Posts tagged genetics

Amyotrophic Lateral Sclerosis (ALS) is a devastating motor neuron disease that rapidly atrophies the muscles, leading to complete paralysis. Despite its high profile — established when it afflicted the New York Yankees’ Lou Gehrig — ALS remains a disease that scientists are unable to predict, prevent, or cure.

Although several genetic ALS mutations have been identified, they only apply to a small number of cases. The ongoing challenge is to identify the mechanisms behind the non-genetic form of the disease and draw useful comparisons with the genetic forms.

Now, using samples of stem cells derived from the bone marrow of non-genetic ALS patients, Prof. Miguel Weil of Tel Aviv University’s Laboratory for Neurodegenerative Diseases and Personalized Medicine in the Department of Cell Research and Immunology and his team of researchers have uncovered four different biomarkers that characterize the non-genetic form of the disease. Each sample shows similar biological abnormalities to four specific genes, and further research could reveal additional commonalities. “Because these genes and their functions are already known, they give us a specific direction for research into non-genetic ALS diagnostics and therapeutics,” Prof. Weil says. His initial findings were reported in the journal Disease Markers.

Giving in to stress

To hunt for these biomarkers, Prof. Weil and his colleagues turned to samples of bone marrow collected from ALS patients. Though more difficult to collect than blood, bone marrow’s stem cells are easy to isolate and grow in a consistent manner. In the lab, he used these cells as cellular models for the disease. He ultimately discovered that cells from different ALS patients shared the same abnormal characteristics of four different genes that may act as biomarkers of the disease. And because the characteristics appear in tissues that are related to ALS — including in muscle, brain, and spinal cord tissues in mouse models of genetic ALS — they may well be connected to the degenerative process of the disease in humans, he believes.

Searching for the biological significance of these abnormalities, Prof. Weil put the cells under stress, applying toxins to induce the cells’ defense mechanisms. Healthy cells will try to fight off threats and often prove quite resilient, but ALS cells were found to be overwhelmingly sensitive to stress, with the vast majority choosing to die rather than fight. Because this is such an ingrained response, it can be used as a feature for drug screening for the disease, he adds.

The hunt for therapeutics

Whether these biomarkers are a cause or consequence of ALS is still unknown. However, this finding remains an important step towards uncovering the mechanisms of the disease. Because these genes have already been identified, it gives scientists a clear direction for future research. In addition, these biomarkers could lead to earlier and more accurate diagnostics.

Next, Prof. Weil plans to use his lab’s high-throughput screening facility — which can test thousands of compounds’ effects on diseased cells every day — to search for drug candidates with the potential to affect the abnormal expression of these genes or the stress response of ALS cells. A compound that has an impact on these indicators of ALS could be meaningful for treating the disease, he says.

Prof. Weil is the director of the new Cell Screening Facility for Personalized Medicine at TAU. The facility is dedicated to finding potential drugs for rare and Jewish hereditary diseases.

(Source: aftau.org)

In a study that could change the way scientists view the process of protein production in humans, University of Chicago researchers have found a single gene that encodes two separate proteins from the same sequence of messenger RNA.

Published online July 3 in Cell, their finding elucidates a previously unknown mechanism in human gene expression and opens the door for new therapeutic strategies against a thus-far untreatable neurological disease.

"This is the first example of a mechanism in a higher organism in which one gene creates two proteins from the same mRNA transcript, simultaneously," said Christopher Gomez, MD, PhD, professor and chairman of the Department of Neurology at the University of Chicago, who led the study. "It represents a paradigm shift in our understanding of how genes ultimately encode proteins."

The human genome contains a similar number of protein-coding genes as the nematode worm (roughly 20,000). This disparity between biological complexity and gene count partially can be explained by the fact that individual genes can encode multiple protein variants via the production of different sequences of messenger RNA (mRNA) — short, mass-produced copies of genetic code that guide the creation of myriad cellular machinery.

Gomez and his team, which included first author Xiaofei Du, MD, discovered a new layer of complexity in this process of gene expression as they studied spinocerebellar ataxia type-6 (SCA6), a neurodegenerative disease that causes patients to slowly lose coordination of their muscles and eventually their ability to speak and stand. Human genetic studies identified its cause as a mutation in CACNA1A — a gene that encodes a calcium channel protein important for nerve cell function — resulting in extra copies of the amino acid glutamine.

However, although the gene, mutation and dysfunction are known, attempts to find the biological mechanism of the disease proved inconclusive. Calcium channel proteins with the mutation still seemed to function normally.

Suspecting another factor at play, Gomez and his team instead focused on α1ACT, a poorly understood, free-floating fragment of the CACNA1A calcium channel protein known to express extra copies of glutamine in SCA6 cells. The researchers first looked at its origin and found that, to their surprise, α1ACT was generated from the same mRNA sequence as the CACNA1A calcium channel.

For the first time, they had evidence of a human gene that coded one strand of mRNA that coded two separate, structurally distinct proteins. This occurred due to the presence of a special sequence in the mRNA known as an internal ribosomal entry site (IRES). Normally found at the beginning of an mRNA sequence, this IRES site sat in the middle, creating a second location for ribosomes, the cellular machines that read mRNA, to begin the process of protein production.

Looking at function, Gomez and his team found that normal α1ACT acted as a transcription factor and enhanced the growth of specific brain cells. Importantly, mutated α1ACT appeared to be toxic to nerve cells in a petri dish, and caused SCA6-like symptoms in an animal model.

The team hopes to discover other examples of human genes with similar IRES sites to better understand the implications of this new class of “bifunctional” genes on our basic biology. For now, they are focused on leveraging their findings toward helping SCA6 patients and already are working on ways to silence mutated α1ACT.

"We discovered this genetic phenomenon in the pursuit of a disease cause and, in finding it, immediately have a potential strategy for developing preclinical tools to treat that disease," Gomez said. "If we can target the IRES and inhibit production of this mutant form of α1ACT in SCA6, we may be able to stop the progression of the disease."

(Source: uchospitals.edu)

Scientists at Karolinska Institutet have identified the neuronal circuits in the spinal cord of mice that control the ability to produce the alternating movements of the legs during walking. The study, published in the journal Nature, demonstrates that two genetically-defined groups of nerve cells are in control of limb alternation at different speeds of locomotion, and thus that the animals’ gait is disturbed when these cell populations are missing.

Most land animals can walk or run by alternating their left and right legs in different coordinated patterns. Some animals, such as rabbits, move both leg pairs simultaneously to obtain a hopping motion. In the present study, the researchers Adolfo Talpalar and Julien Bouvier together with professor Ole Kiehn and colleagues, have studied the spinal networks that control these movement patterns in mice. By using advanced genetic methods that allow the elimination of discrete groups of neurons from the spinal cord, they were able to remove a type of neurons characterized by the expression of the gene Dbx1.

"It was classically thought that only one group of nerve cells controls left right alternation", says Ole Kiehn who leads the laboratory behind the study at the Department of Neuroscience. "It was then very interesting to find that there are actually two specific neuronal populations involved, and on top of that that they each control different aspect of the limb coordination."

Indeed, the researchers found that the gene Dbx1 is expressed in two different groups of nerve cells, one of which is inhibitory and one that is excitatory. The new study shows that the two cellular populations control different forms of the behaviour. Just like when we change gear to accelerate in a car, one part of the neuronal circuit controls the mouse’s alternating gait at low speeds, while the other population is engaged when the animal moves faster. Accordingly, the study also show that when the two populations are removed altogether in the same animal, the mice were unable to alternate at all, and hopped like rabbits instead.

There are some animals, such as desert mice and kangaroos, which only hop. The researchers behind the study speculate that the locomotive pattern of these animals could be attributable to the lack of the Dbx1 controlled alternating system.

(Source: ki.se)

For the first time, scientists from the Florida campus of The Scripps Research Institute (TSRI) have identified small molecules that allow for complete control over a genetic defect responsible for the most common adult onset form of muscular dystrophy. These small molecules will enable scientists to investigate potential new therapies and to study the long-term impact of the disease.

“This is the first example I know of at all where someone can literally turn on and off a disease,” said TSRI Associate Professor Matthew Disney, whose new research was published June 28, 2013, by the journal Nature Communications. “This easy approach is an entirely new way to turn a genetic defect off or on.”

Myotonic dystrophy is an inherited disorder, the most common form of a group of conditions called muscular dystrophies that involve progressive muscle wasting and weakness. Myotonic dystrophy type 1 is caused a type of RNA defect known as a “triplet repeat,” a series of three nucleotides repeated more times than normal in an individual’s genetic code. In this case, a cytosine-uracil-guanine (CUG) triplet repeat binds to the protein MBNL1, rendering it inactive and resulting in RNA splicing abnormalities.

To find drug candidates that act against the defect, Disney and his colleagues analyzed the results of a National Institutes of Health (NIH)-sponsored screen of more than 300,000 small molecules that inhibit a critical RNA-protein complex in the disease.

The team divided the NIH hits into three “buckets”—the first group bound RNA, the second bound protein, and a third whose mechanism was unclear. The researchers then studied the compounds by looking at their effect on human muscle tissue both with and without the defect.

Startlingly, diseased muscle tissue treated with RNA-binding compounds caused signs of the disease to go away. In contrast, both healthy and diseased tissue treated with the protein-binding compounds showed the opposite effect—signs of the disease either appeared (in healthy tissue) or became worse.

The new compounds will serve as useful tools to study the disease on a molecular level. “In complex diseases, there are always unanticipated mechanisms,” Disney noted. “Now that we can reverse the disease at will, we can study those aspects of it.”

In addition, Disney said, with the new discovery, scientists will be able to develop a greater understanding of how to control RNA splicing with small molecules. RNA splicing can cause a host of diseases that range from sickle-cell disease to cancer, yet prior to this study, no tools were available to control specific RNA splicing.

(Source: scripps.edu)

Scientists using sophisticated imaging techniques have observed a molecular protein folding process that may help medical researchers understand and treat diseases such as Alzheimer’s, Lou Gehrig’s and cancer.

The study, reported this month in the journal Cell, verifies a process that scientists knew existed but with a mechanism they had never been able to observe, according to Dr. Hays Rye, Texas A&M AgriLife Research biochemist.

“This is a step in the direction of understanding how to modulate systems to prevent diseases like Alzheimer’s. We needed to understand the cell’s folding machines and how they interact with each other in a complicated network,” said Rye, who also is associate professor of biochemistry and biophysics at Texas A&M.

Rye explained that individual amino acids get linked together like beads on a string as a protein is made in the cell.

“But that linear sequence of amino acids is not functional,” he explained. “It’s like an origami structure that has to fold up into a three-dimensional shape to do what it has to do.”

Rye said researchers have been trying to understand this process for more than 50 years, but in a living cell the process is complicated by the presence of many proteins in a concentrated environment.

"The constraints on getting that protein to fold up into a good ‘origami’ structure are a lot more demanding,” he said. “So, there are special protein machines, known as molecular chaperones, in the cell that help proteins fold.”

But how the molecular chaperones help protein fold when it isn’t folding well by itself has been the nagging question for researchers.

“Molecular chaperones are like little machines, because they have levers and gears and power sources. They go through turning over cycles and just sort of buzz along inside a cell, driving a protein folding reaction every few seconds,” Rye said.

The many chemical reactions that are essential to life rely on the exact three-dimensional shape of folded proteins, he said. In the cell, enzymes, for example, are specialized proteins that help speed biological processes along by binding molecules and bringing them together in just the right way.

“They are bound together like a three-dimensional jigsaw puzzle,” Rye explained.  “And the proteins — those little beads on the string that are designed to fold up like origami — are folded to position all these beads in three-dimensional space to perfectly wrap around those molecules and do those chemical reactions.

“If that doesn’t happen — if the protein doesn’t get folded up right – the chemical reaction can’t be done. And if it’s essential, the cell dies because it can’t convert food into power needed to build the other structures in the cell that are needed. Chemical reactions are the structural underpinning of how cells are put together, and all of that depends on the proteins being folded in the right way.”

When a protein doesn’t fold or folds incorrectly it turns into an “aggregate,” which Rye described as “white goo that looks kind of like a mayonnaise, like crud in the test tube.

“You’re dead; the cell dies,” he said.

Over the past 20 years, he said, researchers have linked that aggregation process “pretty convincingly” to the development of diseases — Alzheimer’s disease, Lou Gehrig’s disease, Huntington’s disease, to name a few. There’s evidence that diabetes and cancer also are linked to protein folding disorders.

“One of the main roles for the molecular chaperones is preventing those protein misfolding events that lead to aggregation and not letting a cell get poisoned by badly folded or aggregated proteins,” he said.

Rye’s team focused on a key molecular chaperone — the HSP60.

“They’re called HSP for ‘heat shock protein’ because when the cell is stressed with heat, the proteins get unstable and start to fall apart and unfold,” Rye said. “The cell is built to respond by making more of the chaperones to try and fix the problem.

“This particular chaperone takes unfolded protein and goes through a chemical reaction to bind the unfolded protein and literally puts it inside a little ‘box,’” Rye said.

He added that the mystery had long been how the folding worked because, while researchers could see evidence of that happening, no one had ever seen precisely how it happened.

Rye and the team zeroed in on a chemically modified mutant that in other experiments had seemed to stall at an important step in the process that the “machine” goes through to start the folding action. This clued the researchers that this stalling might make it easier to watch.

They then used cryo-electron microscopy to capture hundreds of thousands of images of the process at very high resolutions which allowed them to reconstruct from two-dimensional flat images a three-dimensional model. A highly sophisticated computer algorithm aligns the images and classifies them in subcategories.

“If you have enough of them you can actually reconstruct and view a structure as a three-dimensional model,” Rye said.

What the team saw was this: The HSP60 chaperone is designed to recognize proteins that are not folded from the ones that are. It binds them and then has a separate co-chaperone that puts a “lid” on top of the box to keep the folding intermediate in the box. They could see the box move, and parts of the molecule moved to peel the chaperone box away from the bound protein — or “gift” in the box. But the bound protein was kept inside the package where it could then initiate a folding reaction. They saw tiny tentacles, “like a little octopus in the bottom of the box rising up and grabbing hold of the substrate protein and helping hold it inside the cavity.”

"The first thing we saw was a large amount of an unfolded protein inside of this cavity,” he said. “Even though we knew from lots and lots of other studies that it had to go in there, nobody had ever seen it like this before. We can also see the non-native protein interacting with parts of the box that no one had ever seen before. It was exciting to see all of this for the first time. I think we got a glimpse of a protein in the process of folding, which we actually can compare to other structures.”

“By understanding the mechanism of these machines, the hope is that one of the things we can learn to do is turn them up or turn them off when we need to, like for a patient who has one of the protein folding diseases,” he said.

(Source: today.agrilife.org)

Researchers with the UC Davis MIND Institute and Agilent Laboratories have found that Prader-Willi syndrome — a genetic disorder best known for causing an insatiable appetite that can lead to morbid obesity — is associated with the loss of non-coding RNAs, resulting in the dysregulation of circadian and metabolic genes, accelerated energy expenditure and metabolic differences during sleep.

The research was led by Janine LaSalle, a professor in the UC Davis Department of Medical Microbiology and Immunology who is affiliated with the MIND Institute. It is published online in Human Molecular Genetics.

“Prader-Willi syndrome children do not sleep as well at night and have daytime sleepiness,” LaSalle said. “Parents have to lock up their pantries because the kids are rummaging for food in the middle of the night, even breaking into their neighbors’ houses to eat.”

The study found that these behaviors are rooted in the loss of a long non-coding RNA that functions to balance energy expenditure in the brain during sleep. The finding could have a profound effect on how clinicians treat children with Prader-Willi, as well as point the way to new, innovative therapies, LaSalle said.

The leading cause of morbid obesity among children in the United States, Prader-Willi involves a complex, and sometimes contradictory, array of symptoms. Shortly after birth children with Prader-Willi experience failure to thrive. Yet after they begin to feed themselves, they have difficulty sleeping and insatiable appetites that lead to obesity if their diets are not carefully monitored.

The current study was conducted in a mouse model of Prader-Willi syndrome. It found that mice engineered with the loss of a long non-coding RNA showed altered energy use and metabolic differences during sleep.

Prader-Willi has been traced to a specific region on chromosome 15 (SNORD116), which produces RNAs that regulate gene expression, rather than coding for proteins. When functioning normally, SNORD116 produces small nucleolar (sno) RNAs and a long non-coding RNA (116HG), as well as a third non-coding RNA implicated in a related disorder, Angelman syndrome. The 116HG long non-coding RNA forms a cloud inside neuronal nuclei that associates with proteins and genes regulating diurnal metabolism in the brain, LaSalle said.

“We thought the cloud would be activating transcription, but in fact it was doing the opposite,” she said. “Most of the genes were dampened by the cloud. This long non-coding RNA was acting as a decoy, pulling the active transcription factors away from genes and keeping them from being expressed.”

As a result, losing snoRNAs and 116HG causes a chain reaction, eliminating the RNA cloud and allowing circadian and metabolic genes to get turned on during sleep periods, when they should be dampened down. This underlies a complex cycle in which the RNA cloud grew during sleep periods (daytime for nocturnal mice), turning down genes associated with energy use, and receded during waking periods, allowing these genes to be expressed. Mice without the 116HG gene lacked the benefit of this neuronal cloud, causing greater energy expenditure during sleep.

The researchers said that the work provides a clearer picture of why children with Prader-Willi syndrome can’t sleep or feel satiated and may change therapeutic approaches. For example, many such children have been treated with growth hormone because of short stature, but this actually may boost other aspects of the disease.

“People had thought the kids weren’t sleeping at night because of the sleep apnea caused by obesity,” said LaSalle. “What this study shows is that the diurnal metabolism is central to the disorder, and that the obesity may be as a result of that. If you can work with that, you could improve therapies, for example figuring out the best times to administer medications.”

(Source: ucdmc.ucdavis.edu)