Unlocking a Mystery of Human Disease … in Space

Huntington’s disease is a grim diagnosis. A hereditary disorder with debilitating physical and cognitive symptoms, the disease usually robs adult patients of their ability to walk, balance, and speak. More than 15 years ago, researchers revealed the disorder’s likely cause—an abnormal version of the protein huntingtin; however, the mutant protein’s mechanism is poorly understood, and the disease remains untreatable.

Now, a new project led by Pamela Bjorkman, Max Delbrück Professor of Biology, will investigate whether the huntingtin protein can form crystals in microgravity aboard the International Space Station (ISS)—crystals that are crucial for understanding the molecular structure of the protein. The experiment was launched from Cape Canaveral in Florida on Friday, April 18 aboard the SpaceX CRS-3 cargo resupply mission to the ISS. On Sunday, April 20 the station’s robotic arm captured the mission’s payload, which included the proteins for Bjorkman’s experiment—which is the first Caltech experiment to take place aboard the ISS.

In the experiment, the researchers hope to grow a crystal of the huntingtin protein—the crystal would be an organized, latticelike arrangement of the protein’s molecules—which is needed to determine the molecular structure of the protein. However, molecules of the huntingtin protein tend to aggregate, or clump together, in Earth’s gravity. And this disordered arrangement makes it incredibly hard to parse the protein’s structure, says Gwen Owens, a graduate student in Bjorkman’s lab and a researcher who helped design the study.

"We need crystals for X-ray crystallography, the technique we use to study the protein, in which we shoot an X-ray through the protein crystal and analyze the organized pattern of radiation that scatters off of it," Owens says. "That pattern is what we depend on to identify the location of every carbon, nitrogen, and sulfur atom within the protein; if we shoot an X-ray beam at a clumped, aggregate protein—like huntingtin often is—we can’t get any data from it," she says.

Researchers have previously studied small fragments of crystallized huntingtin, but because of its large size and propensity to clumping, no one has ever successfully grown a crystal of the full-length protein large enough to analyze with X-ray crystallography. To understand what the protein does—and how defects in it lead to the symptoms of Huntington’s disease—the researchers need to study the full-length protein.

Looking for a solution to this problem, Owens was inspired by a few previous studies of protein formation on space shuttles and the ISS—studies suggesting that proteins can form crystals more readily in a condition of near-weightlessness called microgravity. “The previous studies looked at much simpler proteins, but we thought we could make a pretty good case that huntingtin would be an excellent candidate to study on the ISS,” Owens says.

They proposed such an experiment to the Center for the Advancement of Science in Space (CASIS), which manages U.S. research on the ISS, and it was accepted, becoming part of the first Advancing Research Knowledge, or ARK1, mission.

Because Owens and Bjorkman cannot travel with their proteins, and staff and resources are limited aboard the ISS, the crystal will be grown with a Handheld High-Density Protein Crystal Growth device—an apparatus that will allow astronauts to initiate growth of normal and mutant huntingtin protein crystals from a solution of protein molecules with just the flip of a switch.

As the crystals grow larger over a period of several months, samples will come back to Earth via the SpaceX CRS-4 return mission. The results of the experiment are scheduled to drop into the ocean just off the coast of Southern California—along with the rest of the return cargo—sometime this fall. At that point, Owens will finally be able to analyze the proteins.

"Our ideal result would be to have large crystals of the normal and mutant huntingtin proteins right away—on the first try," she says. After analyzing crystals of the full-length protein with X-ray crystallography, the researchers could finally determine huntingtin’s structure—information that will be crucial to developing treatments for Huntington’s disease.

Physicists push new Parkinson’s treatment toward clinical trials

The most effective way to tackle debilitating diseases is to punch them at the start and keep them from growing.

Research at Michigan State University, published in the Journal of Biological Chemistry, shows that a small “molecular tweezer” keeps proteins from clumping, or aggregating, the first step of neurological disorders such as Parkinson’s disease, Alzheimer’s disease and Huntington’s disease.

The results are pushing the promising molecule toward clinical trials and actually becoming a new drug, said Lisa Lapidus, MSU associate professor of physics and astronomy and co-author of the paper.

“By the time patients show symptoms and go to a doctor, aggregation already has a stronghold in their brains,” she said. “In the lab, however, we can see the first steps, at the very place where the drugs could be the most effective. This could be a strong model for fighting Parkinson’s and other diseases that involve neurotoxic aggregation.”

Lapidus’ lab uses lasers to study the speed of protein reconfiguration before aggregation, a technique Lapidus pioneered. Proteins are chains of amino acids that do most of the work in cells. Scientists understand protein structure, but they don’t know how they are built – a process known as folding.

Lapidus’ lab has shed light on the process by correlating the speed at which an unfolded protein changes shape, or reconfigures, with its tendency to clump or bind with other proteins. If reconfiguration is much faster or slower than the speed at which proteins bump into each other, aggregation is slow, but if reconfiguration is the same speed, aggregation is fast.

Srabasti Acharya, lead author and doctoral candidate in Lapidus’ lab, tested the molecule, CLR01, which was patented jointly by researchers at the University of Duisburg-Essen (Germany) and UCLA. CLR01 binds to the protein and prevents aggregation by speeding up reconfiguration. It’s like a claw that attaches to the amino acid lysine, which is part of the protein.

This work was preceded by Lapidus’ research involving the spice curcumin. While the spice molecules put the researchers on a solid path, the molecules weren’t viable drug candidates because they cannot cross the blood-brain barrier, or BBB, the filter that controls what chemicals reach the brain.

It’s the BBB, in fact, that disproves the notion that people should simply eat more spicy food to stave off Parkinson’s disease.

Spicy misconceptions notwithstanding, CLR01 mimics curcumin molecules’ ability to prevent aggregation. But unlike the spice, CLR01 can crossover the BBB and treat its targeted site. Not only do they go to the right place, but CLR01 molecules also work even better because they speed up reconfiguration even more than curcumin. Additionally Acharya showed that CLR01 slows the first step of aggregation, and the results from the study map out a clear road map for moving the drug to clinical trials.

Hearing about a nontraditional physics lab that was advancing medicine is what brought Acharya to work with Lapidus.

“I knew I wanted to study physics when I came to MSU, but when I heard Dr. Lapidus’ presentation during orientation, I knew this is what I wanted to do,” Acharya said. “We are using physics to better understand biology to help cure actual diseases.”

To help move the research to the next phase, Gal Bitan, co-author and professor at UCLA, is using crowdsourcing to raise funds for the clinical trials. Log on to the indiegogo.com website for more information.

Common links between neurodegenerative diseases identified

Diseases of the central nervous system are a big burden to society. According to estimates, they cost €800 billion per year in Europe. And for most of them, there is no definitive cure. This is true, for example, for Parkinson disease. Although good treatments exist to manage its symptoms, they become more and more ineffective as the disease progresses. Now, the EU-funded REPLACES project, completed in 2013, which associated scientists with clinicians, has shed light on the abnormal working of a particular brain circuitry related to Parkinson’s disease. The results of the project suggest that these same circuits are implicated in different forms of pathologies. And this gives important insights into the possible common links between neurodegenerative diseases such as Parkinson and intellective disabilities or autism.

Existing treatments for Parkinson are very effective at the beginning. When the disease progresses, however, drugs, such as levodopa and so-called dopamine agonists, produce side effects that are sometimes even worse than the initial symptoms of the condition. In particular, they cause a complication called dyskinesia, characterised by abnormal involuntary movements. Therapies are therefore sought that allow better management of symptoms.

The project focused on the study of a highly plastic brain circuitry, which connects regions of the cerebral cortex with the basal ganglia. It is involved in very important functions such as learning and memory. “This system, based onglutamate as a mean of signalling between neurons, has also been discovered to be damaged in Parkinson disease,” says Monica Di Luca, professor of neuropharmacology at the University of Milan, Italy, and the project coordinator. She adds: “Parkinson’s more well-known and characteristic trait is the selective loss of cells producers of neurotransmitter dopamine.”

Researchers involved into the project studied the function and plasticity of this circuit in different animal models of Parkinson disease, from mice to non-human primates. They found that exactly the same alterations were present and conserved. This makes it an interesting and alternative target for trying to re-establish the correct functioning and reverse the symptoms of the disease.

One expert agrees with the need to target alternative target systems. “What researchers are trying to do is to intervene to modulate other systems that do not involve dopamine and obtain a better symptoms management,” explains Erwan Bezard, a researcher at the Neurodenerative Diseases Institute at the University of Bordeaux, in France. He also works on alternative targets in Parkinson disease. In monkeys, compounds that target glutamate receptors, used in combination with traditional drugs, have previously shown to improve some deficits in voluntary motor control.

But the research has also shed some light into apparently unrelated diseases. It is becoming more and more obvious that the same alterations in the working of the communication systems among neurons are shared among different diseases. “This is why we speak about ‘synaptopathies’: there are common players among Parkinson disease, autism and other forms of intellectual disabilities and even schizophrenia. Several of the mutated genes are the same, and affect the signalling systems through common molecules,” says Claudia Bagni, who works on synaptic plasticity in the context of intellectual disabilities at the University of Leuven, in Belgium and University of Rome Tor Vergata, in Italy. “For example, the glutamatergic system is also affected in the X-fragile syndrome, the most common form of inherited intellectual disability.”

Progress is in sight thanks to a much better understanding of the working of the abnormal synapses in Parkinson disease, and experiments performed in monkeys showing encouraging results. Indeed, “the team studied human primates, the model system closest to humans, and therefore their findings are relevant to human health.” says Bagni. Project researchers hope the door is now opened for the first clinical trials in humans. “We have identified a potential new target for treatment, and tested a couple of molecules in animals,” says Di Luca, the “next step would be to find a partnership with pharmaceutical industries interested in pursuing this research.”

Researchers identify how zinc regulates a key enzyme involved in cell death

Findings may help develop targeted drug interventions and fight cancer and neurodegenerative diseases

The molecular details of how zinc, an essential trace element of human metabolism, interacts with the enzyme caspase-3, which is central to apoptosis or cell death, have been elucidated in a new study led by researchers at Virginia Commonwealth University. The study is featured on the cover of the April issue of the journal Angewandte Chemie’s International Edition.

Dysregulation of apoptosis is implicated in cancer and neurodegenerative disease such as Alzheimer’s disease. Zinc is known to affect the process by inhibiting the activity of caspases, which are important drug targets for the treatment of the above conditions. The findings may help researchers design therapeutic agents that target zinc-caspase interaction to specifically control the activity of caspases, and hence, apoptosis.

“The work is unique in helping to open up a broad new area of research which we call the bioinorganic chemistry of apoptosis – understanding the role of essential metal ions in one of life’s fundamental processes,” said corresponding author Nicholas P. Farrell, Ph.D., member of the Developmental Therapeutics program at VCU Massey Cancer Center and professor of chemistry in the VCU College of Humanities and Sciences.

“Indeed, the zinc inhibition of apoptosis in fact contrasts with the role of its closely related neighbor copper, which is understood to enhance apoptosis,” he said.

In the study, Farrell and his research team, A. Gerard Daniel, Ph.D., and Erica J. Peterson, used conventional enzymology and biophysical techniques combined with state-of-the-art computational methods, to show evidence for a hitherto unrecognized interaction site with caspase-3.

According to Farrell, caspases were discovered in the mid-1990s. There are 11 caspases known humans, and seven of these are involved in cell death. The study suggests a regulatory zinc site that may be common to all caspases. Previous findings have shown other zinc binding sites in caspase-6 and -9. Now, Farrell said, the generality of the team’s observations must be extended and verified in other caspases.

“The [journal] cover epitomizes the contrasting but interdependent roles of the metal ions copper/zinc in the regulation of apoptosis and perfectly captures the duality of this most fundamental of biological processes,” Farrell said.

Bioimaging: Visualizing real-time development of capillary networks in adult brains

The advancement of microscopic photoimaging techniques has enabled the visualization of real-time cellular events in living organs. The brain capillary network exhibits a unique feature that forms a blood-brain barrier (BBB), which is an interface of vascular endothelial cells that control the traffic of substances from the bloodstream into the brain. Damage and disruption to the BBB are implicated in contributing to the pathogenesis and progression of neurodegenerative disorders such as Alzheimer’s and epilepsy. However, the cellular interactions present in the BBB are incredibly difficult to study in vivo, so understanding of these mechanisms in living brains is limited.

Now, Kazuto Masamoto and co-workers at the University of Electro-Communications in Tokyo, National Institute of Radiological Sciences, and Keio University School of Medicine, have used 4D live imaging technology to study the effects of hypoxia (a deprivation of oxygen) on the BBB plasticity in live adult mice.

The team focused their attention on how the BBB plastic changes work against hypoxia, looking in particular at the endothelial cells and their communications to the neighboring astrocytes - interactions which take place in controlling the BBB traffic to fulfill neural demands. Using genetically-modified mice with endothelial cells that express green-fluorescent protein, Masamoto and colleagues imaged the real-time changes of BBBs before and during a three-week period of hypoxia in adult mouse cortex.

Their results showed that the capillaries in the BBB, which prior to hypoxia showed no signs of activity, began to sprout new blood vessels which in places formed new networks together. The neighboring astrocytes reacted quickly to wrap the outside of the new vessels, activity which the researchers believe helps stabilize the BBB traffic and integrity.

Further investigations into the molecular mechanisms that control BBB plasticity are expected to lead to advances in treatment of neurodegenerative disorders and cerebral ischemia, and thus provide an effective way for preventing BBB dysfunction in diabetes, hypertension, and aging.