79G, L141S-nAChR V13 is activated by the ligand PSEM9S, enhancing calcium signaling. (Because there are two different PSEM ligands, PSAMs-PSEMs can also be combined in the same animal.)
When to opt for optogenetics
The single biggest consideration in your choice of, and among, these technologies is the temporal control needed for your experiment. Optogenetics, for example, offers the finest level of control, on the order of milliseconds to seconds. If you’re examining decision-making behaviors, for example, then optogenetics (or electrical stimulation) is for you.
If you’re studying behaviors—such as eating or drinking—or physiological changes that occur over minutes to hours, then either optogenetics, chemogenetics, or both might work.
For long-lasting behaviors being measured over the course of hours to days, a chemogenetic approach such as DREADDs or PSAMs-PSEMs is the clear winner. The ligands linger longer than short light pulses and can even be dissolved into the animals’ drinking water. The chemogenetic approach is also superior for investigating larger swaths of brain, which are challenging to illuminate using optogenetics methods, Roth says.
Researchers have also successfully combined the approaches, typically using an optogenetic approach to turn on neurons and chemogenetic approaches to switch them off in the same animal. The inhibitory DREADD hM4Di targets presynaptic terminals, which could be especially helpful if you’re investigating a region of the brain where long-range projection neurons terminate.
Use DREADDs or PSAMs-PSEMs first?
TWOFERS: In 2015, researchers announced a new DREADD: KORD. Because it is activated by a different ligand than the one for previously developed DREADDs, the engineered receptors can now be combined in the same animal. BRYAN ROTH
Researchers tend to start with DREADDs simply because they have been around longer, Sternson says. But some will turn to PSAMs if DREADDs have been ineffective. “Most cell types will respond to [DREADDs] as they’re supposed to, but not all,” Sternson says. That’s because DREADDs tap into a complex signaling pathway that eventually results in neuronal activation or silencing. In contrast, PSAMs work by controlling the gating of an ion channel. On the other hand, ligand-gated ion channels may affect some types of cells, such as developing neurons, differently than they do adult cells, says Olsen, who coauthored the Neuronpaper describing KORD, the newest DREADD. But in general, Sternson says, “they’re completely complementary methods, and they can in principle be used together in the same animal.”
Another consideration is that PSEMs tend to take slightly longer to work—15 minutes, compared with the DREADD ligand CNO, which takes 5–10 minutes. On the other hand, PSEMs tend to take less time to clear from the body, 1–2 hours (vs. about 2 hours for CNO). Salvinorin B, the ligand of the new KOR-based DREADD, works almost instantaneously, and the effects last less than an hour. Although these differences are minor, they may factor into your experiment.
Experimental procedure
The operational steps are similar for both tools. Most people inject viruses carrying the engineered receptors into the brain area of interest and wait two to three weeks for expression. They then administer the ligand and make their measurements.
If you’ve already performed stereotactically guided brain surgery, there’s nothing new to learn. For newcomers, a Journal of Visualized Experiments protocol describes the surgery and injection of the virally ferried chemogenetic tools (100:e52859, 2015), though it’s best to learn by shadowing someone with experience, Roth says.
Viral constructs for both DREADDs and PSAMs are available from Addgene. For DREADDs, the UNC Vector Core sells high-titer virus stocks. CNO is available for free or at a reduced price for NIH-funded investigators through the National Institute of Drug Abuse’s Drug Supply Program. For PSAMs, you make your own receptor-carrying virus. Sternson provides PSEMs to researchers for their pilot experiments, and they are available for purchase through Apex Scientific for about $15 for 10 mg, he says.
You don’t necessarily need to do surgery if you can afford mutant mice whose DREADDs are under the control of an inducible promoter, such as Cre. Such mice are available through Jackson Labs. In general, just be sure to use validated Cre driver lines, Roth says.
You should make sure the receptor is expressed and working in vitro before you move to whole animals. Expression of both DREADDs and PSAMs is linked to the translation of a fluorescent protein. On his blog, chemogenetic.blogspot.com, Roth gives more specific advice on immunofluorescent staining for visualization of DREADDs.
To make sure that the receptors are actually working involves more-detailed studies, such as the calcium imaging Olsen used to ascertain whether his activating DREADD responded to the ligand, or electrophysiological studies in slices, but the particulars depend on what mechanism your receptor-ligand uses.
“One thing that’s important to know when using these receptors is that they’re not completely off when expressed at high levels,” Conklin says, referring to DREADDs. “[Simply] by expressing them, one cannot be sure.” To get around potential abnormal background activity, you have to include a control without the receptor. Also, having a good label on the receptor is helpful. Using DREADDs in combination with an inducible transcription system, such as Cre, allows you to measure receptor expression before and after inducing it.
Future uses
Although DREADDs and PSAMs-PSEMs are proving to be useful research tools for cell biologists and neurobiologists, both Roth and Sternson are actively developing orthogonal systems for potential clinical use, either as gene-based therapies that would go directly into humans or to be used in stem cell–based therapies.
Chemogenetic tools to interrogate brain functions.
Annu Rev Neurosci. 2014;37:387-407. doi: 10.1146/annurev-neuro-071013-014048. Epub 2014 Jun 16.
Elucidating the roles of neuronal cell types for physiology and behavior is essential for understanding brain functions. Perturbation of neuron electrical activity can be used to probe the causal relationship between neuronal cell types and behavior. New genetically encoded neuron perturbation tools have been developed for remotely controlling neuron function using small molecules that activate engineered receptors that can be targeted to cell types using genetic methods. Here we describe recent progress for approaches using genetically engineered receptors that selectively interact with small molecules. Called “chemogenetics,” receptors with diverse cellular functions have been developed that facilitate the selective pharmacological control over a diverse range of cell-signaling processes, including electrical activity, for molecularly defined cell types. These tools have revealed remarkably specific behavioral physiological influences for molecularly defined cell types that are often intermingled with populations having different or even opposite functions.
A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning.
J Vis Exp. 2015 Jun 22;(100):e52859. doi: 10.3791/52859.
This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.
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