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Special issue of “Neurochemical Research” honors Vittorio Gallo, Ph.D.

March 19, 2020

Investigators from around the world penned manuscripts that were assembled in a special issue of “Neurochemical Research” that honors Vittorio Gallo, Ph.D., for his leadership in the field of neural development and regeneration.

At a pivotal moment early in his career, Vittorio Gallo, Ph.D., was accepted to work with Professor Giulio Levi at the Institute for Cell Biology in Rome, a position that leveraged courses Gallo had taken in neurobiology and neurochemistry, and allowed him to work in the top research institute in Italy directed by the Nobel laureate, Professor Rita Levi-Montalcini.

For four years as a student and later as Levi’s collaborator, Gallo focused on amino acid neurotransmitters in the brain and mechanisms of glutamate and GABA release from nerve terminals. Those early years cemented a research focus on glutamate neurotransmission that would lead to a number of pivotal publications and research collaborations that have spanned decades.

Now, investigators from around the world who have worked most closely with Gallo penned tributes in the form of manuscripts that were assembled in a special issue of “Neurochemical Research” that honors Gallo “for his contributions to our understanding of glutamatergic and GABAergic transmission during brain development and to his leadership in the field of neural development and regeneration,” writes guest editor Arne Schousboe, of the University of Copenhagen in Denmark.

Vittorio Gallo, Ph.D. as a 21-year-old mustachioed graduate student.

“In spite of news headlines about competition in research and many of the negative things we hear about the research world, this shows that research is also able to create a community around us,” says Gallo, chief research officer at Children’s National Hospital and scientific director for the Children’s National Research Institute.

As just one example, he first met Schousboe 44 years ago when Gallo was a 21-year-old mustachioed graduate student.

“Research can really create a sense of community that we carry on from the time we are in training, nurture as we meet our colleagues at periodic conferences, and continue up to the present. Creating community is bi-directional: influencing people and being influenced by people. People were willing to contribute these 17 articles because they value me,” Gallo says. “This is a lot of work for the editor and the people who prepared papers for this special issue.”

In addition to Gallo publishing more than 140 peer-reviewed papers, 30 review articles and book chapters, Schousboe notes a number of Gallo’s accomplishments, including:

He helped to develop the cerebellar granule cell cultures as a model system to study how electrical activity and voltage-dependent calcium channels modulate granule neuron development and glutamate release.
He developed a biochemical/neuropharmacological assay to monitor the effects of GABA receptor modulators on the activity of GABA chloride channels in living neurons.
He and Maria Usowicz used patch-clamp recording and single channel analysis to demonstrate for the first time that astrocytes express glutamate-activated channels that display functional properties similar to neuronal counterparts.
He characterized one of the spliced isoforms of the AMPA receptor subunit gene Gria4 and demonstrated that this isoform was highly expressed in the cerebellum.
He and his Children’s National colleagues demonstrated that glutamate and GABA regulate oligodendrocyte progenitor cell proliferation and differentiation.

Purkinje cells are large neurons located in the cerebellum that are elaborately branched like interlocking tree limbs and represent the only source of output for the entire cerebellar cortex.

Even the image selected to grace the special issue’s cover continues the theme of continuity and leaving behind a legacy. That image of Purkinje cells was created by a young scientist who works in Gallo’s lab, Aaron Sathyanesan, Ph.D. Gallo began his career working on the cerebellum – a region of the brain important for motor control – and now studies with a team of scientists and clinician-scientists Purkinje cells’ role in locomotor adaptive behavior and how that is disrupted after neonatal brain injury.

“These cells are the main players in cerebellar circuitry,” Gallo says. “It’s a meaningful image because goes back to my roots as a graduate student and is also an image that someone produced in my lab early in his career. It’s very meaningful to me that Aaron agreed to provide this image for the cover of the special issue.”

Research team develops new and improved method for studying cardiac function

December 3, 2019

While researching how plastic affects heart function in sensitive populations, such as children born with congenital heart defects, Children’s National researcher Nikki Posnack, Ph.D., led a team that developed a new and improved, replicable method of performing simultaneous dual optical mapping to examine electrical activity and calcium for the study of cardiac function.

Since arriving at the Sheikh Zayed Institute for Pediatric Surgical Innovation, researcher Nikki Gillum Posnack, Ph.D., a principal investigator with the institute and assistant professor of pediatrics at the George Washington University School of Medicine and Health Sciences, has been focused on examining how exposure to plastic affects heart function in sensitive populations, such as children born with congenital heart defects. She performs optical mapping to conduct this research, but the industry standard approaches of either using dual cameras or sequential single cameras were cost prohibitive and technically challenging while also diminishing the quality of the imaging results.

Fast forward to July 2019 when Dr. Posnack and her team published “Plasticizer Interaction With the Heart” in the journal Arrhythmia and Electrophysiology, which used imaging techniques to reveal the impact of plastic chemicals on the electrical activity of the heart. Dr. Posnack’s laboratory has since expanded this technique and revealed a new replicable method of performing simultaneous dual optical mapping to examine electrical activity and calcium handling in the heart.

Sharing a new method for studying cardiac function

This groundbreaking method is itself the focus of a new BMC Biomedical Engineering journal article titled “Lights, camera, path splitter: a new approach for truly simultaneous dual optical mapping of the heart with a single camera.”

The article compares and contrasts the current standard for dual camera simultaneous configurations and single camera sequential configurations to Dr. Posnack’s new single camera simultaneous configuration.

Simultaneous dual mapping systems use two probes and dual dyes – one for electrical voltage and the other for calcium. While dual-dye combinations like Di-4-ANEPPS with Indo-1, Di-2-ANEPEQ and calcium green have been developed to separate fluorescence signals by emission, these dye combinations can have spectral overlap, creating the need for non-ideal emission bandpass to negate spectral overlap and/or the inclusion of a calcium probe with an inferior dissociation constant. Additionally, dual-sensor systems require proper alignment to ensure that fluorescence signals are being analyzed from the same tissue region on each individual detector, which could lead to erroneous results. The dual-camera optical setup is expensive, technically challenging and requires a large physical footprint that is often not feasible for basic science and teaching laboratories conducting critical research.

As an alternative, some researchers use a single camera configuration to sequentially image the voltage and calcium probes using excitation light patterning. This approach also has limitations. These single-sensor designs use dual-dye combinations that require two or more excitation light sources, but share a single emission band. Like the dual camera system, this platform design is also technically challenging since the different excitation light wavelengths require light source triggering, camera synchronization and frame interleaving. Due to timing coordination, decreased frame rates, excitation light ramp up/down times and shutter open/close times, single system setups require shorter exposure times compared to dual sensor setups, diminishing the signal-to-noise quality without offering the same temporal fidelity. There is a cost advantage to the single camera system, however, because the additional camera is often one of the most expensive components.

This new single camera, simultaneous dual optical mapping approach is the first multiparametric mapping system that simultaneously acquires calcium and voltage signals from cardiac preparations, using a commercially available optical path splitter, single camera and single excitation light. Using a large field of view sCMOS sensor that is faster and more sensitive, this configuration separates the two emission bands for voltage and calcium probes and simultaneously directs them to either sides of the single, large camera sensor. This protocol employs a commonly used dual-dye combination (RH237 and Rhod2-AM). In contrast, other protocols may require genetically-encoded indicators or fluorescent probes that are not yet commercially available.

The team validated the utility of the approach by performing high-speed simultaneous dual imaging with sufficient signal-to-noise ratio for calcium and voltage signals and specificity of emission signals with negligible cross-talk. Demonstrating the need for simultaneous electrical and calcium sensors, they found that when ventricular tachycardia is induced, there is spatially discordant calcium alternans present in different regions of the heart even when the electrical alternans remain concordant.

Having eliminated the second camera as well as the need for multiple excitation light sources, light pattering and frame interleaving, this system is more cost effective, simpler, and can be easily setup by various types of researchers, not just those with engineering backgrounds.

With a limited research budget and a background in physiology, Dr. Posnack worked collaboratively with her post-doctoral fellow Rafael Jaimes III, an engineer in the Sheikh Zayed Institute for Pediatric Surgical Innovation, to develop a cost-effective system that would enable her to truly study the effects of plastics on the heart.

Multidisciplinary approach

“We’re fortunate to have a multidisciplinary team in the Sheikh Zayed Institute so that I could work with an engineer to develop the technology and system we needed to propel our research,” said Dr. Posnack. “There are so many researchers who have the science background, but not necessarily the technical aptitude, and they get stymied in their research, so we’re proud that this paper will help other researchers replicate the system to study cardiac function.”

The research paper was funded by a grant from the National Institutes of Health as well as support from the Children’s Research Institute, Children’s National Heart Institute and the Sheikh Zayed Institute for Pediatric Surgical Innovation.

The applications for this optical mapping system are significant and Dr. Posnack has been consulted by other research teams looking to implement it in their labs. Additionally, Dr. Posnack has collaborated with several neuroscience teams at Children’s National Hospital, including one that is investigating the effects of hypoxia on brain and heart development, and another that is interested in using image modalities and data processing to analyze calcium as an indicator of neuron firing.

Dr. Posnack continues to use this new dual optical mapping system to further her research as she anticipates the publication of a new article about age-dependent changes in cardiac electrophysiology and calcium handling.

Molecular gatekeepers that regulate calcium ions key to muscle function

October 31, 2019

Controlled entry of calcium ions into the mitochondria, the cell’s energy powerhouses, makes the difference between whether muscles grow strong or easily tire and perish from injury, according to research published in Cell Reports.

Calcium ions are essential to how muscles work effectively, playing a starring role in how and when muscles contract, tap energy stores to keep working and self-repair damage. Not only are calcium ions vital for the repair of injured muscle fibers, their controlled entry into the mitochondria, the cell’s energy powerhouses, spells the difference between whether muscles will be healthy or if they will easily tire and perish following an injury, according to research published Oct. 29, 2019, in Cell Reports.

“Lack of the protein mitochondrial calcium uptake1 (MICU1) lowers the activation threshold for calcium uptake mediated by the mitochondrial calcium uniporter in both, muscle fibers from an experimental model and fibroblast of a patient lacking MICU1,” says Jyoti K. Jaiswal, MSc, Ph.D., a principal investigator in the Center for Genetic Medicine Research at Children’s National Hospital and one of the paper’s corresponding authors. “Missing MICU1 also tips the calcium ion balance in the mitochondria when muscles contract or are injured, leading to more pronounced muscle weakness and myofiber death.”

Five years ago, patients with a very rare disease linked to mutations in the mitochondrial gene MICU1 were described to suffer from a neuromuscular disease with signs of muscle weakness and damage that could not be fully explained.

To determine what was going awry, the multi-institutional research team used a comprehensive approach that included fibroblasts donated by a patient lacking MICU1 and an experimental model whose MICU1 gene was deleted in the muscles.

Loss of MICU1 in skeletal muscle fibers leads to less contractile force, increased fatigue and diminished capacity to repair damage to their cell membrane, called the sarcolemma. Just like human patients, the experimental model suffers more pronounced muscle weakness, increased numbers of dead myofibers, with greater loss of muscle mass in certain muscles, like the quadriceps and triceps, the research team writes.

“What was happening to the patient’s muscles was a big riddle that our research addressed,” Jaiswal adds. “Lacking this protein is not supposed to make the muscle fiber die, like we see in patients with this rare disease. The missing protein is just supposed to cause atrophy and weakness.”

Patients with this rare disease show early muscle weakness, fluctuating levels of fatigue and lethargy, muscle aches after exercise, and elevated creatine kinase in their bloodstream, an indication of cell damage due to physical stress.

“One by one, we investigated these specific features in experimental models that look normal and have normal body weight, but also show lost muscle mass in the quadriceps and triceps,” explains Adam Horn, Ph.D., the lead researcher in Jaiswal’s lab who conducted this study. “Our experimental model lacking MICU1 only in skeletal muscles responded to muscle deficits so similar to humans that it suggests that some of the symptoms we see in patients can be attributed to MICU1 loss in skeletal muscles.”

Future research will aim to explore the details of how the impact of MICU1 deficit in muscles may be addressed therapeutically and possible implications of lacking MICU1 or its paralog in other organs.

In addition to Jaiswal and Horn, Children’s National Hospital Center for Genetic Medicine Research co-authors include Marshall W. Hogarth and Davi A. Mazala. Additional co-authors include Lead Author Valentina Debattisti, Raghavendra Singh, Erin L. Seifert, Kai Ting Huang, and Senior Author György Hajnóczky, all from Thomas Jefferson University; and Rita Horvath, from Newcastle University.

Financial support for research described in this post was provided by the National Institutes of Health under award numbers R01AR55686, U54HD090257 and RO1 GM102724; National Institute of Arthritis and Musculoskeletal and Skin Diseases under award number T32AR056993; and Foundation Leducq.

Understanding individual and collective mechanisms behind cell membrane repair

August 3, 2018

Signals released during plasma membrane repair initiate tissue repair: Extracellular vesicle signaling, an intracellular calcium increase that initiates plasma membrane repair in injured cells and an increase in cytosolic calcium that stimulates release of ATP by vesicle exocytosis or through plasma membrane channels.

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What’s known

All cells are surrounded by a cell membrane: a double layer of lipids with embedded proteins that separates the inside of the cell from the outside environment. At only 10 nanometers in thickness, this layer is quite fragile. Any breach can be fatal for a cell, causing chemical imbalances by exposing its interior to the extracellular milieu. Consequently, cells have evolved a set of responses to rapidly restore the integrity of the cell membrane in the event of a rupture, coordinating actions spurred by both immediate and longer-term signals. Research is providing a growing understanding of these repair mechanisms, which could go awry in degenerative diseases.

What’s new

Adam Horn, Ph.D., a postdoctoral fellow, and Jyoti K. Jaiswal, Ph.D., a principal investigator at Children’s Center for Neuroscience Research and the Center for Genetic Medicine Research, recently co-authored a literature review article summarizing these cell membrane repair mechanisms and the signals that trigger them. They delve into a variety of resourceful ways that cells fix tears or holes in this membrane, including one akin to blood clotting that stuffs a tear with proteins, organelles or vesicles; another in which the proteins that give a cell structure (the cytoskeleton) disassemble, relaxing tension that helps pull the damaged membrane together; or one in which the damaged portion in the membrane is shed. These repairs are driven by signals that largely rely on a large calcium influx into the cellular fluid, which spurs into action a variety of repair-related proteins. Better understanding each element could help researchers develop new and better ways to treat degenerative diseases in which cells inadequately repair damage.

Questions for future research

Q: How do the different types of signals coordinate individual and collective mechanisms of cell membrane repair?

Q: How is cell membrane repair coordinated among populations of cells at the tissue level?

Source: “Cellular Mechanisms and Signals That Coordinate Plasma Membrane Repair.” A. Horn and J.K. Jaiswal. Published by Cellular and Molecular Life Sciences July 26, 2018.

Antioxidants could thwart muscle repair

September 6, 2017

Science Signaling features a Research Article that describes the pathway by which mitochondria transduce the increase in cytosolic Ca2+ caused by plasma membrane injury into a ROS-dependent repair response. The image shows ROS production and actin polymerization as detected by fluorescent reporters near a plasma membrane injury site in a skeletal myofiber in an intact bicep of an experimental model. Credit: Adam Horn and Jyoti Jaiswal, M.S.C., Ph.D. Children’s National Health System and The George Washington University School of Medicine and Health Sciences

Reactive oxygen species (ROS) are a biological double-edged sword. These atoms, molecules or molecular fragments containing oxygen that is poised for chemical reactions, are a key part of the immune response, used by immune cells to kill potentially dangerous invaders such as bacteria. However, too much ROS – which also are produced as a normal part of cellular metabolism – can cause extreme damage to normal, healthy cells.

Because oxidative damage has been linked with cancer, many people make a concerted effort to consume antioxidants in food and as concentrated supplements. These compounds can neutralize ROS, stemming cellular damage. Taking antioxidants also has been thought to stem the muscle soreness from exercise since ROS are produced in excess during hard physical activity.

However, a new study led by researchers from Children’s National Health System finds that taking antioxidants could thwart the processes that repair muscle fibers. According to the study published Sept. 5, 2017 in Science Signaling and featured on the journal’s cover, oxidative species are crucial signals that start the process of repairing muscle fibers.

Cellular powerhouses known as mitochondria help injured muscle cells (myofibers) repair by soaking up calcium that enters from the site of injury and using it to trigger increased production of reactive oxygen species. Loading up mitochondria with excess antioxidants inhibits this signaling process, blocking muscle repair, exacerbating myofiber damage and diminishing muscle strength.

“Our results suggest a physiological role for mitochondria in plasma membrane repair in injured muscle cells, a role that highlights a beneficial effect of reactive oxygen species,” says Jyoti K. Jaiswal, M.S.C., Ph.D., principal investigator in the Center for Genetic Medicine Research at Children’s National Health System, associate professor of genomics and precision medicine at The George Washington University School of Medicine and Health Sciences and senior study author. “Our work highlights the need to take a nuanced view of the role of reactive oxygen species, as they are necessary when they are present at the right place and right time. Indiscriminate use of antioxidants actually could harm an adult with healthy muscles as well as a child with diseased muscle.”

Antioxidants are widely used by Baby Boomers with muscles that ache from a grueling workout or newborns diagnosed with muscular dystrophy. Jaiswal and Children’s National colleagues understand that their results buck conventional wisdom that antioxidants generally benefit muscle recovery.

“It is still a common belief within the fitness community that taking antioxidant supplements after a workout will help your muscles recover better. That’s what people think; that’s what I thought,” says Adam Horn, lead study author, a graduate student at The George Washington University who works with Jaiswal at Children’s National. “What we’ve done is figure out that mitochondria need to produce a very specific oxidative signal in response to muscle damage in order to help injured muscles repair.” Jyoti Jaiswal and Adam Horn

The oxidative signals produced by mitochondria are delicately balanced by the antioxidant defenses in healthy cells. This balance can be disrupted in diseases such as Duchenne muscular dystrophy, which is caused by the lack of a muscle-specific protein, dystrophin. Lack of dystrophin makes the muscle cell plasma membrane more vulnerable to injury. In an experimental model of Duchenne muscular dystrophy, the muscles at birth are seemingly normal but, within weeks, show obvious muscle damage and progressive weakness.

“What changes? One of the things that changes in the third and fourth week of life of this experimental model is mitochondrial functionality,” Jaiswal adds. “They end up with many dysfunctional mitochondria, which compromise repair of injured myofibers. This permits chronic and excessive oxidation of the myofibers and disruption of the proper oxidant-antioxidant balance.”

In this case, a dose of antioxidants may restore that proper balance and help to reverse muscle damage and progressive weakness.

As a next step, the research team is examining oxidation in healthy and diseased muscle to understand how the oxidant-antioxidant balance is disrupted and how it could be restored efficiently by using existing supplements. In one such study funded by the National Institutes of Health, the team is looking at the potential benefit of vitamin E supplements for patients with muscular dystrophy.

“Antioxidant supplements are made from extracts of bark, sap, chocolate and other compounds so they’re all different,” Jaiswal says. “Knowing which ones can restore balance under a specific circumstance has the potential to help the body maintain proper cellular signaling ability, which will keep muscles healthy and working properly.”

The response of actin protein following injury to a pair of muscle fibers in an intact biceps muscle.