A University of California, San Diego School of Medicine research team has provided new information about a well-known protein that provides the switch for cells to become neurons. This protein is part of a regulatory circuit that can push an immature neural cell to become a functional neuron.
Postdoctoral fellow Chih-Hong Lou and his colleagues worked with principal investigator Miles F. Wilkinson, who is a professor in the Department of Reproductive Medicine, and is also a member of the UC San Diego Institute for Genomic Medicine. These data were published in the February 13 online issue of the journal Cell Reports. These data may also elucidate a still poorly understood process – neuron specification – and might significantly accelerate the development of new therapies for specific neurological disorders, such as autism and schizophrenia.
Wilkinson, Lou and others discovered that the conversion of immature cells to neurons is controlled by a protein called UPF1. UPF1 works in a pathway called the “nonsense-mediated RNA decay” or NMD pathway. The NMD pathway provides a quality control mechanism that eliminates faulty messenger RNA (mRNA) molecules.
mRNA molecules are synthesized from DNA in the nucleus of cells and are exported to the cytoplasm where they are translated by ribosomes into protein. All proteins are encoded by stretches of DNA known as genes and the synthesis of an RNA copy of this stretch of DNA is called transcription. After the transcription of a messenger RNA molecule, is goes to the cytoplasm and is used as the template for the synthesis of a specific protein. Occasionally, mistakes are made in the transcription of mRNAs, and such aberrant mRNAs will either be translated into junk protein, or are so damaged that they cannot be recognized by ribosomes. Such junk mRNAs will gum up the protein synthesis machinery, but cells have the NMD pathway that degrades junk mRNAs to prevent the collapse of the protein synthesis machinery.
A second function for the NMD pathway is to degrade a specific group of normal mRNAs to prevent the production of particular proteins. This NMD function is physiologically important, but until now it had not been clear why it is important.
Wilkinson and others have discovered that UPF1, in combination with a particular class of microRNAs, acts as a molecular switch to determine when immature (non-functional) neural cells take the plunge and differentiate into non-dividing (functional) neurons. In particular, UPF1 directs the degradation of a specific mRNA that encodes for a protein in the TGF-beta signaling pathway, which promotes neural differentiation. The destruction of this mRNA prevents the proper functioning of the TGF-beta signaling pathway and neural differentiation fails to occur. Therefore, Wilkinson, Lou and co-workers identified, for the first time, a molecular pathway in which NMD drives a normal biological response.
NMD also promotes the decay of mRNAs that encode proliferation inhibitors, which Wilkinson said might explain why NMD stimulates the proliferative state characteristic of stem cells. There are many potential clinical ramifications for these findings,” Wilkinson said. “One is that by promoting the stem-like state, NMD may be useful for reprogramming differentiated cells into stem cells more efficiently.
Wilkinson continued: “Another implication follows from the finding that NMD is vital to the normal development of the brain in diverse species, including humans. Humans with deficiencies in NMD have intellectual disability and often also have schizophrenia and autism. Therapies to enhance NMD in affected individuals could be useful in restoring the correct balance of stem cells and differentiated neurons and thereby help restore normal brain function.”
Co-authors on this paper include Ada Shao, Eleen Y. Shum, Josh L. Espinoza and Rachid Karam, from the UCSD Department of Reproductive Medicine; and Lulu Huang, from Isis Pharmaceuticals.
Funding for this research came, in part, from National Institutes of Health (grant GM-58595) and the California Institute for Regenerative Medicine.
Everyone has a brain that has the tools to develop Alzheimer’s disease. Why therefore do some people develop Alzheimer’s disease (AD) while others do not? An estimated five million Americans have AD – a number projected to triple by 2050– the vast majority of people do not and will not develop the devastating neurological condition. What is the difference between those whole develop AD and those who do not?
Subhojit Roy, associate professor in the Departments of Pathology and Neurosciences at the University of California, San Diego School of Medicine, asked this exact question. In a paper published in the journal Neuron, Roy and colleagues offer an explanation for this question. As it turns out, in most people there is a critical separation between a protein and an enzyme that, when combined, trigger the progressive cell degeneration and death characteristic of AD.
“It’s like physically separating gunpowder and match so that the inevitable explosion is avoided,” says principal investigator Roy, a cell biologist and neuropathologist in the Shiley-Marcos Alzheimer’s Disease Research Center at UC San Diego. “Knowing how the gunpowder and match are separated may give us new insights into possibly stopping the disease.”
Neurologists measure the severity of AD by the loss of functioning neurons. In terms of pathology, there are two tell-tale signs of AD: a) clumps of a protein called beta-amyloid “plaques” that accumulate outside neurons and, b) threads or “tangles” of ‘tau” protein found inside neurons. Most neuroscientists believe AD is caused by the accumulation of aggregates of beta-amyloid protein, which triggers a sequence of events that leads to impaired cell function and death. This so-called “amyloid cascade hypothesis” puts beta-amyloid protein at the center of AD pathology.
Creating beta-amyloid requires the convergence of a protein called amyloid precursor protein (APP) and an enzyme that cleaves APP into smaller toxic fragments called beta-secretase or BACE.
“Both of these proteins are highly expressed in the brain,” says Roy, “and if they were allowed to combine continuously, we would all have AD.”
It sounds inexorable, but it doesn’t always happen. Using cultured hippocampal neurons and tissue from human and mouse brains, Roy and Utpal Das, a postdoctoral fellow in Roy’s lab who was the first author of this paper, and other colleagues, discovered that healthy brain cells largely segregate APP and BACE-1 into distinct compartments as soon as they are manufactured, which ensures that these two proteins do not have much contact with each other.
“Nature seems to have come up with an interesting trick to separate co-conspirators,” says Roy.
What then brings APP and BACE together? Roy and his team found that those conditions that promote greater production of beta-amyloid protein also increase the convergence of APP and BACE. Specifically, an increase in neuronal electrical activity, which is known to increase the production of beta-amyloid, also increased the convergence of APP and BACE. Post-mortem examinations of AD patients have shown that the cellular locations of APP and BACE overlap, which lends credence to the pathophysiological significance of this phenomenon in human disease.
Das says that their findings are fundamentally important because they elucidate some of the earliest molecular events triggering AD and show how a healthy brain naturally avoids them. In clinical terms, they point to a possible new avenue for ultimately treating or even preventing the disease.
“An exciting aspect is that we can perhaps screen for molecules that can physically keep APP and BACE-1 apart,” says Das. “It’s a somewhat unconventional approach.”
Disclaimer: I am reporting on this experiment because of its significance for people with spinal cord-injuries even though I remain appalled at the manner in which the stem cells were acquired.
An international research team has reported that a single set of injections of human neural stem cells had provided significant neuronal regeneration and improvement of function in rats impaired by acute spinal cord injury.
Dr. Martin Marsala, who is professor of anesthesiology at the University of California, San Diego, with colleagues from academic institutions in Slovakia, the Czech Republic, and the Netherlands, used neural stem cells derived from an aborted human fetus to treat spinal cord-injured rats.
Sprague-Dawley rats received spinal cord injuries at the level of the third lumbar vertebra by means of compression. Such injuries render the rats incapable of using their hind legs. They cannot climb a ladder, walk a catwalk or perform other tasks that require the effective use of their hind legs.
The stem cells that were transplanted into the spinal cords of these rats were NSI-566RSC cells, which were provided by the biotechnology company Neuralstem. These cells were initially isolated from the spinal cord of an eight-week old human fetus whose life was terminated through elective abortion. These cells have been grown in culture and split many times. They are a neural stem cell culture that has the capacity to form neurons and glia.
The rats were broken into six groups, and four of these groups received spinal cord injuries. One of these spinal cord-injured groups received injections of were injured NSI-566RSC cells (12 injections total, about 20,000 cells per microliter of fluid injected), another received injections of only fluid, and the third group received no injections. The final spinal cord-injured group of rats received injections of NSI-566RSC cells that had been genetically engineered to express a green glowing protein. Another group of rats were operated on, but no spinal cord injury was given to these animals, and the final group of rats were never operated on.
All rats that received injections of cells were administered powerful drugs to prevent their immune systems from rejecting the administered human cells before the injections (methylprednisolone acetate for those who are interested at 10 mg / kg), and after the stem cell injections (tacrolimus at 1.5 mg / kg).
The results were significant and exciting. In the words of Marsala, “The primary benefits were improvement in the positioning and control of paws during walking tests and suppression of muscle spasticity.” Spasticity refers to an exaggerated muscle tone or uncontrolled spasms of muscles. Spasticity is a serious and common complication of traumatic injury. It can cause severe cramping and uncontrolled contractions of muscles, which increases the patient’s pain and decreases their control.
First, it is clear from several control experiments that the injection procedure did not affect the spinal cord function of these animals, since the sham injected rats had perfectly normal use of their hind limbs and normal sensory function of their limbs. Thus the injection procedure is innocuous. Also, the use of the drugs to suppress the immune response were also equally unimportant when it came to the spinal cord health of the rats.
Two months after the stem cell injections, the rats were subjected to the “catwalk test,” in which the animals walked a narrow path and their paw position was assessed. As you can see in the figure below, the stem cell-injected rats have a paw position that is far more similar to the normal rats than to the spinal cord injured rats.
Secondly, when muscle spasticity was measured, the stem cell-injected rats showed definite decreases in muscle spasticity. The spinal cord-injured rats that received no stem cell injections showed no such changes.
Sensory assessments also showed improvements in the stem cell-treated rats, but the improvements were not stellar. Nevertheless, the stem cell-treated rats progressively improved in their sensory sensitivity whereas the non-treated spinal cord-injured rats consistently showed no such improvement.
What were the implanted cells doing? To answer this question, Marsala and his co-workers examined tissue sections of spinal cords from the rats implanted with the glowing green stem cells. According to Marsala, the implanted neural stem cells are stimulating host neuron regeneration and partially replacing the function of lost neurons.
Marsala explained: “Grafted spinal stem cells are a rich source of different growth factors which can have a neuroprotective effect and can promote sprouting of nerve fibers of host neurons. We have demonstrated that grafted neurons can develop contacts with the host neurons and, to some extent, restore the connectivity between centers, above and below the injury, which are involved in motor and sensory processing.”
The implanted neural stem cells definitely showed extensive integration with the spinal nerves of the host rats. Again Marsala, “In all cell-grafted animals, there was a robust engraftment and neuronal maturation of grafted human neurons was noted.” Marsala continued: “Importantly cysts or cavities were not present in any cell-treated animal. The injury-caused cavity was completely filled by grafted cells.”
Marsala’s goal is to used a neuronal stem cell line derived from a patient-specific induced pluripotent stem cell line in a clinical trial. For now, the UC San Diego Institutional Review Board or IRB is reviewing a small phase 1 clinical trial to test the safety and efficacy of this neural stem cell line in patients with spinal cord injuries who have no feeling or motor function below the level of the spinal cord injury.
Spinal cord injuries represent one of the most intractable problems for regenerative medicine. When the spinal cord is injured, a tissue that is normally isolated from the bloodstream, now comes into contact with a variety of inflammatory factors and cells that increase the destruction of the original lesion. The spinal responds with a glial scar that plugs the lesion and prevents further exposure of the spinal cord to damaging inflammation, but the scar is also filled with molecules that repel neuronal axon growth cones. This spells curtains for neuronal regeneration, and finding a cell type that can negotiate around the glial scar and find the original muscle is a genuine tour de force.
Given this to be the case, there have been many experiments in rodents to examine the efficacy of various stem cell populations to as treatments for spinal cord injuries. A recent paper in Stem Cell Research and Therapy (van Gorp et al., 2013, 4:57) has examined human fetal spinal cord-derived neural stem cells (HSSCs) and their ability to restore motor function in rats with spinal cord injuries to the lower back. Because this group examined movement and spinal cord tissue samples, this paper contributes something significant to our knowledge of HSSC-mediate healing of spinal cord injuries.
The HSSC line used in this paper is neural stem cell line NSI566RSC, which was extracted from the spinal cord of an 8-week old “fetus.” I have placed fetus in quotes because at eight weeks, the fetus is actually a very old embryo, since the end of the eighth week is end of embryonic development. I realize that these types of age calculations have room for error, and therefore, the baby might very well have been at the early fetal stage. However, the baby’s mother terminated her pregnancy (yes it was an abortion and no I am not cool with that) and donated the dead baby’s tissue to UC San Diego for research purposes.
Sprague-Daley rats were subjected to spinal cord injuries at the level of the third lumbar vertebra. Three days later, half of the rats were given saline injections into their spinal cord and the other half were given HSSC injections into their spinal cords. The animals were evaluated for two months after the treatments on a daily basis. After two months, the rats were sacrificed (put down) and the spinal cord tissue was extensively analyzed.
Of the 35 animals employed in this study, 3 were excluded because of paw injuries or drug toxicity. Eight weeks after the cells were implanted, the rats were tested with a CatWalk apparatus to determine their gait. The rats injected with HSSCs showed a much more normal gait than those injected with saline. To give you some idea of the improvement, the rats that were not injured had a RCHPP or rostro-caudal hind paw positioning score of 0+/- 1.7mm, and the saline injected animals had an average RCHPP of -18 +/- 3.1 mm, and those injected with HSSCs had an RCHPP of -9.0 +/- 1.9 mm.
Despite these improvements, there were no significant differences in ladder climbing, stride length, overall coordination, or single-frame motion.
Next, Marsala and colleagues showed that the muscle spasms associated with spinal cord injury were slightly decreased by the implantation of HSSCs and not by injection of saline. To measure spasticity, the ankle or front paw is rotated and the electromyograph of the muscle is measured. The electromyograph or EMG measures the electrical activity of the muscle showed modest improvements in the HSSC-injected animals
Sensory sensitivity was improved in the HSSC-injected animals, and this improvement was progressive. When the rats were prodded below the level of the injury, where they should have no feeling, the HSSC-injected rats showed better response to the stimulation. This was the case with mechanical stimulation and thermal stimulation.
Post-mortem analysis also showed something interesting. When the fluid-filled cavity of the damaged spine was examined, the HSSC-injected animals had a significantly small cavity. Because the injected cells had been labeled with green fluorescent protein, they glowed under UV light and any neuronal cells derived from the injected HSSCs glowed green too. The lesioned areas in the HSSC-injected mice were repopulated with cells. Motorneurons, interneurons and glial cells were detected.
What to make of this study? The repopulation of the spinal cord and the growth of spinal nerve elements within the fluid-filled cavity is remarkable, but the lack of better motor function is disappointing. The recovery of sensory ability is significant, especially, since it is pretty clearly not due to spinal hypersensitivity.
There are two possibilities for the low motor recovery. First, there is a possibility that the these experiments were not conducted for as long a time period as they needed to be. Since the sensory ability improvement was progressive, maybe the motor recovery was too, perhaps? Secondly, maybe the grow and connection of motor neurons had trouble with the glial scar. Why the sensory nerves did not have such a problem and the motor neurons would is inexplicable at this time. However, another possibility is that the muscular targets of motor neurons are not as obvious in adult animals as they are in a developing animal. Finding ways to “paint” the muscles might be a way to increase motor neuron innervation in the future.
Thus, this cell line, NSI-566 RSC is certainly a potential treatment for spinal cord patients. A phase I trial is in the works.
Scientists from the University of California, San Diego School of Medicine, have created stem cell-derived, in vitro models of sporadic and hereditary Alzheimer’s, using induced pluripotent stem cells from patients with the neurodegenerative disorder. This experiment provides the ability to study the precise abnormalities present in neurons that cause the pathology of this neurodegenerative disease.
Senior study author Lawrence Goldstein, PhD, professor in the Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute investigator and director of the UC San Diego Stem Cell Program, noted that the production of highly purified, functional human Alzheimer’s neurons in culture has never been done before. Goldstein said: “It’s a first step. These aren’t perfect models. They’re proof of concept. But now we know how to make them.”
This experiment represents a new method for studying the causes of Alzheimer’s disease. These living cells provide a tool for developing and testing drugs to treat the disorder. According to Goldstein, “We’re dealing with the human brain. You can’t just do a biopsy on living patients. Instead, researchers have had to work around, mimicking some aspects of the disease in non-neuronal human cells or using limited animal models. Neither approach is really satisfactory.”
Goldstein and colleagues extracted skin cells called fibroblasts from skin tissues from two patients with familial Alzheimer’s disease. They also used fibroblasts from two patients with sporadic Alzheimer’s disease, and two persons with no known neurological problems. They reprogrammed the fibroblasts into induced pluripotent stem cells (iPSCs) that then differentiated them into working neurons. These iPSC-derived neurons from the Alzheimer’s patients exhibited normal electrophysiological activity, formed functional synaptic contacts and displayed tell-tale indicators of Alzheimer’s disease. Also, they possessed higher-than-normal levels of proteins associated with Alzheimer’s disease.
With cultured neurons from Alzheimer’s patients, scientists can more deeply investigate how Alzheimer’s disease begins and chart the biochemical processes that eventually destroy brain cells and produce degeneration of elemental cognitive functions like memory. Currently, Alzheimer’s research depends heavily upon autopsies performed after the patient has died and the damage has been done. Goldstein added, “The differences between a healthy neuron and an Alzheimer’s neuron are subtle. It basically comes down to low-level mischief accumulating over a very long time, with catastrophic results.”
Neurons derived from one of the two patients with sporadic Alzheimer’s disease showed biochemical changes possibly linked to the disease. Thus there may be sub-categories of the disorder and, in the future, potential therapies might be targeted to specific groups of Alzheimer’s patients.