Researchers at Brigham and Women’s Hospital have found two new potential drug targets for treating arterial diseases such as atherosclerosis. By using proteomics to screen a vast number of molecules, the researchers identified PARP9 and PARP14 – two members of the PARP family of proteins – as regulators of macrophage activation, which has been linked to arterial disease by systems biology.

Though the mechanisms that activate macrophages, a type of digestive white blood cell that targets foreign cells, remain incompletely understood, previous research shows that macrophages play an important role in the development of atherosclerosis and its thrombotic complications. Masanori Aikawa, MD, PhD, director of the Center for Interdisciplinary Cardiovascular Sciences (CICS) at the Brigham, his research fellow Hiroshi Iwata, MD, PhD, and colleagues studied atherosclerosis on the protein-level to determine which molecules were most involved in the regulation of macrophages.

Once Aikawa and his colleagues narrowed down their search to these two proteins, they silenced each gene in cultured macrophages and found that tamping down PARP14 increased macrophage activation while tamping down PARP9 had the opposite effect.

Aikawa founded CICS and hopes that this hypothesis-generating method can be used to streamline the lengthy process of drug development. Aikawa and CICS are using a more systematic approach which hinges on network analysis; this analysis predicts which pathways are most likely to control their studied effect so that they can prioritize these pathways. Ideally, this process would take a fraction of the time in comparison to searching through each individual pathway unaware of their likelihood of affecting their studied effect.

Aikawa and his colleagues plan to augment these findings to develop targeted therapeutics for atherosclerosis and other diseases.

‘Macrophage activation plays a role in not only vascular disorders but also various inflammatory and autoimmune diseases,’ said Aikawa. ‘These results could provide important information about the mechanisms of these diseases and help to develop much needed new therapeutics.’

EurekAlert www.eurekalert.org/pub_releases/2016-10/bawh-aot102516.php

Researchers at UMass Medical School have identified a new molecular pathway critical for maintaining the smooth muscle tone that allows the passage of materials through the digestive system. This finding, based on studying calcium ion-controlled pathways in mice, may lead to new treatments for a host of digestive disorders ranging from common gastroesophageal reflux disease (GERD), to swallowing disorders, incontinence and pancreatitis.
“We are excited about the potential to target identified genes to treat disorders such as reflux and incontinence,” said Ronghua ZhuGe, PhD, associate professor of microbiology and physiological systems and a senior author of the study. “Knowing how these muscles stay contracted for such long periods of time will allow us to develop potential new treatments for these diseases. The next step is to see whether this molecular mechanism in mice also operates in humans.”

The human body, and those of other mammals, contains a number of ring-shaped structures made of smooth muscle encircling openings in hollow organs such as the intestines and bladder called sphincters. Smooth muscle is involuntarily controlled, unlike the muscles we use to walk, for example, so that we don’t need to consciously move digested food from stomach to small intestine. Dysfunction in the sphincters, either structurally or functionally, can have severe consequences leading to diseases that impair the ability of the muscle to contract or relax. This can lead to achalasia, which makes it difficult to swallow; gastroesophageal reflux disease (GERD), which allows stomach acid to enter the oesophagus or incontinence of the bowels.

“A healthy sphincter opens transiently but remains closed most of the time, maintaining a basal tone. This basal tone requires constant generation of force produced by the contraction of smooth muscle cells that make up the sphincters,” said Dr. ZhuGe. “However, the genetics governing how the sphincter smooth muscle stays contracted for such long periods of time remains unknown.”

Smooth muscle operates by generating force as the muscle motor protein myosin and actin filaments move past each other. This happens after a molecule called the myosin regulatory light chain (MLC) is turned on through a common molecular transformation called phosphorylation. How much phosphorylation takes place is controlled by the relative amounts of two enzymes; calcium dependent MLC kinase (MLCK), which promotes phosphorylation, and calcium independent MLC phosphatase (MLCP), which reverses phosphorylation. Through this process, contraction and relaxation of the muscle is achieved.

To understand the molecular mechanism responsible for the involuntary and continuous contraction of the sphincter muscle, Dr. ZhuGe and colleagues examined the internal anal sphincter that controls bowel continence in mice. They showed that genetic deletion of the MLCP enzyme in the smooth muscle had no effect on the basal tone of the mouse sphincter, but deletion of MLCK essentially abolishes the basal tone and mice become incontinent as a result.

“Although previous biochemical studies suggested that lower MLCP activity may be related to the basal tone of this sphincter, our genetic study indicates this doesn’t seem to be the case. It turns out MLCK is essential for the tone formation,” said ZhuGe. “This prompted us to look for specific calcium signals that regulate MLCK.”

Co-author Lawrence Lifshitz, PhD, associate professor of molecular medicine, said, “Calcium signalling is our favourite subject. We originally hypothesized that localized releases of calcium inside the cell, near target ion channels, might do the trick, as we knew that such releases can regulate the contraction of smooth muscle in blood vessels and airways.”

But experiments showed that these local calcium releases have no direct role in muscle tone. Instead, three types of ion channels act in concert to generate a rise in cytosolic calcium which eventually results in MLCK activation and muscle tone.

To test this hypothesis, ZhuGe’s group teamed up with Minsheng Zhu, PhD, at Nanjing University in China, to generate a line of mice in which one of these channels could be turned off in smooth muscle only. “These mice were a powerful tool for establishing our hypothesis,” said ZhuGe. “They helped us identify the Tmem16a (also called Ano1) gene as a critical component for basal tone formation and fecal continence. When we were able to turn off the TMEM16A channels in these mice, they lost the majority of the basal tone and became incontinent. ”

UMass Medical School www.umassmed.edu/news/news-archives/2016/04/umms-scientists-identify-genes-that-control-smooth-muscle-contraction-in-digestive-system/

Scientists at the Stowers Institute for Medical Research have reported a detailed description of how function-impairing mutations in polr1c and polr1d genes cause Treacher Collins syndrome (TCS), a rare congenital craniofacial development disorder that affects an estimated 1 in 50,000 live births.
Collectively the results of the study reveal that a unifying cellular and biochemical mechanism underlies the etiology and pathogenesis of TCS and its possible prevention, irrespective of the causative gene mutation.

Loss-of-function mutations in three human genes, TCOF1, POLR1C and POLR1D, have been implicated in TCS and are thought to be responsible for about 90 percent of the diagnoses of this congenital craniofacial condition.
The clinical manifestations of TCS include facial anomalies such as small jaws and cleft palate, hearing loss, and respiratory problems. Patients with TCS typically undergo multiple surgeries, but rarely are they fully corrective. By uncovering a mechanism of action common to all three genes, Stowers scientists have advanced scientific understanding of TCS etiology and pathogenesis and identified possible new avenues for preventing or treating the birth defect. This latest study from the laboratory of Stowers Investigator Paul Trainor, Ph.D., focused on Polr1c and Polr1d, whose roles as a genetic cause of TCS were revealed in a 2011 study of a small group of patients who had been diagnosed with TCS but who did not have the TCOF1 mutation. Unlike POLR1C and POLR1D, TCOF1 has been long recognized as a causative gene in TCS and as a result has been more extensively investigated.
“Before we began the study, nothing was known about the role of Polr1c and Polr1d in craniofacial development,” said Kristin Watt, Ph.D., lead author of the PLoS Genetics paper and postdoctoral scientist in the Trainor Lab. “Using zebrafish as our animal model, we set out to explore the functional roles of polr1c and polr1d during embryogenesis and more specifically in craniofacial development.”
Trainor, Watt and their collaborators compared the results of their findings on polr1c and polr1d with their and other labs’ previous research results on Tcof1. In all three loss-of-function models, the researchers found that the chain of cellular events that led to the TCS phenotype of abnormal craniofacial development originated in ribosomes, the cellular components that translate messenger RNA into proteins. Like the Tcof1 gene, polr1c and polr1d mutations were found to perturb ribosome biogenesis, or production of ribosomes, which affects the generation and survival of progenitor neural crest cells, the precursors of craniofacial bone, cartilage and connective tissue.
In animal models of all three causative genes, the scientists determined that deficient ribosome biogenesis triggered a p53-dependent cell death mechanism in progenitor neural crest cells. As a result of the activation of the p53 gene, developing embryos no longer made the quantity of neural crest cells needed to properly form the craniofacial skeleton.
However, in the polr1c and polr1d models as in the Tcof1 models, Stowers scientists found that by experimentally blocking p53 activation, they could restore the neural crest cell population and thereby rescue the animal models’ cranioskeletal cartilage.
Despite the rescue effect, Trainor said that he does not view the “guardian of the genome,” as the p53 gene is often called due to its ability to suppress cancer, as the basis of a potential therapy to prevent or reduce TCS during embryonic development. The p53 gene’s association with cancer makes inhibiting its function too risky, he said.
A less risky and perhaps more effective target for the prevention or treatment of TCS could be enhancing ribosomes, Trainor said, because the loss-of-function mutations in all three causative genes involve ribosome RNA (rRNA) transcription. Polr1c and Polr1d, for example, are subunits of RNA polymerases I and III that are essential for ribosome biogenesis.
“Rather than blocking p53, a better approach may be to try to prevent TCS by treating the problem in ribosome biogenesis that triggers the activation of p53 and the loss of neural crest cells,” said Trainor.
In their research with zebrafish embryos, Trainor and collaborators also determined that polr1c and polr1d are spatiotemporally and dynamically expressed, particularly during craniofacial development. Furthermore, zebrafish embryos with the polr1c and polr1d loss-of-function mutations develop abnormalities in craniofacial cartilage development that mimic the clinical manifestations of TCS in patients. Trainor said that he and his fellow researchers were surprised that mutations in polr1c and polr1d as well as Tcof1 specifically affected craniofacial development, because ribosome biogenesis occurs in every cell of the body. The mutation of a gene that is part of the ribosome complex would be expected to be detrimental to each of these cells, he said. However, in the zebrafish models, the mutation appears to primarily affect progenitor neural crest cells. Trainor said that he and his team theorize that progenitor neural crest cells may be particularly sensitive to deficiencies in ribosome biogenesis during embryogenesis.
Thus, the study revealed new animal models for TCS: zebrafish with polr1c and polr1d loss-of-function mutations. Moreover, the existence of a common mechanism of action may simplify the research, particularly the search for a therapy to prevent or treat TCS. Because of the similarities among the three causative genes, “we may be able to develop creative ways of preventing TCS that will prove effective in at-risk individuals who have one of the gene mutations,” said Trainor, who has investigated the molecular origins and development of TCS and related craniofacial developmental disorders for 10 years.

Stowers Institute for Medical Research www.stowers.org/media/news/jul-22-2016

A strong association between a genetic mutation and a rare kind of heart muscle disease has been discovered by researchers at the University of Colorado Anschutz Medical Campus.

“There are many kinds of cardiomyopathies that can lead to heart failure so this is a serious problem,” said Teisha J. Rowland, PhD, a post-doctoral fellow in the lab of Luisa Mestroni, MD, and Matthew R. G. Taylor, MD, PhD, at the University of Colorado School of Medicine and first author of the study.

The Mestroni and Taylor lab sequenced nearly 5,000 genes in 335 patients with a family history of heart muscle disease, looking for mutations that could cause a variety of cardiomyopathies.

“Many kinds of heart disease are caused by genetics. When that happens, the disease is often more severe and happens at an earlier age,” said Rowland, who studies genetics and cardiology. “So we look at the DNA in entire families to see what sort of genetic variants those with the illness have in common.”

They found that several people with left ventricular noncompaction (LVNC) had a mutation in a gene called Obscurin. Obscurin is part of the sarcomere, the basic unit of striated muscles that pull and glide past each other when muscles contract.  That includes the heart muscle. If there is a mutation in Obscurin that process may not function properly.

 “We found a strong association between this gene, which has not been studied much, and this rare form of genetic heart disease,” Rowland said. “Left ventricular noncompaction is thought to happen during early human development. It would be interesting to see if mutated Obscurin affects heart formation during development.”

Rowland said the findings point to areas warranting further attention.

“We expect this will ultimately improve our understanding of the disease,” she said.

University of Colorado www.cuanschutztoday.org/researchers-find-association-gene-mutation-rare-heart-disease/

A small stretch of ribonucleic acid called microRNA could make the difference between a healthy adult brain and one that’s prone to disorders including schizophrenia.

Scientists at the Salk Institute discovered that miR-19 guides the placement of new neurons in the adult brain, and the molecule is disrupted in cells from patients with schizophrenia. The findings pave the way toward a better understanding of how the adult brain controls the growth of new neurons and how it can go wrong.

“This is one of the first links between an individual microRNA and a specific process in the brain or a brain disorder,” says senior author Rusty Gage, professor in Salk’s Laboratory of Genetics and holder of the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Disease.

While most RNA molecules contain the instructions for making proteins—the physical workhorses of cells—microRNAs don’t encode proteins. Instead, they’re active themselves, binding to other strands of RNA to block them from creating proteins. Previously, scientists have shown that levels of microRNA molecules are altered in brain disorders but not which microRNAs are responsible.

“People have broadly studied microRNAs in the brain quite a bit,” says Jinju Han, a senior research associate at Salk and first author of the new paper. “But there are more than 2,000 microRNAs and only a few have been looked at in any depth.”

In a few discrete areas of the human brain, new cells can emerge during adulthood. Gage, Han and their colleagues found that levels of miR-19 changed more than levels of any other microRNA when precursors to new brain cells in these areas (called neural progenitor cells) were coaxed to become neurons in the adult brain.

“The microRNA miR-19 has been implicated in cancer and people never thought it was related to the brain,” says Han. “But we saw that its levels changed quite dramatically when stem cells differentiated into neurons.”

The researchers went on to show that when miR-19 was blocked in neural progenitor cells, levels of RNA corresponding to a gene called Rapgef2 were altered. Moreover, new neurons did not migrate to the correct areas of the brain.

Because the incorrect migration of new brain cells has been implicated in neuropsychiatric disorders like schizophrenia, Gage’s group next analysed the levels of miR-19 and Rapgef2 in neural progenitor cells that had been created by reprogramming skin cells from schizophrenic patients. Although the patients had no mutations in the gene for Rapgef2, they had high levels of miR-19 that corresponded with low levels of both the RNA and protein for Rapgef2. The team is now studying the role of miR-19 in mouse models of schizophrenia, as well as looking at cells from broader cohorts of human patients.

Because miR-19 has been linked to cancers—including breast cancer, prostate cancer and B cell lymphoma—researchers have already been working to develop drugs that block the molecule. But the new results, Han says, suggest that such drugs could have an effect on the brain. “This means that if miR-19 is being targeted in cancer, effects on the brain need to be carefully considered,” she says. “But it also means that people might use these therapies to treat neuropsychiatric disorders.” More work is needed, though, to see whether the results hold true in humans.

Salk Institute www.salk.edu/news-release/small-molecule-keeps-new-adult-neurons-from-straying-may-be-tied-to-schizophrenia/