What’s New in Research: April 2016
Highly-Sensitive Detection Method Makes Close Monitoring of HDL Kinetics Possible
High-density lipoprotein (HDL) is often referred to as good cholesterol: high levels of HDL are associated with lower risk of cardiovascular disease. But many clinical outcome trials for drugs that raise HDL levels have failed to show significant benefits for trial participants. However, current HDL detection methods usually measure only total HDL cholesterol – a more sensitive detection method could allow investigators to measure the subfractions of HDL, and more precisely pinpoint which of these subfractions should be raised to help protect against cardiovascular events. In a paper published in the April 1 print edition of The Journal of Lipid Research, Brigham and Women’s Hospital investigators and collaborators at the Harvard T.H. Chan School of Public Health describe a mass spectromeric approach that has allowed them to identify HDL subfractions of various sizes and distribution. The new technique for monitoring HDL kinetics has helped reveal new lipid biology and may help pharmaceutical companies better design and test lipid modulators in the future.
Using this technique for proteomic analysis, the multidisciplinary team led by Masanori Aikawa, MD, PhD, Director of the Center for Interdisciplinary Cardiovascular Sciences at BWH and Associate Professor at Harvard Medical School, and Frank Sacks, MD, Professor at the Harvard T.H. Chan School of Public Health was able to identify 58 proteins in HDL that were shared among three humans. Co-first authors Sasha A. Singh, PhD, Director of Proteomics at CICS, and Allison B. Andraski, PhD student at HSPH, followed up on seven of these proteins, monitoring their kinetics to better understand apolipoprotein metabolism and the formation of HDL particles. Their results suggest that the traditional view of the role of HDL in reverse cholesterol transport may oversimplify the roles and contributions of various components of HDL.
“Our study demonstrates the feasibility of closer monitoring of HDL kinetics. We believe that establishing new, high-resolution methods that can monitor HDL kinetics is critical to examine the desired effects of new drugs,” said Aikawa.
Sacks, co-senior author, also underscored the importance of this technique for future drug development. “This approach not only revealed novel evidence for the formation of HDL particles, but also found that each HDL subfraction has a unique proteome, which may help to discover new therapeutic targets.”
Paper cited: Singh SA, Andraski AB, et al. “Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring” Journal of Lipid Research DOI: 10.1194/jlr.D061432
New Insights into the Cause of Neurological Symptoms in Mitochondrial Diseases
Mitochondrial diseases – which affect 1 in 5,000 people – encompass a spectrum of disorders with an array of symptoms. Many patients with a mitochondrial disease experience neurological symptoms, including intellectual disability, childhood epilepsy and autism spectrum disorder, but why dysfunctional mitochondria – the powerhouses of cells –lead to these sorts of symptoms has been unclear.
In a paper published on March 31 in Cell Reports, BWH investigators shed light on what may be the root cause of these neurological symptoms by tracing the development of interneurons. Interneurons, also known as connector neurons, must migrate long distances during the brain’s development, taking a circuitous path to travel from their neural stem cell origins in the ventral forebrain to their location in the dorsal neocortex. The new study indicates that mitochondria, which provide energy for cells, may play a vital role in this migration. Using preclinical models, the team determined that interneurons have higher energetic requirements than other neurons and that properly working mitochondria move about inside interneurons rapidly during migration. When Ant1, a gene known to play a role in mitochondrial disease, was disrupted, mitochondria did not move about in the same patterns and interneurons migrated more slowly and shorter distances.
The new work suggests that correcting epilepsy or reversing other neurological symptoms in patients with mitochondrial diseases will not be as simple as replacing dysfunctional mitochondria at any time. Instead, these symptoms may be the result of brain developmental defects that took place during embryonic development. Further studies will be needed to validate these results in tissue samples from humans.
“We need to rethink how we strategize caring for patients. Historically, patients have been recalcitrant to therapy, and this new work may explain why,” said Jeffrey Golden, MD, chair of BWH’s Department of Pathology and a co-corresponding author of the study. “This work also provides an insight into rethinking therapeutic strategies.”
Paper cited: Lin-Hendel EG et al. “Differential mitochondrial requirements for radially and non-radially migrating cortical neurons: Implications for mitochondrial disorders” Cell Reports DOI: 10.1016/j.celrep.2016.03.024
Unique Repertoires of DNA Binding Activities
With the advent of genomic sequencing, scientists now know that lots of genetic variation affects the coding sequences of human transcription factors (TF). Transcription factors are proteins that bind to DNA and control the rate at which genetic information is transcribed. Genetic variants that alter transcription factors and, in turn, transcription levels of targeted genes, have been associated with human disease risk, and many Mendelian diseases are attributable to mutations in TFs. Martha Bulyk, PhD, and her colleagues developed a computational, structure-based approach to assess individual TF composition on a large scale. The BWH-led team found that most individuals have a unique repertoire of TF alleles predicted to have altered DNA binding activities, which may contribute to phenotypic variation, or differences in observable traits. The researchers’ results are published in Science.
Bulyk and her team computationally analyzed genotype data from more than 64,000 individuals of African, Asian and European ancestries, and then biochemically analyzed a subset of the identified TF alleles using a novel DNA microarray-based in vitro technology, called protein binding microarrays (PBMs). This technology allowed the researchers to rapidly characterize DNA binding site sequence specificities of TFs.
“Our work suggests that individuals have a unique repertoire of TF alleles with a distinct landscape of DNA binding activities,” said Bulyk. “Continued work in this area will likely provide insights into how genetic variation in TFs leads to phenotypic differences among people.”
Paper cited: Barrera LA et al. “Survey of variation in human transcription factors reveals prevalent DNA binding changes.” Science DOI: 10.1126/science.aad2257
A Boost in MicroRNA may Protect Against Obesity and Diabetes
Obesity, which is associated with low-grade inflammation, is an important contributor in the development of diabetes and cardiovascular disease. While the role of several organs including adipose tissue have been implicated in this process, the cell types and factors driving this process have not been clear. Using a pre-clinical model of obesity, researchers have discovered that a small, non-coding RNA molecule called miR-181b is an important determinant of obesity-induced changes in adipose tissue by controlling the function of the vessels in adipose tissue. The findings could point toward new targets for the development of treatment or obesity and diabetes. The study is published in the March 4 edition of Circulation Research.
The researchers identified that the expression of miR-181b was lower in adipose tissue endothelial cells, but not adipocytes, after just one week of high-fat feeding in mice. The team hypothesized that reconstituting this microRNA in obese mice might improve the development of insulin resistance/diabetes. Indeed, they found that injections of a miR-181b mimic into obese mice markedly improved insulin sensitivity, glucose levels and reduced inflammation in adipose tissue.
The team found that the protein phosphatase PHLPP2 is a direct target of miR-181b, and that suppression of the protein also improved insulin sensitivity, glucose levels and inflammation in mice, providing an additional new target for therapy.
Finally, the team noted that levels of PHLPP2 were higher in endothelial cells from diabetic patients than healthy patients, suggesting the new findings in mice are relevant to human disease.
“We have discovered a microRNA that functions to dampen the inflammatory response in the vasculature of adipose tissue by targeting endothelial cells that surround adipocytes and a pathway that leads to increased nitric oxide production,” said senior author Mark W. Feinberg, an associate physician at BWH. “The beneficial role of this microRNA in obesity is likely the tip of the iceberg since excessive inflammation is a pervasive finding in a wide-range of chronic inflammatory diseases.”
An accompanying editorial in the journal notes that “using microRNAs to modulate adipocyte-endothelial cell axis in adipose tissue may offer new tools to combat the growing epidemic of obesity and its associate comorbidities.”
Paper cited: Sun X et al. “MicroRNA-181b Improves Glucose Homeostasis and Insulin Sensitivity by Regulating Endothelial Function in White Adipose Tissue” Circulation Research DOI: 10.1161/CIRCRESAHA.115.308166
Zebrafish Offer a Window into the Immune System
Zebrafish – tiny, bony fish that are popular in home aquariums and laboratories alike – have an immune system that has many similarities to our own. Like humans, zebrafish possess a diverse population of T-cells, which can respond to a wide variety of potential threats. However, in zebrafish the T-cell population is one to ten million times smaller than the T-cell population in mice. This makes them an ideal model for studying the T-cell receptor (TCR) repertoire.
Francisco Quintana, PhD, a principal investigator in BWH’s Ann Romney Center for Neurologic Diseases, and colleagues set out to comprehensively sequence T-cells within zebrafish, allowing them to test the complete TCR repertoire in a vertebrate for the first time. The team sequenced cells from adult zebrafish that had been exposed to self and foreign antigens. In total, they sequenced all the T-cells from more than 500 individual zebrafish. Their findings appear in Cell Reports.
The team found that all zebrafish examined had more than 10 percent of their T-cells in common. These common or “public elements” may provide a mechanism for rapid response to foreign invaders, but may also trigger auto-immune reactions. Quintana and colleagues hope that these public elements could be used to develop better strategies for training the immune system to not only fight infections but also tumors.
Paper cited: Covacu R et al. “System-wide Analysis of the T Cell Response” Cell Reports DOI: 10.1016/j.celrep.2016.02.056
Improving Diagnoses of Pediatric Brain Tumors
Keith Ligon, MD, PhD, Rameen Beroukhim, MD, PhD, and a large collaborative research team have discovered the genetic cause of angiocentric glioma, a type of brain tumor found in children and young adults. The tumor causes seizures, but is generally benign and can be treated with surgery. Until now, however, it was diagnostically difficult to differentiate these tumors from more aggressive ones that require debilitating treatment. The researchers, including teams from Dana-Farber Cancer Institute, the Children’s Hospital of Philadelphia and BWH, identified the DNA rearrangement of MYB-QKI that triggers three tumor-forming mechanisms. This discovery has also helped Dr. Azra Ligon, PhD, to develop two clinical diagnostic tests for these tumors by Dr. SA, which are only available at BWH. The study’s findings are published in Nature Genetics.
The researchers describe a single DNA rearrangement that forms a new fusion gene, in this case one that activates a cancer causing gene (MYB) and inactivates a tumor suppressing gene (QKI). This in turn prompts abnormally enhanced expression of the fusion gene. The researchers found this to be a defining event in angiocentric glioma, which will help clinicians distinguish and diagnose these tumors more accurately in the future.
“Having a reliable way to diagnose tumors confidently helps young patients avoid unnecessary treatment,” said Ligon. “Our findings that one rearrangement contributes to tumor development through multiple mechanisms may also be applicable to a large number of pediatric tumors.”
Paper cited: Bandopadhayay, P. et al. (2016). MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism. Nature Genetics, 48(3), 273-282.
Alopecia Areata Linked to Lower Risk of Skin Cancer
Patients with alopecia areata (AA) have a decreased risk of skin cancer, according to a new study by Arash Mostaghimi, MD, MPA, MPH, Kathie Huang, MD, and their colleagues. The BWH-led team found that having AA – an autoimmune disease characterized by patches of hair loss – is associated with a reduced risk of nonmelanoma skin cancer and a trend toward decreased risk of melanoma. Genetics likely account for the lower risk since patients showed a decreased risk of skin cancer even in areas without hair loss. These results are published in Cancer Epidemiology.
The researchers retrospectively analyzed data from the Research Patient Data Repository, a Partners Healthcare System registry that includes information from 6.5 million patients. Similarities between AA and vitiligo (which causes the loss of skin color in blotches) prompted this investigation, since vitiligo has already been linked to decreased risk of skin cancer. Studies like these may help researchers further elucidate a molecular basis for the development and treatment of skin cancer.
“These results suggest that patients with AA have heightened protection against skin cancer,” said the authors. “These findings allow us to improve our counseling of patients with AA and reinforce the hypothesis of a role for anti-melanocyte autoantigens in patients with AA.”
Paper cited: Mostaghimi, A., Qureshi, S., Joyce, C., Guo, Y., & Huang, K. P. (2016). Reduced incidence of skin cancer in patients with alopecia areata: A retrospective cohort study. Cancer Epidemiology, 41, 129-131.