Brain Disorder: Parkinson’s Disease

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.

 

Alan Jasanoff

Next Generation Brain Imaging

One of the greatest challenges of modern neuroscience is to relate high-level operations of the brain and mind to well-defined biological processes that arise from molecules and cells. The Jasanoff lab is creating a suite of experimental approaches designed to achieve this by permitting brain-wide dynamics of neural signaling and plasticity to be imaged for the first time, with molecular specificity. These potentially transformative approaches use novel probes detectable by magnetic resonance imaging (MRI) and other noninvasive readouts. The probes afford qualitatively new ways to study healthy and pathological aspects of integrated brain function in mechanistically-informative detail, in animals and possibly also people.

Michale Fee

Song Circuits

Michale Fee studies how the brain learns and generates complex sequential behaviors, focusing on the songbird as a model system. Birdsong is a complex behavior that young birds learn from their fathers and it provides an ideal system to study the neural basis of learned behavior. Because the parts of the bird’s brain that control song learning are closely related to human circuits that are disrupted in brain disorders such as Parkinson’s and Huntington’s disease, Fee hopes the lessons learned from birdsong will provide new clues to the causes and possible treatment of these conditions.

Ann Graybiel

Probing the Deep Brain

Ann Graybiel studies the basal ganglia, forebrain structures that are profoundly important for normal brain function. Dysfunction in these regions is implicated in neurologic and neuropsychiatric disorders ranging from Parkinson’s disease and Huntington’s disease to obsessive-compulsive disorder, anxiety and depression, and addiction. Graybiel’s laboratory is uncovering circuits underlying both the neural deficits related to these disorders, as well as the role that the basal ganglia play in guiding normal learning, motivation, and behavior.

Plugging into the brain

by Sabbi Lall |

Driven by curiosity and therapeutic goals, Anikeeva leaves no scientific stone unturned in her drive to invent neurotechnology.

The audience sits utterly riveted as Polina Anikeeva highlights the gaps she sees in the landscape of neural tools. With a background in optoelectronics, she has a decidedly unique take on the brain.

“In neuroscience,” says Anikeeva, “we are currently applying silicon-based neural probes with the elastic properties of a knife to a delicate material with the consistency of chocolate pudding—the brain.”

A key problem, summarized by Anikeeva, is that these sharp probes damage tissue, making such interfaces unreliable and thwarting long term brain studies of processes including development and aging. The state of the art is even grimmer in the clinic. An avid climber, Anikeeva recalls a friend sustaining a spinal cord injury. “She made a remarkable recovery,” explains Anikeeva, “but seeing the technology being used to help her was shocking. Not even the simplest electronic tools were used, it was basically lots of screws and physical therapy.” This crude approach, compared to the elegant optoelectronic tools familiar to Anikeeva, sparked a drive to bring advanced materials technology to biological systems.

Outside the box

As the group breaks up after the seminar, the chatter includes boxes, more precisely, thinking outside of them. An associate professor in material sciences and engineering at MIT, Anikeeva’s interest in neuroscience recently led to a McGovern Institute appointment. She sees her journey to neurobiology as serendipitous, having earned her doctorate designing light-emitting devices at MIT.

“I wanted to work on tools that don’t exist, and neuroscience seemed like an obvious choice. Neurons communicate in part through membrane voltage changes and as an electronics designer, I felt that I should be able to use voltage.”

Comfort at the intersection of sciences requires, according to Anikeeva, clarity and focus, also important in her chief athletic pursuits, running and climbing. Through long distant running, Anikeeva finds solitary time (“assuming that no one can chase me”) and the clarity to consider complicated technical questions. Climbing hones something different, absolute focus in the face of the often-tangled information that comes with working at scientific intersections.

“When climbing, you can only think about one thing, your next move. Only the most important thoughts float up.”

This became particularly important when, in Yosemite National Park, she made the decision to go up, instead of down, during an impending thunderstorm. Getting out depended on clear focus, despite imminent hypothermia and being exposed “on one of the tallest features in the area, holding large quantities of metal.” Polina and her climbing partner made it out, but her summary of events echoes her research philosophy: “What you learn and develop is a strong mindset where you don’t do the comfortable thing, the easy thing. Instead you always find, and execute, the most logical strategy.”

In this vein, Anikeeva’s research pursues two very novel, but exceptionally logical, paths to brain research and therapeutics: fiber development and magnetic nanomaterials.

Drawing new fibers

Walking into Anikeeva’s lab, the eye is immediately drawn to a robust metal frame containing, upon closer scrutiny, recognizable parts: a large drill bit, a motor, a heating element. This custom-built machine applies principles from telecommunications to draw multifunctional fibers using more “brain-friendly” materials.

“We start out with a macroscopic model, a preform, of the device that we ultimately want,” explains Anikeeva.

This “preform” is a transparent block of polymers, composites, and soft low-melting temperature metals with optical and electrical properties needed in the final fiber. “So, this could include
electrodes for recording, optical channels for optogenetics, microfluidics for drug delivery, and one day even components that allow chemical or mechanical sensing.” After sitting in a vacuum to remove gases and impurities, the two-inch by one-inch preform arrives at the fiber-drawing tower.

“Then we heat it and pull it, and the macroscopic model becomes a kilometer-long fiber with a lateral dimension of microns, even nanometers,” explains Anikeeva. “Take one of your hairs, and imagine that inside there are electrodes for recording, there are microfluidic channels to infuse drugs, optical channels for stimulation. All of this is combined in a single miniature form
factor, and it can be quite flexible and even stretchable.”

Construction crew

Anikeeva’s lab comprises an eclectic mix of 21 researchers from over 13 different countries, and a range of expertises, including materials science, chemistry, electrical and mechanical engineering, and neuroscience. In 2011, Andres Canales, a materials scientist from Mexico, was the second person to join Anikeeva’s lab.

“There was only an idea, a diagram,” explains Canales. “I didn’t want to work on biology when I arrived at MIT, but talking to Polina, seeing the pictures, thinking about what it would entail, I became very excited by the methods and the potential applications she was thinking of.”

Despite the lack of preliminary models, Anikeeva’s ideas were compelling. Elegant as the fibers are, the road involved painstaking, iterative refinement. From a materials perspective, drawing a fiber containing a continuous conductive element was challenging, as was validation of its properties. But the resulting fiber can deliver optogenetics vectors, monitor expression, and then stimulate neuronal activity in a single surgery, removing the spatial and temporal guesswork usually involved in such an experiment.

Seongjun Park, an electrical engineering graduate student in the lab, explains one biological challenge. “For long term recording in the spinal cord, there was even an additional challenge as the fiber needed to be stretchable to respond to the spine’s movement. For this we developed a drawing process compatible with an elastomer.”

The resulting fibers can be deployed chronically without the scar tissue accumulation that usually prevents long-term optical manipulation and drug delivery, making them good candidates for the treatment of brain disorders. The lab’s current papers find that these implanted fibers are useful for three months, and material innovations make them confident that longer time periods are possible.

Magnetic moments

Another wing of Anikeeva’s research aims to develop entirely non-invasive modalities, and use magnetic nanoparticles to stimulate the brain and deliver therapeutics.

“Magnetic fields are probably the best modality for getting any kind of stimulus to deep tissues,” explains Anikeeva, “because biological systems, except for very specialized systems, do not perceive magnetic fields. They go through us unattenuated, and they don’t couple to our physiology.”

In other words, magnetic fields can safely reach deep tissues, including the brain. Upon reaching their tissue targets these fields can be used to stimulate magnetic nanoparticles, which might one day, for example, be used to deliver dopamine to the brains of Parkinson’s disease patients. The alternating magnetic fields being used in these experiments are tiny, 100-1000 times smaller than fields clinically approved for MRI-based brain imaging.

Tiny fields, but they can be used to powerful effect. By manipulating magnetic moments in these nanoparticles, the magnetic field can cause heat dissipation by the particle that can stimulate thermal receptors in the nervous system. These receptors naturally detect heat, chili peppers and vanilla, but Anikeeva’s magnetic nanoparticles act as tiny heaters that activate these receptors, and, in turn, local neurons. This principle has already been used to activate the brain’s reward center in freely moving mice.

Siyuan Rao, a postdoc who works on the magnetic nanoparticles in collaboration with McGovern Investigator Guoping Feng, is unhesitating when asked what most inspires her.

“As a materials scientist, it is really rewarding to see my materials at work. We can remotely modulate mouse behavior, even turn hopeless behavior into motivation.”

Pushing the boundaries

Such collaborations are valued by Anikeeva. Early on she worked with McGovern Investigator Emilio Bizzi to use the above fiber technology in the spinal cord. “It is important to us to not just make these devices,” explains Anikeeva, “but to use them and show ourselves, and our colleagues, the types of experiments that they can enable.”

Far from an assembly line, the researchers in Anikeeva’s lab follow projects from ideation to deployment. “The student that designs a fiber, performs their own behavioral experiments, and data analysis,” says Anikeeva. “Biology is unforgiving. You can trivially design the most brilliant electrophysiological recording probe, but unless you are directly working in the system, it is easy to miss important design considerations.”

Inspired by this, Anikeeva’s students even started a project with Gloria Choi’s group on their own initiative. This collaborative, can-do ethos spreads beyond the walls of the lab, inspiring people around MIT.

“We often work with a teaching instructor, David Bono, who is an expert on electronics and magnetic instruments,” explains Alex Senko, a senior graduate student in the lab. “In his spare time, he helps those of us who work on electrical engineering flavored projects to hunt down components needed to build our devices.”

These components extend to whatever is needed. When a low frequency source was needed, the Anikeeva lab drafted a guitar amplifier.

Queried about difficulties that she faces having chosen to navigate such a broad swath of fields, Anikeeva is focused, as ever, on the unknown, the boundaries of knowledge.

“Honestly, I really, really enjoy it. It keeps me engaged and not bored. Even when thinking about complicated physics and chemistry, I always have eyes on the prize, that this will allow us to address really interesting neuroscience questions.”

With such thinking, and by relentlessly seeking the tools needed to accomplish scientific goals, Anikeeva and her lab continue to avoid the comfortable route, instead using logical routes toward new technologies.

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.

Do you have a question for The Brain? Ask it here.

Polina Anikeeva

Probing the Mind

Polina Anikeeva designs, synthesizes, and fabricates optoelectronic and magnetic devices to advance fundamental understanding and treatment of disorders of the nervous system. Anikeeva’s lab designs probes that are compatible with delicate neural tissue, but match the signaling complexity of neural circuits. In addition, her group develops magnetic nanoparticles for non-invasive neural stimulation. Most recently, Anikeeva is exploring the pathways connecting the brain to other body organ systems with the goal of advancing therapies and predictive diagnostics to achieve healthy minds in healthy bodies.

Ed Boyden

Engineering Matter and Mind

Ed Boyden develops new tools for probing, analyzing, and engineering brain circuits. He uses a range of approaches, including synthetic biology, nanotechnology, chemistry, electrical engineering, and optics to develop tools capable of revealing fundamental mechanisms underlying complex brain processes.

Boyden may be best known for pioneering the development of optogenetics, a powerful method that enables neuronal activity to be controlled with light. He also led the team that invented expansion microscopy, in which a specimen is embedded in a gel that swells as it absorbs water, thereby expanding nanoscale features to a size where they can be seen using conventional microscopes. He is now seeking to systematically integrate these technologies to create detailed maps and models of brain circuitry.

Virtual Tour of Boyden Lab

Chronic neural implants modulate microstructures in the brain with pinpoint accuracy

Anne Trafton |

Post by Windy Pham

The diversity of structures and functions of the brain is becoming increasingly realized in research today. Key structures exist in the brain that regulate emotion, anxiety, happiness, memory, and mobility. These structures can come in a huge variety of shapes and sizes and can all be physically near one another. Dysfunction of these structures and circuits linking them are common causes of many neurologic and neuropsychiatric diseases. For example, the substantia nigra is only a few millimeters in size yet is crucial for movement and coordination. Destruction of substantia nigra neurons is what causes motor symptoms in Parkinson’s disease.

New technologies such as optogenetics have allowed us to identify similar microstructures in the brain. However, these techniques rely on liquid infusions into the brain, which prepare the regions to be studied to respond to light. These infusions are done with large needles, which do not have the fine control to target specific regions. Clinical therapy has also lagged behind. New drug therapies aimed at treating these conditions are delivered orally, which results in drug distribution throughout the brain, or through large needle-cannulas, which do not have the fine control to accurately dose specific regions. As a result, patients of neurologic and psychiatric disorders frequently fail to respond to therapies due to poor drug delivery to diseased regions.

A new study addressing this problem has been published in Proceedings of the National Academy of Sciences. The lead author is Khalil Ramadi, a medical engineering and medical physics (MEMP) PhD candidate in the Harvard-MIT Program in Health Sciences and Technology (HST). For this study, Khalil and his thesis advisor, Michael Cima, the David H. Koch Professor of Engineering within the Department of Materials Science and Engineering and the Koch Institute for Integrative Cancer Research, and associate dean of innovation in the School of Engineering, collaborated with Institute Professors Robert Langer and Ann Graybiel, an Investigator at the McGovern Institute of Brain Research to tackle this issue.

The team developed tools to enable targeted delivery of nanoliters of drugs to deep brain structures through chronically implanted microprobes. They also developed nuclear imaging techniques using positron emission tomography (PET) to measure the volume of the brain region targeted with each infusion. “Drugs for disorders of the central nervous system are nonspecific and get distributed throughout the brain,” Cima says. “Our animal studies show that volume is a critical factor when delivering drugs to the brain, as important as the total dose delivered. Using microcannulas and microPET imaging, we can control the area of brain exposed to these drugs, improving targeting accuracy double time comparing to the traditional methods used today.”

The researchers were also able to design cannulas that are MRI-compatible and implanted up to one year in rats. Implanting these cannulas with micropumps allowed the researchers to remotely control the behavior of animals. Significantly, they found that varying the volume infused alone had a profound effect on behavior induced, even if the total drug dose delivered stayed constant. These results show that regulation of volume delivery to brain region is extremely important in influencing brain activity. This technology could potentially enable precise investigation of neurological disease pathology in preclinical models, and more effective treatment in human patients.