Quinn Eastman

DNA copying problems explain muscular dystrophy mutations

Geneticist Madhuri Hegde and her colleagues have a paper in the journal Genome Researchthat addresses the question: where do copy number variations come from?

Madhuri Hegde, PhD

Copy number variations (CNVs), which are deletions or duplications of small parts of the genome, have been the subject of genetic research for a long time. But only in the last few years has it become clear that copy number variations are where the action is for complex diseases such as autism and schizophrenia. Geneticists studying these diseases are shifting their focus from short, common mutations (often, single nucleotide polymorphisms or SNPs) to looking at rarer variants such as CNVs. A 2009 discussion of this trend with Steve Warren and Brad Pearce can be found here.

Hegde is the Scientific Director of the Department of Human Genetics’ clinical laboratory. Postdoctoral fellow Arun Ankala is the first author. In the new paper, Ankala and Hegde examine rearrangements in patients’ genomes that arose in 62 clinical cases of Duchenne’s muscular dystrophy and several other diseases. Mutations in the DMD gene are responsible for Duchenne’s muscular dystrophy.

The pattern of the rearrangement hints what events took place in the cell beforehand, and hint that a problem took place during replication of the DNA. The signature is a tandem duplication of a short segment next to a large deletion, indicating how the DNA was repaired.

The authors note that the DMD locus is especially prone to these types of problems because it is much larger than other gene loci. The gene is actually the longest human gene known on the DNA level, covering 2.4 megabases (0.08 percent of the genome.)

Replication origins are where the DNA copying machinery in the cell starts unwinding and copying the DNA. Bacterial circular chromosomes have just one replication origin. In contrast, humans have thousands of replication origins spread across our chromosomes. In the discussion, the authors suggest that DNA copying problems may also explain duplications and historically embedded rearrangements of the genome.

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A twist on epigenetic therapy vs cancer

Epigenetic therapies against cancer have attracted considerable attention in recent years. But many of the drugs currently being studied as epigenetic anticancer therapies may have indiscriminate effects. A recent paper in Cancer Research from brain cancer researcher Erwin Van Meir’s laboratory highlights a different type of target within cancer cells that may be more selective. Postdoctoral fellow Dan Zhu is the first author of the paper.

Erwin Van Meir, PhD

The basic idea for epigenetic therapy is to focus on how cancer cells’ DNA is wrapped instead of the mutations in the DNA. Cancer cells often have aberrant patterns of methylation or chromatin modifications. Methylation is a punctuation-like modification of DNA that usually shuts genes off, and chromatin is the term describing DNA when it is clothed by proteins such as histones, a form of packaging that determines whether a gene is on or off.

In contrast to mutations that are hard-wired in the DNA, changes in cancer cells’ methylation or chromatin may be reversible with certain drug treatments. But a puzzle remains: if a drug wipes away methylation indiscriminately, that might turn on an oncogene just as much as it might restore a tumor suppressor gene.

The ability of an inhibitor of methylation to treat cancer may depend on cell type and context, explains chromatin/methylation expert and co-author Paula Vertino. She points out that one well-known methylation inhibitor, azacytidine (Vidaza), is a standard treatment for myelodysplastic syndrome, but the strategy of blanket-inhibition of methylation can’t be expected to work for all cancers. A similar challenge exists for agents that target histone acetylation in a global fashion.

Epigenetic therapies seek to modify how DNA is packaged in the cell.

Van Meir’s laboratory has been studying a tumor suppressor protein called BAI1 (brain angiogenesis inhibitor 1), which prevents tumor and blood vessel growth. BAI1 is produced by brain cells naturally, but is often silenced epigenetically in glioblastoma cells. His team found that azacytidine de-represses the BAI1 gene.

Methylation won’t turn a gene off without the help of a set of proteins that bind preferentially to methylated DNA. These proteins are what recognize the methylation state of a given gene and recruit repressive chromatin. Zhu and colleagues in Van Meir’s group found that one particular methyl-binding protein, MBD2, is overproduced in glioblastoma and is enriched on the BAI1 gene.

“Taken together, our results suggest that MBD2 overexpression during gliomagenesis may drive tumor growth by suppressing the anti-angiogenic activity of a key tumor suppressor. These findings have therapeutic implications since inhibiting MBD2 could offer a strategy to reactivate BAI1 and suppress glioma pathobiology,” the authors write.

By itself, MBD2 appears to be dispensable, since mice seem to be able to develop and survive without it. Not having it even seems to push back against tumor formation in the intestine, for example. Targeting MBD2 may represent an alternative way to steer away from cancer cells’ altered state.

Van Meir cautions: “We need to have a better understanding of all the genes that are turned on or off by silencing MBD2 in a given cancer before we can envision to use this approach for therapy.”

Vertino and Steven Hunter, both at Emory, are co-authors on the paper. The work was supported by grants from the NIH and the Southeastern Brain Tumor Foundation and the Emory University Research Council.

Posted on by Quinn Eastman in Cancer 1 Comment

Scientists identify trigger for glowing plankton

Have you ever waded or paddled through ocean water in dim light, and found that your actions caused the water to light up?

Susan Smith, PhD

Single-celled plankton called dinoflagellates are responsible for this phenomenon. Almost 40 years ago, scientists studying bioluminescence (light emitted by living things) proposed a mechanism by which physical deformation of the cell could lead to a trigger of the flash.

Susan M.E. Smith, a research assistant professor in David Lambeth’s laboratory in Emory’s Department of Pathology and Laboratory Medicine, recently was first author on a paper in PNAS identifying a molecule that scientists have long believed to be the key to this mechanism. The paper is the result of a collaboration with Tom DeCoursey’s laboratory at Rush University in Chicago.

The mechanism for the trigger, first envisioned by co-author Woody Hastings, works like this. It is known that acidic conditions activate luciferase, the enzyme that generates the light. Part of the dinoflagellate cell, the vacuole, is about as acidic as orange juice. Normally the acidity within the vacuole is kept separate from the luciferase, which is found in pockets on the outside of the vacuole called scintillons.

Proton channels are needed to trigger bioluminescence. Illustration courtesy of the National Science Foundation, which supported Smith's research

Now something is needed to let acidity (that is, protons) pass from the vacuole to the scintillons. That something is a proton channel: a protein that acts as a gate in the membrane, opening in response to electrical changes in the cell. Smith and her collaborators identified a proton channel called kHV1 that has unique properties: it lets protons flow in the right direction for the trigger to work! They studied kHV1 by inserting the dinoflagellate gene that encodes it into mammalian cells and probing its electrochemical properties, which are distinct from other proton channels.

The authors write: “Whereas other proton channels apparently evolved to extrude acid from cells, kHV1 seems to be optimized to enable proton influx.”

The gene they found actually comes from a type of dinoflagellate that does not flash: K. veneficum, which feeds on algae and sometimes forms harmful blooms that kill fish. They propose that it uses acid influx to aid in capturing or digesting its prey.

“Hastings’ prediction led us to look for this kind of channel, we found it in a related organism, and it had the right properties to fit the prediction,” Smith says, and adds that her team has since found a similar gene in flashing dinoflagellates. She says studying the proton channel may give clues to ways to control harmful dinoflagellates, as well as help scientists understand how plankton respond to greater ocean acidity.

Proton channels are found in humans too. In fact, the same kind of molecule that triggers plankton flashing in the ocean helps human white blood cells produce a bacteria-killing burst of bleach. They are also involved in allergic reactions and in sperm maturation.

Smith is co-author on a paper that is in the journal Nature this week, exploring the selectivity of the human version of kHV1. Smith says that her interest in proton channels grew out of her work on Nox enzymes (which produce the bacteria-killing bleach) with Lambeth.

“I got interested in the proton channel because its function is necessary for peak Nox performance in human phagocytes. We started a little side project on the human proton channel that kind of blossomed,” she says. Her collaboration with DeCoursey uses “evolutionary information to get at the function of these channels in general.”

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COX-2 and epilepsy: it’s complicated

How much is the development of epilepsy like arthritis?

More than you might expect. Inflammation, or the overactivation of the immune system, appears to be involved in both. In addition, for both diseases, inhibiting the enzyme COX-2 initially looked like a promising approach.

Ray Dingledine, PhD

COX-2 (cyclooxygenase 2) is a target of traditional non-steroid anti-inflammatory drugs like aspirin and ibuprofen, as well as more selective drugs such as Celebrex. With arthritis, selectively inhibiting COX-2 relieves pain and inflammation, but turns out to have the side effect of increasing the risk of heart attack and stroke.

In the development of epilepsy, inhibiting COX-2 turns out to be complicated as well. Ray Dingledine, chair of pharmacology at Emory, and colleagues have a new paper showing that COX-2 has both protective and harmful effects in mice after status epilepticus, depending on the timing and what cells the enzyme comes from. Status epilepticus is a period of continuous seizures leading to neurodegeneration, used as a model for the development of epilepsy.

Postdoc Geidy Serrano, now at the Banner Sun Health Research Institute in Arizona, is first author of the paper in Journal of Neuroscience. She and Dingledine were able to dissect COX-2’s effects because they engineered mice to have a deletion of the COX-2 gene, but only in some parts of the brain.
They show that deleting COX-2 in the brain reduces the level of inflammatory molecules produced by neurons, but this is the reverse effect of deleting it all over the body or inhibiting the enzyme with drugs.

Four days after status epilepticus, fewer neurons are damaged (bright green) in the neuronal COX-2 knockout mice.

Dingledine identified two take-home messages from the paper:
First, COX-2 itself is probably not a good target for antiepileptic therapy, and it may be better to go downstream, to prostaglandin receptors like EP2.
Second, the timing of intervention will be important, because the same enzyme has opposing actions a few hours after status epilepticus compared to a couple days later.

More of Dingledine’s thinking about inflammation in the development of epilepsy can be found in a recent review.

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Drug discovery: shifting from brain growth factors to insulin

Earlier this year, the FDA put limitations on some anti-diabetic drugs because of their cardiovascular risks. The prevalence of diabetes in the United States continues to increase and is now above 8 percent of the population, so the need for effective therapies remains strong.

Keqiang Ye, PhD

Pathologist Keqiang Ye and colleagues have a paper in the Journal of Biological Chemistry describing their identification of a compound that mimics the action of insulin. This could be the starting point for developing new anti-diabetes drugs.

The new research is an extension of the Ye laboratory’s work on TrkA and TrkB, which are important for the response of neurons to growth factors. Ye and Sung-Wuk Jang, a remarkably productive postdoc who is now an assistant professor at Korea University, developed an assay that allowed them to screen drug libraries for compounds that directly activate TrkA and TrkB. This led them to find a family of growth-factor-mimicking compounds that could treat conditions such as Parkinson’s disease, depression and stroke.

Since TrkA/B and the insulin receptor are basically the same kind of molecule — receptor tyrosine kinases– and use some of the same cellular circuitry, Ye and Jang’s assay could also be used with the insulin receptor. Kunyan He and Chi-Bun Chan are the first two authors on the new paper. They report that the compound DDN can make cells more sensitive to insulin and improve their ability to take up glucose. They show that DDN (5,8-diacetyloxy-2,3-dichloro-1,4- naphthoquinone) can lower blood sugar, both in standard laboratory mice and in obese mice that serve as a model for type II diabetes.

Ye reports that he and his colleagues are working with medicinal chemists to identify related compounds that may have improved efficacy and potency.

“I hope in the near future we may have something that could replace insulin for treating diabetes orally,” he says.

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Redirecting beta-amyloid production in Alzheimer’s

Pharmacologist Thomas Kukar is exploring a strategy to subtly redirect the enzyme that produces beta-amyloid, which makes up the plaques appearing in the brains of Alzheimer’s patients.

Thomas Kukar, PhD

Preventing beta-amyloid production could be an ideal way to head off Alzheimer’s, but the reason why a subtle approach is necessary was illustrated last year by disappointing results from a phase III clinical trial. The experimental drug semagacestat was designed to block the enzyme gamma-secretase, which “chomps” on the amyloid precursor protein (APP), usually producing an innocuous fragment but sometimes producing toxic beta-amyloid.

Gamma-secretase also is involved in processing a bunch of other vital proteins, such as Notch, central to an important developmental signaling pathway. Scientists suspect that this is one of the reasons why trial participants who received semagacestat did worse on cognitive/daily function measures than controls and saw an increase in skin cancer, leading watchdogs to halt the study.

While a postdoc at Mayo Clinic Jacksonville and working with Todd Golde and Edward Koo, Kukar identified compounds – gamma-secretase modulators or GSM’s — that may offer an alternative.

“We are looking at a strategy that’s different from global gamma-secretase inhibition,” he says. “The approach is: don’t inhibit the enzyme overall, but instead modify its activity so that it makes less toxic products.”

Gamma-secretase chomps on amyloid precursor protein, and how it does so determines whether toxic beta-amyloid is produced. It also processes several other proteins important for brain function.

This line of inquiry started when it was discovered that some anti-inflammatory drugs also could reduce beta-amyloid production. Then, the crosslinkable probes Kukar was using to identify which part of the gamma-secretase fish was doing the chomping ended up binding the bait (APP). This suggested that drugs might be able to change how the enzyme acts on one protein, APP, but not others.

Now an assistant professor at Emory, he is examining in greater detail how gamma-secretase modulators work. Two recent papers he co-authored in Journal of Biological Chemistry show 1) how the proteins that gamma-secretase chews up are “anchored” in the membrane and 2) how selective GSM’s can be on amyloid precursor protein.

Although clinical studies of a “first generation” GSM, tarenflurbil, were also stopped after negative results, Kukar says GSM’s still haven’t really been tested adequately, since researchers do not know if the drugs are really having an effect on beta-amyloid levels in the brain. Newer compounds coming through the pharmaceutical pipeline are more potent and more able to get into the brain. While looking for more potent GSM’s is critical, Kukar says it’s equally as important to understand how gamma-secretase works to understand its biology.

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Blue pill or red pill? Brains need both for memory consolidation

In the 1999 film The Matrix, the character Neo is offered a choice between a blue pill (to forget) and a red pill (to remember). If only neuroscience was that simple! It may be that neurons need both red and blue, possibly an elaborate dance of molecules, for a fragile memory to lodge itself in the brain.

Neuroscientists Kimberly Maguschak and Kerry Ressler provide a glimpse into this process with their recent paper in the Journal of Neuroscience.

Ressler is both a psychiatrist and a Howard Hughes Medical Institute-supported researcher with a laboratory at Yerkes National Primate Research Center. Maguschak completed her doctorate at Emory and is now a postdoc with Guoping Feng at MIT.

The research is a follow-up on their work probing the role of beta-catenin in fear memory formation. We previously described this protein as acting “like a Velcro strap”, attaching cells’ internal skeletons to proteins on their external membranes that help them adhere to other cells. If brain cells need to change shape and form new connections for memories to be consolidated, we can see how this kind of molecule would be important.

Beta-catenin is also central to a signaling circuit that maintains stem cells and prods an embryo to separate into front and back or top and bottom. This circuit is called “Wnt” (the name is a fusion of the fruit fly gene wingless and a cancer-promoting gene discovered in mice, originally called Int-1).

Maguschak and Ressler wanted to assess the role Wnt signals play in learning and memory. The model system was the same as in their previous work: if mice are electrically shocked just after they hear a certain tone, they gradually learn to fear that tone, and they show that fear by freezing.

Kerry Ressler, MD, PhD

Maguschak saw that in the amygdala, a part of the brain important for fear responses, Wnt genes are turned down during the learning process temporarily but then come back on. If the mice only hear the tone or only get the shock, the genes’ activities don’t change significantly.

She then introduced proteins that perturb Wnt signaling directly into the amygdala. Extra Wnt injected before training, while it didn’t stop the mice from learning to fear the tone, made that training less likely to “stick.” Two days later, the mice that received Wnt didn’t seem to fear the tone as much.

Here’s the possibly confusing part: a Wnt inhibitor also impaired fear memory consolidation. In effect, both blue and red pills actually interfered with how well memories endured. The authors suggest this is because Wnt signals have to be turned down during fear memory formation but then turned back up so those memories can solidify. The Wnt signals seem to go along with the adhesive interactions of beta-catenin. It looks like beta-catenin’s stickiness also needs to be tuned down and then back up.

The off-then-on-again requirement Maguschak and Ressler observe is reminiscent of results from cell biologist James Zheng’s lab. He and his colleagues saw that the actin cytoskeleton needed to be weakened and then stabilized during long-term potentiation, an enhancement of connections between neurons thought to lie behind learning and memory.

Several laboratories have identified potential drugs that modify beta-catenin/Wnt. These new results suggest that the timing of when and how to use such drugs to enhance memory may critically important to consider, Ressler says.

“To interfere with memory formation after trauma or enhance memory formation in people with dementia, researchers will clearly need to attend to the full complexity of the dynamics of synaptic plasticity and memory,” he says.

A nifty link to an animation of Wnt signaling


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Magnanimous magnolias keep on giving

Honokiol, the versatile compound found by Emory dermatologist Jack Arbiser in the cones of magnolia trees, makes a surprise appearance in a recent paper in Nature Medicine.

Jack Arbiser, MD, PhD, and colleagues originally isolated honokiol from magnolia cones. It can also be found in herbal teas.

The paper, from Sabrina Diano, Tamas Horvath and colleagues at Yale, probes the role of reactive oxygen species (ROS) in the hypothalamus, a part of the brain that regulates appetite. In the paper, Horvath’s laboratory uses honokiol as a super-antioxidant, mopping up ROS that suppress appetite. Arbiser initiated the collaboration with Horvath after finding, while working with Emory free radical expert Sergei Dikalov, how effective honokiol is at neutralizing ROS.

The paper is intriguing partly because it’s an example of a situation where ROS, often thought to be harmful because of their links to aging and several diseases, are actually beneficial. In this case, they provide a signal to stop eating. A recent paper from Andrew Neish’s lab at Emory provides another example, where probiotic bacteria stimulate production of ROS, which promote healing of the intestine.

Arbiser notes that since honokiol can increase appetite, the compound may be helpful in situations where doctors want patients to eat more.

“This might be particularly valuable in patients who are nutritionally deficient due to chemotherapy and provides a rationale for adding honokiol to chemotherapy regimens,” he writes.

Satiety producing neurons in the hypothalamus

A note of caution: in the Nature Medicine paper, honokiol is infused directly into the brain.

Honokiol has been shown to counteract inflammation and slow the growth of blood vessels (important in fighting cancer). Collaborating with Arbiser, Emory endocrinologist Neale Weitzmann has recently found that honokiol stimulates osteoblasts, the cells that build bone, suggesting that it could reduce bone loss in osteoporosis.

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Neuroinflammation: a different way to look at Parkinson’s disease

Emory physiologist Malu Tansey and her colleagues are using recent insights into the role of inflammation in Parkinson’s disease to envision new treatments. One possible form this treatment strategy could take would be surprisingly simple, and comparable to medications that are approved for rheumatoid arthritis.

Malu Tansey, PhD

Understanding the role of inflammation in Parkinson’s requires a shift in focus. Many Parkinson’s researchers understandably emphasize the neurons that make the neurotransmitter dopamine. They’re the cells that are dying or already lost as the disease progresses, leading to tremors, motor difficulties and a variety of other symptoms.

But thinking about the role of inflammation in Parkinson’s means getting familiar with microglia, the immune system’s field reps within the brain. At first, it was thought that the profusion of microglia in the brains of Parkinson’s patients was just a side effect of neurodegeneration. The neurons die, and the microglia come in to try to clean up the debris.

Now it seems like microglia and inflammation might be one of the main events, if not the initiating event.

“Something about the neurons’ metabolic state, whether it’s toxins, oxidative stress, unfolded proteins, or a combination, makes them more sensitive. But inflammation, sustained by the presence of microglia, is what sends them over the edge,” Tansey says.

She says that several recent studies have led to renewed attention to this area:

  1. In vivo PET imaging using a probe for microglia has allowed scientists to see inflammation starting early in the progression of Parkinson’s (see figure below)
  2. Epidemiology studies show that taking ibuprofen regularly is linked to lower incidence of Parkinson’s
  3. Experiments with animal models of genetic susceptibility demonstrate that inflammatory agents like endotoxin can accelerate neurodegeneration
  4. Genomics screens have identified HLA-DR, an immune system gene, as a susceptibility marker for Parkinson’s (Emory’s Stewart Factor was a co-author on this paper)

Popping a few ibuprofen pills everyday for prevention and possibly damaging the stomach along the way is probably not going to work well, Tansey says. It should be possible to identify a more selective way to inhibit microglia, which may be able to inhibit disease progression after it has started.

Activated microglia in the midbrain and striatum of a Parkinson's patient

Targeting TNF (tumor necrosis factor), an important inflammatory signaling molecule, may be one way to go. Anti-TNF agents are already used to treat rheumatoid arthritis and inflammatory bowel disease. This January, Tansey and her co-workers published a paper showing that a gene therapy approach using decoy TNF can reduce neuronal loss in a rat model of Parkinson’s. More recently, her lab has also shown that targeting the gene RGS10 is another way to inhibit microglia and reduce neurodegeneration in the same models.

It is important to note that in the rat studies, they do surgery and put the gene therapy viral vector straight into the brain. She says it might possible to perform peripheral gene therapy with the microglia, or even anti-TNF medical therapy. In terms of mechanism, decoy (technically, dominant negative) TNF is more selective and may avoid the side effects, such as opportunistic infections, of existing anti-TNF agents.

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Excitement building over potential for universal flu vaccine

Francis Collins, director of the National Institutes of Health, made a splash last week predicting the arrival of a universal flu vaccine in the next five years.

Francis Collins told USA Today he is "guardedly optimistic" about the possibility of long-term vaccination that could replace seasonal flu shots.

His prediction came at the same time as a report in Science identifying an antibody that can protect against several strains of the flu virus. Taking a look at the Science paper, how the scientists found the “super antibody” seems remarkably similar to how Emory’s Jens Wrammert, Rafi Ahmed and colleagues found a similar broadly protective antibody. Their results were published in the Journal of Experimental Medicine in January.

In both cases, the researchers started with someone who had been infected with the 2009 H1N1 swine origin flu virus, sifted through the antibodies that person produced and found some that reacted against several varieties of the flu virus. There must be something special about that 2009 pandemic strain!

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