Adult Mammalian CNS Research Articles

Mechanical trauma induces rapid astroglial activation of ERK/MAP kinase: Evidence for a paracrine signal.

Astrogliosis is a prominent and ubiquitous reaction of astrocytes to many forms of CNS injury, often implicated in the poor regenerative capacity of the adult mammalian CNS. Transmembrane signals that rapidly trigger and maintain astroglial responses to injury are largely undefined. Several candidate inducers of astrogliosis, including growth factors and neuropeptides, act via the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. We previously observed chronically activated ERK/MAPK in human reactive astrocytes. To investigate mechanisms of pathway activation in a defined in vitro model, primary cultured astroglial monolayers were subjected to focal mechanical injury. Within 2-10 min, ERK/MAPK was activated, but only in cells near the wound edge. By 30 min, the entire monolayer showed activation, which persisted for 4 to 8 h. ERK/MAPK activation was specifically blocked by application of the MEK inhibitors, PD98059 and U0126. Cell-cell contact was not necessary for intercellular spread of ERK/MAPK activation, and ERK/MAPK-stimulating activity was found in the injury-conditioned medium. The activating factor was shown to have a native size of 50-100 kD and did not signal through the classical EGF receptor. Injury-induced signaling to ERK/MAPK required Ras, as demonstrated by specific blockade after transient transfection with a dominant negative Ha-RasN17 construct. Finally, we demonstrated that focal lesioning of adult rat cortex induces a rapid activation and spreading of astroglial ERK/MAPK, suggesting that similar mechanisms may operate in astroglial activation following acute brain injury.

The astrocyte/meningeal cell interface--a barrier to successful nerve regeneration?

Following injuries to the adult mammalian CNS meningeal cells migrate into the lesion cavity, forming a fibrotic scar and accessory glia limitans. This infiltration re-establishes the meningeal layer that normally surrounds the CNS, and so reforms the barrier between the CNS and external environment, thus protecting the damaged region from events outside it. However, the newly formed meningeal layer and glia limitans may impede subsequent nerve regeneration through the injured region. This structure can be modelled in vitro using an astrocyte/meningeal co-culture system. We have examined patterns of neurite outgrowth on such cultures, and we find that axons cross readily from meningeal cells to astrocytes, but are unwilling to cross in the other direction. The distribution of cell surface and matrix molecules on these cultures is described, and the effect of various pharmacological interventions which can affect axon growth between the two cell types is summarised in this review.

BDNF Increases the Number of Axotomized Rat Retinal Ganglion Cells Expressing GAP-43, L1, and TAG-1 mRNA—A Supportive Role for Nitric Oxide?

The death of neurons and the limited ability to activate growth-associated genes prevent the restoration of lesioned fiber tracts in the adult mammalian CNS. Here, we characterized the effects of the survival-promoting neurotrophin brain-derived neurotrophic factor (BDNF) on mRNA expression of GAP-43, L1, TAG-1, and SC-1 in axotomized and regenerating rat retinal ganglion cells (RGCs). BDNF led to de novo upregulation of TAG-1 mRNA in axotomized RGCs and to a threefold increase in the number of GAP-43 and L1 mRNA-expressing RGCs. SC-1 expression remained unchanged. However, BDNF did not improve long-distance axon regeneration into a peripheral nerve graft. Surprisingly, potentiating BDNF-mediated neuroprotection by simultaneous administration of a spin trap or a NOS inhibitor counteracted the BDNF-induced growth-associated gene expression. This led us to hypothesize that the BDNF effects on GAP-43, L1, and TAG-1 mRNA expression are mediated by a NO-dependent mechanism. In summary, our data support the idea that survival and axon regeneration of lesioned CNS neurons can be regulated independently.

The response of adult oligodendrocyte progenitors to demyelination in EAE

Cells with the phenotypic characteristics of oligodendrocyte progenitors (NG2+/PDGF alpha R+/O4+) are found throughout the adult mammalian CNS in numbers similar to microglia. They are a reactive glial cell population and respond to demyelination by increasing in number, thereby repopulating the lesion site with cells capable of differentiating into remyelinating oligodendrocytes. Direct evidence that they differentiate into remyelinating cells is missing, although this is the most likely scenario. Cells with the same phenotype are found in normal human CNS tissue and also in chronic MS lesions. Further studies on this intriguing cell type are necessary in order to understand the molecular signals involved in their reaction to injury, particularly in multiple sclerosis.

Neural stem cells are uniquely suited for cell replacement and gene therapy in the CNS.

In recent years, it has become evident that the developing and even the adult mammalian CNS contain a population of undifferentiated, multipotent cell precursors, neural stem cells, the plastic properties of which might be of advantage for the design of more effective therapies for many neurological diseases. This article reviews the recent progress in establishing rodent and human clonal neural stem cell lines, their biological properties, and how these cells can be utilized to correct a variety of defects, with prospects for the near future to harness their behaviour for neural stem cell-based treatment of diseases in humans.

Putting the spinal cord together again.

Demonstration of successful spinal cord regeneration in experimental models is nontrivial. It requires unambiguous proof that injured and not neighboring intact neurons have grown, that novel functional synapses have been established, and that these are responsible for any improvement in functional recovery and that any such recovery is not accompanied by maladaptive function. Very few studies fulfill all these criteria, and consequently there is often uncertainty as to how much regeneration has occurred and its functional significance. The rate of recent progress indicates, though, that we are likely to learn soon why regeneration fails in the adult CNS and may be able to devise technical approaches to use this knowledge to further enhance regeneration.The therapeutic strategies necessary to achieve successful spinal cord regeneration are shown in Table 1Table 1. Given the complexity and diverse response of the CNS to injury, it is extremely unlikely that a single approach will suffice. Multiple interventions will be required at coordinated times to initiate, maintain, and guide regeneration of injured neurons from their site of injury to the appropriate target neurons, and to reestablish functional synaptic contact. Although cautious optimism is beginning to be appropriate, there are great scientific challenges that still need to be met before it is time to focus on or initiate clinical therapies.*To whom correspondence should be addressed (e-mail: woolf.clifford@mgh.harvard.edu).Table 1Strategies for Promoting Spinal Cord RegenerationObjectivesneuronal death/atrophyglial cell reaction/extracellular matrix formationdecrease inhibitory environment provide permissive environmentincrease growth response decrease response to inhibitory influences provide axon guidance cues ensure contact with appropriate target neuron promote synaptic differentiation prevent maladaptive connectivityTechniquesanti-inflammatory agents—methyl prednisoloneneuroprotection—glutamate receptor antagonists/ion channel blockers/growth factorstarget glial cell reactionvaccination against myelin proteins neutralizing antibodies/receptor bodies targeted at inhibitory proteins (Nogo, etc.) receptor antagonistsabort growth cone collapse—intracellular signal transduction modifierssupporting cell bridges—peripheral nerve grafts embryonic tissue/stem cell grafts Schwann cells/olfactory ensheathing glia acellular matrix conduitsadminister molecules that increase intrinsic growth capacity activate growth cone signal transduction cascades potassium channel blockers—optimizing conduction replace neurons—stem cell transplantsprovide guidance cues and ensure specific target recognition

Transplantation of human olfactory ensheathing cells elicits remyelination of demyelinated rat spinal cord.

Human olfactory ensheathing cells (OECs) were prepared from adult human olfactory nerves, which were removed during surgery for frontal base tumors, and were transplanted into the demyelinated spinal cord of immunosuppressed adult rats. Extensive remyelination was observed in the lesion site: In situ hybridization using a human DNA probe (COT-1) indicated a similar number of COT-1-positive cells and OEC nuclei within the repaired lesion. The myelination was of a peripheral type with large nuclei and cytoplasmic regions surrounding the axons, characteristic of Schwann cell and OEC remyelination. These results provide evidence that adult human OECs are able to produce Schwann cell-like myelin sheaths around demyelinated axons in the adult mammalian CNS in vivo.

Lysophosphatidylcholine induces rapid recruitment and activation of macrophages in the adult mouse spinal cord

Lysophosphatidylcholine (LPC) can induce rapid breakdown and removal of myelin from the adult mammalian CNS. In this paper we report the detailed characterization of the immune cell response as well as changes in the expression of cell adhesion molecules and the permeability of the blood-brain barrier after microinjection of LPC into the adult mouse spinal cord. T cells and neutrophils were seen in the spinal cord 6-12 h after LPC injection, but not in PBS-injected mice. Mac-1+ monocytes were also seen at 6 h and 12 h in the white and gray matter of mice injected with LPC and PBS but were significantly greater in the white matter after LPC injections. At later time points LPC induced an increase in the number of activated Mac-1+ macrophages that displayed a variety of morphologies in the white and gray matter. These cells were not present in PBS-injected control mice. LPC also induced widespread microglial activation in the white and gray matter. The number of these Mac-1+ microglia reduced drastically at 96 h after LPC injection suggesting that they may have transformed into Mac-1+ phagocytic cells with a different morphology. These LPC-induced changes in immune cells were accompanied by significant increases in VCAM-1+ and ICAM-1+ blood vessels in the spinal cord. In addition, LPC induced a rapid and widespread disruption of the blood-brain barrier, as compared to PBS injected mice. Therefore, LPC can induce an early and transient T cell and neutrophil response in the CNS. These cells likely promote the rapid influx of monocytes followed by widespread and effective activation of macrophages that mediate rapid phagocytosis of myelin debris.

Glial inhibition of nerve regeneration in the mature mammalian CNS

The lack of axonal regeneration in the adult mammalian CNS is due to both unfavorable environmental glial factors and the intrinsic neuronal state. Inhibitors associated with myelin and the glial scar have been extensively studies and it has been shown that neutralizing at least some of the inhibitors can lead to improved growth. Meanwhile, important advances have also been made towards our understanding of the neuronal intrinsic state, particularly the intracellular levels of cyclic nucleotide, that influence the capacity of mature CNS neurons to initiate and maintain a regrowth response. It is well recognized that successful regeneration may only be achieved by application of a combination of strategies that both block glial inhibitors and enhance the intrinsic neuronal growth capacity.

NG2-expressing cells in the central nervous system: are they oligodendroglial progenitors?

Antibodies against the chondroitin sulphate proteoglycan, NG2, are increasingly being used to identify the widespread population of oligodendrocyte progenitor cells in the adult mammalian CNS. However, the specificity of this marker and the role of NG2-expressing cells in CNS function are still open to question. In this review we consider the evidence that NG2(+) cells in the CNS are part of the oligodendrocyte lineage and whether they can give rise to new oligodendrocytes following demyelination. In both the developing and mature rodent CNS, NG2(+) cells express the established oligodendrocyte lineage marker PDGF-alphaR and from P7, the late progenitor antigen O4, which persists in immature oligodendrocytes. They do not express markers of other CNS populations, such as OX42 or GFAP, at any developmental age. NG2(+) cells represent the major cycling cell population in the normal adult rat CNS, suggesting they have stem cell-like properties. NG2 immunoreactivity is upregulated as a result of physical, viral, excitotoxic and inflammatory insults to the CNS. Following demyelination NG2(+) cell number increases in the immediate vicinity of the lesion and rapid remyelination ensues. NG2 expression has also been investigated in human tissue. Multi-process bearing cells, which morphologically resemble those identified with antibodies against O4, persist in chronically demyelinated multiple sclerosis lesions.

Regeneration and Proliferation of Embryonic and Adult Rat Hippocampal Neurons in Culture

Adult mammalian CNS neurons appear to be terminally differentiated and postmitotic. However, this conclusion may be due to nonpermissive conditions in the brain or in culture media. If embryonic rat hippocampal neurons are cultured in Neurobasal/B27 with FGF2, nearly all neurons proliferated until a maximum density was reached. Similarly, adult neurons can be cultured that fire action potentials and display immunoreactivity for neurofilament, MAP2, tau, and glutamate. Seventy percent of the 3000 isolated adult cells per milligram of brain tissue began to proliferate after 3 days in culture and incorporated BrdU. By 4 days of regeneration in culture, virtually all neuron-like cells with asymmetric processes were glutamate positive and immunoreactive for neurofilament. Immunoreactivity of the intermediate filament stem cell marker nestin increased in adult cells to levels present in freshly isolated embryonic neurons. These are the first studies to demonstrate that over 50% of adult CNS cells with neuron-like characteristics retain regenerative and proliferative potential.

Transplants of Fibroblasts Genetically Modified to Express BDNF Promote Regeneration of Adult Rat Rubrospinal Axons and Recovery of Forelimb Function

Adult mammalian CNS neurons do not normally regenerate their severed axons. This failure has been attributed to scar tissue and inhibitory molecules at the injury site that block the regenerating axons, a lack of trophic support for the axotomized neurons, and intrinsic neuronal changes that follow axotomy, including cell atrophy and death. We studied whether transplants of fibroblasts genetically engineered to produce brain-derived neurotrophic factor (BDNF) would promote rubrospinal tract (RST) regeneration in adult rats. Primary fibroblasts were modified by retroviral-mediated transfer of a DNA construct encoding the human BDNF gene, an internal ribosomal entry site, and a fusion gene of lacZ and neomycin resistance genes. The modified fibroblasts produce biologically active BDNF in vitro. These cells were grafted into a partial cervical hemisection cavity that completely interrupted one RST. One and two months after lesion and transplantation, RST regeneration was demonstrated with retrograde and anterograde tracing techniques. Retrograde tracing with fluorogold showed that approximately 7% of RST neurons regenerated axons at least three to four segments caudal to the transplants. Anterograde tracing with biotinylated dextran amine revealed that the RST axons regenerated through and around the transplants, grew for long distances within white matter caudal to the transplant, and terminated in spinal cord gray matter regions that are the normal targets of RST axons. Transplants of unmodified primary fibroblasts or Gelfoam alone did not elicit regeneration. Behavioral tests demonstrated that recipients of BDNF-producing fibroblasts showed significant recovery of forelimb usage, which was abolished by a second lesion that transected the regenerated axons.

Enhanced Neurotrophin-Induced Axon Growth in Myelinated Portions of the CNS in Mice Lacking the p75 Neurotrophin Receptor

Axonal growth in the adult mammalian CNS is limited because of inhibitory influences of the glial environment and/or a lack of growth-promoting molecules. Here, we investigate whether supplementation of nerve growth factor (NGF) to the CNS during postnatal development and into adulthood can support the growth of sympathetic axons within myelinated portions of the maturing brain. We have also asked whether p75(NTR) plays a role in this NGF-induced axon growth. To address these questions we used two lines of transgenic mice overexpressing NGF centrally, with or without functional expression of p75(NTR) (NGF/p75(+/+) and NGF/p75(-/-) mice, respectively). Sympathetic axons invade the myelinated portions of the cerebellum, beginning shortly before the second week of postnatal life, in both lines of NGF transgenic mice. Despite the presence of central myelin, these sympathetic axons continue to sprout and increase in density between postnatal days 14 and 100, resulting in a dense plexus of sympathetic fibers within this myelinated environment. Surprisingly, the growth response of sympathetic fibers into the cerebellar white matter of NGF/p75(-/-) mice is enhanced, such that both the density and extent of axon ingrowth are increased, compared with age-matched NGF/p75(+/+) mice. These dissimilar growth responses cannot be attributed to differences in cerebellar levels of NGF protein or sympathetic neuron numbers between NGF/p75(+/+) and NGF/p75(-/-) mice. Our data provide evidence demonstrating that growth factors are capable of overcoming the inhibitory influences of central myelin in the adult CNS and that neutralization of the p75(NTR) may further enhance this growth response.

Activity-Dependent Activation of TrkB Neurotrophin Receptors in the Adult CNS

In this paper we have investigated the hypothesis that neural activity causes rapid activation of TrkB neurotrophin receptors in the adult mammalian CNS. These studies demonstrate that kainic acid-induced seizures led to a rapid and transient activation of TrkB receptors in the cortex. Subcellular fractionation demonstrated that these activated Trk receptors were preferentially enriched in the synaptosomal membrane fraction that also contained postsynaptic glutamate receptors. The fast activation of synaptic TrkB receptors could be duplicated in isolated cortical synaptosomes with KCl, presumably as a consequence of depolarization-induced BDNF release. Importantly, TrkB activation was also observed following pharmacological activation of brain-stem noradrenergic neurons, which synthesize and anterogradely transport BDNF; treatment with yohimbine led to activation of cortical TrkB receptors within 30 min. Pharmacological blockade of the postsynaptic alpha1-adrenergic receptors with prazosin only partially inhibited this effect, suggesting that the TrkB activation was partially due to a direct effect on postsynaptic cortical neurons. Together, these data support the hypothesis that activity causes release of BDNF from presynaptic terminals, resulting in a rapid activation of postsynaptic TrkB receptors. This activity-dependent TrkB activation could play a major role in morphological growth and remodelling in both the developing and mature nervous systems.

Regeneration of dorsal column fibers into and beyond the lesion site following adult spinal cord injury.

Regeneration is abortive following adult mammalian CNS injury. We have investigated whether increasing the intrinsic growth state of primary sensory neurons by a conditioning peripheral nerve lesion increases regrowth of their central axons. After dorsal column lesions, all fibers stop at the injury site. Animals with a peripheral axotomy concomitant with the central lesion show axonal growth into the lesion but not into the spinal cord above the lesion. A preconditioning lesion 1 or 2 weeks prior to the dorsal column injury results in growth into the spinal cord above the lesion. In vitro, the growth capacity of DRG neurite is also increased following preconditioning lesions. The intrinsic growth state of injured neurons is, therefore, a key determinant for central regeneration.

Multipotent Neurospheres Can Be Derived from Forebrain Subependymal Zone and Spinal Cord of Adult Mice after Protracted Postmortem Intervals

The adult mammalian CNS harbors a population of multipotent stem/progenitor cells that can be induced to grow as proliferative neurospheresin vitro.We demonstrate here that neurosphere-generating cells can be isolated from adult mouse spinal cord and forebrain subependymal zone after postmortem intervals of up to 140 h, when kept at 4°C, and up to 30 h when kept at room temperature. Although there was an inverse relationship between postmortem interval and the number of neurospheres generated, neurospheres derived under these conditions were proliferative and could give rise to both neurons and glia.

Regeneration and sprouting of chronically injured corticospinal tract fibers in adult rats promoted by NT-3 and the mAb IN-1, which neutralizes myelin-associated neurite growth inhibitors.

Myelin-associated inhibitors of neurite growth play an important role in the regenerative failure after injury in the adult mammalian CNS. The application of the mAb IN-1, which efficiently neutralizes the NI-250/35 inhibitory proteins, alone or in combination with neurotrophin-3 (NT-3), has been shown to promote axonal regeneration when applied in acute injury models. To test whether IN-1 application can induce axonal growth also in a chronic injury model, we treated rats with IN-1 and NT-3 starting 2 or 8 weeks after injury. Rats underwent bilateral dorsal hemisection of the spinal cord at the age of 5-6 weeks. Regeneration of corticospinal (CST) fibers into the caudal spinal cord was observed in three of eight of those animals with a 2-week delay between lesion and treatment. CST fibers regenerated for 2-11.4 mm. In the control group sprouting occurred rostral to the lesion but no long-distance regeneration occurred. In animals where treatment started at 8 weeks after injury the longest fibers observed grew up to 2 mm into the caudal spinal cord. The results show that transected corticospinal axons retain the ability to regenerate at least for a few weeks after injury. Functional analysis of these animals showed a slight improvement of functional recovery.

GABA-dependent firing of glutamate-evoked action potentials at AMPA/kainate receptors in developing hypothalamic neurons.

Although it plays a major inhibitory role in the adult mammalian CNS, gamma-aminobutyric acid (GABA) may have an excitatory function in developing neurons. The present study focuses on the dependence of glutamate on GABA to generate action potentials in developing hypothalamic neurons. Under conditions where glutamate by itself could not evoke an action potential, GABA facilitated glutamate-mediated depolarization to fire action potentials. This facilitation had a broad time window during the decaying phase of the GABA-mediated depolarization in developing neurons in culture. The glutamate-mediated depolarization was shunted only during the peak of GABA-mediated depolarization, but was facilitated after that. Similar results were obtained in the presence of 2-amino-5-phosphonopentanoic acid (AP5), indicating that GABA can facilitate glutamate responses independent of relief of the Mg2+ block of the N-methyl-D-aspartate (NMDA) receptor. This novel interaction between GABA- and glutamate-mediated excitation could play a role in strengthening neuronal circuits during early development and would exert a maximal effect if GABA and glutamate receptors were activated after a slight temporal delay.

Chapter 17 Cellular and molecular correlates of the regeneration of adult mammalian CNS axons into peripheral nerve grafts

Studies of the regeneration of CNS axons into peripheral nerve grafts have provided information crucial to our understanding of the regenerative potential of CNS neurons. Injured axons in the thalamus and corpus striatum produce regenerative sprouts within a few days of graft implantation, apparently in response to living cells in the grafts. The regenerating axons often grow directly towards the grafts, and enter Schwann cell columns where they elongate surrounded by Schwann cell processes. The regenerating CNS axons, and the Schwann cell processes along which they grow, initially express the cell adhesion molecules NCAM, and L1. The axons also express polysialic acid and, unlike regenerating peripheral axons, bind tenascin-C derived from Schwann cells. Wherever peripheral nerve grafts are implanted into the CNS they appear to promote the differential regeneration of CNS axons. Most of the axons which grow into grafts in the thalamus are derived from the thalamic reticular nucleus (TRN), whereas grafts in the striatum promote regeneration of axons from the substantia nigra pars compacta (SNpc) and grafts in the cerebellum promote regeneration from deep cerebellar nuclei (DCN) and brainstem precerebellar neurons. In contrast most thalamocortical projection neurons, striatal projection neurons and Purkinje cells in the cerebellar cortex are poor at regenerating. There are patterns to the expression of regeneration-related molecules by axons injured by nerve grafts in the CNS. Most neurons which regenerate well (e.g. TRN and DCN neurons) upregulate GAP-43, L1 and the transcription factor c-jun in response to a graft, whereas those neurons which do not regenerate well (e.g. Purkinje cells, thalamocortical and striatal projection neurons) do not upregulate these molecules. These observations suggest that some classes of CNS neurons may be intrinsically unable to regenerate axons and the repair of injuries in the brain and spinal cord may consequently require some form of gene therapy for axotomised neurons.

Expression of LIM Protein GenesLmo1, Lmo2,andLmo3in Adult Mouse Hippocampus and Other Forebrain Regions: Differential Regulation by Seizure Activity

The LIM domain is a zinc-binding amino acid motif that characterizes various proteins which function in protein-protein interactions and transcriptional regulation. Expression patterns of several LIM protein genes are compatible with roles in vertebrate CNS development, but little is known about the expression, regulation, or function of LIM proteins in the mature CNS. Lmo1, Lmo2, and Lmo3 are LIM-only genes originally identified as putative oncogenes that have been implicated in the control of cell differentiation and are active during CNS development. Using in situ hybridization for mRNA and immunohistochemical detection of reporter protein expression in transgenic mice, we found that Lmo1, Lmo2, and Lmo3 show individually unique but partially overlapping patterns of expression in several regions of the adult mouse forebrain, including hippocampus, caudate putamen, medial habenula, thalamus, amygdala, olfactory bulb, hypothalamus, and cerebral cortex. In the hippocampal formation, Lmo1, Lmo2, and Lmo3 show different combinatorial patterns of expression levels in CA pyramidal and dentate granule neurons, and Lmo1 is present in topographically restricted subpopulations of astrocytes. Kainic acid-induced limbic seizures differentially regulated Lmo1, Lmo2, and Lmo3 mRNA levels in hippocampal pyramidal and granule neurons, such that Lmo1 mRNA increased, whereas Lmo2 and Lmo3 mRNAs decreased significantly, with maximal changes at 6 hr after seizure onset and return to baseline by 24 hr. These findings show that Lmo1, Lmo2, and Lmo3 continue to be expressed in the adult mammalian CNS in a cell type-specific manner, are differentially regulated by neuronal activity, and may thus be involved in cell phenotype-specific regulatory functions.