Brain repair after injury and Alzheimer’s disease

Researchers at Penn State University have developed an innovative technology to regenerate functional neurons after brain injury, and also in model systems used for research on Alzheimer’s disease. The scientists have used supporting cells of the central nervous system, glial cells, to regenerate healthy, functional neurons, which are critical for transmitting signals in the brain.

Gong Chen, a professor of biology, the Verne M. Willaman Chair in Life Sciences at Penn State, and the leader of the research team, calls the method a breakthrough in the long journey toward brain repair. “This technology may be developed into a new therapeutic treatment for traumatic brain and spinal cord injuries, stroke, Alzheimer’s disease, Parkinson’s disease, and other neurological disorders,” Chen said. The research will be posted online by the journal Cell Stem Cell on 19 December 2013.

When the brain is harmed by injury or disease, neurons often die or degenerate, but glial cells become more branched and numerous. These “reactive glial cells” initially build a defense system to prevent bacteria and toxins from invading healthy tissues, but this process eventually forms glial scars that limit the growth of healthy neurons. “A brain-injury site is like a car-crash site,” Chen explained. “Reactive glial cells are like police vehicles, ambulances, and fire trucks immediately rushing in to help — but these rescue vehicles can cause problems if too many of them get stuck at the scene. The problem with reactive glial cells is that they often stay at the injury site, forming a glial scar and preventing neurons from growing back into the injured areas,” he explained.

So several years ago, Chen’s lab tested new ways to transform glial scar tissue back to normal neural tissue. “There are more reactive glial cells and fewer functional neurons in the injury site,” Chen said, “so we hypothesized that we might be able to convert glial cells in the scar into functional neurons at the site of injury in the brain. This research was inspired by the Nobel prize-winning technology of induced pluripotent stem cells (iPSCs) developed in Shinya Yamanaka’s group, which showed how to reprogram skin cells into stem cells,” Chen recalled.

Chen and his team began by studying how reactive glial cells respond to a specific protein, NeuroD1, which is known to be important in the formation of nerve cells in the hippocampus area of adult brains. They hypothesized that expressing NeuroD1 protein into the reactive glial cells at the injury site might help to generate new neurons — just as it does in the hippocampus. To test this hypothesis, his team infected reactive glial cells with a retrovirus that specifies the genetic code for the NeuroD1 protein. “The retrovirus we used is replication-deficient and thus cannot kill infected cells like other viruses found in the wild,” Chen said. “More importantly, a retrovirus can infect only dividing cells such as reactive glial cells, but it does not affect neurons, which makes it ideal for therapeutic use with minimal side effect on normal brain functions.”

In a first test, Chen and his team investigated whether reactive glial cells can be converted into functional neurons after injecting NeuroD1 retrovirus into the cortex area of adult mice. The scientists found that two types of reactive glial cells — star-shaped astroglial cells and NG2 glial cells — were reprogrammed into neurons within one week after being infected with the NeuroD1 retrovirus. “Interestingly, the reactive astroglial cells were reprogrammed into excitatory neurons, whereas the NG2 cells were reprogrammed into both excitatory and inhibitory neurons, making it possible to achieve an excitation-inhibition balance in the brain after reprogramming,” Chen said. His lab also performed electrophysiological tests, which demonstrated that the new neurons converted by the NeuroD1 retrovirus could receive neurotransmitter signals from other nerve cells, suggesting that the newly converted neurons had successfully integrated into local neural circuits.

In a second test, Chen and his team used a transgenic-mouse model for Alzheimer’s disease, and demonstrated that reactive glial cells in the mouse’s diseased brain also can be converted into functional neurons. Furthermore, the team demonstrated that even in 14-month-old mice with Alzheimer’s disease — an age roughly equivalent to 60 years old for humans — injection of the NeuroD1 retrovirus into a mouse cortex can still induce a large number of newborn neurons reprogrammed from reactive glial cells. “Therefore, the conversion technology that we have demonstrated in the brains of mice potentially may be used to regenerate functional neurons in people with Alzheimer’s disease,” Chen said.

To ensure that the glial cell-to-neuron conversion method is not limited to rodent animals, Chen and his team further tested the method on cultured human glial cells. “Within 3 weeks after expression of the NeuroD1 protein, we saw in the microscope that human glial cells were reinventing themselves: they changed their shape from flat sheet-like glial cells into normal-looking neurons with axon and dendritic branches,” Chen said. The scientists further tested the function of these newly converted human neurons and found that, indeed, they were capable of both releasing and responding to neurotransmitters.

"Our dream is to develop this in vivo conversion method into a useful therapy to treat people suffering from neural injury or neurological disorders," Chen said. "Our passionate motivation for this research is the idea that an Alzheimer’s patient, who for a long time was not able to remember things, could start to have new memories after regenerating new neurons as a result of our in vivo conversion method, and that a stroke victim who could not even move his legs might start to walk again."

Dogs recognize familiar faces from images

So far the specialized skill for recognizing facial features holistically has been assumed to be a quality that only humans and possibly primates possess. Although it’s well known, that faces and eye contact play an important role in the communication between dogs and humans, this was the first study, where facial recognition of dogs was investigated with eye movement tracking.

Main focus on spontaneous behavior of dogs

Typically animals’ ability to discriminate different individuals has been studied by training the animals to discriminate photographs of familiar and strange individuals. The researchers, led by Professor Outi Vainio at the University of Helsinki, tested dogs’ spontaneous behavior towards images – if the dogs are not trained to recognize faces are they able to see faces in the images and do they naturally look at familiar and strange faces differently?

“Dogs were trained to lie still during the image presentation and to perform the task independently. Dogs seemed to experience the task rewarding, because they were very eager to participate” says professor Vainio. Dogs’ eye movements were measured while they watched facial images of familiar humans and dogs (e.g. dog’s owner and another dog from the same family) being displayed on the computer screen. As a comparison, the dogs were shown facial images from dogs and humans that the dogs had never met.

Dogs preferred faces of familiar conspecifics

The results indicate that dogs were able to perceive faces in the images. Dogs looked at images of dogs longer than images of humans, regardless of the familiarity of the faces presented in the images. This corresponds to a previous study by Professor Vainio’s research group, where it was found that dogs prefer viewing conspecific faces over human faces.

Dogs fixed their gaze more often on familiar faces and eyes rather than strange ones, i.e. dogs scanned familiar faces more thoroughly.

In addition, part of the images was presented in inverted forms i.e. upside-down. The inverted faces were presented because their physical properties correspond to normal upright facial images e.g. same colors, contrasts, shapes. It’s known that the human brain process upside-down images in a different way than normal facial images. Thus far, it had not been studied how dogs gaze at inverted or familiar faces. Dogs viewed upright faces as long as inverted faces, but they gazed more at the eye area of upright faces, just like humans.

This study shows that the gazing behavior of dogs is not only following the physical properties of images, but also the information presented in the image and its semantic meaning. Dogs are able to see faces in the images and they differentiate familiar and strange faces from each other. These results indicate that dogs might have facial recognition skills, similar to humans.

Brain Area Attacked by Alzheimer’s Links Learning and Rewards

One of the first areas of the brain to be attacked by Alzheimer’s disease is more active when the brain isn’t working very hard, and quiets down during the brain’s peak performance.

The question that Duke University graduate student Sarah Heilbronner wanted to resolve was whether this brain region, called the posterior cingulate cortex, or PCC, actively dampens cognitive performance, say by allowing the mind to wander, or is instead monitoring performance and trying to improve it when needed.

If the PCC were monitoring and improving performance, increased activity there would be the result of poor performance, not the cause of it.

The PCC connects to both learning and reward systems, Heilbronner said, and is a part of the “default mode network.” It lies along a mid-line between the ears, where many structures related to rewards can be found. “It’s kind of a nexus for multiple systems,” said Heilbronner, who is currently a postdoctoral researcher in neuroanatomy at the University of Rochester.

"As this area begins to deteriorate, people begin to show the early signs of cognitive decline — problems learning and remembering things, getting lost, trouble planning — that ultimately manifest as outright dementia," said Michael Platt, director of the Duke Institute for Brain Sciences, who supervised Heilbronner’s 2012 dissertation. Their findings appear Dec. 18 in the journal Neuron.

Heilbronner’s experiment to better understand the PCC’s role in learning and remembering relied on two rhesus macaque monkeys fitted with electrodes to read out the activity of individual neurons in their brains. Their task was akin to playing video games with their eyes. The monkeys were shown a series of photographs each day marked with dots at the upper left and lower right corners. To get a rewarding squirt of juice, they had to move their gaze to the correct target dot on a photo, and they learned by trial and error which dot would yield the reward for each photo.

Each day, they were shown up to 12 photos from an assortment of Heilbronner’s vacation snaps  at Yellowstone National Park and the Grand Canyon. Some of each day’s images were familiar with a known reward target, and others were new. As the monkeys responded with their gaze, the researchers watched the activity of dozens of neurons in each monkey’s brain immediately following correct and incorrect responses. They also altered the amount of juice dispensed in some cases, creating a sense of high-reward and low-reward answers.

If the PCC actively dampened performance, the researchers would expect to see it active before a choice is made or the feedback is received. Instead, they saw it working after the feedback, lasting sometimes until the next image was presented. Neurons in the PCC responded strongly when the monkeys needed to learn something new, especially when they made errors or didn’t earn enough reward to keep motivated.

The researchers also ran the task after administering a drug, muscimol, that impaired the function of the PCC temporarily during testing. With the center inactivated by the drug, the monkeys could recall earlier learning regardless of the size of the reward. Learning a new item was still possible when the reward was large, but the monkeys couldn’t learn anything new when rewards were small. “Maybe it didn’t seem worth it,” Heilbronner said.

The dampening experiment also reinforced what the researchers had seen in the timing of the PCC’s response. If this center’s role is to let the mind wander, performance should have improved when the muscimol was administered, but the opposite was true.

Heilbronner concludes that the PCC summons more resources for a challenging cognitive task. So rather than being the cause of poor performance on a task, PCC actually steps in during a challenge to improve the situation.

"This study tells us that a healthy PCC is required for monitoring performance and keeping motivated during learning, particularly when problems are challenging," Platt said.

Heilbronner  is now interested in finding out whether the PCC is more important to learning than it is to recall, and how motivation interacts with PCC abnormalities seen in Alzheimer’s disease.

Study provides new insights into cause of human neurodegenerative disease

A recent study led by scientists from the National University of Singapore (NUS) opens a possible new route for treatment of Spinal Muscular Atrophy (SMA), a devastating disease that is the most common genetic cause of infant death and also affects young adults. As there is currently no known cure for SMA, the new discovery gives a strong boost to the fight against SMA.

SMA is caused by deficiencies in the Survival Motor Neuron (SMN) gene. This gene controls the activity of various target genes. It has long been speculated that deregulation of some of these targets contributes to SMA, yet their identity remained unknown.

Using global genome analysis, the research team, led by Associate Professor Christoph Winkler of the Department of Biological Sciences at the NUS Faculty of Science and Dr Kelvin See, a former A*STAR graduate scholar in NUS who is currently a Research Fellow at the Genome Institute of Singapore (GIS), found that deficiency in the SMN gene impairs the function of the Neurexin2 gene. This in turn limits the neurotransmitter release required for the normal function of nerve cells. The degeneration of motor neurons in the spinal cord causes SMA. This is the first time that scientists establish an association between Neurexin2 and SMA.

Preliminary experimental data also showed that a restoration of Neurexin2 activity can partially recover neuron function in SMN deficient zebrafish. This indicates a possible new direction for therapy of neurodegeneration.

Collaborating with Assoc Prof Winkler and the NUS researchers are Dr S. Mathavan and his team at GIS, as well as researchers from the University of Wuerzburg in Germany. The breakthrough discovery was first published in scientific journal Human Molecular Genetics last month.

Small zebrafish provides insights into human neurodegenerative disease

SMA is a genetic disease that attacks a distinct type of nerve cells called motor neurons in the spinal cord. The disease has been found to be caused by a defect in the SMN gene, a widely used gene that is responsible for normal motor functions in the body.

To study how defects in SMN cause neuron degeneration, the scientists utilised a zebrafish model, as the small fish has a relatively simple nervous system that allows detailed imaging of neuron behaviour.

In laboratory experiments, the researchers showed when SMN activity in zebrafish was reduced to levels found in human SMA patients, Neurexin2 function was impaired. This novel disease mechanism was also discovered in other in vivo models, suggesting that it is applicable to mammals and possibly human patients.

When the scientists measured the activity of nerve cells in zebrafish using laser imaging, they found that nerve cells deficient for Neurexin2 or SMN could not be activated to the same level as healthy nerve cells. This impairment consequently led to the reduction of muscular activity. Interestingly, preliminary data showed that a restoration of Neurexin2 activity can partially recover neuron function in SMN deficient zebrafish.

Further studies

Assoc Prof Winkler, who is also with the NUS Centre for Biolmaging Sciences, explained, “These findings significantly advance our understanding of how the loss of SMN leads to neurodegeneration. A better understanding of these mechanisms will lead to novel therapeutic strategies that could aim at restoring and maintaining functions in deficient nerve cells of SMA patients.”

Dr See added, “Our study provides a link between SMN deficiency and its effects on a critical gene important for neuronal function. It would be interesting to perform follow up studies in clinical samples to further investigate the role of Neurexin2 in SMA pathophysiology.”

Moving forward, the team of scientists will conduct further research to determine if Neurexin2 is an exclusive mediator of SMN induced defects and hence can be used as a target for future drug designs. They hope their findings will contribute towards treatment of neurodegeneration.

Cells from the eye are inkjet printed for the first time

A group of researchers from the UK have used inkjet printing technology to successfully print cells taken from the eye for the very first time.

The breakthrough, which has been detailed in a paper published today, 18 December, in IOP Publishing’s journal Biofabrication, could lead to the production of artificial tissue grafts made from the variety of cells found in the human retina and may aid in the search to cure blindness.

At the moment the results are preliminary and provide proof-of-principle that an inkjet printer can be used to print two types of cells from the retina of adult rats―ganglion cells and glial cells. This is the first time the technology has been used successfully to print mature central nervous system cells and the results showed that printed cells remained healthy and retained their ability to survive and grow in culture.

Co-authors of the study Professor Keith Martin and Dr Barbara Lorber, from the John van Geest Centre for Brain Repair, University of Cambridge, said: “The loss of nerve cells in the retina is a feature of many blinding eye diseases. The retina is an exquisitely organised structure where the precise arrangement of cells in relation to one another is critical for effective visual function”.

“Our study has shown, for the first time, that cells derived from the mature central nervous system, the eye, can be printed using a piezoelectric inkjet printer. Although our results are preliminary and much more work is still required, the aim is to develop this technology for use in retinal repair in the future.”

The ability to arrange cells into highly defined patterns and structures has recently elevated the use of 3D printing in the biomedical sciences to create cell-based structures for use in regenerative medicine.

In their study, the researchers used a piezoelectric inkjet printer device that ejected the cells through a sub-millimetre diameter nozzle when a specific electrical pulse was applied. They also used high speed video technology to record the printing process with high resolution and optimised their procedures accordingly.

“In order for a fluid to print well from an inkjet print head, its properties, such as viscosity and surface tension, need to conform to a fairly narrow range of values. Adding cells to the fluid complicates its properties significantly,” commented Dr Wen-Kai Hsiao, another member of the team based at the Inkjet Research Centre in Cambridge.

Once printed, a number of tests were performed on each type of cell to see how many of the cells survived the process and how it affected their ability to survive and grow.

The cells derived from the retina of the rats were retinal ganglion cells, which transmit information from the eye to certain parts of the brain, and glial cells, which provide support and protection for neurons.

“We plan to extend this study to print other cells of the retina and to investigate if light-sensitive photoreceptors can be successfully printed using inkjet technology. In addition, we would like to further develop our printing process to be suitable for commercial, multi-nozzle print heads,” Professor Martin concluded.