Glymphatics is a new subdiscipline in neuroscience that was essentially discovered by a Danish neuroscientist named Maiken Nedergaard. Dr. Nedergaard gave a fine seminar on this subject on Sunday.
Glymphatics consists of the system that removes waste products from the brain. Dr. Nedergaard showed movies that showed how the cerebrospinal fluid that bathes the periphery of the brain pulsates as it moves over the brain. When die molecules are injected into the cerebrospinal fluid, these dyes wend up in the blood system. How does this happen?
Nedergaard reasoned that diffusion of the fluid was far too slow for the dye to get to the blood system as fast as it does. Instead, she suspected that fluid moves by means of a “convection current.” How does this work? The blood vessels that feed the brain are surrounded by cells known as astrocytes. These astrocytes prevent molecules from entering the brain unless they can properly negotiate their way across these astrocytes, and this forms the basis for the blood-brain barrier. Cerebrospinal fluid moves across the cells of the brain and is removed by the astrocyte-surrounded vessels. This sink for the cerebrospinal fluid essentially pulls the cerebrospinal fluid across the brain cells and serves as the means by which the brain is cleansed of waste products.
This system, however, is subject to regulation, since the flow of fluid from the cerebrospinal fluid depends on the size of the spaces between brain cells. As it turns out, the spaces between brain cells in larger during sleep than when we are awake. Therefore, sleep seems to be the means by which our bodies clear the rubbish from our brains.
The molecule that controls the space between brain cells is norepinephrine. How it does that remains uncertain, but this is the molecule that is released during sleep to help clear out the garbage in the brain.
Since Alzheimer’s disease, Parkinson’s disease, other neurodegenerative diseases include the accumulation of protein aggregates in the brain, the removal of waste products in the brain would seem to be a rather important process. Also, when there is a head injury, surgeons sometimes leave the skull cap open while the brain heals. This, however, hamstrings the glymphatic system and surgeons should replace the skull cap so that the glymphatic system can do its job. Secondly, if norepinephrine can regulate this system, then this might be a way to increase clearance of waste products from the brain to reduce or delay the accumulation of protein aggregates in the brain.
Several studies in laboratory animals have shown that transplanted neural stem cells have remarkable ability to differentiate into brain and spinal cord cells and replace dead cells. A few clinical trials have also shown that neural stem cells have a great deal of promise to treat neurological diseases.
Unfortunately, getting stem cells into the spinal cord requires injections directly into the spinal cord by highly skilled neurosurgeons using special equipment. Such a procedure is highly invasive and risky. It would be much safer and easier if intravenously administered cells could find their way to the spinal cord.
Giacomo Comi and Stefanie Corti from the University of Milan may have found a way to do just that. With their coworkers, Comi and Corti made neural stem cells from human induced pluripotent stem cells, but they selected their cells in a very unique way. They screened differentiating induced pluripotent stem cells that expressed high levels of an enzyme called aldehyde dehydrogenase, scattered light in a particular way, and expressed the cell adhesion molecule VLA4. Previous experiments showed that neural stem cells made from induced pluripotent stem cells that expressed high levels of aldehyde dehydrogenase with low side scattering of light grew well in the spinal cords of rodents with a neurodegenerative disease, differentiated into nerve cells and relieved symptoms (see Corti S., et al. Hum. Mol. Genet. 2006;15:167–187 and Corti S., et al. J. Clin. Invest. 2008;118:3316–3330). Additional work in other laboratories have shown that cells that express the VLA4 protein on their cell surfaces can enter the central nervous system from the general circulation (see Pluchino S., et al. Nature. 2005;438:266–271; and Winkler E.A., et al. Acta Neuropathol.2013;125:111–120). Thus, Comi and Corti sought to make neural stem cells from induced pluripotent stem cells that had all the qualities they had previously relied upon, but also expressed the cell adhesion molecule VLA4 to determine if such cells could enter the nervous system from the general circulation.
After establishing their desired neural stem cell lines in culture, Comi and Corti and their coworkers transplanted these cells into the spinal cords of mice that suffered from an experimental form of Amyotrophic Lateral Sclerosis (ALS). The implanted cells had previously been labeled with a green-glowing protein, and the presence of green-glowing cells in the spinal cord of the rats was confirmed. However, another set of ALS rats were given these same cells intravenously, and once again green-glowing cells were found in the spinal cord of the ALS rats. Donor cells also reached the brain and were detected in the cortical and subcortical areas of the brain. Even more remarkably, no adverse effects, including tumor formation, abnormal cell growth or inflammation, were detected in any of the recipient animals.
Neural stem cells administered in either manner increased survival in the recipient mice and reduced the loss of neurons and their connections with other cells. Also, the levels of nerve growth factors were increased in the spinal cords of transplanted animals.
Likewise, transplanted animals did not display the massive proliferation of cells known as astrocytes that is so characteristic of ALS spinal cords. As it turns out, the administered neural stem cells prevented the astrocyte explosion by activating an astrocyte cell surface protein called TRPV1. The activation of this cell surface protein prevented the astrocytes from dividing and cluttering up the spinal cord.
These remarkable experiments show, first of all, that neural stem cells can be made that express the VLA4 protein and such cells do not need to be injected into the spinal cord. Instead they can be given intravenously and they will enter the spinal cord on their own, which is a much safer mode of administration. Secondly, neural stem cells made from induced pluripotent stem cells are notorious for being able to cause tumors, but these cells, and the screening method used to select them from differentiating induced pluripotent stem cells, produced cells that apparently do not cause readily cause tumors in laboratory animals. Of course, more intense screening is required to establish the safety of this line, but the initial observations appear hopeful. Thirdly, this shows that we do not need to rip the spinal cords from 10-week old fetuses to make therapeutically useful neural stem cell lines; induced pluripotent stem cell technology will provide the means to do this.
A gaggle of laboratory animal experiments and clinical studies in human patients have established that stem cell injections into the spinal cord after spinal cord injury promote functional recovery (see Beattie, M. S., et al., Exp. Neurol. 148(2):453‐463; 1997; Bennett, D. L., et al., J. Neurosci. 20(1):427‐437; 2000; Kim HK, et al., PLos One 4(3): e4987 2009; Lu, P.; Tuszynski, M. H. Exp. Neurol. 209(2):313‐320; 2008; McTigue, D. M., et al., J. Neurosci. 18(14):5354-5365; 1998; Widenfalk, J.; Lundströmer, K. J. Neurosci. 21(10):3457‐3475; 2001; also see Salazar DL, et al., PLoS ONE, August 2010; Hooshmand M, et al., PLoS ONE, June 2009; Cummings BJ, et al., Neurological Research, July 2006; and Cummings BJ, et al., PNAS, September 19, 2005). Stem Cell, Inc., for example, has conducted several tests with human patients using their HuCNS-SC human neural stem cell line, and transplantation of these stem cells promotes functional recovery in human patients who have suffered spinal cord injury.
However, one factor that has yet to be properly determined is the best site for stem cell injection. Previous work by scientists at the Keio University School of Medicine in Japan has shown that injection of neural stem cells and neural progenitor cells (NS/PCs) into non-injured sites by either intravenous or intrathecal (introduced directly into the space under the arachnoid membrane of the brain or spinal cord) administration failed to produce sufficient engraftment of stem cells at the site of injury.
Instead cells were trapped in the lungs and kidneys, and many mice even developed fatal lung conditions as a result of intravenous administration (see Takahashi Y., et al., Cell Transplant. 2011;20(5):727-39). These data convinced them that intralesional application of the stem cells (injections directly into the damaged site of the spinal cord) might be the most effective and reliable method for NS/PC tranplantations.
A new study by the Keio group has attempted to ascertain the efficacy of the intralesional injections. Mice with spinal cord injuries were injected with NS/PCs that had been derived from mice that expression glowing proteins. This allowed the injected cells to be tracked with bio-luminescence imaging (BLI).
The principal investigator of this research is Masaya Nakamura from the Department of Orthopedic Surgery at the Keio University School of Medicine. Dr. Nakamura and his team gave mice spinal contusions at the level of the tenth thoracic vertebra. Then some mice were given low doses and others high doses of NS/PCs that were derived from fetal mice (for those who are interested, low dose – 250,000 cells per mouse; high dose – 1 million cells per mouse) nine days after spinal cord injury. These mice were further divided into two groups: those injected at the lesion epicenter (E), those injected at sites at the front and back of the lesion (RC for “rostral/caudal”). Thus there were four groups total: High dose E, High dose RC, Low dose E, and Low dose RC.
All four groups showed better functional recovery than the control group, which was injected with phosphate buffered saline. BLI showed that the number of cells that survived in each of the four cell-transplanted groups was about the same across these groups. Thus injecting more cells does not lead to greater numbers of surviving neural stem cells. This makes sense, since the damaged spinal cord in very inhospitable place for transplanted cells.
However, when the mice were examined for the expression of particular brain-derived neurotropic factors, the expression of such genes was higher in the RC-injected mice than in the E-injected mice. These results seems to explain why the transplanted NS/PCs differentiated more readily into neurons in the RC-injected mice rather than a type of glial cell known as an astrocyte, as was the case in the E-injected mice.
Nakamura and his team interpreted these results to mean that the environments of the E and RC sites can both support the survival of transplanted NS/PCs during the sub-acute phase of spinal cord injury. The authors conclude with a practical note: “Therefore, we conclude that it is optimal to graft a certain threshold number of NS/PCs into the epicenter lesion during the sub-acute phase of SCI, and thereby avoid causing further iatrogenic injury to the intact RC regions of the spinal cord.”
Hopefully Nakamura’s work will be translated into further human clinical trials. One feature of this study is that a particular threshold of stem cells survive when injected into the spinal cord and injecting larger numbers of cells does not increase the number of surviving cells. Injecting more cells might only contribute to the cell debris in the spinal cord. This is certainly a good thing to know when conducting clinical trials with neural stem cells in spinal cord-injured patients.
A stroke results when the brain suffers from “ischemia” or a lack of blood flow for an extended period of time. Blockage in the small vessels that feed blood to the brain can cause a trans-ischemic attack (TIA) or stroke. The lack of oxygen causes localized death of brain cells. The dying cells dump a whole gaggle of molecules into the spaces surrounding nearby brain cells, and these cell-derived molecules can actually poison surrounding cells, thus increasing the area that dies as a result of a stroke.
New work from by Henry Ford Hospital researchers in Detroit, Michigan suggests that some of the molecules released by brain cells during a stroke might actually help the brain heal after a stroke. Small RNA molecules or microRNAs that are packaged into lipid-bound vesicles in cells known as exosomes are released by stem cells after a stroke and seem to contribute to neurological recovery.
Exosomes are secreted vesicles that were first discovered nearly 30 years ago. They were, at first, considered little more than garbage cans whose job was to discard unwanted cellular components. However, once cell biologists began to study these little structures, evidence began to accumulate that these dumpsters also act as messengers that convey information to distant tissues. Exosomes contain cell-specific payloads of proteins, lipids, and genetic material that are transported to other cells, where they alter function and physiology.
Therefore, it is little wonder that exosomes can also transport microRNAs. In this present study from the laboratory of Michael Chopp, rats were given experimentally induced strokes, and then the neurological recovery of the rats was examined at the molecular level.
Chopp and his colleagues first isolated mesenchymal stem cells (MSCs) from the bone marrow of their laboratory rats. Then they genetically engineered these MSCs to release exosomes laden with specific microRNAs; in particular miR-133b.
MicroRNAs are a class of post-transcriptional regulators. Since they are usually only about 22 base pairs in length, they are far too short to encode anything. microRNAs usually bind to complementary sequences in the 3’ untranslated region of messenger RNAs, and this binding silences the RNA, which simply means that the RNA cannot be recognized by ribosomes and will not be translated into protein, or that the RNA is degraded by special enzymes that target RNAs bound by microRNAs. Single microRNAs target hundreds genes at a time, and some 60% of all genes are regulated by microRNAs. MicroRNAs are abundantly present in all human cells. They are also highly conserved in organisms ranging from the unicellular algae Chlamydomonas reinhardtii to mitochondria in vertebrates, which suggest that they are a vital part of genetic regulation throughout the plant and animal kingdoms.
The microRNA known as miR-133b has been shown to enhance the death of prostate cancer cells when they are delivered to them (see Patron JP, Fendler A, Bild M, Jung U, Müller H, et al. (2012) MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis. PLoS ONE 7(4): e35345. doi:10.1371/journal.pone.0035345). However, because different cell types show different responses to the same reagents, exposing brain cells to this microRNA after a stroke might elicit a very different response.
By raising or lowering the amount of miR-133b in MSCs, Chopp and his colleagues were able to determine the effects of miR-133b on brains cells after a stroke. Chopp and others injected their genetically engineered MSCs into the bloodstream of rats 24 hours after inducing a stroke in these animals. When the exosomes of the MSCs were enriched in miR-133b, the neurological recovery in the rats was amplified, but when injected MSCs were deprived of miR-133b, the neurological recovery was substantially less.
To measure neurological recovery, researchers separated the disabled rats into several groups and injected each groups with saline, nongenetically-engineered MSCs, MSCs with low levels of miR-133b, and MSCs with high levels of miR-133b. The rats were given behavioral tests 3, 7, and 14 days after treatment. These tests measured the gait of the animals on a grid to determine if the rats could walk on an unevenly spaced grid (foot-fault test). The second test determined how long it took the rats to remove a piece of adhesive tape that was stuck to their front paws.
in every test, the rats injected with miR-133b-enriched MSCs showed superior levels of neurological recovery. Autopsies of these same animals revealed that the rats treated with miR-133b-enriched MSCs had enhanced rewiring of the brain and axonal outgrowth. In the areas of the brain adversely affected by the stroke, the rats showed increased axonal plasticity and neurite remodeling.
Most stroke victims recover some ability to use their hands and other body parts on a voluntary basis, but almost half of all stroke victims are left with some weakness on one side of their body and many are permanently disabled by the stroke.
No treatment presently exists for improving or restoring this lost motor function in stroke patients, mainly because of mysteries about how the brain and nerves repair themselves.
Chopp said, “This study may have solved one of these mysteries by showing how certain stem cells play a role in the brain’s ability to heal itself to differing degrees after stroke or other trauma. Chopp also serves as the scientific director of the Henry Ford Neuroscience Institute.
Diseases that are hard to study, such as Alzheimer’s, schizophrenia, and autism can be examined more safely and effectively thanks to an innovative new method for making mature brain cells from reprogrammed skin cells. Gong Chen, the Verne M. William Chair in Life Sciences and professor of biology at Penn State University and the leader of the research team that designed this method said this: “The most exciting part of this research is that it offers the promise of direct disease modeling, allowing for the creation, in a Petri dish, of mature human neurons that behave a lot like neurons that grow naturally in the human brain.”
Chen’s method could lead to customized treatment for individual patients that are based on their own genetic and cellular profile. Chen explained it this way: “Obviously we do not want to remove someone’s brain to experiment on, so recreating the patient’s brain cells in a Petri dish is the next best thing for research purposes and drug screening.”
In previous work, scientists at the University of Wisconsin in James Thomson’s laboratory and in Shinya Yamanaka’s laboratory at Kyoto University in Kyoto, Japan discovered a way to reprogram adult cells into pluripotent stem cells. Such stem cells are called induced pluripotent stem cells or iPSCs. To make iPSCs, scientists infect adult cells with genetically engineered viruses that introduce four specific genes (OCT4, SOX2, KLF4 and cMYC for those who are interested). These genes encode transcription factors, which are proteins that bind to DNA or to the machinery that directly regulates gene expression. These transcription factors turn on those genes (e.g., OCT4, NANOG, REX1, DNMT3β and SALL4, and OCT4) that induce pluripotency, which means the ability to form any adult cell type. Once in the pluripotent state, iPSCs can be cultured and grown life embryonic stem cells and can differentiate into adult cell types and tissues.
As Chen explained, “A pluripotent stem cell is a kind of blank slate.” Chen continued, “During development, such stem cells differentiate into many diverse specialized cell types, such as a muscle cell, a brain cell, or a blood cell. So, after generating iPSCs from skin cells, researchers then can culture them to become brain cells, or neurons, which can be studies safely in a Petri dish.”
Chen’s team invented a protocol to differentiate iPSCs into mature human neurons much more effectively than previous protocols. This generates cells that behave neurons in our own brains and can be used to model the individualized disease of a single patient.
In the brain, neurons rarely work alone, but instead are usually in close proximity to star-shaped cells called astrocytes. Astrocytes are very abundant cells and they assist neuron function and mediate neuronal survival. “Because neurons are adjacent to astrocytes in the brain, we predicted that this direct physical contact might be an integral part of neuronal growth and health,” said Chen. To test this hypothesis, Chen and his colleagues began by culturing iPSCs-derived neural stem cells, which are stem cells that have the potential to become neurons. These cells were cultured on top of a one-cell-thick layer of astrocytes sop that the two cell types were physically touching each other.
“We found that these neural stem cells cultured on astrocytes differentiated into mature neurons much more effectively,” Chen said. This contrasts Chen’s method with other neural stem cells that were cultured alone in a Petri dish. As Chen put it, the astrocytes seems to be “cheering the stem cells on, telling them what to do, and helping them to fulfill their destiny to become neurons.”
While this sounds a little cheesy, it is undeniable that the astrocyte layer increases the efficiency of neuronal differentiation of iPSCs. Personalized medicine is moving beyond the gene level, to the level of cellular organization and tissue physiology, and iPSCs are showing the way.
A study from Columbia University and Harvard University has uncovered a complex interplay between neurons and support cells known as astrocytes that contributes to the pathology of ALS. Such an intricate interplay complicates regenerative therapies for this disease.
In the spinal cord, a group of neurons called motor neurons extend their axons to skeletal muscles and provide the neural signals for the muscles to contract, which allows movement. Motor neurons also have associated support cells known as glial cells, and a specific group of glial cells known as astrocytes associate with motor neurons in the spinal cord.
Astrocytes are star-shaped cells that surround neurons in the brain and spinal cord, and they outnumber neurons 50:1. Astrocytes are very active in the central nervous system, and serve to maintain, support, and repair the nervous tissue that they serve, and are responsible, in large part, for the plasticity of the nervous system.
Motor neurons die off during the course of ALS, which leads to paralysis and death within two to fives years of diagnosis. ALS also affects neurons in the brain and it completely robs the individual of the ability to initiate movement or even breathe. There is, at present, no cure and no life-prolonging treatment for ALS.
Data from the ALS Association group suggests that astrocytes in ALS patients go from supporting neurons to strangling them. According to Lucie Bruijn, the chief scientist at the ALS Association in Washington D.C.,, these results seem to “strengthen the case that astrocytes are central to the ALS disease process.” She continued: “Furthermore, the results are based on an exciting new disease model system, one that will allow us to test important hypotheses and search for new therapeutic targets.”
In a cell culture system of ALS, in which neurons derived from embryonic cells were co-cultured with normal and ALS astrocytes, Bruijn’s team found that gene expression patterns in those neurons associated with ALS astrocytes were abnormal. In this experiment, neurons derived from embryonic stem cells with co-cultured with normal and ALS affected astrocytes. In a time course experiment in which gene expression profiles were analyzed from the neurons after specific amounts of time, the gene expression patterns from the normal astrocytes co-cultured with neurons were compared with those of the ALS-affected astrocytes co-cultured with neurons. From these experiments, it became clear that the ALS-affected astrocytes did not communicate properly with the nearby neurons.
Even though neurons communicated with each other by means of the release of neurotransmitters, astrocytes and other glial cells also communicate with each other by means of the release of various molecules. This astrocyte-neuron communication maintains healthy neuron function. However, in the case of ALS, the neuron-astrocyte communication is “profoundly disrupted” and is disruption is not neuron dependent, since in this experiment, the neurons were normal. Without proper communication with their astrocytes, motor neurons the spinal cords of ALS patients are not able to function properly.
According to Bruijn, “This study points out several potential points for treatment intervention.” The protection of motor neurons is the goal, since the astrocytes seem to be doing little to protect and support the neurons and also might be hurting them.
An added bonus to this study is that when spinal cords from mice with a disease that shows some similarities to ALS have their gene expression profiles compared to these gene expression profiles observed in the cultured neurons, the results are remarkably similar. This shows that culture system does recapitulate what goes on in the spinal cord.
The next step is to show that the molecular abnormalities discovered in this system mimics those that occur in human disease. This publication utilized mouse cells, and the human disease, while similar, is not exactly the same.