Taming suspect gene reverses schizophrenia-like abnormalities in mice

Scientists have reversed behavioral and brain abnormalities in adult mice that resemble some features of schizophrenia by restoring normal expression to a suspect gene that is over-expressed in humans with the illness. Targeting expression of the gene Neuregulin1, which makes a protein important for brain development, may hold promise for treating at least some patients with the brain disorder, say researchers funded by the National Institutes of Health.

Like patients with schizophrenia, adult mice biogenetically-engineered to have higher Neuregulin 1 levels showed reduced activity of the brain messenger chemicals glutamate and GABA. The mice also showed behaviors related to aspects of the human illness. For example, they interacted less with other animals and faltered on thinking tasks.

“The deficits reversed when we normalized Neuregulin 1 expression in animals that had been symptomatic, suggesting that damage which occurred during development is recoverable in adulthood,” explained Lin Mei, M.D., Ph.D.External Web Site Policy , of the Medical College of Georgia at Georgia Regents University, a grantee of NIH’s National Institute of Mental Health (NIMH).

Mei, Dong-Min Yin, Ph.D., Yong-Jun Chen, Ph.D., and colleagues report on their findings May 22, 2013 in the journal Neuron.

“While mouse models can’t really do full justice to a complex brain disorder that impairs our most uniquely human characteristics, this study demonstrates the potential of dissecting the workings of intermediate components of disorders in animals to discover underlying mechanisms and new treatment targets,” said NIMH Director Thomas R. Insel, M.D. “Hopeful news about how an illness process that originates early in development might be reversible in adulthood illustrates the promise of such translational research.”

Schizophrenia is thought to stem from early damage to the developing fetal brain, traceable to a complex mix of genetic and environmental causes. Although genes identified to date account for only a small fraction of cases, evidence has implicated variation in the Neuregulin 1 gene. For example, postmortem studies have found that it is overexpressed in the brain’s thinking hub, or prefrontal cortex, of some people who had schizophrenia. It codes for a chemical messenger that plays a pivotal role in communication between brain cells, as well as in brain development.

Prior to the new study, it was unclear whether damage caused by abnormal prenatal Neuregulin 1 expression might be reversible in adulthood. Nor was it known whether any resulting behavioral and brain deficits must be sustained by continued errant Neuregulin 1 expression in adulthood.

To find out, the researchers engineered laboratory mice to mimic some components of the human illness by over-expressing the Neuregulin 1 gene in the forebrain, comparable to the prefrontal cortex in humans. Increasing Neuregulin 1 expression in adult animals was sufficient to produce behavioral features, such as hyperactivity, social and cognitive impairments, and to hobble neural communications via the messenger chemicals glutamate and GABA.

Unexpectedly, the abnormalities disappeared when the researchers experimentally switched off Neuregulin 1 overexpression in the adult animals. Treatment with clozapine, an antipsychotic medication, also reversed the behavioral abnormalities. The researchers traced the glutamate impairment to an errant enzyme called LIMK1, triggered by the overexpressed Neuregulin 1 — a previously unknown potential pathological mechanism in schizophrenia.

The study results suggest that even if their illness stems from disruptions early in brain development, adult patients whose schizophrenia is rooted in faulty Neuregulin 1 activity might experience a reduction in some of the symptoms following treatments that target overexpression of the protein, say the researchers.

Reducing caloric intake delays nerve cell loss

Activating an enzyme known to play a role in the anti-aging benefits of calorie restriction delays the loss of brain cells and preserves cognitive function in mice, according to a study published in the May 22 issue of The Journal of Neuroscience. The findings could one day guide researchers to discover drug alternatives that slow the progress of age-associated impairments in the brain.

Previous studies have shown that reducing calorie consumption extends the lifespan of a variety of species and decreases the brain changes that often accompany aging and neurodegenerative diseases such as Alzheimer’s. There is also evidence that caloric restriction activates an enzyme called Sirtuin 1 (SIRT1), which studies suggest offers some protection against age-associated impairments in the brain.

In the current study, Li-Huei Tsai — director of the Picower Institute for Learning and Memory and Picower Professor of Neuroscience at MIT — along with postdoc Johannes Gräff and others at MIT tested whether reducing caloric intake would delay the onset of nerve cell loss that is common in neurodegenerative disease, and if so, whether SIRT1 activation was driving this effect. The group not only confirmed that caloric restriction delays nerve cell loss, but also found that a drug that activates SIRT1 produces the same effects.

“There has been great interest in finding compounds that mimic the benefits of caloric restriction that could be used to delay the onset of age-associated problems and/or diseases,” says Dr. Luigi Puglielli, who studies aging at the University of Wisconsin, Madison, and was not involved in this study. “If proven safe for humans, this study suggests such a drug could be used as a preventive tool to delay the onset of neurodegeneration associated with several diseases that affect the aging brain.”

In the study, Tsai’s team first decreased the normal diets of mice genetically engineered to rapidly undergo changes in the brain associated with neurodegeneration by 30 percent. Following three months on the diet, the mice completed several learning and memory tests. “We not only observed a delay in the onset of neurodegeneration in the calorie-restricted mice, but the animals were spared the learning and memory deficits of mice that did not consume reduced-calorie diets,” Tsai says.

Curious if they could recreate the benefits of caloric restriction without changing the animals’ diets, the scientists gave a separate group of mice a drug that activates SIRT1. Similar to what the researchers found in the mice exposed to reduced-calorie diets, the mice that received the drug had less cell loss and better cellular connectivity than the mice that did not receive the drug. Additionally, the mice that received the drug treatment performed as well as normal mice in learning and memory tests.

“The question now is whether this type of treatment will work in other animal models, whether it’s safe for use over time, and whether it only temporarily slows down the progression of neurodegeneration or stops it altogether,” Tsai says.

Drugs found to both prevent and treat Alzheimer’s disease in mice

Researchers at USC have found that a class of pharmaceuticals can both prevent and treat Alzheimer’s Disease in mice.

The drugs, known as “TSPO ligands,” are currently used for certain types of neuroimaging.

"We looked at the effects of TSPO ligand in young adult mice when pathology was at an early stage, and in aged mice when pathology was quite severe," said lead researcher Christian Pike of the USC Davis School of Gerontology. "TSPO ligand reduced measures of pathology and improved behavior at both ages."

The team’s findings were published online by the Journal of Neuroscience on May 15. Pike’s coauthors include USC postdoctoral scientists Anna M. Barron, Anusha Jayaraman and Joo-Won Lee; as well as Donatella Caruso and Roberto C. Melcangi of the University of Milan and Luis M. Garcia-Segura of the Instituto Cajal in Spain.

The most surprising finding for Pike and his team was the effect of TSPO ligand in the aged mice. Four treatments—once per week over four weeks—in older mice resulted in a significant decrease of Alzheimer’s-related symptoms and improvements in memory – meaning that TSPO ligands may actually reverse some elements of Alzheimer’s disease.

"Our data suggests the possibility of drugs that can prevent and treat Alzheimer’s," Pike said. "It’s just mouse data, but extremely encouraging mouse data. There is a strong possibility that TSPO ligands similar to the ones used in our study could be evaluated for therapeutic efficacy in Alzheimer’s patients within the next few years."

Next, the team will next focus on understanding how TSPO ligands reduce Alzheimer’s disease pathology. Building on the established knowledge that TSPO ligands can reduce inflammation—shielding nerve cells from injury and increasing the production of neuroactive hormones in the brain—the team will study which of these actions is the most significant in fighting Alzheimer’s disease so they can develop newer TSPO ligands accordingly.

Animals in research: zebrafish

Zebrafish are probably not the first creatures that come to mind when it comes to animals that are valuable for medical research.

You might struggle to imagine you have much in common with this small tropical freshwater fish, though you may be inclined to keep a few “zebra danios” in your home aquarium, given they are hardy, undemanding animals that cost only a few dollars each.

Yet each year more and more scientists are turning to zebrafish to unravel the mechanisms underlying their favourite genetic or infectious disease, be it muscular dystrophy, schizophrenia, tuberculosis or cancer.

My (conservative) estimate is that zebrafish research is now carried out in at least 600 labs worldwide, including 20 in Australia.

So what is it about zebrafish that has taken them from the freshwater rivers and streams of Southeast Asia, beyond the pet shops and into universities and research institutes the world over?

A short history of zebrafish

A scientist called George Streisinger, working at the University of Oregon in Eugene, USA in the 1970s and 80s, recognised the vast potential of this organism for developmental biology and genetics research.

In contrast to fruit flies and worms, the other simple model organisms established at the time, zebrafish are vertebrates.

They have a backbone, brain and spinal cord as well as several other organs, including a heart, liver and pancreas, kidneys, bones and cartilage, which makes them much more similar to humans than you may have otherwise thought.

But as a vertebrate model, could they be as useful as mice?

Several things captured Streisinger’s imagination.

Most famously, zebrafish embryos, unlike mouse embryos, develop outside the mother’s body and are transparent throughout the first few days of life.

This provides unparallelled opportunities for researchers to scrutinise the fine details of embryonic vertebrate development without first having to resort to invasive procedures or killing the mother.

But this advantage is enhanced by the fact zebrafish reproduce profusely (each pair can produce 200-300 fertilised eggs every week); an ideal attribute for genetic studies. Again, the large, external embryos are a critical part of this success.

When just one or two cells old, zebrafish embryos can be easily microinjected with mRNA or DNA corresponding to genes of interest; undeterred, they then they go on to grow and reproduce, handing down the injected gene to the next generation.

From zebrafish to humans

A paper published last month in Nature unveiled the long-awaited sequence of the zebrafish genome, revealing that zebrafish, mice and human have 12,719 genes in common.

Put another way, 70% of human genes are found in zebrafish.

But even more notable is the finding that 84% of human disease-causing genes are found in zebrafish.

Perhaps not surprisingly then, when these genes are injected into zebrafish embryos, the growing animals are doomed to acquire the same diseases.

And while zebrafish are still used widely to answer fundamental questions of developmental biology, much current research is directed towards combining their many attributes in studies that are designed to improve human health.

This is especially true for cancer research where the expression of cancer-causing genes (oncogenes) can be directed to specific organs, virtually at will.

This process, known as transgenesis, is very straightforward in zebrafish and has allowed researchers to produce zebrafish models of liver, pancreatic, skeletal muscle, blood and skin cancers, to name but a few.

And when the genomic make-up of these zebrafish tumours is deciphered using the latest DNA sequencing technology, the patterns of mutations, or “gene signatures”, are found to overlap substantially with those in the corresponding human tumours.

Trialling cancer drugs

These parallels have encouraged researchers to exploit zebrafish in drug development – in particular for high throughput approaches such as chemical/small molecule screens.

Here, the ability to generate tens of thousands of zebrafish embryos harbouring the same disease-causing mutations is crucial.

Then, as the tumours grow in the synchronously developing larvae, the fish are transferred to small volumes of water containing chemicals that may stop the growth, or better still, kill the cancer cells.

Large collections of drugs can be screened relatively quickly for anti-cancer efficacy in this way.

One drug, Leflunomide, identified in such a screen is now in early phase clinical trials to kill melanoma cells.

The only other drug from a zebrafish chemical screen currently in clinical trials is dimethyl-prostaglandin E2 (dmPGE2).

There, the intent is not to kill cancer cells but rather to make mainstream leukaemia treatment more effective.

Studies of dmPGE2 increased the number of blood stem cells in zebrafish embryos and it is being trialled now as a way to expand the number of stem cells in human cord blood samples.

Human cord blood samples are a valuable commodity to restore bone marrow in leukaemia patients after high dose chemotherapy when a matched bone marrow transplant is unavailable.

But the success of this approach is currently limited by the scant number of stem cells in individual cord blood samples, requiring the use of two precious samples for each patient.

Tumour growth

As well as the transgenic zebrafish models of cancer described above, researchers are also transplanting cells derived from human tumours into zebrafish embryos and watching them grow and spread.

The creation of a transparent (non-striped) version of adult zebrafish (called casper, after the cartoon ghost) means the behaviour of tumour cells inside these living organisms can be followed for days at a time.

Coupled with the advent of high resolution live-imaging techniques, the birth, growth and spread of tumours can be scrutinised in movies that can be played over and over again.

These experiments are usually conducted in zebrafish that have been genetically modified to express genes that glow in specific body compartments, giving researchers the ability to pinpoint potentially critical connections between “host” cells and tumour cells that may determine whether the latter survive or die.

This type of experiment is revealing a complex interplay of potentially beneficial and detrimental components.

While the proximity of immune cells may instigate mechanisms capable of destroying the tumour, the stimulation of new blood and lymphatic vessel growth towards the tumour is more insidious, since it delivers the tumour with both the nutrients it needs to survive and a network to spread throughout the body.

These processes, once properly understood, are likely to provide opportunities for therapeutic intervention in the future.

The future of zebrafish

Cancer research is just one part of the zebrafish story. In Australia alone, investigators are also using zebrafish to study metabolic disorders such as:

• diabetes
• muscle diseases, including muscular dystrophy
• neurodegenerative disease
• the response of the host innate immune system to bacterial and fungal infections

Excitingly, research is also underway in this country to unravel the genetic mechanisms controlling heart, skeletal muscle and nervous tissue regeneration in zebrafish, in the hope that these processes can be one day recapitulated in humans to address the burgeoning socioeconomic problem of tissue degeneration in our ageing population.

So next time you peer into someone’s home aquarium, imagine the biomedical possibilities inherent in this lively and amiable little fish!

Advance in tuberous sclerosis brain science

By manipulating the timing of disease-causing mutations in the brains of developing mice, Brown University researchers have found that early genetic deletions in the thalamus may play an important role in course and severity of the developmental disease tuberous sclerosis complex. Findings appear in the journal Neuron.

Doctors often diagnose tuberous sclerosis complex (TSC) based on the abnormal growths the genetic disease causes in organs around the body. Those overt anatomical structures, however, belie the microscopic and mysterious neurological differences behind the disease’s troublesome behavioral symptoms: autism, intellectual disabilities, and seizures. In a new study in mice, Brown University researchers highlight a role for a brain region called the thalamus and show that the timing of gene mutation during thalamus development makes a huge difference in the severity of the disease.

TSC can arise in humans and mice alike when both alleles (the one from mom and the one from dad) of the TSC1 gene are deleted. One bad gene is often inherited and the other accumulates a mutation some time during embryonic development. This happens to one in 6,000 people.

“We don’t know when during development the mutations are occurring in the patients,” said Elizabeth Normand, a Brown neuroscience graduate student and lead author of the paper in the journal Neuron. “That’s why we chose to look at the timing. It can give us some insight into the role of genes during embryonic development.”

Normand and adviser Mark Zervas, assistant professor of biology, not only wanted to assess the timing but also to probe the role the thalamus might have in contributing to the neurological symptoms of the disease. To do both, their team genetically engineered a clever mouse model in which they could, with a dose of the drug tamoxifen, delete both alleles exclusively in thalamus neurons at the developmental stage of their choosing.

Their interest in the thalamus comes from its role in forging strong but intricate links to the cortex, which is where most other TSC researchers have focused. As for timing, they tested the effect of controlling allele deletions on day 12 of gestation in some mice and day 18 (just before birth) in others. Still other mice were left healthy as experimental controls.

Significant symptoms

Overall, the researchers found they could indeed generate TSC-like behavioral symptoms in the mice, such as seizures, by deleting TSC1 alleles in developing cells of the thalamus. They also found that the timing of the deletion mattered tremendously to the extent of the disease in the brain, the degree of abnormality, and the severity of TSC-like symptoms.

The mice whose alleles were deleted on embryonic day 12 fared much worse behaviorally than the mice whose alleles were deleted on embryonic day 18.

At two months of age, the mice with the embryonic day 12 deletion exhibited excessive self-grooming to the point where they experienced lesions. Among those mice, 10 of 11 experienced seizures at an average rate of more than three per hour.

The mice with the embryonic day 18 deletion, on the other hand, fared better without any over-grooming. By eight months of age, however, four of 17 of the mice did exhibit rare seizures.

These behavioral differences traced to differences in the the way the mice’s brains became wired. A comparison of brain tissue from adult mice — some of which had the early TSC1 deletions and some of which didn’t — revealed differences in the connections between the thalamus and the cortex and in the electrical and physical properties of thalamus cells.

“We’re building off the core idea of the thalamus playing an important role in brain function and showing that if you disrupt the way that the thalamic neurons develop that you can get some of these behavioral consequences such as overgrooming or seizures,” said Zervas, who is affiliated with the Brown Institute for Brain Science.

The extent of mutant neurons was much more severe in the mice with the embryonic day 12 versus day 18 mutations. In embryonic day 12 deleted mice, for example, the deletion disrupted the growth-regulating “mTOR” pathway in 70 percent of neurons versus only 29 percent of neurons in the embryonic day 18 deleted mice. The disruptions occurred in more areas of the thalamus in embryonic day 12 than in day 18 mice as well. The overactivity of mTOR in TSC is what produces the unusual growths around the body, though these new findings indicate additional roles for the mTOR pathway in brain development and function, Zervas said.

In future work, the team plans to study the effects of deleting the TSC1 allele at other days during development as well as to understand whether there is a threshold of mutant neurons with mTOR disruption at which TSC-like symptoms begin to emerge.

Experience leads to the growth of new brain cells

A new study examines how individuality develops

The DFG-Center for Regenerative Therapies Dresden - Cluster of Excellence at the TU Dresden (CRTD), the Dresden site of the German Center for Neurodegenerative Diseases (DZNE), and the Max Planck Institute for Human Development in Berlin played a pivotal role in the study.

The adult brain continues to grow with the challenges that it faces; its changes are linked to the development of personality and behavior. But what is the link between individual experience and brain structure? Why do identical twins not resemble each other perfectly even when they grew up together? To shed light on these questions, the scientists observed forty genetically identical mice that were kept in an enclosure offering a large variety of activity and exploration options.

"The animals were not only genetically identical, they were also living in the same environment," explains principal investigator Gerd Kempermann, Professor for Genomics of Regeneration, CRTD, and Site Speaker of the DZNE in Dresden. "However, this environment was so rich that each mouse gathered its own individual experiences in it. Over time, the animals therefore increasingly differed in their realm of experience and behavior."

New neurons for individualized brains

Each of the mice was equipped with a special micro-chip emitting electromagnetic signals. This allowed the scientists to construct the mice’s movement profiles and to quantify their exploratory behavior. The result: Despite a common environment and identical genes the mice showed highly individualized behavioral patterns. They reacted to their environment differently. In the course of the three-month experiment these differences increased in size.

"Though the animals shared the same life space, they increasingly differed in their activity levels. These differences were associated with differences in the generation of new neurons in the hippocampus, a region of the brain that supports learning and memory," says Kempermann. "Animals that explored the environment to a greater degree also grew more new neurons than animals that were more passive."

Adult neurogenesis, that is, the generation of new neurons in the hippocampus, allows the brain to react to new information flexibly. With this study, the authors show for the first time that personal experiences and ensuing behavior contribute to the „individualization of the brain.” The individualization they observed cannot be reduced to differences in environment or genetic makeup.

"Adult neurogenesis also occurs in the hippocampus of humans," says Kempermann. "Hence we assume that we have tracked down a neurobiological foundation for individuality that also applies to humans."

Impulses for discussion across disciplines

"The finding that behavior and experience contribute to differences between individuals has implications for debates in psychology, education science, biology, and medicine," states Prof. Ulman Lindenberger, Director of the Center for Lifespan Psychology at the Max Planck Institute for Human Development (MPIB) in Berlin. "Our findings show that development itself contributes to differences in adult behavior. This is what many have assumed, but now there is direct neurobiological evidence in support of this claim. Our results suggest that experience influences the aging of the human mind."

In the study, a control group of animals housed in a relatively unattractive enclosure was also examined; on average, neurogenesis in these animals was lower than in the experimental mice. „When viewed from educational and psychological perspectives, the results of our experiment suggest that an enriched environment fosters the development of individuality,” comments Lindenberger.

Interdisciplinary teamwork

The study is also an example of multidisciplinary cooperation — it was made possible because neuroscientists, ethologists, computer scientists, and developmental psychologists collaborated closely in designing the experimental set-up and applying new data analysis methods. Biologist Julia Freund from the CRTD Dresden and computer scientist Dr. Andreas Brandmaier from the MPIB in Berlin share first authorship on the article. In addition to the DZNE, CRTD, and the MPIB, the German Research Center for Artificial Intelligence in Saarbrücken and the Institute for Geoinformatics and the Department of Behavioural Biology at the University of Münster were also involved in this project.

Original publication

"Emergence of Individuality in Genetically Identical Mice", Julia Freund, Andreas M. Brandmaier, Lars Lewejohann, Imke Kirste, Mareike Kritzler, Antonio Krüger, Norbert Sachser, Ulman Lindenberger, Gerd Kempermann, Science

(Image: Dr Jonathan Clarke, Wellcome Images)

Researchers Use Nasal Lining to Breach the Blood-Brain Barrier, Widening Treatment Options for Neurodegenerative and Central Nervous System Disease

Neurodegenerative and central nervous system (CNS) diseases represent a major public health issue affecting at least 20 million children and adults in the United States alone. Multiple drugs exist to treat and potentially cure these debilitating diseases, but 98 percent of all potential pharmaceutical agents are prevented from reaching the CNS directly due to the blood-brain barrier.

Using mucosa, or the lining of the nose, researchers in the department of Otology and Laryngology at the Massachusetts Eye and Ear/Harvard Medical School and the Biomedical Engineering Department of Boston University have demonstrated what may be the first known method to permanently bypass the blood-brain barrier, thus opening the door to new treatment options for those with neurodegenerative and CNS disease. Their study is published on PLOS ONE.
Many attempts have been made to deliver drugs across the blood-brain barrier using methods such as osmotic disruption and implantation of catheters into the brain; however these methods are temporary and prone to infection and dislodgement.

"As an endoscopic skull base surgeon, I and many other researchers have helped to develop methods to reconstruct large defects between the nose and brain using the patient’s own mucosa or nasal lining," said Benjamin S. Bleier, M.D., Otolaryngologist at Mass. Eye and Ear and HMS Assistant Professor.

Study co-author Xue Han, Ph.D., an assistant professor of Biomedical Engineering at Boston University, said, “The development of this model enables us to perform critical preclinical testing of novel therapies for neurological and psychiatric diseases.”

Inspired by recent advances in human endoscopic transnasal skull based surgical techniques, the investigators went to work to develop an animal model of this technique and use it to evaluate transmucosal permeability for the purpose of direct drug delivery to the brain.

In this study using a mouse model, researchers describe a novel method of creating a semi-permeable window in the blood-brain barrier using purely autologous tissues to allow for higher molecular weight drug delivery to the CNS. They demonstrated for the first time that these membranes are capable of delivering molecules to the brain which are up to 1,000-times larger than those excluded by the blood-brain barrier.

"Since this is a proven surgical technique which is known to be safe and well tolerated, this data suggests that these membranes may represent the first known method to permanently bypass the blood-brain barrier using the patient’s own tissue," Dr. Bleier said. "This method may open the door for the development of a variety of new therapies for neurodegenerative and CNS disease.
Future studies will be directed towards developing clinical trials to test this method in patients who have already undergone these endoscopic surgeries.”

(Image: iStockphoto)