Scientists have identified a master regulator gene for early embryonic development of the pancreas and other organs, putting researchers closer to coaxing stem cells into pancreatic cells as a possible cure for type1 diabetes.

Researchers at Cincinnati Children’s Hospital Medical Center report their findings in the July 21 Developmental Cell.

Besides having important implications in diabetes research, the study offers new insights into congenital birth defects involving the pancreas and biliary system by concluding both organs share a common cellular ancestry in the early mouse embryo.

This discovery reverses a long standing belief that the biliary system’s origin is connected to early embryonic formation of the liver, the researchers said. The pancreas regulates digestion and blood sugar, and the biliary system is vital for digestion. If the organs do not form properly during fetal development, it can be fatal.

The study reports that one gene, Sox17 (a transcription factor that controls which genes are turned on or off in a cell) is the key regulator for giving instruction to cells in early mouse embryos to become either a pancreatic cell or part of the biliary system.

The first author on the paper is Jason Spence, Ph.D., a research fellow in the lab of the study’s senior investigator, James Wells, Ph.D., a researcher in the Division of Developmental Biology at Cincinnati Children’s and associate professor of pediatrics at the University of Cincinnati College of Medicine.

“We show that Sox17 acts like a toggle or binary switch that sets off a cascade of genetic events,” said Dr. Wells. “In normal embryonic development, when you have an undecided cell, if Sox17 goes one way the cell becomes part of the biliary system. If it goes the other way, the cell becomes part of the pancreas.”

The finding advances ongoing research by Dr. Wells and his team to guide embryonic stem cells to become pancreatic beta cells, which scientists believe could be used to treat or cure type1 diabetes. The disease occurs when the immune system attacks insulin producing beta cells in the pancreas, usually destroying them beyond repair before the illness is diagnosed.

“With this study showing us that turning one gene on or off in a mouse embryo instructs a cell to become pancreatic or biliary, now we’ll see if that same gene, Sox17, can be used to direct an embryonic stem cell to become a biliary cell instead of a pancreatic cell. This might be used one day to replace a diseased pancreas or bile duct in people,” said Dr. Wells.

The study explains that Sox17 initially works in conjunction with two other genes (the transcription factors Pdx1 and Hes1) to decide which organ fate ventral foregut progenitor cells will take. Researches demonstrated that Sox17’s key role begins when the mouse embryo is 81/2 days old. If Sox17 toggles one way, with its expression repressed by its interaction with Hes1, then Pdx1 more or less takes over to prompt formation of the ventral pancreas. If Sox17 toggles the other way to increases its expression, the gene helps set off formation of the biliary system.

Dr. Wells and his colleagues are also using data from the current study to conduct experiments that should reveal what other genes are turned on or off along molecular cascade set into motion by Sox17.

“Although Sox17 is the master switch, it triggers a molecular cascade of switches, and a defect in any of those can cause the whole thing to go wrong, resulting in congenital defects of the pancreas and biliary system,” Dr. Wells said.

Jeffrey Whitsett, M.D., executive director of the Cincinnati Children’s Perinatal Institute and one of the current study’s authors, said the research provides important clues for clinicians managing congenital birth defects of the pancreas and biliary system, which includes the bile ducts and gall bladder. Malformations in this region of the gastrointestinal tract can cause blockage of bile ducts or the intestines. One of the common defects is a condition called biliary atresia, in which the bile ducts are blocked, causing bile to accumulate, back up and leading to potential damage of the pancreas or liver.

“Babies in neonatal intensive care frequently are born with medically challenging birth defects. The present studies help unravel the complex genetic systems controlling the formation of the gastrointestinal tract and provide the framework for future therapies of disease affecting the formation and function of the pancreas, liver, and bile ducts,” Dr. Whitsett said.

Funding support for the study came from the Juvenile Diabetes Research Foundation and National Institutes of Health.

Other Cincinnati Children’s researchers participating in the study include Alex W. Lange, Ph.D., and Suh-Chin J. Lin, Ph.D.

About Cincinnati Children’s

Cincinnati Children’s Hospital Medical Center is one of 10 children’s hospitals in the United States to make the Honor Roll in U.S. News and World Report’s 2009-10 America’s Best Children’s Hospitals issue. It is #1 ranked for digestive disorders and is also highly ranked for its expertise in respiratory diseases, cancer, neonatal care, heart care, neurosurgery, diabetes, orthopedics, kidney disorders and urology. One of the three largest children’s hospitals in the U.S., Cincinnati Children’s is affiliated with the University of Cincinnati College of Medicine and is one of the top two recipients of pediatric research grants from the National Institutes of Health.

President Barack Obama in June 2009 cited Cincinnati Children’s as an “island of excellence” in health care. For its achievements in transforming health care, Cincinnati Children’s is one of six U.S. hospitals since 2002 to be awarded the American Hospital Association-McKesson Quest for Quality Prize for leadership and innovation in quality, safety and commitment to patient care. The hospital is a national and international referral center for complex cases. Additional information can be found at www.cincinnatichildrens.org.

Scientists have a better understanding of what causes an abnormal number of chromosomes in offspring, a condition called aneuploidy that encompasses the most common genetic disorders in humans, such as Down syndrome, and is a leading cause of pregnancy loss.

To pinpoint what goes awry in these cases, researchers at the U.S. Department of Energys Lawrence Berkeley National Laboratory and the University of Tennessee, Knoxville studied mice. They found that if a mothers egg cell has a mutation in just one copy of a gene, called Bub1, then she is more likely to have fewer offspring that survive to birth.

Ordinarily, both copies of a gene in a chromosome must carry the same mutation in order for an organism to be adversely effected.

But we found that a mutation in a single copy of the Bub1 gene can have an impact and this is not the case with most genes. With Bub1, if you have one bad gene and one healthy gene, theres a problem, says Francesco Marchetti of Berkeley Labs Life Sciences Division. He worked with Sundaresan Venkatachalam of the University of Tennessee and other scientists on the research. Their findings appear in the online early edition of the Proceedings of the National Academy of Sciences the week of July 13.

The importance of their discovery is underscored by the fact that its rare for humans to have mutations in both copies of a gene, while it is quite common to have a mutation in only one copy. Usually, the healthy gene overrides the mutated gene - but not in Bub1, at least in mice.

more here: Berkeley Labs

Amsterdam, The Netherlands: One-step screening for both genetic and chromosomal abnormalities has come a stage closer as scientists announced that an embryo test they have been developing has successfully screened cells taken from spare embryos that were known to have cystic fibrosis.

They told a news briefing at the 25th annual meeting of the European Society of Human Reproduction and Embryology in Amsterdam today (Tuesday) that, as a result, they would be able to offer clinical trials to couples seeking fertility treatment later this year.

The researchers based in the USA and the UK have been able to prove that the technique, known as genome-wide karyomapping, was capable of not only detecting diseases caused by a specific gene mutation, in this case cystic fibrosis, but that it was also capable of detecting aneuploidy (an abnormal number of any of the 23 pairs of chromosome) at the same time. This is the first time they have been able to demonstrate that the test can work in cells taken from embryos that have already been diagnosed with the cystic fibrosis gene mutation using conventional preimplantation genetic diagnosis (PGD).

Gary Harton, PGD scientific director of the Genetics & IVF Institute in Fairfax, Virginia (USA) told a news briefing: “Karyomapping is a universal method for analysing the inheritance of genetic defects in the preimplantation embryo without any prior patient or disease specific test development, which often delays patient treatment. For the first time, the inheritance of both single gene defects and chromosomal abnormalities can be detected simultaneously at the single cell level. Unlike other methods, this is achieved entirely by analysing the DNA sequence at over 300,000 locations genome-wide in parents and appropriate family members, often children already affected by a disease, and comparing their sequence with that inherited by the embryo. This can be achieved very rapidly using current microchip technology known as microarray.”

With karyomapping it is not necessary to know the exact DNA mutation that is being sought; the scientists just need to take the relevant chunk of DNA from the parent that carries the mutation somewhere along its length, and if it matches a chunk of DNA from the embryo, then they know the embryo has inherited the mutation. As karyomapping involves analysing chromosomes, it also detects the existence of aneuploidy at the same time.

“The range of applications is broad and includes single gene defects, abnormal chromosome number, structural chromosome abnormalities and HLA [human leukocyte antigen] matching in ’saviour sibling’ cases,” said Mr Harton.

Karyomapping was developed by Professor Alan Handyside of the London Bridge Fertility Gynaecology and Genetics Centre in London (UK), and Mr Harton has been providing samples and DNA information in order to test the method and validate it for use in the clinic.

“The hope is that clinicians will be able to test embryos for specific genetic diseases and know that, with one test, they are transferring chromosomally normal embryos. This will be a step forward from current technology that is mostly limited to choosing one test or the other,” explained Prof Handyside.

Karyomapping would also be quicker and cheaper. Currently, developing a PGD test for a single gene defect can take weeks or months, as scientists have to identify the exact patient or disease-specific genetic mutation first before screening for it, which is labour-intensive and costly. By contrast, karyomapping can be carried out without such extended pre-test development; at present, it takes about three days, but Mr Harton and his colleagues believe this could be reduced to 18-24 hours.

In this most recent stage of their research they examined cells from five embryos that had been donated for medical research by a couple who had received successful fertility treatment, including PGD for cystic fibrosis. The embryos had developed to the blastocyst stage, which is about five days after fertilisation. Conventional PGD had already identified which embryos were unaffected, affected or were carriers of the disease. Karyomapping of cells from the donated embryos confirmed these diagnoses, but, in addition, it was able to identify which parent carried the affected chunk of DNA. Karyomapping also revealed two aneuploidies in two embryos, which had not been detected by the earlier PGD.

Mr Harton said: “This demonstrates that karyomapping, following genome-wide analysis of a single cell biopsied from embryos at the blastocyst stage, can provide highly accurate analysis for cystic fibrosis, combined with the detection of chromosomal aneuploidy. Now that vitrification [an improved method of embryo freezing] has improved embryo survival after thawing, it should be possible to vitrify embryos at the blastocyst stage, either before or after biopsy, and analyse the embryos for virtually any genetic disease and screen for aneuploidy of all 23 pairs of chromosomes simultaneously. This approach could make PGD by karyomapping less expensive than conventional single disease PGD because fewer embryos will be biopsied, more embryos will be chromosomally normal following growth to the blastocyst stage, and there is no need to custom develop tests for each disease or couple interested in PGD.”

Prof Handyside concluded: “These tests have helped us to learn everything we can before we start to treat actual patients. I am confident that we will be offering a clinical trial to patients using karyomapping some time this year.”

European Society for Human Reproduction and Embryology

Missing DNA segments may suggest future drug targets

Pediatric researchers have identified hundreds of gene variations that occur more frequently in children with attention-deficit hyperactivity disorder (ADHD) than in children without ADHD. Many of those genes were already known to be important for learning, behavior, brain function and neurodevelopment, but had not been previously associated with ADHD.

“Because the gene alterations we found are involved in the development of the nervous system, they may eventually guide researchers to better targets in designing early intervention for children with ADHD,” said lead author Josephine Elia, M.D., a psychiatrist and ADHD expert at The Children’s Hospital of Philadelphia.

The study appeared online today in the journal Molecular Psychiatry.

Unlike changes to single DNA bases, called SNPs or “snips,” the alterations examined in the current study are broader changes in structure. Called copy number variations (CNVs), they are missing or repeated stretches of DNA. CNVs have recently been found to play significant roles in many diseases, including autism and schizophrenia Everyone has CNVs in their DNA, but not all of the variations occur in locations that affect the function of a gene. The current study is the first to investigate the role of CNVs in ADHD.

Individually, each CNV may be rare, but taken together, a combination of changes in crucial regions may interact to raise an individual’s risk for a specific disease. “When we began this study in 2003, we expected to find a handful of genes that predispose a child to ADHD,” said study co-leader Peter S. White, Ph.D., a molecular geneticist and director of the Center for Biomedical Informatics at Children’s Hospital. “Instead, there may be hundreds of genes involved, only some of which are changed in each person. But if those genes act on similar pathways, you may end up with a similar resultADHD. This may also help to explain why children with ADHD often present clinically with slightly different symptoms.”

ADHD is the most common neuropsychiatric disorder in children, affecting an estimated 1 in 20 children worldwide. It may include hyperactive behavior, impulsivity and inattentive symptoms, with impaired skills in planning, organizing, and maintaining focus. Its cause is unknown, but it is known from family studies to be strongly influenced by genetics.

Drawing on DNA samples from the Children’s Hospital pediatric network, the researchers analyzed genomes from 335 ADHD patients and their families, compared to more than 2,000 unrelated healthy children. The team used highly automated gene-analyzing technology at the Center for Applied Genomics at Children’s Hospital, directed by Hakon Hakonarson, M.D., Ph.D., a co-leader of this study.

The study team found a similar quantity of CNVs in both groups. However, distinct patterns emerged. Among 222 inherited CNVs found in ADHD families but not in healthy subjects, a significant number were in genes previously identified in other neurodevelopmental disorders, including autism, schizophrenia and Tourette syndrome. The CNVs found in ADHD families also altered genes important in psychological and neurological functions such as learning, behavior, synaptic transmission and nervous system development.

“We took a systems biology approach, grouping genes into groups with common functions,” said White. “We found that the sets of genes more likely to be changed in ADHD patients and families affected functions that made sense biologically.” For instance, said White, the team found four deletions of DNA in a gene recently linked to restless legs syndrome, a type of sleep disorder common in adults with ADHD.

Another deletion occurred in a gene for a glutamate receptor. Glutamate is a neurotransmitter, a protein that carries signals in the brain. While ADHD medications act on dopamine and serotonin, which are also neurotransmitters, this new finding may suggest an important role for glutamate as well, at least for some ADHD patients.

“As we delve into the genetics of very complex diseases such as ADHD, we find many contributing genes, often differing from one family to another,” added White. “Studying the functions of different genes allows us to identify biological pathways that may be involved in this neuropsychiatric disorder.”

Some of the biological pathways involved in ADHD may also be common to other neurological conditions, say the researchers. Likewise, there is some overlap among the CNVs found in ADHD that also occur in autism, schizophrenia and other neurological disorders. This overlap was not surprising, said Elia, because ADHD patients frequently also have one of more of these disorders. However, as researchers learn more about specific genes in neurological conditions, the hope is that researchers might in the future personalize treatments to a patient’s own genetic profile, to achieve more targeted, specific therapies.

Elia and White stressed that much further work must be done before genetic findings lead to ADHD treatments.

The National Institutes of Health provided grant support for the study, as did the University of Pennsylvania, the Pennsylvania Department of Health, the Cotswold Foundation and the ADHD: Climbing to a Cure Foundation. Elia, White and Hakonarson all are faculty members of the University of Pennsylvania School of Medicine (Penn). Xiaowu Gai, Ph.D., of the Center for Biomedical Informatics at Children’s Hospital, was a co-first author with Elia. Other collaborators were from Children’s Hospital and Penn.

Elia et al, “Rare Structural Variants Found in Attention-Deficit Hyperactivity Disorder Are Preferentially Associated with Neurodevelopmental Genes,” Molecular Psychiatry, published online, June 23, 2009.

About The Children’s Hospital of Philadelphia

The Children’s Hospital of Philadelphia was founded in 1855 as the nation’s first pediatric hospital. Through its long-standing commitment to providing exceptional patient care, training new generations of pediatric healthcare professionals and pioneering major research initiatives, Children’s Hospital has fostered many discoveries that have benefited children worldwide. Its pediatric research program is among the largest in the country, ranking second in National Institutes of Health funding. In addition, its unique family-centered care and public service programs have brought the 430-bed hospital recognition as a leading advocate for children and adolescents. For more information, visit http://www.chop.edu.

Hemophilia A is an inherited bleeding disease caused by a lack of the blood clotting protein Factor VIII. It had been hoped that gene therapy would provide a breakthrough in treatment, but the most common gene therapy approach has had little clinical success. However, a team of researchers, at the University of Minnesota Medical School, Minneapolis, has now developed a new approach to target genes specifically to mouse liver sinusoidal endothelial cells (the cells that are the main source of Factor VIII) and used it to provide long-term expression of Factor VIII in hemophilia A mice, markedly reducing their disease. They hope that their data might prove to be a step toward successful human clinical trials in individuals with hemophilia A.

The team, led by Betsy Kren and Clifford Steer, coated nanoparticles with hyaluron so that they targeted liver sinusoidal endothelial cells. To test the efficacy of gene delivery, the hyaluron-coated nanoparticles were engineered to contain a therapeutic gene (Factor VIII) together with a genetic element known as Sleeping Beauty, which helps the therapeutic gene insert into the genome of the targeted cells (i.e, the liver sinusoidal endothelial cells). Even 50 weeks after hemophilia A mice were injected with these nanoparticles, levels of Factor VIII in the blood were the same as in the blood of normal mice and bleeding times were also similar to those of normal mice. The authors hope that this combination of technologies, the cell-specific nanocapsule delivery system and the Sleeping Beauty genetic element, will prove to be a viable gene therapy.

TITLE: Nanocapsule-delivered Sleeping Beauty mediates therapeutic Factor VIII expression in liver sinusoidal endothelial cells of hemophilia A mice

Journal of Clinical Investigation

Gene transfer technology may lead to an HIV vaccine

A research team may have broken the stubborn impasse that has frustrated the invention of an effective HIV vaccine, by using an approach that bypasses the usual path followed by vaccine developers. By using gene transfer technology that produces molecules that block infection, the scientists protected monkeys from infection by a virus closely related to HIVthe simian immunodeficiency virus, or SIVthat causes AIDS in rhesus monkeys.

“We used a leapfrog strategy, bypassing the natural immune system response that was the target of all previous HIV and SIV vaccine candidates,” said study leader Philip R. Johnson, M.D., chief scientific officer at The Children’s Hospital of Philadelphia. Johnson developed the novel approach over a ten-year period, collaborating with K. Reed Clark, Ph.D., a molecular virologist at Nationwide Children’s Hospital in Columbus, Ohio.

The study appeared today in the online version of Nature Medicine.

Johnson cautioned that many hurdles remain before the technique used in this animal study might be translated into an HIV vaccine for humans. If the technique leads to an effective HIV vaccine, such a vaccine may be years away from realization.

Most attempts at developing an HIV vaccine have used substances aimed at stimulating the body’s immune system to produce antibodies or killer cells that would eliminate the virus before or after it infected cells in the body. However, clinical trials have been disappointing. HIV vaccines have not elicited protective immune responses, just as the body fails on its own to produce an effective response against HIV during natural HIV infection.

The approach taken in the current study was divided into two phases. In the first phase, the research team created antibody-like proteins (called immunoadhesins) that were specifically designed to bind to SIV and block it from infecting cells. Once proven to work against SIV in the laboratory, DNA representing SIV-specific immunoadhesins was engineered into a carrier virus designed to deliver the DNA to monkeys. The researchers chose adeno-associated virus (AAV) as the carrier virus because it is a very effective way to insert DNA into the cells of a monkey or human.

In the second part of the study, the team injected AAV carriers into the muscles of monkeys, where the imported DNA produced immunoadhesins that entered the blood circulation. One month after administration of the AAV carriers, the immunized monkeys were injected with live, AIDS-causing SIV. The majority of the immunized monkeys were completely protected from SIV infection, and all were protected from AIDS. In contrast, a group of unimmunized monkeys were all infected by SIV, and two-thirds died of AIDS complications. High concentrations of the SIV-specific immunoadhesins remained in the blood for over a year.

Further studies need to be conducted if this technique is to become an actual preventive measure against HIV infection in people, Johnson said. “To ultimately succeed, more and better molecules that work against HIV, including human monoclonal antibodies, will be needed,” he and his co-authors conclude. Finally, added Johnson, their approach may also have potential use in preventing other infectious diseases, such as malaria.

Grants from the National Institute of Allergic and Infectious Diseases of the National Institutes of Health supported this study. Johnson’s collaborators, in addition to Clark, were Jianchao Zhang, of Nationwide Children’s Hospital, Columbus, Ohio; Eloisa Yuste and Ronald C. Desrosiers of the New England Primate Research Center and Harvard Medical School; and Bruce C. Schnepp, Mary J. Connell, and Sean M. Greene, of Children’s Hospital and the University of Pennsylvania School of Medicine. Johnson also is on the University of Pennsylvania faculty.

Johnson et al, “Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys,” Nature Medicine, published online May 17, 2009. (http://dx.doi.org/10.1038/nm.1967)

About The Children’s Hospital of Philadelphia

The Children’s Hospital of Philadelphia was founded in 1855 as the nation’s first pediatric hospital. Through its long-standing commitment to providing exceptional patient care, training new generations of pediatric healthcare professionals and pioneering major research initiatives, Children’s Hospital has fostered many discoveries that have benefited children worldwide. Its pediatric research program is among the largest in the country, ranking second in National Institutes of Health funding. In addition, its unique family-centered care and public service programs have brought the 430-bed hospital recognition as a leading advocate for children and adolescents. For more information, visit http://www.chop.edu.

Children’s Hospital of Philadelphia

Researchers at Duke University Medical Center and the University of North Carolina have discovered in mice how a single disrupted gene can cause a form of severe mental retardation known as Angelman syndrome.

In a study published in the journal Nature Neuroscience, they found that the gene, UBE3A, is needed so that neurons in the brain can form and adjust their connections to other neurons for storing sensory information. They also made a promising discovery: When the mice were deprived of sensory stimulation, the brain connections could be recovered, a finding that indicated a pharmaceutical or behavioral treatment might be possible in the future.

The scientists undertook this project because of the developmental-onset period seen in Angelman syndrome, typically when children are between one and two years old. It is during this time in humans that the cortex, the sheet of convoluted folds at the surface of the brain, undergoes profound rearrangements driven by sensory experiences the experience of seeing reorganizes the visual cortex, for example, during the same time period when the deficits are becoming obvious in Angelman syndrome, part of the autism spectrum of disorders.

“We wanted to look at an animal model to learn if this experience-dependent reorganization of the cortex was abnormal in animals that were missing the gene,” said Michael Ehlers, M.D., Ph.D., a Duke professor of neurobiology and co-senior author of the study. “We looked at the visual cortex, because in this well-studied model, we could precisely control the sensory stimulus and study the mice in the light or the dark. We speculated that similar deficits may be happening in areas of the cortex that are important for language, cognition and emotion, all of which are quite abnormal in Angelman syndrome patients.”

The authors found that brains cells in Angelman syndrome mice lacked the ability to appropriately strengthen or weaken in the cortex, an area of the brain important for cognitive abilities. Angelman syndrome is one among a small family of single gene, autism-related, neurodevelopmental disorders. Children with the condition appear to respond normally to stimuli during their first year, but around 12-18 months, they start missing milestones of cognitive development and language, typically learning only a 2-3 words over their lifetime.

“When we have experiences, connections between brain cells are modified so that we can learn,” said Ben Philpot, Ph.D., a University of North Carolina professor in Cell and Molecular Physiology and co-senior author of the study. “By strengthening and weakening appropriate connections between brain cells, a process termed synaptic plasticity, we are able to constantly learn and adapt to an ever-changing environment.”

“It is difficult to study how experiences lead to changes in the brain in models of mental retardation,” said Koji Yashiro, Ph.D., a former University of North Carolina graduate student and lead author of the study. “Instead of studying a complex learning model, we studied how connections between brain cells change in visual areas of mice exposed to light or kept in darkness. This approach revealed that brain cells in normal mice can modify their connections in response to changes in visual experiences, while the brain cells in Angelman syndrome mice could not.”

The inability of brain cells to encode information from experiences in the Angelman syndrome model suggested that this is the basis for the profound learning difficulties in these patients.

The scientists didn’t expect to find that the plasticity of the cellular connections could be restored in visual areas of the brain after brief periods of visual deprivation.

“By showing that brain plasticity can be restored in Angelman syndrome model mice, our findings suggest that brain cells in Angelman syndrome patients maintain a latent ability to express plasticity. We are now collaborating to find a way to tap into this latent plasticity, as this could offer a treatment, or even a cure, for Angelman syndrome,” Philpot said.

Ehlers, who is also a Howard Hughes Medical Investigator, said that perhaps some of these developmental brain disorders are a form of social and cognitive blindness. In a condition known as amblyopia, or cortical blindness, the eye can function normally, but past a critical period, the brain cannot process the sensory input correctly.

“We think that children with Angelman syndrome may have a condition in which sensory experience dampens down plasticity and affects learning,” Ehlers said. “One important aspect of our findings is that sensory manipulations recovered plasticity, suggesting that the underlying substrates for plasticity are intact in mice. If the same thing holds true for the human disease, there may be a chance to improve brain function.”

Other authors included Kathryn Condon, Duke Department of Neurobiology; Thorfinn Riday, Adam Roberts, Danilo Bernardo, and Rohit Prakash of the Curriculum in Neurobiology, Neuroscience Center, Department of Cell and Molecular Physiology, and the Neurodevelopmental Disorders Research Center at the University of North Carolina, Chapel Hill; and Richard Weinberg, Department of Cell and Developmental Biology, University of North Carolina. This work was supported by grants from the National Institutes of Health, the Howard Hughes Medical Institute, the Angelman Syndrome Foundation, and the Simons Foundation.

Duke University Medical Center

A discovery made by researchers at Wake Forest University School of Medicine while studying mice may help explain how some people without a genetic predisposition to epilepsy can develop the disorder.

In a study published this month in the Journal of Neuroscience, senior researcher Dwayne W. Godwin, Ph.D., a professor of neurobiology and anatomy, and colleagues, report discovering that a gene, already known to predispose people who inherit an active form of it to certain forms of epilepsy, can actually be “switched on” in animals that do not appear to have inherited the active form, and therefore a genetic predisposition, to the condition. The gene codes a calcium channel in the brain that underlies seizures, so the finding may reveal a mechanism by which epilepsy develops in those with no apparent genetic predisposition to it.

“Epilepsy is a terrible disorder that affects millions of kids and adults all over the world,” Godwin said. “There are many different forms of epilepsy with different symptoms. We don’t know why some people acquire epilepsy the cause isn’t always clear from the person’s genetic makeup. We do know that in some forms of epilepsy, once someone has a seizure they tend to have more. Our findings from this study suggest that something about the brain changes that can lead to this increased tendency to have a seizure. Our study shows that an important change occurs in calcium channels that help to transmit this abnormal activity throughout the brain.”

Calcium channels come in a variety of forms throughout the body and are responsible for several key functions, depending on their placement and quantity. The calcium channels in the brain are normally embedded within the membrane of brain cells, where they allow passage of calcium ions into the cell and are responsible for the electrical activity of the brain. The passage of calcium ions into cells determines how excitable the cells are, and how easily abnormal activity spreads through the brain.

If, as in epilepsy, a particular channel shows up where it is not supposed to or appears in too many or too few numbers, the function that channel is responsible for can become abnormal. Researchers know that during epileptic seizures, these calcium channels in the brain, responsible for generating electrical brain rhythms, become highly active.

For the study, researchers used a mouse model to observe changes in tissue from regions of the brain that are involved in seizures, the hippocampus and the thalamus. They measured these changes at different time intervals as the mice developed epilepsy. The researchers found that after an initial seizure, more of this particular kind of calcium channel begins to be expressed where it wasn’t before, and the presence of the channel caused brain activity to become increasingly abnormal and epileptic.

“Calcium channels underlie valuable functions,” Godwin said. “But in the wrong place, at the wrong time, or in the wrong amount, their presence can be disruptive. In the context of brain circuits, the brain cells that have too many copies of the channel get over excited and respond abnormally.”

While the hippocampus is usually targeted in studies of epilepsy, the new channels were being made in a region of the brain called the thalamus. The thalamus is connected to the hippocampus and is involved in the spread of seizures throughout the brain.

“Certain kinds of channels are normal and expected in the thalamus, but after an initial seizure more copies of a channel that isn’t normally found in this brain region begin to appear,” explained graduate student John Graef, the first author on the study. “The brain activity then becomes dominated by the new copies of this channel. It helps explain how seizures can develop and spread.”

The particular gene that codes for the misplaced channel has been called a “susceptibility gene” within the research community because it shows up in the genetic makeup of some individuals with epilepsy. In other individuals, there is no genetic indication that they are capable of making extra copies of the channel.

“What we’ve shown is that this gene can be switched on in individuals who don’t appear to have inherited the susceptibility,” Godwin said.

The good news is that certain drugs can inhibit calcium channels, so, if researchers can determine that the over-expression of this calcium channel is solely responsible for seizure activity, future studies could look into the possibility of selectively inhibiting the channel with drugs, or even nutritional changes. Godwin explained that this study provided vital information but that more work needs to be done to translate the findings to human patients.

Other co-authors on the study, funded by Citizens United for Research in Epilepsy, the National Eye Institute and the National Institute on Alcohol Abuse and Alcoholism, are Brian Nordskog, Ph.D., and Walter Wiggins, a medical student, both of Wake Forest University School of Medicine.

Researchers discover that gene switches on during development of epilepsyWake Forest University Baptist Medical Center

Turns model parasite into genetic workhorse

Hanover, NH–In a genetic leap that could help fast track vaccine and drug development to prevent or tame serious global diseases, DMS researchers have discovered how to destroy a key DNA pathway in a wily and widespread human parasite. The feat surmounts a major hurdle for targeting genes in Toxoplasma gondii, an infection model whose close relatives are responsible for diseases that include malaria and severe diarrhea.

“This opens a wide window on a complex parasite family and can help accelerate the development of safe and effective genetically modified vaccines and drug therapies,” says team leader David Bzik, PHD, professor of microbiology and immunology. The work is reported in the April issue of Eukaryotic Cell with Barbara Fox, senior research associate of microbiology and immunology who is the lead author and innovator of the study.

Parasites steal shamelessly from their hosts, co-opting resources to survive and infect. T gondii, however is a clever contrarian: it invites destruction and goes underground.

“Most parasites, along with bacteria and viruses, are shape shifters, so the immune system can’t catch up with them; but T. gondii actually wants to be destroyed,” says Bzik. “It has a unique strategy to elicit an immune response that stops the actively growing parasite and something in that response drives it to a latent stage which is necessary for its transmission.”

The food borne parasite, often transmitted from cats, can be serious, even fatal for immune deficient people or newborns of mothers infected in pregnancy. While the T. gondii infection is harmless in most people, the parasite does takes up permanent residence inside its host. Its virulent cousins include Plasmodium, which causes lethal malaria and Cryptosporidium, a common source of waterborne diarrhea that can be severe or intractable in children or those with HIV.

“There is an amazing immune response hard-wired into this parasite to deliver life-long immunity to T. gondii,” Fox says. “So our work has been recently focused at creating safe, attenuated (weakened), and genetically defined T. gondii strains that also piggyback antigens to deliver sorely needed vaccines for malaria, cryptosporidiosis, tuberculosis, HIV/AIDS, or even cancer. This finding overcomes the bottleneck for quickly developing multiple manipulated and completely safe strains where each genetic manipulation is precisely defined and irreversible.”

T. gondii is easy to grow in the lab and has other amenable attributes that have made it a leading model for understanding intracellular pathogens. It belongs to the Apicomplexan family of protozoa, along with its other medically important relatives. Family members share numerous genes, but many are unique to Apicomplexa, making it difficult to predict or determine gene functions.

Employing a cut and paste genetic engineering technique, scientists can knock out or replace a gene to determine or change its functions. Most model organisms rejoin the manipulated pieces at the location of their proper and predictable sequence.

The dominant pathway in T. gondii, however, is random insertion. The parasite uses a pathway of nonhomologous end joining (NHEJ), which is also used to repair DNA in broken chromosomes, and arbitrarily reinserts targeting DNA segments at incorrect locations. That makes isolating strains with defined and targeted gene knockouts a difficult, time consuming and painstaking adventure.

Using a strategy Fox devised, the DMS team disrupted and killed a parasite gene called KU80 that is involved in the NHEJ DNA repair pathway. Their success effectively turned the parasite into a dependable genetic workhorse for all the diverse organisms in the Apicomplexa phylum. It permits a direct approach to determine gene function by examining mutants lacking a specific gene.

“The KU80 knockout strain holds much genetic magic,” says Bzik. “Remarkably, it exhibits 100 percent homologous recombination and gene targeting efficiency compared to the parent strain. This also provides the first biological proof of a functional NHEJ DNA repair pathway in a protozoan.”

The work makes T. gondii an effective model for understanding a globally significant parasite family and holds promise for speeding up new therapies. “To create safe, genetically modified products or vaccines to put into people, we need to be able to efficiently and reliably target strains for genetic manipulation,” Fox explains.

“Fundamentally, all possible growth and virulence factors as well as the potential for transmission must be first genetically deleted; then key protective antigens or genes from other sources must be introduced in a precisely defined way. We needed to be able to do this efficiently, reliably and, cleanly. Now we can.”

Coauthors of the study (in Vol. 8, No. 4: 520-529) are Jessica Ristuccia, a former research assistant now at Tufts School of Dental Medicine, and Jason Gigley, a former student now at George Washington University. The work was funded by grants from the NIH National Institute of Allergy and Infectious Diseases.

Dartmouth Medical School gene targeting discovery opens door for vaccines and drugsDartmouth Medical School