A gene linked to autism spectrum disorders that was manipulated in two lines of transgenic mice produced mature adults with irreversible deficits affecting either learning or social interaction.
The findings have implications for potential gene therapies but they also suggest that there may be narrow windows of opportunity to be effective, says principal investigator Philip Washbourne, a professor of biology and member of the University of Oregon’s Institute of Neuroscience.
The research, reported by an 11-member team from three universities, targeted the impacts of alterations in the gene neuroligin 1 — one of many genes implicated in human autism spectrum disorders — to neuronal synapses in the altered mice during postnatal development and as they entered adulthood. One group over-expressed the normal gene, the other a mutated version.
Mice with higher-than-normal levels of the normal gene after a month had skewed synapses at maturity. Many were larger, appearing more mature, than normal. In these mice, Washbourne said, there were clear cognitive problems. ‘Behaviour was just not normal. They didn’t learn very well, and they were slower to learn, but their social behaviour was not impacted.’
Mice over-producing a mutated version of the gene reached adulthood with structurally immature synapses. ‘They were held back in development and behaviour — the way they behave in terms of learning and memory, in terms of social interaction,’ he said. ‘These were adult mice, three months old, but they behaved like normal mice at four weeks old. We saw arrested development. Learning is a little bit better, they are more flexible just like young mice, they learn faster, but their social interaction is off. To us, this looked more like Asperger’s syndrome.
‘So with the same gene, doing two different manipulations — over-expressing the normal form or over-expressing a mutated form — we’ve gone to two different ends of the autism spectrum,’ said Washbourne, whose lab focuses on basic synapse formation and what goes wrong in relationship to autism. Work has been done in both mice and zebra fish.
‘We made these mice so that we can turn the genes on and off as we want,’ Washbourne said. ‘Using an antibiotic, doxycycline, it turns off these altered genes that we inserted into their chromosomes. While on doxycycline, the mice are absolutely normal.’
However, if the inserted gene was turned off after the completion of development, mice still showed altered synapses and behaviour. This result suggests that any kind of gene therapy may have to be applied to individuals with autism early on.
Effects seen in the social behaviour of mice with the mutated gene, he said, are not unlike observations reported by parents of many autistic children. While normal mice prefer to engage with new mice entering their world rather than familiar others, or even a new inanimate object, these mice split their time equally. ‘It’s not a deficit in memory regarding which mouse is which, it’s more a weighting of their interaction. Does that mean they are autistic? I don’t know, but if you talk to parents of autistic children, one of the frustrating things they report is that their children treat complete strangers in exactly the same way that they treat them.’
While the findings provide new insights, Washbourne said, any translation into treatment could be decades away. ‘A problem with autism is there are many different genes potentially involved. It could be that some day, if you are diagnosed with autism, a mouth swab might allow for the identification of the exact gene that is mutated and allow for targeted therapy,’ he said. ‘Genome sequencing already has turned up subtle mutations in lots of genes. Autism might be like cancer, with hundreds of potential combinations of faulty genes.’ University of Oregon

Tiny biomolecular chambers called nanopores that can be selectively heated may help doctors diagnose disease more effectively if recent research by a team at the National Institute of Standards and Technology (NIST), Wheaton College, and Virginia Commonwealth University (VCU) proves effective. Though the findings may be years away from application in the clinic, they may one day improve doctors’ ability to search the bloodstream quickly for indicators of disease—a longstanding goal of medical research.
The team has pioneered work on the use of nanopores—tiny chambers that mimic the ion channels in the membranes of cells—for the detection and identification of a wide range of molecules, including DNA. Ion channels are the gateways by which the cell admits and expels materials like proteins, ions and nucleic acids. The typical ion channel is so small that only one molecule can fit inside at a time.
Previously, team members inserted a nanopore into an artificial cell membrane, which they placed between two electrodes. With this set-up, they could drive individual molecules into the nanopore and trap them there for a few milliseconds, enough to explore some of their physical characteristics.
‘A single molecule creates a marked change in current that flows through the pore, which allows us to measure the molecule’s mass and electrical charge with high accuracy,’ says Joseph Reiner, a physicist at VCU who previously worked at NIST. ‘This enables discrimination between different molecules at high resolution. But for real-world medical work, doctors and clinicians will need even more advanced measurement capability.’
A goal of the team’s work is to differentiate among not just several types of molecules, but among the many thousands of different proteins and other biomarkers in our bloodstream. For example, changes in protein levels can indicate the onset of disease, but with so many similar molecules in the mix, it is important not to mistake one for another. So the team expanded their measurement capability by attaching gold nanoparticles to engineered nanopores, ‘which provides another means to discriminate between various molecular species via temperature control,’ Reiner says.
The team attached gold nanoparticles to the nanopore via tethers made from complementary DNA strands. Gold’s ability to absorb light and quickly convert its energy to heat that conducts into the adjacent solution allows the team to alter the temperature of the nanopore with a laser at will, dynamically changing the way individual molecules interact with it.
‘Historically, sudden temperature changes were used to determine the rates of chemical reactions that were previously inaccessible to measurement,’ says NIST biophysicist John Kasianowicz. ‘The ability to rapidly change temperatures in volumes commensurate with the size of single molecules will permit the separation of subtly different species. This will not only aid the detection and identification of biomarkers, it will also help develop a deeper understanding of thermodynamic and kinetic processes in single molecules.’ EurekAlert

Using the Department of Defense Serum Repository (DoDSR), University of Cincinnati researchers have identified a number of biomarkers for inflammatory bowel disease (IBD), which could help with earlier diagnosis and intervention in those who have not yet shown symptoms.
The DoDSR is a biological repository operated by the U.S. Department of Defense and contains over 50 million human serum specimens, collected primarily from applicants to and members of the U.S. uniformed services.
‘With collaborators from Walter Reed, we were able to identify all of the active duty service men and women who developed IBD and then used the repository to go back and look at various biomarkers to see what each person had in common,’ says Yacyshyn, a professor of medicine at the UC College of Medicine and UC Health gastroenterologist.
IBD is a group of inflammatory conditions of the colon and small intestine. The main types of IBD are Crohn’s disease and ulcerative colitis; inflammatory bowel diseases are considered autoimmune diseases in which the body’s own immune system attacks elements of the digestive system.
In this study, researchers used the repository to identify 50 cases of Crohn’s disease and 50 cases of ulcerative colitis. They analysed proteins from three samples per case—two taken before and one after diagnosis—using a statistical analysis format.
Certain proteins were found in elevated levels in samples from patients who developed IBD.
‘The selection of proteins we chose to analyse was based on a prior study conducted at UC,’ Yacyshyn says. ‘Although the presence of proteins in those who develop Crohn’s disease varies from those present in ulcerative colitis patients, we were able to show that there were elevated levels of certain proteins in patients who developed IBD.’
‘Future large validation studies are needed to confirm the presence of biomarkers to guide in diagnosis, prevention and management of these patients,’ he adds. UC Academic Health News

Researchers at Karolinska Institutet in Sweden have identified a new so-called pseudogene that regulates the tumour-suppressing PTEN gene. They hope that this pseudogene will be able to control PTEN to reverse the tumour process, make the cancer tumour more sensitive to chemotherapy and to prevent the development of resistance.
The development of tumours coincides with the activation of several cancer genes as well as the inactivation of other tumour-suppressing genes owing to damage to the DNA and to the fact that the cancer cells manage to switch off the transcription of tumour-suppressor genes. To identify what might be regulating this silencing, the researchers studied PTEN, one of the most commonly inactivated tumour-suppressor genes. It has long been believed that the switching-off process is irreversible, but the team has now shown that silenced PTEN genes in tumour cells can be ‘rescued’ and re-activated by a ‘pseudogene’, a type of gene that, unlike normal genes, does not encode an entire protein.
　
‘We identified a new non-protein encoding pseudogene, which determines whether the expression of PTEN is to be switched on or off,’ says research team member Per Johnsson, doctoral student at Karolinska Institutet’s Department of Oncology-Pathology. ‘What makes this case spectacular is that the gene only produces RNA, the protein’s template. It is this RNA that, through a sequence of mechanisms, regulates PTEN. Pseudogenes have been known about for many years, but it was thought that they were only junk material.’
　
No less than 98 per cent of human DNA consists of non-protein encoding genes (i.e. pseudogenes), and by studying these formerly neglected genes the researchers have begun to understand that they are very important and can have an effect without encoding proteins. Using model systems, the team has shown that the new pseudogene can control the expression of PTEN and make tumours more responsive to conventional chemotherapy.
　
‘This means that we might one day be able to re-programme cancer cells to proliferate less, become more normal, and that resistance to chemotherapy can hopefully be avoided,’ says Per Johnsson. ‘We also believe that our findings can be very important for the future development of cancer drugs. What we’re seeing here is just the tip of the iceberg. The human genome conceals no less than 15,000 or so pseudogenes, and it’s not unreasonable to think that many of them are relevant to diseases such as cancer.’ Karolinska Institutet

Researchers discover that the extra chromosome inherited in Down syndrome impairs learning and memory because it leads to low levels of SNX27 protein in the brain.

What is it about the extra chromosome inherited in Down syndrome—chromosome 21—that alters brain and body development? Researchers have new evidence that points to a protein called sorting nexin 27, or SNX27. SNX27 production is inhibited by a molecule encoded on chromosome 21. The study shows that SNX27 is reduced in human Down syndrome brains. The extra copy of chromosome 21 means a person with Down syndrome produces less SNX27 protein, which in turn disrupts brain function. What’s more, the researchers showed that restoring SNX27 in Down syndrome mice improves cognitive function and behaviour.

‘In the brain, SNX27 keeps certain receptors on the cell surface—receptors that are necessary for neurons to fire properly,’ said Huaxi Xu, Ph.D., Sanford-Burnham professor and senior author of the study. ‘So, in Down syndrome, we believe lack of SNX27 is at least partly to blame for developmental and cognitive defects.’
Xu and colleagues started out working with mice that lack one copy of the snx27 gene. They noticed that the mice were mostly normal, but showed some significant defects in learning and memory. So the team dug deeper to determine why SNX27 would have that effect. They found that SNX27 helps keep glutamate receptors on the cell surface in neurons. Neurons need glutamate receptors in order to function correctly. With less SNX27, these mice had fewer active glutamate receptors and thus impaired learning and memory.
Then the team got thinking about Down syndrome. The SNX27-deficient mice shared some characteristics with Down syndrome, so they took a look at human brains with the condition. This confirmed the clinical significance of their laboratory findings—humans with Down syndrome have significantly lower levels of SNX27.

Next, Xu and colleagues wondered how Down syndrome and low SNX27 are connected—could the extra chromosome 21 encode something that affects SNX27 levels? They suspected microRNAs, small pieces of genetic material that don’t code for protein, but instead influence the production of other genes. It turns out that chromosome 21 encodes one particular microRNA called miR-155. In human Down syndrome brains, the increase in miR-155 levels correlates almost perfectly with the decrease in SNX27.

Xu and his team concluded that, due to the extra chromosome 21 copy, the brains of people with Down syndrome produce extra miR-155, which by indirect means decreases SNX27 levels, in turn decreasing surface glutamate receptors. Through this mechanism, learning, memory, and behaviour are impaired.

If people with Down syndrome simply have too much miR-155 or not enough SNX27, could that be fixed? The team explored this possibility. They used a non-infectious virus as a delivery vehicle to introduce new human SNX27 in the brains of Down syndrome mice.

‘Everything goes back to normal after SNX27 treatment. It’s amazing—first we see the glutamate receptors come back, then memory deficit is repaired in our Down syndrome mice,’ said Xin Wang, a graduate student in Xu’s lab and first author of the study. ‘Gene therapy of this sort hasn’t really panned out in humans, however. So we’re now screening small molecules to look for some that might increase SNX27 production or function in the brain.’ Sanford-Burnham Institute