Microscopes allow researchers to peer into tiny cells and tissues by making the objects appear larger. However, visualizing extremely tiny cellular structures requires specialized microscopes that are very expensive, and even the most advanced microscopes are limited in how much they can magnify objects. Using a paradigm-shifting technique called expansion microscopy (ExM), Pioneer and Transformative Research Awardee Dr. Edward Boyden is taking the novel approach of actually making the samples themselves larger. Using a material commonly found in baby diapers, Dr. Boyden and colleagues at the Massachusetts Institute of Technology (MIT) are able to expand tissues to about 4.5 times their normal size, enabling visualization of tiny cellular structures and proteins with ordinary microscopes. This material, called sodium acrylate, holds proteins in place while it swells in the presence of water, resulting in minimal distortion of the sample’s structure. Using ExM, researchers could image a 3-dimensional slice of brain tissue and could zoom in to see the tiny synapse where neurons communicate with each other. By eliminating the need for costly, specialized equipment, ExM has the potential to enable many more scientists to examine structures otherwise unobservable in the cell.
(Photo: Dr. Edward Boyden presenting ExM at the Common Fund's High Risk, High Reward Research Symposium in December 2014)
Listen to Dr. Boyden’s talk at the Common Fund’s High Risk, High Reward Research Symposium (starts at 1 hour, 33 minutes)
Read the press release from MIT
Chen F, Tillberg PW, and Boyden ES. Expansion Microscopy. Science, Jan 2014, DOI: 10.1126/science.1260088.
The maintenance of cognitive ability during the aging process has become a significant medical challenge of our time. Alzheimer’s disease (AD), currently having no treatment, is one of the leading causes of death in the United States. A host of other neurodegenerative diseases cause a decline in mental ability that is capable of interfering with daily life. The reasons for the onset of these diseases are still being examined. Earlier studies suggest that neuronal loss was a normal consequence of brain aging; however, neuronal cells are preserved in the aging brain and decline only in the presence of neurodegenerative disease. Much of the research into the causes of diseases resulting in dementia has focused on the abnormal proteins that appear in the brains of people with neurodegenerative diseases; however some people with these abnormal protein clumps show little or no signs of cognitive decline. Why is it that some individuals, even those presenting dementia pathology, age with their cognitive function intact while others develop dementia?
A new study, led by Pioneer awardee, Dr. Bruce Yankner of Harvard Medical School, begins to answer this question. Dr. Yankner and colleagues have discovered a mechanism involving repressor element 1-silencing transcription factor (REST), a repressor of neuronal genes during embryonic development that is down regulated after this stage. The researchers show that REST returns later in life to protect aging neurons from various stresses and regulates a network of genes that mediate cell death, stress resistance and dementia pathology. In an example involving AD, in its early stages, REST disappears from the nucleus, causing the gene network to lose regulation. Further experiments in the study indicate that REST protects neurons from age-related toxic insults and that the elevated REST levels in aging humans are associated with the preservation of cognitive function as well as increased longevity. Additionally, the presence of REST increases the expression of other transcription factors and enzymes that act to resist oxidative stress. REST is also shown to protect against a model of a different dementia disease, Parkinson’s disease, indicating that it might be involved in a stress response that is protective of neurons in a range of dementia diseases. So, even when harboring the abnormal protein clumps associated with dementia diseases, when neuronal REST levels are high, these individuals do not proceed to dementia, suggesting that dementia protein presence may not be enough and that failure of the brain’s stress response system may also be necessary to present cognitive inability. This finding introduces new possibilities for therapeutic intervention.
To read more about this research, please see the publication in Nature.
Please also see the related press release from Dr. Yankner’s institution, Harvard Medical School.
Two NIH Common Fund High Risk – High Reward Researchers named as 2013 Allen Distinguished Investigators
The Paul G. Allen Family Foundation, co-founded by Paul G. Allen (Microsoft co-founder) and his sister, Jo Lynn Allen, with a goal of “transforming lives and strengthening communities by fostering innovation, creating knowledge, and promoting social progress” through its philanthropic efforts. In 2010 the Foundation created the Allen Distinguished Investigators Awards to support high-risk, high-reward ideas in science. The program awards grants to ambitious scientists, who are pioneering innovative early-stage research. Two NIH High Risk – High Reward Investigators, Jeff Gore, a 2012 New Innovator from MIT, and Markus Covert, a 2009 Pioneer from Stanford University, were selected as 2013 Allen Distinguished Investigators. Dr. Gore will use the $1.5 million prize to investigate decision making and the evolutionary origins or cooperation using yeast as a model. Dr. Covert will also receive $1.5 million and plans to use this grant to study whole-cell models of higher organisms, including human cells. These awards will be funded over the course of three years and the Foundation hopes these grants will enable the awardees to unlock fundamental questions in biology that are essential to achieving world-changing breakthroughs.
Two NIH Common Fund High Risk-High Reward Awardees were named as winners of The Brain Prize for 2013, which is awarded by the Grete Lundbeck European Brain Research Foundation. Americans, Karl Deisseroth, a 2005 Pioneer Awardee, and 2012 Transformative Research Awardee, and Edward S. Boyden, a 2007 New Innovator, and 2012 Transformative Research Awardee and four European scientists were awarded the 2013 prize for their contributions to the development of “optogenetics.” Optogenetics is a technique that merges the fields of optics and genetics using light to specifically control the activity of genetically selected neurons. This revolutionary technique has allowed scientists to begin to gain a better understanding of the way circuits of neurons carry out complex functions. This technique has broad application potential in research from studying fundamental activities, such as memory formation and breathing, to more complex disorders and diseases, such as addiction, psychiatric disorders, and Alzheimer’s disease.
The Brain Prize, the world’s largest brain research prize, is awarded to scientists who have made an outstanding contribution to European brain research, and is awarded to scientists who have conducted research in Europe, or to scientists who have conducted research in collaboration with European scientists.
Miguel A. L. Nicolelis, a 2010 NIH Director’s Pioneer Awardee from Duke University, and his colleagues, have connected the brains of rat pairs through the internet and shown that the rats can share sensory information, acting as a dyad rather than strictly as individuals. Dr. Nicolelis’ lab is well known for their work on brain-to-machine interface (BTM), the current paradigm used to help develop technology that allows an amputee’s brain to control a prosthetic limb. The team of researchers took the brain interfacing concept further and connected pairs of rat brains using a new paradigm they termed brain-to-brain interface (BTBI). This BTBI enabled the rat pairs to act as a dyad exchanging sensorimotor information to achieve simple behavioral goals. The researchers implanted microelectrode bundles in the cortical area in the brains of rats. These microelectrodes allowed the researchers to record the neural activity of an “encoder” rat acting out a specific behavior and directly transmit this recorded signal to a “decoder” rat’s brain using intracortical microstimulation (ICMS), the stimulation of individual nerve cells using a small electric current, to inform the decoder rat to complete a specific activity.
The encoder rat was trained to press one of two levers in response to a light stimulus above one lever. While the encoder rat pressed this lever, the neural activity was recorded and transformed into ICMS which was then applied to the brain of the decoder rat informing the decoder rat of which lever to press. If the decoder rat pressed the correct lever, both rats received a reward. If the decoder rat pressed the incorrect lever, neither rat received a reward. The researchers took this experiment a step further and were able to perform this experiment in real time, with the encoder rat located in Natal, Brazil, and the decoder rat located at Duke University in North Carolina, transmitting the ICMS over the internet. In a second set of experiments, the scientists trained the encoder rat to determine the width of an opening (wide or narrow) using its whiskers and to push either the right or left lever, respectively. Again, the neural activity of the encoder rat’s brain during this task was recorded and transmitted by ICMS to the decoder rat informing the decoder of which lever to press. In both activities, the decoder rats pressed the correct lever significantly more often than rats who were not receiving neural activity information via ICMS. Further, the encoder rat changed both its behavior and its neural signals in response to feedback from the decoder rat’s behavior, indicating that the rat pairs were acting as dyads indicating a more complex system of communication. The researchers also mechanically stimulated both the encoder and decoder rat’s whiskers and recorded the neural response to this stimulation. They then stimulated the encoder rat’s whiskers, recorded the activity and sent it the decoder rat’s brain using ICMS and recorded the neural activity of the decoder rats brain in response. Passive whisker stimulation in either the encoder or decoder rats induced significant neural activity in the decoder rat’s brain.
Through this set of experiments the researchers demonstrated that tactile and motor information can be recorded in real-time from a rat’s brains and transmitted directly into another rat’s brain using BTBI. In this BTBI rat dyad, the decoder rat relied exclusively on the neural patterns generated by the encoder rat in order to reproduce the encoder rat’s behavioral choice. The ICMS patterns reflecting the neural activity in the encoder rat’s brain was sufficient to inform decoder rats to perform both tactile and motor activities significantly above chance in real-time. The potential impact of this research is not limited to behavioral information exchange of animal dyads, but could be expanded to create a multi-brain system where groups of interconnected brain networks could communicate through large scale BTBI.
Read the Duke University Press Release here
M. Pais-Vieira, M. Lebedev, C. Kunicki, J. Wang, M. A. L. Nicolelis, A Brain-to-Brain Interface for Real-Time Sharing of Sensorimotor Information. Sci. Rep. 3, (2013). PMID: 23448946
More than 60,000 patients in the United States undergo general anesthesia for surgery every day. Currently, doctors use indirect measures of the brain state, such as heart rate and blood pressure, along with drug pharmacokinetics, pharmacodynamics and exhaled anesthetic gas typically are converted to a simple index score to indicate if a patient is adequately anesthetized during surgery. These indices are neither entirely reliable nor very informative. Inadequately administered anesthesia can result in intraoperative awareness and post-operative delirium. Drs. Emery N. Brown and Patrick L. Purdon, former NIH Director’s Pioneer Awardee and current NIH Director’s New Innovator Awardee, respectively, have studied brain activity during propofol-induced anesthesia using electroencephalogram (EEG) technology, which measures scalp electrical potentials, to better understand the mechanism of unconsciousness and to establish a direct method of tracking the transition between consciousness and unconsciousness during general anesthesia. In this study, the researchers gradually administered and reduced the anesthesia drug propofol in patients, while monitoring brain activity and behavioral loss of consciousness by asking patients to respond to auditory stimuli. This gradual induction and emergence from anesthesia allowed the researchers to precisely define an EEG brain signature, as well as behavioral markers that are associated with consciousness, unconsciousness, and the transition between these two states in patients undergoing propofol-induced general anesthesia. This research has provided a deeper understanding of the mechanism of propofol-induced loss of consciousness, and could be used to determine the EEG brain signatures of other anesthetics. Additionally, these results could be used to develop more direct and reliable methods for monitoring brain states of patients undergoing general anesthesia and could lead to new insights into the tailoring of drug dosage in real-time.
P. L. Purdon et al., Electroencephalogram signatures of loss and recovery of consciousness from propofol. Proceedings of the National Academy of Sciences, (March 4, 2013, 2013).
Shortening the time required to treat tuberculosis (TB) is key to reducing the development of drug resistance and lowering worldwide rates of TB infection and mortality. Reducing treatment duration however depends on: (1) understanding how Mycobacterium tuberculosis (Mtb), the bacteria that causes TB in humans, becomes tolerant to antibiotics; and (2) devising ways to prevent or overcome drug tolerance. NIH Director’s Pioneer Award recipient Dr. Lalita Ramakrishnan and colleagues at the University of Washington, in collaboration with Dr. Paul Edelstein at the University of Pennsylvania, report in a study appearing in the April 1, 2011 issue of Cell, that the development of drug tolerance in Mtb is due in part to the activity of “efflux” pumps in the cell membrane that presumably flush away any antibiotic agents that penetrate the Mtb cells. The authors found that these pumps are stoked into action by host cells called macrophages that are, ironically, part of the body’s frontline defenses against foreign invaders. Based on these findings, the study authors suggest that expanding treatment to include medications that inhibit these pumps could dramatically shorten the time needed to cure infection. While Mtb can infect many body tissues and organs, it primarily attacks the lungs. Once inside the lungs, the bacteria infect the aforementioned macrophages. The macrophages and other types of immune cells respond by aggregating into structures called granulomas which are thought to contain the spread of persistent pathogens. Having taken up residence within macrophages, some populations of Mtb cells quickly become tolerant to anti-TB drugs. Nearly all models of Mtb drug tolerance postulate that this temporary resistance to antibiotics arises when the bacterial cells enter a dormant state in which they stop replicating. Because most antibiotics are only effective against bacterial cells that are reproducing, dormant cells are effectively resistant to antimicrobial agents. In their latest paper however, Dr. Ramakrishnan and colleagues report finding multi-drug tolerant Mtb populations that were actively growing and reproducing inside of host macrophages suggesting that residence within macrophages rapidly induces tolerance. The study authors also found that, having infected macrophages, the Mtb cells deploy efflux pumps that are essential for the Mtb cells to grow within the macrophages and may be used by the bacterial cells to remove toxic substances such as antimicrobial agents. In addition to expanding our understanding of the pathogenesis of TB and drug tolerance, these findings suggest that inhibiting the activity of macrophage-induced bacterial efflux pumps using currently available drugs such as verapamil may be an effective means of reducing the duration of TB treatment. Shorter treatment is likely to translate into increased adherence which will in turn slow the development of multi-drug resistance, reduce transmission of infection to new hosts, and reduce TB-associated mortality around the world.
Adams KN, Takaki K, Connolly LE, Wiedenhoft H, Winglee K, Humbert O, Edelstein PH, Cosma CL, Ramakrishnan L. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell, 2011 April 1; 145(1): 39-53. PMID: 21376383.
While simple blood tests can accurately and efficiently screen for some diseases, such as diabetes, the lack of blood tests for the majority of diseases can result in delayed or incorrect diagnoses. To overcome this diagnostic obstacle, Dr. Thomas Kodadek, a researcher at The Scripps Research Institute and funded in part by an NIH Director’s Pioneer Award, has developed a novel screening method that could detect disease-associated proteins in the blood of patients with a variety of conditions. In the January 7, 2011 edition of the journal Cell, Dr. Kodadek and colleagues describe their technique for using a collection of synthetic molecules to detect the presence of unique proteins in the blood of diseased individuals that do not appear in the blood of healthy individuals. By screening blood samples from mice with multiple sclerosis, the researchers identified several molecules in their synthetic collection that would only bind to proteins in the blood of the diseased mice, and could distinguish between “patient” mice and normal, healthy mice. Importantly, the researchers went on to demonstrate that in samples from humans, they could use the same technique to identify different molecules that bound to proteins uniquely present in the blood of patients with Alzheimer’s disease. This same binding did not occur in samples from healthy people of similar ages or in patients with another neurodegenerative disorder, Parkinson’s disease. These promising results suggest that this type of blood test has the potential to screen for a wide variety of different diseases, including diseases that currently lack a reliable diagnostic test.
Reddy MM, Wilson R, Wilson J, Connell S, Gocke A, Hynan L, German D, Kodadek T. Identification of candidate IgG biomarkers for Alzheimer’s disease via combinatorial library screening. Cell, 2011 Jan 7; 144(1): 132-42. PMID: 21215375.
Repeated use of the antibiotic ciprofloxacin (Cipro) leads to persistent changes in the beneficial microbes of the gut, according to a study by David Relman, a researcher at Stanford University and recipient of an NIH Director's Pioneer Award. While ciprofloxacin usually does not cause gastrointestinal side effects normally associated with disturbance of gut-dwelling bacteria, this research demonstrates the occurrence of more subtle changes in gut microbe composition, such as replacement of some bacterial species with closely related species or eradication of some sub-sets of bacteria, particularly when multiple courses of antibiotics are administered. These long-term, persistent changes in microbe composition raise concerns about the evolution of antibiotic-resistant bacteria as well as chronic changes in pathogen-host interactions in the gut, regulation of host immunity, energy balance, or metabolism.
Dethlefsen L, Relman DA. Incomplete recovery and individualized responses of the human distal gut microbiota to repeated antibiotic perturbation. Proceedings of the National Academy of Sciences, 2011 Mar 15; 108 Suppl 1: 4554-61. Epub 2010 Sep 16. PMID: 20847294.
Neurons in the brain communicate with each other using a combination of carefully regulated chemical and electrical signals. NIH Director’s Pioneer Award recipient Dr. James Eberwine and colleagues at the University of Pennsylvania and Sequenom, Inc. have discovered a novel method by which neurons selectively express proteins that control their electrical properties , reported in the December 7, 2010 issue of the Proceedings of the National Academy of Sciences. In neurons, electrical signals are controlled by channel proteins that create a pore in the cell membrane to allow positively or negatively charged ions to flow in and out of the cell. One of these channel proteins, called BKCa, helps regulate electrical signals in the hippocampus, an area of the brain that is important for learning and memory. Interestingly, the hippocampus is also the focus of many epileptic seizures, which result from disturbances in the electrical firing of neurons. The BKCa channel protein has several different variations. These protein variations arise during a cellular process called “splicing,” which is analogous to the process of cutting an undesired segment out of an audiotape and joining the resulting ends together. To make a protein, a cell must identify the piece of DNA that contains the instructions for the protein (called a gene), copy the DNA into another form of genetic material, called RNA, and then use the information encoded in the RNA to make the protein. Often, the instructions in the DNA for making a protein are not in a continuous stretch, but contain intervening sequences or “introns.” After the DNA is copied into RNA, these introns are removed and the flanking coding sequences, or “exons,” are connected to each other. It is the sequence of merged exons that tells the cell how to make a protein. Since the RNA can be spliced in different ways, one gene sequence can be used to make several different protein variants, depending on which exons are included. Normally splicing takes place in the nucleus of a cell, the cell’s control center where the DNA is stored. However, previous work from Dr. Eberwine’s lab has shown that splicing can also occur outside the nucleus, in a neuron’s cytoplasm. Dr. Eberwine’s latest paper demonstrates for the first time that one of the introns removed during cytoplamic splicing can regulate which form of BKCa channel protein is made, which in turn affects important electrical properties of the neuron. While introns used to be considered “junk DNA,” a number of studies, including Dr. Eberwine’s papers, are demonstrating that introns play crucial regulatory roles. Studying the process of splicing and intron removal in the production of BKCa channels may provide new insights about disorders of neuronal misfiring, such as epilepsy.
Bell TJ, Miyashiro KY, Sul JY, Buckley PT, Lee MT, McCullough R, Jochems J, Kim J, Cantor CR, Parsons TD, Eberwine JH. Intron retention facilitates splice variant diversity in calcium-activated big potassium channel populations. Proceedings of the National Academy of Sciences, 2010 Dec 7; 107(49): 21152-7. PMID: 21078998.