PTEN deletion makes healthy, happy neurons. One more step along the path.

BY Steward Lab
Healthy happy neurons with PTEN deletion
Healthy, happy neurons with PTEN deletion. The image shows neurons that give rise to the corticospinal tract (CST) immunostained for a molecule that reflects activation of a growth-promoting pathway (the phosphorylated form of ribosomal protein S6).
We’ve been reporting regularly on our progress in the CST/PTEN project to develop interventions to regenerate nerve connections (axons) after spinal cord injury (SCI). The latest advance is described in a paper by Erin Gutilla, an M.D./Ph.D. student in Os Steward’s lab, reporting that long-term deletion of PTEN in neurons not only isn’t harmful; it causes neurons to grow and...well, look young again.

PTEN negatively regulates an intracellular signaling pathway called AKT/mTOR. In early development, the pathway is turned on by growth factors acting through receptors, which stimulates cell division and cell growth. As organisms mature to adulthood, PTEN turns on and shuts down AKT/mTOR. So, deleting PTEN takes the brakes off of the growth-promoting AKT/mTOR pathway, which enables nerve cells to regenerate their axons after spinal cord injury. In essence, what PTEN deletion seems to do is turn back the developmental clock to make neurons into early teenagers in a growth spurt.

Like almost every powerful therapy, however, there are potential concerns. Turning nerve cells into early teenagers primes them for a growth spurt, but as we all know, teenagers can be difficult. Parents sometimes may wonder whether an alien has taken over a teenager’s body. I could go on, but enough on that…

One concern was that deleting PTEN might increase the risk of tumors. PTEN is a tumor suppressor gene. Mutations in PTEN have been identified in many cancers, and mutating PTEN in early development can cause overgrowth of the brain, called macrocephaly. For these reasons, it was important to determine whether there were negative consequences of deleting PTEN in nerve cells in the way that was needed to induce regeneration.

In the study that just came out (Gutilla et al., 2016) we deleted PTEN in 1 day old mice and then examined their brains when they were up to 1.5 years old. The lifespan of mice is a bit over 2 years, so 1.5 years is the equivalent of retirement age. Importantly, there was no evidence of any neuropathology or tumors. Instead, the neurons lacking PTEN actually were larger and more healthy-looking than neurons in other parts of the brain (Figure), and looked like neurons in young adult mice. It was as if the aging clock had been turned back.

The lack of any detectable neuropathology due to PTEN deletion is very good news, suggesting that long-term deletion of PTEN selectively in neurons may have relatively low risk. Of course, there will need to be more safety testing but this is an encouraging assessment.

The broader implication of these findings is that targeting PTEN may be a way to reduce neuronal atrophy and death in neurodegenerative diseases like Parkinson’s disease, Alzheimer’s Disease, and ALS and even reverse “normal” age related deterioration of neurons. Erin Gutilla will be completing her dissertation research in the next year by exploring these questions.

Mark Tuszynski Awarded the Reeve-Irvine Research Medal

BY Oswald Steward
Mark Tuszynski awarded the Reeve-Irvine Research Medal
Dr. Oswald Steward (left) RIRC Director and James Swinden (right) of the Joan Irvine Smith & Athalie R. Clarke Foundation award Mark Tuszynski the Reeve-Irvine Research Medal.
The Reeve-Irvine Medal Symposium celebrated Mark Tuszynski as the recipient of the Reeve-Irvine Research Medal. In 1996 well known philanthropist Joan Irvine Smith established an annual award originally named the “Christopher Reeve Research Medal”, with Christopher’s blessing became the “Reeve-Irvine Research Medal”. The medal recognizes an individual who has made highly meritorious scientific contributions in the area of spinal cord repair, and whose research has stood the test of time and scrutiny. The medal and a $50,000 cash award is provided through the generosity of the Joan Irvine Smith and Athalie R. Clarke Foundation, whose kindness has made it possible to continue to recognize the work of pioneering investigators whose research has brought us closer to cures for afflictions affecting the spinal cord.

Mark Tuszynski is a physician-scientist exploring the topics of spinal cord injury, degenerative disorders of the nervous system, and fundamental mechanisms underlying learning and memory. Dr. Tuszynski obtained his bachelor of science and M.D. degrees at the University of Minnesota, and completed residency training in neurology at Cornell University Medical Center / The New York Hospital. He then earned a Ph.D. in neuroscience at the University of California-San Diego. He has been a faculty member at the University of California-San Diego since 1991, and is currently the Director of the Center for Neural Repair and Founding Director of the UCSD Translational Neuroscience Institute.

Dr. Tuszynski has published over 190 research articles and 3 books. The overarching goal of his research is to develop effective therapies for untreatable neurological disorders. Dr. Tuszynski performed the first human clinical trial of gene delivery in the adult central nervous system: Nerve Growth Factor gene therapy for has received 15 awards for his research. His research is supported by the NIH, the Veterans Administration and has received 15 awards for his research.

2015 Annual Symposium

Nervous System Regeneration: Molecular & Cellular Mechanisms

BY Oswald Steward
On November 5th the RIRC hosted the Reeve-Irvine Research Medal symposium titled “Nervous System Regeneration: Molecular and Cellular Mechanisms” focused in axonal regeneration, neural repair and plasticity. We brought in experts who have made significant contributions in these areas. Many of our speakers have been involved in pre-clinical trials that have great potential for clinical applications in the very near future. The target audience for the symposium consisted of MD’s and RN’s in Neurological Surgery, Emergency Medicine, Physical Medicine and Rehabilitation and Orthopedic Surgery. Additionally the target audience included Ph.D.’s in Neurology, Anatomy & Neurobiology, Bioengineering and Translational Sciences.

The objective of the symposium:
• Identify terminology related to axonal growth and the limitations of experimental methods.
• Determine the benefits and issues with the study of central nervous system regeneration.
• Describe the biological phenomena of regeneration and how this research can be applied to regeneration-based therapies.

Course Director

Dr. Oswald Steward
Dr. Oswald Steward, Ph.D.
Director, Reeve-Irvine Research Center
Professor, Anatomy and Neurobiology,
University of California, Irvine


Distinguished Speakers

Mark Tuszynski, M.D., Ph.D Mark Tuszynski, M.D., Ph.D
Director, Center for Neural Repair
Professor, Department of Neurosciences
University of California, San Diego
Regeneration after spinal cord injury

Simone di Giovanni, Ph.D Simone di Giovanni, Ph.D
Chair, Restorative Neuroscience
Brain Sciences
Imperial College London
Axonal injury signaling and epigenietic cross talk for nerve regeneration

Daniel Geschwind, M.D., Ph.D Daniel Geschwind, M.D., Ph.D
Gordon and Virginia McDonald, Distinguished Professor
Department of Neurology, Psychiatry and Human Genetics, University of California, Los Angeles
Imperial College London
Integrative genomic investigation of neural repair

Murray Blackmore, Ph.D Murray Blackmore, Ph.D
Assistant Professor
Department of Biomedical Sciences
Marquette University
Transcriptional interventions to promote axon regeneration in the injured spinal cord

James Fawcett, Ph.D James Fawcett, Ph.D
Head of Department of Neuroscience
John van Geest Centre for Brain Repair
University of Cambridge
How can we enhance the regerative ability of CNS axons?

Armin Blesch, Ph.D Armin Blesch, Ph.D
Stark Neuroscience Research Institute
Department of Neurological Surgery
Indiana University, School of Medicine
Neural activity, regeneration and pain in spinal cord injury

Paul Lu, Ph.D Paul Lu, Ph.D
Associate Research Scientist
Center for Neural Repair
University of California, San Diego
Long-term human neural stem cell transplant after spinal cord injury: neurogenesis, gliogenesis and axon persistence


RIRC undergraduate research stars present at UCI’s Undergraduate Research Opportunity Symposium

BY Dr. Oswald Steward
RIRC undergraduate research stars present at UCI’s
Undergraduate Research Opportunity Symposium
Undergraduate researchers are an important part of our RIRC team, and two of them presented posters on their research in the Undergraduate Research Opportunity Symposium on May 14. Sara Jahangiri has been working with Erin Gutilla on a study of whether it is possible to activate the AKT/mTOR pathway by electrical stimulation of the cortex. The AKT/mTOR pathway is the one activated by deleting PTEN, and the idea here is that it might be possible to enhance regeneration of cortical axons by stimulation. Walter Guerrero has been working with Zach Gallaher on a study demonstrating that deleting PTEN and another gene called SOCS3 enhances regeneration of peripheral nerve axons. Walter is graduating in June and is applying for graduate programs. Congratulations to Sara and Walter and their excellent mentors Erin Gutilla and Zach Gallaher.

The 2016 Meet the Scientists Forum...

Highlighted by Special Guests Dr. Zoran Nenadic and An Do

BY Dr. Oswald Steward
2016 Meet the Scientists Forum with guest speakers Drs. Zoran Nenadic and An Do
For 16 years the Reeve-Irvine Research Center (RIRC) has been holding an annual “Meet the Scientists” forum, where community members and the public can come to hear about the progress RIRC members are making in spinal cord injury (SCI) research. The goal of this forum is to help educate and inform the community on new discoveries relevant to SCI. It is also an opportunity for people living with SCI and their family members to talk directly with our researchers and clinicians. This event is also important to our researchers as it has helped us target research toward the needs of the spinal cord injury population. What many may not realize is that the forum initially stemmed from a special request from Christopher Reeve to meet with SCI researchers to hear scientific updates. After his passing the event has continued on and is now opened up to the public and has been a valuable exchange of information.

This year, Drs. Zoran Nenadic and An Do demonstrated their Brain Computer interface study, a novel brain-computer-interface (BCI) technology created by the University of California, Irvine researchers that has allowed a man with paraplegia to walk for a short distance, with body weight support. This study received much attention from the media. We were excited to have them here in the iMOVE laboratory to personally talk to our guests at the event. Dr. David Reinkesmeyer also demonstrated his music glove study to help improve hand and arm function.

Other Reeve-Irvine Faculty members were also present to discuss areas of research including regeneration of connections in the injured spinal cord (Dr. Os Steward), pain following SCI (Drs. David Luo and Catherine Cahill) and exciting new ways to manipulate molecules that block regeneration (Dr. Melanie Cocco).

Drs. Reinkensmeyer and Sharp are recruiting volunteers for this study. Click this link to see the ad for more information on enrolling as a participant.

Look for information on next year’s Meet the Scientists here on our website and be sure to join our Facebook page. For additional information contact Tania Jope at (949) 824-5925 or tania.jope@uci.edu

UCI Brain-Computer Interface Enables Paralyzed Man to Walk

Proof-of-concept study shows possibilities for mind-controlled technology.

BY iMove Lab
UCI Brain-Computer Interface Enables Paralyzed Man to Walk
A novel brain-computer interface technology created by University of California, Irvine researchers has allowed a paraplegic man to walk for a short distance.

In the preliminary proof-of-concept study, led by UCI biomedical engineer Zoran Nenadic and neurologist An Do, a person with complete paralysis in both legs due to spinal cord injury was able – for the first time – to take steps without relying on manually controlled robotic limbs.

The male participant, whose legs had been paralyzed for five years, walked along a 12-foot course using an electroencephalogram-based system that lets the brain bypass the spinal cord to send messages to the legs. It takes electrical signals from the subject’s brain, processes them through a computer algorithm, and fires them off to electrodes placed around the knees that trigger movement in the leg muscles.

Study results appear in the open-access Journal of NeuroEngineering & Rehabilitation. See the video at news.uci.edu/feature/to-walk-again/. Even after years of paralysis, the brain can still generate robust brain waves that can be harnessed to enable basic walking, said Nenadic, an associate professor of biomedical engineering. We showed that you can restore intuitive, brain-controlled walking after a complete spinal cord injury. This noninvasive system for leg muscle stimulation is a promising method and is an advance of our current brain-controlled systems that use virtual reality or a robotic exoskeleton.

Months of mental training to reactivate the brain’s walking ability and physical therapy were needed for the study participant to reach the stage where he could take steps. Wearing an EEG cap to read his brain waves, he was first asked to think about moving his legs. The brain waves this created were processed through a computer algorithm Nenadic had formulated to isolate those related to leg movement. The subject later was trained to control an avatar in a virtual reality environment, which validated the specific brain wave signals produced by the algorithm.
“We showed that you can restore intuitive, brain-controlled walking after a complete spinal cord injury.” -Zoran Nenadic
This training process yielded a custom-made system, Nenadic said, so that when the participant sought to initiate leg movement, the computer algorithm could process the brain waves into signals that could stimulate his leg muscles.

To make this work, the subject required extensive physical therapy to recondition and strengthen his leg muscles. Then, with the EEG cap on, he practiced walking while suspended 5 centimeters above the floor, so he could freely move his legs without having to support himself. Finally, he translated these skills to the ground, wearing a body-weight support system and pausing to prevent falls. Since this proof-of-concept study involved a single patient, Do said, further research is needed to establish whether the results can be duplicated in a larger population of individuals with paraplegia.

Once we’ve confirmed the usability of this noninvasive system, we can look into invasive means, such as brain implants, said Do, an assistant clinical professor of neurology. We hope that an implant could achieve an even greater level of prosthesis control because brain waves are recorded with higher quality. In addition, such an implant could deliver sensation back to the brain, enabling the user to feel his legs.

Christine King, Po Wang, Colin McCrimmon and Cathy Chou of UCI contributed to the study, which received support from the National Science Foundation (grant 1160200).

Trials and Tribulations: Update on Stem Cell Trials for Spinal Cord Injury

BY Dr. Oswald Steward
Recent news for stem cell trials for spinal cord injury is decidedly mixed. On the one hand, the trials have proceeded without any reports of serious adverse effects, and there have been encouraging reports of improvements. On the other, it’s sad to report that the “Pathway” trial by the company Stem Cells Inc. has been terminated and the company announced it would wind down its operations. Also, another company sponsoring a stem cell trial for SCI (Neuralstem) announced a strategic reorganization to re-focus its priorities on small molecule therapies.

As background, most readers of “Spinal Connections” know there have been 3 ongoing trials of stem cell therapies for spinal cord injury. The first to be launched was the trial of oligodendrocyte precursor cells (OPCs) by the company Geron. The OPC trial was based on research by Hans Keirstead at the RIRC. Geron discontinued the trial but it was re-launched by the company Asterias. Next was the trial by the company “Stem Cells Inc,” which began in Switzerland, and was later extended to the United States and Canada. This trial involved transplantation of neural stem cells (NSCs) that differentiate into nerve cells, and glial cells. The foundation for the trial was research by Aileen Anderson and Brian Cummings at the RIRC. A third trial was launched by the company “Neuralstem” and is being run out of the University of California San Diego. This is an extension to spinal cord injury of an approach initially tested for amyotrophic lateral sclerosis (ALS). This trial involves a different type of proprietary neural stem cell. The OPC product of Asterias is derived by differentiating human embryonic stem cells (HESCs), whereas both of the NSC products were originally derived from cells harvested from aborted human fetuses.

The Asterias “SCiStar” trial-OPCs: In the Phase 1 clinical trial launched by Geron, five patients with neurologically complete, thoracic spinal cord injury received two million AST-OPC1 cells at the spinal cord injury site 7-14 days post-injury. Based on the lack of serious adverse events in this safety trial, Asterias launched the SCiStar trial, which will test three sequential escalating doses of “AST-OPC1” cells (20 million cells in the final cohort). Subjects are individuals who have suffered SCI at cervical level 5-7 and are neurologically complete (Asia A). Subjects will receive OPC transplants 14 to 30 days post-injury and will be followed by neurological exams and imaging procedures. A news release in October, 2015 announced a positive safety profile and absence of serious adverse events for the first 3 subjects who received the low dose, so the next cohort of 5 subjects will receive 10 million cells. According to the press release, the first patient who received OPCs at Shepherd Center in Atlanta exhibited neurological improvement from ASIA Impairment Scale (AIS) A to an AIS C at the 3 month assessment.

The Stem Cells Inc “Pathway Study”, NSCs: This was a trial involving the company’s proprietary human NSC line “HuCNS-SC®” for subjects with cervical SCI. Things seemed to be going well for this trial earlier in the spring of 2016. At the 2016 American Spinal Injury Association (ASIA) annual meeting in Philadelphia in April, Dr. Stephen Huhn, Chief Medical Officer and VP of Clinical Research at Stem Cells Inc. gave an update. The 6-month results from Cohort I (6 subjects) revealed improved muscle strength in 5/6 subjects, improved dexterity and fine motor skill in 4/6 patients and no serious side effects. On this basis, Cohort II had been launched, which was to be a randomized, single-blinded study of 40 AIS-B subjects. By late May, 11 subjects had received transplants in Cohort II.

But then suddenly on May 31 Stem Cells Inc. announced that it would terminate the study and close out operations. The 6,9, and 12-month results from Cohort I revealed encouraging patterns of improvements, but the effect was smaller at the 12 month time point. Because of this, the Company conducted an interim analysis of data from Cohort II. There were differences in motor strength that favored the treatment group, but the effect size was small and the company concluded that the study would be unlikely to achieve the primary endpoint objective.

For perspective regarding the issue of “effect size”, it’s important to emphasize the difference between preclinical studies in animals and human clinical trials. In most studies with animals, scientists make every effort to be sure that injuries are comparable and everything except the treatment is similar between groups. This practice of “minimizing variability” is so that it is possible to detect treatment effects. The problem is that human SCIs are highly variable, so if “effect size” is small, it’s harder to achieve a statistically significant result in a clinical trial.

Of course there is major disappointment that the “Pathway” trial is terminated. There will be a lot to learn from the Stem Cells Inc. experience, both in terms of the science and the economics of developing therapies for SCI. This is another practical lesson that limited financial resources make it difficult for small companies to continue when it becomes clear that the path to a therapy is longer than expected.

Neuralstem NSI-566/cSCI: The trial by Neuralstem is a phase I trial in which 5 subjects received six injections in, or around, the injury site, using the same cells and similar procedure as the company’s ALS trials. Subjects also receive physical therapy and immunosuppressive therapy. The 5th subject received the cells in July 2015. At the time of this writing, there are no updates on the results of the Neuralstem trial except that there have been no reports of adverse events.

However, on May 20, 2016 Neuralstem announced a “Plan of reorganization to further align business with strategic intent”. According to the press release: “The company's refocused strategy emphasizes its commitment to prioritize the small molecule platform, undertake business development efforts to secure alternative funding and partnerships for its stem cell assets.... Accordingly, the corporate reorganization includes a workforce reduction across all divisions that will result in significantly lower operating expenses while retaining the expertise needed to implement the company's refocused strategy”. It remains to be seen how this reorganization will affect any further trials of Neuralstem’s product for spinal cord injury.

CST Regeneration in the Chronic Injury Setting

BY Steward Lab
CST Regeneration
Most of our readers know that the "Corticospinal Tract Regeneration Project" has been the focus of the Steward Research Group for years, and for the last 5 years has involved a multi-investigator collaboration that has been following up on a paper the team published in 2010. The original paper, published in Nature Neuroscience, showed that it was possible to induce regeneration of the corticospinal tract (CST) following spinal cord injury by deleting a gene called phosphatase and tensin homolog (PTEN). The latest papers from the Steward lab, published in 2014 and earlier this year, showed that CST regeneration was accompanied by recovery of motor function. (See next article: PTEN Deletion Post SCI Regeneration and Recovery by Sam Maddox)

In a new breakthrough, our collaborator Dr. Kai Liu, who was first author on the Nature Neuroscience paper and now has his own lab at Hong Kong University, published a very important paper showing that is possible to induce CST regeneration in the chronic setting either 4 months or 1 year after a spinal cord injury!
the Journal of Neuroscience The paper appeared in the July issue of the flagship journal of our field, the Journal of Neuroscience, and was featured on the cover. In the study, Dr. Liu used the same genetic deletion strategy that was used the original paper. Mice received spinal cord injuries and then 1 year later, received an injection of a viral -based vector into their cortex, which produces a protein that deletes PTEN from the nerve cells that give rise to the CST. After deleting PTEN, CST axons regenerated past the injury and formed new connections. The discovery that regenerative ability can be rebooted even one year after an injury greatly extends the window of opportunity for regenerating connections in the injured spinal cord. Next steps will be to determine if the regeneration is accompanied by reversal of paralysis.

PTEN Deletion Post SCI Regeneration and Recovery

BY Sam Maddox
Over the last few years we have written several times about a nifty genetic manipulation that reboots key nerves in the spinal cord to regenerate in a big way. Starting back in 2008, Zhigang He, at Harvard, got damaged optic nerves going again by deleting PTEN, a tumor suppressor gene that cancer researchers had come across back in the 1990s; this enzyme acts as a sort of brake when axons attempt to regenerate after injury.

In the world of cancer, of course, the idea is to keep the growth brake on; in spinal cord repair, it's just the opposite. Anyway, in later experiments, when PTEN was removed, the corticospinal tract (CST) regenerated in lab animals - important because the CST comprises crucial wiring for arm and hand function.

The most recent research, from Camilla Danilov and Oswald Steward at the Reeve-Irvine Research Center at the University of California, Irvine, takes a big step toward clinical relevance. In all previous experiments, animals used in the tests had PTEN deleted before they were spinal cord injured. This time, they took the PTEN brakes off after animals had been paralyzed.

The paper, out in the journal Experimental Neurology, is titled "Conditional genetic deletion of PTEN after a spinal cord injury enhances regenerative growth of CST axons and motor function recovery in mice."
Improved recovery of hand function in rats due to PTEN deletion: red=animals with PTEN deletion; blue and green=two untreated control groups
From the abstract: Previous studies indicate that conditional genetic deletion of phosphatase and tensin homolog (PTEN) in neonatal mice enhances the ability ofaxons to regenerate following spinal cord injury (SCI) in adults. Here, we assessed whether deleting PTEN in adult neurons post-SCI is also effective, and whether enhanced regenerative growth is accompanied by enhanced recovery of voluntary motor function. These results indicate that PTEN deletion in adult mice shortly post-SCI can enhance regenerative growth of CST axons and forelimb motor function recovery.

The new study used a gene delivery vector to delete PTEN 20 minutes after a moderate contusion injury at C5. The animals without PTEN could grip and grasp much better than control (injured but untreated) animals.

Why this injury model? From the paper: ... the injury model used in this study was a moderate cervical contusion at C5 centered on the midline of the spinal cord that produced bilateral tissue damage and bilateral function deficits. We chose this injury model for its human relevance. More than 50 percent of spinal cord injuries are at the cervical level, impairing both lower and upper extremities and the most common type of injury in humans is the contusive type. Another thing the scientists wanted to know is if this technique would give the CST axons a boost. The answer is yes, although for reasons that are not clear, CST growth is less than in previous experiments. From the paper: The regenerative growth seen here resembles what has been previously reported following spinal cord injury ... The extent of the regenerative growth appears less extensive, however, although direct comparisons are difficult because the site and nature of the injury are different (C5 contusion vs. T8 dorsal hemisection or crush). Further studies will be required to address this issue.

In conclusion, the present study demonstrates enhanced recovery of forepaw gripping and grasping function and enhanced regenerative growth of injured CST axons with conditional genetic deletion of PTEN in adult mice shortly after a spinal cord injury. These results suggest that manipulations of PTEN or the downstream mTOR pathway may be a viable target for therapeutic interventions to promote axon regeneration after spinal cord injury.

Induced Pluripotent Stem Cells: A recent discovery that could lead to personalized medicine for SCI

BY Steward Lab
IPS cells have the potential to make any cell in the adult body, including the cells that make up the spinal cord and brain.
My name is Joe Bonner and I am a postdoctoral fellow in the lab of Dr. Steward at the Reeve-Irvine Research Center and I study the use of human neural stem cells for the treatment of spinal cord injury and my work is currently funded by the California Institute for Regenerative Medicine (CIRM) through a training grant administered at the Bill and Sue Gross Stem Cell Center here at UCI. Stem cells are a special type of cell that have the potential to repair tissue by replacing cells that were lost due to spinal cord injury. Stem cells are present in the brain and spinal cord and they contribute to the normal function of the nervous system throughout life. In the event of a spinal cord injury, resident stem cells respond and make an attempt to repair the damage but there are simply not enough stem cells to fix such serious injuries. In my research, I transplant neural stem cells in an effort to improve tissue repair and to restore the neural connections between the brain and the body. In addition to transplants of stem cells I also administer "growth factors" to improve the function of the transplanted stem cells and at the same time I also manipulate the function of the PTEN/mTOR pathway, which Dr. Steward has previously shown can give the injured nerve cells in the spinal cord a regenerative boost. The goal of my work is to understand how stem cells can contribute to the repair of spinal cord injury and to ultimately create a treatment for people who have suffered a spinal cord injury.

The human neural stem cells that I use in my research are made from a relatively new kind of stem cell called induced pluripotent stem cells, or IPS cells. IPS cells were first described in 2006 by Shinya Yamanaka, who won the Nobel Prize for the discovery in 2012. In the short time IPS cells have been around scientists have begun to understand how they could be applied to treat illness and injury. IPS cells have many characteristics that make them an interesting resource in our search for a treatment for spinal cord injury.
Induced Pluripotent Stem Cells: A Recent Discovery that Could Lead to Personalized Medicine for SCI
IPS cells are made by "reprogramming" or "inducing" an adult cell to become a stem cell. This means that stem cell scientists are able to take a common cell found in an adult, such as a skin cell, and to change that skin cell into a stem cell. This procedure eliminates many of the difficulties that are associated with embryonic stem cells while producing a stem cell with many of the same characteristics. The most intriguing benefit of IPS cells is that they could be made for each individual patient. If an IPS cell therapy was designed to use a patient's own cells then there would be no need for the patient to take additional medications to prevent an immune reaction or transplant rejection.

IPS cells have the potential to make any cell in the adult body, including the cells that make up the spinal cord and brain. IPS cells can be grown for a very long time in a culture dish. This means that cells can be expanded from a small number into enough cells to conduct experiments and to eventually treat human patients. Before IPS cells were discovered, the only type of cell that had these characteristics were embryonic stem cells. Although embryonic stem cells (ESCs) may prove useful for treatment of spinal cord injury, ECS's are not "personalized" so patients who receive ESC transplants have to take immunosuppressant drugs to prevent rejection.

I have been ableto demonstrate that human neural stem cells derived from IPS cells can survive in the injured spinal cord and make nerve cells (neurons) and support cells (glia) after transplantation. My continuing research seeks to determine if these transplanted stem cells can communicate with injured nerve cells in the injured spinal cord and if those lines of communication can improve the recovery of function in an animal model of spinal cord injury.

Chronic Pain Causes Brain Inflammation. Why Should You Care?

BY Cahill Lab
Chronic Pain Causes Brain Inflammation
Research Findings: Pain is a multidimensional experience, and how much the pain is 'bothersome' significantly impacts the quality of life of the sufferer. In fact, the emotional component of pain has been argued to be a great metric in measurements of quality of life. There is extensive interaction between systems involved in pain processing and brain regions responsible for mood. Notably, chronic pain co-exists with depression, which is thought to involve imbalance in a brain system that uses the neurochemical dopamine. Dr. Cahill recently published a paper in Journal of Neuroscience (available as a free open access article on their website at The Journal of Neuroscience ) that neuro-inflammation, as the result of ongoing pain caused by nerve damage (neuropathic pain), occurs in brain regions important for reward and mood. Reward circuitry is important for how we feel (mood), our motivation, and our ability to feel pleasure. Her research team identified that non-neuronal brain cells, called microglia, alter brain activity important for producing reward. This research identifies a pathway from microglia to neurons that causes suppression of the neurochemical dopamine and thus likely contributes to many of the devastating sequelae of chronic pain, including the poor response many pain patients have to their opioid prescription medications, debilitating affective disorders, and substance abuse (Figure 1). Her research is supported by the recent clinical trial that reported minocycline, a microglial inhibitor, did not alter pain intensity, but diminished how bothersome pain is in patients with neuropathic pain. This publication was recently picked up by multiple media sites including the OC Register (Wednesday, June 17th) as well as various websites including: MD magazine, Free Press Joumal, Medical News Today, Neurology Advisor and Psychiatry Advisor.

Why is this research important? Approximately, 20-25% of Americans suffer from chronic pain making it the most common form of chronic illness under the age of 60. The Institute of Medicine estimates that it costs our society $635 billion spent per year. Treating non-cancer chronic pain is a challenge for physicians because many patients do not respond to typical analgesics, such as ibuprofen-like drugs or opioids such as morphine. Additionally, treating patients with opioids long term has been come controversial because of the high incidence of addiction to pain medications, and there are studies questioning the effectiveness of opioids for treating certain types of chronic pain, especially those of nerve injury origin. Dr. Cahill's study imposes a paradigm shift in the field of chronic pain because it identifies that chronic pain can cause neuro-inflammation in brain regions important for reward experiences. The work identifies novel signaling and biophysical mechanisms by which microglia alter neuron activity in reward circuits, important for mood, motivation, and pleasure. The impact of our research is not restricted to the problem of pain, but is broadly relevant to mood disorders such as depression and substance abuse. Both of these medical conditions are associated with disruption of reward circuitry. Chronic pain patients are more than twice as likely to suffer anxiety and depression, and co-existence of pain with mood disorders is reported to be 30-100% depending on the causes of chronic pain. Importantly, chronic pain is second only to bipolar disorder as the major cause of suicide among all medical illnesses. Dr. Cahill's study suggests that treatments that target neuroinflammation will be an important novel therapy to alleviate many of the troubling emotional sequelae of chronic pain. Our study also has implications for why some chronic pain patients become addicted to their opioid analgesics used for managing chronic pain.

Next steps: There are many directions for this research. First, we would like to establish that the circuitry changes account for affective-like behaviors. We have a novel compound from Dr. Yves De Koninck that has the potential to restore function and normalize reward-like behavior. Once these compounds get approval for clinical trials, we will test whether restoring reward function is therapeutic in a chronic pain population. We would also like to follow this up with clinical research pursuing imaging studies to identify whether chronic pain patients also show the same disruption in reward circuitry.

Take home message: Chronic pain is a devastating disease that negatively affects a person's quality of life. Not only do patients have to live with pain everyday; they also commonly suffer from depression. Our studies show that chronic pain changes your brain in areas important for mood. Importantly, we provide proof that the prevalence of depression in chronic pain patients is likely an organic disease and is not psychological. The work indicates that changes in the brain caused by pain likely cause this mood disorder; depression is not simply a consequence of their state of mind from having to live with pain. We have identified multiple proteins thet can be targeted for translational medicine to determine their effectiveness in improving quality of life for those suffering from chronic pain, as well as potentially for Major Depressive Disorders. Our research identifies a pathway that likely contributes to many of the devastating sequelae of chronic pain, which include the poor therapeutic response of oploid analgesics in a subset of chronic pain patients, debilitating affective disorders and substance abuse.

The Legacy of Frank Freed

Fellowship
The Legacy of Frank Freed
It is difficult to say goodbye to our dear friend and supporter of our Center. On April 22, 2015 Frank Holston Freed who was born on September 17, 1924 passed away due to congestive heart failure. He is survived by his wife Evelyn Freed who has generously supported the RIRC since its inception. Frank enlisted in the Army in 1942 and was a soldier in World War II losing his arm in France. Frank graduated from Wheaton College in 1948 with a BA Degree in Philosophy and from Fuller Theological Seminary with a BD Degree in Theology. He was the pastor of Bethany Baptist Church in West Covina, California from 1951-1957. He received an MA Degree in History from Stanford University in 1958, and was a founding Pastor of Valley Church in Cupertino, California from 1959-1969. Frank received a Secondary Teaching Credential in History at USC in California in 1970, and then went to Fuller Theological Seminary School of Psychology where he earned a Ph.D. Degree in Psychology in 1974. Frank was a Clinical Psychologist in private practice in Orange County, ending his career as Director of Counseling at the Crystal Cathedral for ten years until his retirement. In 1997 Harold Shaw Publishers published Frank's book "Breaking Free When You're Feeling Trapped". He also published another book titled "Breaking Free" about bettering your life through attitude. He was credited with making a positive impact on many people's lives with his loving spirit and educational counsel. The generosity and support that Evelyn and Frank have given to our Center over the years has made a lasting impact on spinal cord injury research efforts. Frank has left a very large footprint and will be sorely missed by us all at the Reeve-Irvine Research Center.
FRANK FREED MEMORY FELLOWSHIP: Please consider helping us with a donation to establish the Frank Freed Memory Fellowship. The name is a double entendre; the fellowship is to honor Frank Freed via a fellowship for research on memory.
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Using Stem Cells to Improve Bladder Function After a Low Spinal Cord Injury

BY Havton Laboratory
Using Stem Cells to Improve Bladder Function after a Low Spinal Cord Injury
Stem cells have the potential to become any cell of the body and after a spinal cord injury, researchers have used them to replace everything from the supporting cells of the nervous system to the actual cells that transmit information from the brain to different parts of the body. The Havton lab at the Reeve-Irvine Research Center and the Sue & Bill Gross Stem Cell research Center at UC Irvine is utilizing stem cells to replace cells located in the low lumbar and sacral regions of the spinal cord that are injured after a trauma to the lumbosacral spinal cord and nerve roots. Physicians often refer to these injuries as conus medullaris or cauda equina injuries. After a low spinal cord injury the bladder may become underactive because spinal cord motor neurons that normally signal the bladder to contract are either dead or dying. As a result, the bladder tends to fill up more than it should, because the affected individual is unable to sense the fullness of the bladder or empty the bladder voluntarily. Such over-filling of the bladder can lead to urinary tract infections, kidney disease or overflow incontinence. Ongoing research in the Havton lab focuses on the nerve cells in the spinal cord that normally contract the bladder and cause voiding. For this purpose, stem cells are used in laboratory models to replace the injured and lost motor neurons. The goal of the study is for cell replacement therapy to improve bladder function and the quality of life of people living with a low spinal cord injury.
In collaboration with Drs. Harley Kornblum and Bennett Novitch at UCLA, the Havton lab has developed a method to convert stem cells into a mix of motor neurons and support cells in the dish and transplant the cells into the spinal cord of rats. Ongoing studies are using a cauda equina injury model and the cell transplantation strategies to determine feasibility of this approach to reverse functional deficits of the bladder. Special attention is paid to see whether the transplanted cells may survive over long periods of time after transplantation and reconnect with the peripheral targets in the pelvis to restore bladder function. The collaborative and translational studies have received support from California Institute of Regenerative Medicine (CIRM). Dr. Arthi Amin, a post-doctoral fellow in the Havton lab, will present results from this ongoing study in November at the annual meeting for the Society for Neuroscience in Washington, D.C.

Chronic Pain Changes Your Brain in Areas Important for Reward and Emotion

BY Cahill Lab
Chronic Pain Changes Your Brain In Areas Important For Reward And Emotion
The most effective class of drugs available for treating moderate to severe pain is opioid drugs, such as morphine, hydrocodone (Vicodin®) and oxycodone. Opioids have been used (and abused) for centuries with uses ranging from religious rituals to treatment of dysentery, however, their prominent use in treating moderate to severe pain in modern medicine will undoubtedly continue for the foreseeable future, due to the lack of alternative choices. Most experts would agree that despite the fears of addiction and the plethora of side effects that may limit use, opioid drugs are superior analgesics for the treatment of post-operative pain, traumatic injury-related pain, and cancer pain. However, their long-term use in management in chronic non-malignant pain is now being challenged, where concerns about safety and efficacy are debated.
Over the past 2 decades there has been a tremendous effort by basic scientists, biotechnology companies and the pharmaceutical industry to develop new drugs to alleviate pain. Various drug candidates made it from preclinical testing to clinical trials, only to have failed and consequently many companies have now abandoned their pain programs. Those new drugs that have made it to market are primarily restricted to spinal delivery, limited to use in terminal cancer patients, or are based on success of drugs with similar chemical structure (e.g. pregabalin, which was developed after the success of off-label use of gabapentin which rose to over 1 billion annual sales in the USA). Some analysts blame the lack of drug development for pain drugs in the trial design and/or the patient cohort (type of pain patient recruited for the trial). However, there is also a debate about whether the preclinical testing can capture pain.
Pain is a multidimensional experience comprised of sensory, cognitive, and affective (emotional) components, which are processed within discrete but interacting brain structures. It is well accepted that many chronic pain states, including those that result from spinal cord injury, are accompanied by dramatic sensory disturbances that result in pain hypersensitivity (allodynia – painful experience to something normally not painful and hyperalgesia – exaggerated response to something normally painful) and tonic (unprovoked) ongoing pain. Preclinical animal pain model testing captures the sensory component by measuring the time to respond to a painful stimulus. However, the emotional affective component, or how much the pain is ‘bothersome’ or unpleasant significantly impacts the quality of life of the sufferer. Most animal models of chronic pain typically rely on sensory/threshold measures of pain, but the emotional component of pain has been argued to be a greater measure of quality of life than its sensory component, and there is now a concerted effort to develop assays to capture this aspect of pain with the goal of better predictability of drug candidates. Capturing this aspect of pain is a challenge because you can’t ask a mouse or rat how it feels.
Future directions - How do we proceed? There is fascinating new research indicating that the “bothersome” unpleasant component of chronic pain engages parts of the brain called the limbic system. Within the limbic system are brain regions important for being able to experience something we like (rewarding) or something we try to avoid (aversion). The key point is that pain and reward are opposite processes, but are processed within overlapping or interacting brain structures - see Figure 1. This is important because stimuli that are rewarding such as food and pleasurable music DECREASE pain, and conversely pain can impair reward processing (pleasure). So chronic pain can cause an “anhedonic state” in which a person can’t experience pleasure from activities usually found enjoyable (e.g., exercise, hobbies, music, sexual activities or social interactions). In fact, chronic pain is second only to bipolar disorder as the major cause of suicide among all medical illnesses, further highlighting how devastating this condition is. The interplay between reward pathways and pain validate the importance of these brain areas, not only in why acute pain becomes chronic, but also the minimal effectiveness of opioid analgesics in treating many types of chronic pain (including that of neuropathic origin).

On-going Studies: Dr. Cahill and her research team recently identified that there is dysfunction of reward circuitry in an animal model of neuropathic pain. So, pain changes reward circuits. Reward (pleasure) decreases pain perception, so disrupting reward circuits may increase pain because it makes it more unpleasant. This is a major discovery, and the Cahill lab is now following up with research aimed at understanding the mechanisms responsible for the dysfunction of brain circuitry involved in emotion and reward in models of chronic pain. They are also preparing a proposal to seek pilot data for a clinical study aimed at novel therapies that will alleviate the emotional, affective component of pain. This is early stage research—the follow up to a fundamental discovery, so additional data are needed before a clinical study can be launched. The Cahill lab is seeking federal funding, but this is where private contributions could make a HUGE difference, allowing these scientists to collect the critical preliminary data that would allow a clinical study of novel therapies to address chronic pain.

News Reports of Recovery After Transplants of Olfactory Cells with Peripheral Nerve Bridges

BY Dr. Oswald Steward
Dr. Oswald Steward
Many of you will have seen news stories reporting functional regeneration of connections that enable movement in a man that received transplants of olfactory cells. This approach is a clinical realization of decades of research by a scientist who received our Reeve-Irvine Research Medal in 2005 (Geoff Raisman). The findings are provocative, and the overall study reflects a huge effort by a skilled team of researchers and medical doctors. Unfortunately, though, the situation is not as simple as implied in most of the news stories. So, what is this all about, and what does it mean for those of you living with paralysis?

First the science: the overall approach builds upon years of research on regeneration of nerve connections from the nose to the brain. The ability to smell is mediated by nerve cells in the nose called olfactory receptors, which are embedded in a structure called the nasal mucosa, which is made up of receptor cells and mucosal cells. The olfactory receptors respond to chemicals in the air, and communicate via connections (axons) that project into the brain. The olfactory receptors in the nose turn over throughout life (that is, the cells die and others are born), which is different than almost any other part of the nervous system. When new cells are born, they have to grow their axons from the nasal mucosa in the nose into the olfactory bulbs, which are inside the skull. The olfactory bulb is connected to the brain by the olfactory nerve. To grow from the olfactory mucosa to the brain, the axons grow through the bony structure between the nose and the brain called the cribriform plate, and there is lots of evidence that growth is enabled by a special population of glial cells there called olfactory ensheathing cells (OECs). Dr. Raisman and other scientists have provided strong experimental evidence that OECs have special properties in terms of supporting axon growth, and many scientists have tried different ways of transplanting either OECs or the entire nasal mucosa into the injured spinal cord to promote regeneration.

Based on the studies in animals, quite a few people throughout the world have received a highly experimental therapy involving transplants of olfactory mucosa into the spinal cord. This experimental therapy is not available in the United States, but has been offered in Portugal and elsewhere, and some Americans have traveled to other countries to receive the transplants. These weren’t clinical trials, however; they were experimental therapies without controls or followup testing, so there’s no scientific data on outcomes. Also, there are concerns about the approach of transplanting olfactory mucosa because of the recent finding of a tumor that developed in the spinal cord in a patient that received such a transplant (see our Spring 2014 newsletter).

It’s important to emphasize that this new study involves cells that were isolated from the olfactory mucosa, not the olfactory mucosa itself, so the risk of tumor formation is hopefully less. Another good thing about this new study is that it was actually a clinical trial with an experimental and control group with extensive pre- and post-operative testing, so there is a lot of data. The patients were a total of 6 men between the ages of 22 and 26; 3 patients received the treatment, 3 did not, and all were tested extensively. The study reported outcomes 1 year after cell transplantation. The treatment in this new study, which was carried out in Warsaw Poland, was to transplant OECs that were harvested from the patient (autologous transplants) along with small pieces of peripheral nerves that were inserted to provide a bridge across the injury site. The first step was a complicated surgery to remove the olfactory mucosa.

The olfactory mucosa was then dissociated (meaning broken up so that individual cells are in a mixture—imagine rice mixed with water) and the cells were transferred to a tissue culture dish for several days. The cells were characterized extensively while growing in culture, and at the end of the cell culture period contained OECs as well as other cell types. Then, each patient received another surgical procedure to open the spine and visualize the spinal cord so that the transplant could be done. Then, there was a lot of rehabilitation and testing.

Of the 3 people who received transplants, 2 exhibited improved function on the ASIA scale, the third did not improve on the ASIA scale, but did experience some improvement in sensation just below the injury. The main recovery in all patients was between 6 and 12 months post-transplant. For the man who was featured in the news reports who exhibited the greatest recovery, the first signs of recovery were 6 months following the treatment, and involved tingling sensations below the injury. By 8 months, the patient recovered some ability to feel touch below the injury. By 1 year, the patient had …a slight voluntary flexion of the right hip which qualified for conversion of the ASIA grade from A to C.

It’s important to emphasize how the authors summarized the results: … patients who underwent the operation of OEC transplantation combined with intense pre- and postoperative neurorehabilitation showed modest neurological signs of recovery. This highlights the fact that was a carefully done and carefully interpreted study with a lot of details, although even with that, some of the claims are provocative. The title of the article claims “functional regeneration” (meaning growth of axons across the injury site). This claim was based largely on neurophysiological studies involving stimulation and recording of muscle responses (motor evoked potentials). This would be extraordinary if true. The authors were actually a lot more conservative in the paper itself, noting that even when injuries are functionally complete, there are usually some spared connections that could recover the ability to transmit. In fact, the authors conclude that the neurological recovery in transplanted patients may have been …a combination of remyelination of spared demyelinated axons, stimulation of regeneration of lesioned axons towards the target host neurons, and reactivation or sprouting of surviving axons.

It’s also important to note that the man who was featured in the news reports became paralyzed as a result of a stab injury, which caused extensive but incomplete laceration of the spinal cord leading to paralysis of the legs. This is a different situation than occurs with most spinal cord injuries that result from blunt force trauma like car accidents, diving injuries and falls, which cause contusion injuries. It is possible that the greater recovery in this patient is due to the type of injury.

So, the important thing is that this study was carefully done and produced a lot of scientific data that will form the basis for future studies. The surgical and cell culture procedures used here are complicated, and even in the best case, the neurological recovery was modest. The cost of the therapy would likely be several hundred thousand dollars per patient because of two surgical procedures, the lab work to grow the cells, and intense rehabilitation that extended for months. Only time will tell whether this could ever be adopted as a practical and effective therapy.
This was published in an open access journal, so you can download the original article (http://www.ingentaconnect.com/search/article?option2=author&value2=tabakow&pageSize=10&index=1)
Reference: Tabakow, P., G. Raisman, W. Fortuna, M. Czyz, J. Huber, D. Li, P. Szewczyk, S. Okurowski, R. Miedzybrodzki, B. Czapiga, B. Salomon, A. Halon, Y. Li, J. Lipiec, A. Kulczyk and W. Jarmundowicz (2014). "Functional regeneration of supraspinal connections in a patient with transected spinal cord following transplantation of bulbar olfactory ensheathing cells with peripheral nerve bridging." Cell Transplant

Neural Stem Cell Clinical Trial

BY NEURALSTEM INC.
Chronic Pain Changes Your Brain In Areas Important For Reward And Emotion
In early October, the company Neuralstem Inc. announced that the first patient had been treated in their new Phase I clinical trial involving “Neural Stem Cells” (NSCs). In this trial, four patients with chronic thoracic level injuries (1-2 years post injury) will receive NSC transplants into the injury site. This trial is under the direction of Dr. Joseph Ciacci, MD, UC San Diego School of Medicine and neurosurgeon at UC San Diego Health System. Much of the pre-clinical work with the NSI-566 cells in spinal cord injury was conducted at UC San Diego School of Medicine by Martin Marsala, MD, professor in the Department of Anesthesiology.
As described in our previous newsletters (see Spinal Connections #22, winter 2012), NSCs are stem cells that are already specified to a neural lineage. They are able to multiply and differentiate into all of the cell types of the nervous system including neurons, astrocytes, and oligodendrocytes. The goal of the treatment being tested in this trial is to inject NSCs into the injury site, where it is hoped that they will develop into a tissue bridge that will allow axons to grow across the injury.
The goal here is a bit different from other ongoing trials of stem cells for spinal cord injury. For example, in the Asterias trial, which continues the one started by Geron, the rationale was to use oligodendrocyte precursor cells (OPCs) derived from embryonic stem cells, which are injected near the site of injury in the hope that they will restore myelin and/or provide growth factors to help repair. In the ongoing trial by Stem Cells Inc., their proprietary NSCs are injected near the injury in the hope that they will develop into nerve cells and glial cells and/or provide growth factors to promote repair.
Neuralstem’s proprietary NSC line is the one that was used for the ongoing ALS trial, in which 30 patients received transplants. This was primarily a safety trial, and no adverse events have been reported so far. A follow-up trial with larger numbers of patients to test efficacy for ALS is being planned.
One cautionary note: Dr. Steward has also been studying NSCs as a means of bridging lesion sites. One surprising discovery this year involves NSCs that were treated with high concentrations of growth factors to promote their survival and expansion. In an experiment in rats, Dr. Steward discovered that about half the rats developed ectopic colonies of cells at long distances from the transplant. For example, following transplants into lesion sites at the thoracic level, colonies of stem cells were found in the cervical spinal cord and the brain. This work was first published in the journal “Cell” in early 2014 and a follow-up article was published in the Journal of Neuroscience in early fall. Dr. Steward is carrying out further studies to determine whether these colonies are harmful or not.