The Neurobiology of Rehabilitation

Courtesy of Eric D. Laywell SPHERES OF PROMISE These neurospheres, clusters of cells in culture derived from the CNS of mice, are stained with antibodies against a neuronal protein (red), and a astrocyte protein (green). They have a nuclear counterstain (blue). The brain and spinal cord were once considered mitotic dead ends, a division of neurons dwindling with toddlerhood, with memory and learning the consequence of synaptic plasticity, not new neurons. But the discovery of neural stem cells (NSCs) in the human adult central nervous system (CNS) has raised the possibility of reawakening neurogenesis in the adult to treat neurodegenerative diseases, such as Parkinson, Alzheimer, and Huntington diseases, and spinal cord injuries. "Does the human CNS self-repair? Of course it does! We live 90 years. It is unreasonable to think that there is no turnover, like in every other organ," says Fred Gage, of Salk Institute for Biological Studies, La Jolla, Calif., who led the team that discovered neural stem cells in the human brain in 1998. "Can we turn endogenous cells into neurons in a disease setting? Can we activate our own systems? We are beginning to unravel cell fates and choices, to distinguish intrinsic properties of cells versus local environment cues. For cells, it is not who you are, but where you are, that counts." Researchers must answer many questions before basic neuro-biology can have an effect on rehabilitative medicine. Recapitulating embryonic neurogenesis is especially challenging, because the number and physical relationships of cells in the nervous system changes. Replacing blood, for example, is straightforward--an adult can even replenish the supply with stem cells from an umbilical cord. But the organization of prenatal neurons and glia is unlike that in the adult, where new neural connections must infiltrate dense existing circuitry. Options, and space, are limited. "Adult neurogenesis, and neural repair, might reproduce developmental programs and use developmental mechanisms, or they might not, and instead use a combination of developmental and unique mechanisms," says Jeffrey Macklis, associate professor of neurology and neuroscience, and director of the MGH-HMS (Massachusetts General Hospital-Harvard Medical School) Center for Nervous System Repair. Using injured mice, Macklis' group was the first to report recruitment of new neurons from endogenous precursors.1 DIRECTING CNS REPAIR Disproving the "no new neurons" paradigm that had dominated neurobiology for years has inspired a wave of research that has yet to crest, with clinical applications palpable. Gage sums up the new view of human neurogenesis: "Dividing cells in the spinal cord and outside the hippocampus give rise to glia only. In vitro, they give rise to all lineages. When exposed to a permissive environment, they become neurons as well. This suggests that there are reserves of stem cells throughout the CNS, with equal potentials, whose fates depend on local environments. There must be something different about the regions where neurogenesis occurs." Identifying nurturing combinations of environmental factors will be critical to the efficacy of implanting precursor cells or stimulating endogenous renewal. Either way, it is a complex chore-ography. "These cells must migrate, differentiate into the type of neuron that has been lost to disease or injury, and integrate and connect with existing circuitry with precision," says Macklis. Directing neurogenesis is a question of balance, of replicating the changing milieu of neurotrophins, the intrinsic factors that control them, and inhibitory factors associated with the extracellular matrix and myelin. Experiments mimic permissive conditions. For example, Mark Tuszynski, director of the Center for Neural Repair, University of California, San Diego, uses gene therapy to introduce nerve growth factor (NGF) and neurotrophin-3 (NT-3) genes into fibroblasts, which are then used to patch transected spinal cords in rats. The encouraging reaction, he hypothesizes, reflects a recapitu-lation of responses from certain neurons to a developmental stage when neuro-trophins guided growth. "NGF stimulates axons to go into the injury site, whereas NT-3 stimulates axons to go through remaining healthy tissue. There is a modest degree of functional recovery; the [rat's] forepaws can grab a horizontal ladder," he reports. The boosted cells attract cells that remyelinate axons. The goal: fibroblast "bridges" to help damaged neurons extend their axons past the injury site. Tuszynski also manipulates extra- cellular matrix components and cellular adhesion molecules to favor growth of axons. He presented his work at the recent Experimental Biology meeting. GUINEA PIGS, CANARIES, AND BrdU-POSITIVE CELLS The road to identifying human neural stem cells began with guinea pigs in the 1960s.1 Unlike small-brained, shut-eyed, helpless pink lumps that are baby rats and mice, guinea pigs are born fully furred, mobile, with eyes open and brains developed. Joseph Altman and Gopal Das at the Massachusetts Institute of Technology hypothesized that guinea pigs wouldn't need the burst of brain neurogenesis after birth that rodents need. They injected newborn and six-day-old guinea pigs with tritiated thymidine to mark dividing cells, and then used autoradiography to examine brain cells after six hours, 12 days, and 30 days. They saw dividing brain cells in the dentate gyrus region of the hippocampus, and also adjacent to the lateral ventricles. The implications were clear: If even the independent guinea pig pups require new brain neurons, then so must the human infant, a helpless pink lump at birth. The next clue came from bird brains. Birds sing to communicate about mating and territory. When certain birds learn their songs, their forebrains add neurons to a region called the vocal control nucleus, near the ventricles. Steven Goldman and Fernando Nottebohm at Rockefeller University first described this phenomenon in canaries.2 "Canary neurons die and their precursor cells generate new neurons every fall and spring. They relearn songs using new neurons. This was a strong biological precedent not initially recognized by many in the mammalian world," says Jeffrey Macklis, associate professor of neurology and neuroscience and director of the MGH-HMS (Massachusetts General Hospital- Harvard Medical School) Center for Nervous System Repair.3 "Canaries are one of the nicest organisms in which to study adult neurogenesis," says Arturo Alvarez-Buylla, professor of neurological surgery at the University of California, San Francisco. "Two hot spots next to the ventricles yield many cells in mammals only during development," he adds. His group investigates radial glial cells in birds, which give rise to neuronal progenitors. Other researchers have found dividing cells after probing the brains of tree shrews, marmosets, and rhesus monkeys.4,5 Meanwhile, a team led by Fred Gage at the Salk Institute for Biological Studies was using bromodeoxyuridine (BrdU) to label dividing cells in adult rodent brains. Gage relates the "Eureka!" moment when the idea struck: how to extend research to humans. "We were ... reminiscing. In the mid-1960s, the idea of neurogenesis was relevant in a bigger context: Was it just in birds and rodents, not in primates? Then, we thought, this experiment has already been done!" Gage and postdoc Peter Eriksson recalled that patients with cancer are given BrdU to monitor tumor growth. "We [contacted] pathologists who had stored tissue. Several had paraffin-fixed sections from the brains of people who had died from tumors. They sent them to us. And we could see the BrdU staining," Gage recalls. But, they needed fresh tissue to better discern the cell types. Eriksson learned of a clinical trial using BrdU in patients with terminal larynx and tongue cancer. The participants agreed to donate their brains. "Over the next 2 1/2 years, we got brains from five patients. Peter and the nurses at the hospital dissected ... the tissue, and we started working on it," continues Gage. They looked at the cortex, the subventricular zone, and the corpus callosum. "We found BrdU-positive cells everywhere! In every section!"6 References 1. J. Altman, G. Das, "Postnatal neurogenesis in the guinea-pig," Nature, 214:1098-101. 2. S. Goldman, F. Nottebohm, "Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain," Proc Natl Acad Sci, 80:2390-4, 1983. 3. A. Alvarez-Buylla, F. Nottebohm, "Migration of young neurons in adult avian brain," J Neurosci, 335:353-4, 1988. 4. E. Gould et al., "Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation," J Neurosci, 17:2492-8, 1997. 5. H. Cohen, "Creature comforts," The Scientist, 17[9]:22-5, May 5, 2003. 6. P.S. Eriksson et al., "Neurogenesis in the adult human hippocampus," Nat Med, 4:1313-7, 1998. Stuart Lipton, director, Del E. Webb Center for Neurosciences and Aging, Burnham Institute, La Jolla, works with a different molecule that promotes neurogenesis, a variant of the transcription factor myocyte enhancement factor 2 (MEF2). His team transfected murine ES cells with the MEF2 gene linked to the promoter for nestin, a marker of neural stem cells. Cells exposed to retinoic acid expressed the nestin gene, triggering MEF2 synthesis, which guided differentiation into neurons that resist apoptosis. "Neural development is classically defined as a default program. For example, if you don't become a glial cell, you become a neuron. But we are testing if the antiapoptotic and potentially neurogenic transcription factor MEF2 is instructive rather than permissive," Lipton says. The researchers will use MEF2-boosted cells to treat murine models of neuro-degenerative disease. Boosting may be necessary, because experimental evidence suggests that natural neurogenesis in response to injury is limited. Macklis' group showed that following apoptosis induced in a layer of the cerebral cortex in mice, only 1% to 2% of dividing cells had neuronal markers. Olle Lindvall, Zaal Kokaia, and colleagues at the Wallenberg Neuroscience Center of Lund University Hospital in Sweden demonstrated that only 0.2% of cells in the striatum divide in a rat model of stroke.2 And in yet another experiment, Michelle Monje, Theo Palmer, and colleagues at Stanford University and UC-San Francisco irradiated rat brains and discovered a decreased number of neurons in the hippocampus.3 Reprinted with permission from Nature © Macmillan Magazines Ltd. Induction of neurogenesis in an adult mouse's neocortex. Targeted apoptosis of corticothalamic projection neurons and subsequent recruitment of new neurons from endogenous neural precursors. On the left is an intact neocortex. When corticothalamic projection neurons are induced (right) to undergo synchronous targeted apoptosis, new migratory neuroblasts are born from endogenous precursors. Adapted from Nature, 405:892-4, 2000; re: Nature, 405:951-955, 2000. SPINAL CORD INJURY A spinal cord injury triggers a chain reaction of devastation. At the site of the crush, blood vessels hemorrhage and swelling blocks oxygen and nutrient delivery, as sheared axons and endothelium release toxic factors that wash the area in glutamate. Free radicals destroy myelin, and inflammation continues for days. "The cord is rarely severed, but fibers are denuded," says Gage. "The axons could transmit information if they were insulated. So that will be the first rational approach to treatment--activating endogenous remyelination, tapping into the existing system." Experiments with rodents are promising. The first made headlines in 1998, when researchers used macrophages exposed to peripheral nerve explants to bridge transected spinal cords, enabling some rats to move their hindlegs.4 A year later, John McDonald and coworkers at Washington University School of Medicine in St. Louis restored hindleg movement to rats given murine embryonic stem cells.5 Work with stem cells continues. At Johns Hopkins University, assistant professor of neurology Douglas Kerr and coworkers are using human embryonic germ-cell derivatives to heal rats whose spinal cords are damaged with sindbis virus.6 "We infused hundreds of thousands of human cells into the hindbrain," explains obstetrics and gynecology professor and team member John Gearhart. "If there is injury in the cord, the cells migrate to the ventral horn, where they differentiate into human motor neurons, making cytokines, telling the animals to regrow their own motor neurons." Some of the treated rats walked. Kerr attributes the recovery to transforming growth factor-alpha and brain-derived neurotrophic factor that the implanted cells secrete. Also partially successful is a polymer scaffold infiltrated with murine neural stem cells.7 But some research results are confusing. Three papers in the May 2003 issue of Neuron give three different outcomes for knockout mice that cannot produce Nogo-A, a protein that the myelin sheath normally produces to block axons from extending erratically into injury sites. Several mice in Stephen Strittmatter's group at Yale enjoyed some regeneration and improved gait,8 as did a few in Martin Schwab's lab at the University of Zurich.9 But the rodents in Marc Tessier-Lavagne's facility at Stanford had no such luck.10 The researchers are collaborating to discover differences in their protocols that might explain the discordant results. Farther down the road, a challenge to neurorehabilitation will be to turn off induced repair, because overgrowth can be just as devastating as degeneration. Medical researcher Dennis Steindler, University of Florida, Gainesville, and colleagues found that implanted murine astrocytes, which serve as neural stem cells, homed in to the lateral ventricles, where they formed spheres and invaded surrounding tissue, as a tumor would.11 "In autologous transplants in adult rodents, when progenitors are expanded ex vivo with excess growth factors, you change the biochemistry, and see some hyperplasia," Steindler says. Adds colleague Bryon Peterson, assistant professor of pathology, "The ultimate rule we must follow is to do no harm in bringing about a cure. You can't treat a patient with neural stem cells and [then] cause a glioblastoma." Gage puts the problem into perspective. "How to control overgrowth? I'd love to be faced with that dilemma! We're just not at that point." But those working on animal models today can see the seeds of tomorrow's treatments for people. Says Steindler: "I'm optimistic. Eventually we will rearm cells to surmount obstacles to reconstitute nervous system function." Ricki Lewis (rickilewis@nasw.org) is a freelance writer in Scotia, NY. References 1. S.S. Magavie et al., "Induction of neurogenesis in the neocortex of adult mice," Nature, 405:951-5, 2000. 2. A. Arvidsson et al., "Neuronal replacement from endogenous precursors in the adult brain after stroke," Nat Med, 8:963-70, 2002. 3. M.L. Monje et al., "Irradiation induces neural precursor-cell dysfunction," Nat Med, 8:955-62, 2002. 4. O. Rapalino et al., "Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats," Nat Med, 4:814-21, 1998. 5. J.W. McDonald et al., "Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord," Nat Med, 5:1410-2, 1999. 6. D.A. Kerr et al., "Human embryonic germ cell derivatives facilitate motor recovery of rats with diffuse motoneuron injury," J Neurosci, (in press), 2003. 7. Y. Teng et al., "Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells," Proc Natl Acad Sci, 99:3024-9, 2002. 8. J.-E. Kim et al., "Axon regeneration in young adult mice lacking nogo-A/B," Neuron, 38: 187-199, April 24, 2003. 9. M. Simonen et al., "Systemic deletion of the myelin-associated outgrowth inhibitor nogo-A improves regenerative and plastic responses after spinal cord injury," Neuron, 38: 201-11, April 24, 2003. 10. B. Zheng et al., "Lack of enhanced spinal regeneration in nogo-deficient mice," Neuron, 28:213-24, April 24, 2003. 11. T. Zheng et al., "Transplantation of an indigenous neural stem cell population leading to hyperplasia and atypical integration," Cloning Stem Cells, 4:3-8, 2002. FROM HONEY TO MODIFIED GUANOSINE ANALOG A 5,000-year-old papyrus, thought to be the first surgical guide, refers to spinal cord injury as "an ailment not to be treated." Nonetheless, Hippocrates tried doing so with an elixir of donkey milk, honey, and Egyptian white wine. Although treatments are still limited, those in development are more neurobiology-based than the remedies of times past. Acute approaches minimize the waves of inflammation and apoptosis that immediately follow the injury. In 1997, methylprednisolone became the de rigeur treatment within the first eight hours after damage. When administrated in high doses, this steroid considerably limits inflammation. Long-term treatments, which are still experimental, take several approaches to recreate the milieu of the peripheral nervous system to stimulate axon extension; or to recreate the conditions in the embryo, to jumpstart neurogenesis; or to coax glia to remyelinate stripped axons. Controversial treatments include transposing the omentum (vascular tissue overlying the intestines) to the spinal cord, using the peripheral nerves as bridges in various places, and transplanting shark embryo cells. Treatments found not to help include fetal pig neural stem cell transplants. Here are some treatments, according to company Web sites, that are currently in preclinical or clinical trials: ACUTE Proneuron Biotechnologies, in both Los Angeles and Israel, is removing a patient's macrophages, exposing them to peripheral nerves, and placing them at the injury site. This approach is in Phase 1 and 2 clinical trials. NeoTherapeutics of Davis, Calif., is in Phase 2 trials for AIT-082 (Neotrofin), an orally administered, modified guanosine analog. It is designed to cross the blood-brain barrier and induce nerve growth factors to stimulate axon sprouting and neural stem cell proliferation near the injury. LONG TERM Acorda Therapeutics, in Hawthorne, NY, is in Phase 3 for 4-aminopyridine (Fampridine), which is designed to block potassium channels that are exposed on demyelinated axons, improving conduction. Alexion Pharmaceuticals of Cheshire, Conn., is testing UniGraft-SCI transgenic pig glia in a primate model. The grafted cells stimulate remyelin-ation, restoring some function. Biogen of Cambridge, Mass., is developing Ng-R, a drug candidate that blocks Nogo receptors. Nogo proteins in the myelin sheath normally prevent axons from extending into an injury site. Many investigators are developing human fetal olfactory ensheathing glial cells, which normally surround axons of the first cranial nerve, to remyelinate damaged spinal cord axons. LIVING WITH A SPINAL CORD INJURY Neurodegenerative diseases attack stealthily, slowly sapping the ability to think, remember, move, or stay still. Not so with the sudden devastation of a spinal cord injury. For 52-year-old George Medford, a chemist at General Electric Silicones in Waterford, NY, it happened July 9, 2002, at South Beach on Martha's Vineyard. "It was so simple, yet so catastrophic. There were just small, wind-driven waves, and I was hit from behind as I was walking out of the water. I was pushed into the sand, and I heard a crack. I knew I'd broken my neck," Medford recounts from his home in Clifton Park, NY, his voice fading away in a regular cadence as the ventilator ends each breath. His wife Diane, their teenage son Jimmy, a passerby, and a lifeguard lifted George out of the water. Diane recalls, "He couldn't talk, couldn't breathe. He turned blue and I thought he was dead." If not for a nurse at the beach, he would have been. Medford was injured at the first and second cervical vertebrae, paralyzing him from the chin down, rendering him unable to breathe on his own. He flat-lined three times at a Boston hospital. He remembers only tubes, blinking the alphabet to communicate, nightmares, and a Frankenstein-like contraption screwed into his head. "I still have dents!" he says. He endured convulsions, high fever, and the neurogenic pain that is still with him, from the errant firings of damaged axons. After six months in a rehabilitation center, Medford went home. A variety of devices now stand in for normal function: breath-operated wheelchairs, swaying beds that prevent pressure sores, and molded templates to keep hands supple. But the best medicine, says Medford, is a positive attitude. "You have to look at what Christopher Reeve has done. He's totally focused on getting better. It never entered my mind that I would not survive. Your mind dictates whether you survive or not. 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The Neurobiology of Rehabilitation

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