Seeing is believing: Identifying remyelination in the central nervous system.

Remyelination is the regenerative process by which lost myelin sheaths are restored to demyelinated axons. It is a key target in the treatment of chronic demyelinating disorders such as multiple sclerosis (MS), in which inflammation results in destruction of myelin. In the central nervous system (CNS), remyelination typically requires the differentiation of oligodendrocyte progenitor cells (OPCs) into the myelinating oligodendrocytes (OL). Following successes in preclinical studies, several putative pro-regenerative therapies aimed at enhancing remyelination are under clinical investigation. However, there is a translational barrier in identifying successful outcomes: preclinical measures of remyelination do not translate well to clinical studies, and the paraclinical measures currently deployed in trials are challenging to apply to small rodent models of remyelination. Here, we describe the current approaches to identifying remyelination both in preclinical and clinical settings and highlight exciting translational candidates, which may help to bridge the current impasse.

Polyornithine-based polyplexes to boost effective gene silencing in CNS disorders.

Gene silencing therapies have successfully suppressed the translation of target proteins, a strategy that holds great promise for the treatment of central nervous system (CNS) disorders. Advances in the current knowledge on multimolecular delivery vehicles are concentrated on overcoming the difficulties in delivery of small interfering (si)RNA to target tissues, which include anatomical accessibility, slow diffusion, safety concerns, and the requirement for specific cell uptake within the unique environment of the CNS. The present work addressed these challenges through the implementation of polyornithine derivatives in the construction of polyplexes used as non-viral siRNA delivery vectors. Physicochemical and biological characterization revealed biodegradability and biocompatibility of our polyornithine-based system and the ability to silence gene expression in primary oligodendrocyte progenitor cells (OPCs) effectively. In summary, the well-defined properties and neurological compatibility of this polypeptide-based platform highlight its potential utility in the treatment of CNS disorders.

Oligodendrocyte progenitors: adult stem cells of the central nervous system?

Oligodendrocyte progenitors (OPs) are a major proliferating cell population within the adult CNS. In response to myelin loss or increasing demand, OPs have the capacity to differentiate into mature, myelinating oligodendrocytes. The name 'oligodendrocyte progenitor' suggests restriction to the oligodendrocyte cell lineage. However, with growing evidence of the lineage plasticity of OPs both in vitro and in vivo, we discuss whether they have potential beyond that expected of dedicated progenitor cells, and hence may justify categorization as adult stem cells.

Remyelination: the true regeneration of the central nervous system.

The myelin sheath, generated by oligodendrocytes in the central nervous system (CNS), is crucial to neuronal function, enabling rapid propagation of nerve impulses and providing trophic support to the axon. Remyelination is the default response to myelin damage. Oligodendrocyte precursor cells, distributed throughout both the grey and white matter of the CNS, are activated in response to myelin injury, undergoing proliferation, migration to the site of damage and differentiation into mature myelinating oligodendrocytes. The end result is complete reconstruction of the area of myelin loss. However, this remarkable regenerative capacity of the CNS becomes less efficient with age and can show clinically significant failure in diseases such as multiple sclerosis. Without the myelin sheath, neuronal function and survival is compromised, leading to axonal degeneration and progressive deterioration in neurological function. Therapies to enhance remyelination could offer a means to prevent the neurological decline of chronic demyelinating disease. In order to develop such therapies, a detailed understanding of the process of remyelination, the major cellular players involved and the mechanisms of remyelination failure is needed. As the intricacies of remyelination continue to be unravelled, effective remyelination therapies are ever closer to becoming a reality.

Efficient derivation of NPCs, spinal motor neurons and midbrain dopaminergic neurons from hESCs at 3% oxygen.

This protocol has been designed to generate neural precursor cells (NPCs) from human embryonic stem cells (hESCs) using a physiological oxygen (O(2)) level of 3% (previously termed hypoxia) and chemically defined conditions. The first stage involves suspension culture of hESC colonies at 3% O(2), where they acquire a neuroepithelial identity over a period of 2 weeks. This timescale is comparable to that observed at 20% O(2), but survival is enhanced. Sequential application of retinoic acid and purmorphamine (PM), from day 14 to day 28, directs differentiation toward spinal motor neurons. Alternatively, addition of fibroblast growth factor-8 and PM generates midbrain dopaminergic neurons. OLIG2 (encoding oligodendrocyte lineage transcription factor 2) induction in motor neuron precursors is twofold greater than that at 20% O(2), whereas EN1 (encoding engrailed homeobox 1) expression is enhanced fivefold. NPCs (at 3% O(2)) can be differentiated into all three neural lineages, and such cultures can be maintained long term in the absence of neurotrophins. The ability to generate defined cell types at 3% O(2) should represent a significant advancement for in vitro disease modeling and potentially for cell-based therapies.

Derivation of neural precursor cells from human ES cells at 3% O(2) is efficient, enhances survival and presents no barrier to regional specification and functional differentiation.

In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O(2), demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O(2) can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O(2) environment results in significant cell death, while maintenance in 3% O(2) is protective. Together these findings support 3% O(2) as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury.

Debris clearance by microglia: an essential link between degeneration and regeneration.

Microglia are cells of myeloid origin that populate the CNS during early development and form the brain's innate immune cell type. They perform homoeostatic activity in the normal CNS, a function associated with high motility of their ramified processes and their constant phagocytic clearance of cell debris. This debris clearance role is amplified in CNS injury, where there is frank loss of tissue and recruitment of microglia to the injured area. Recent evidence suggests that this phagocytic clearance following injury is more than simply tidying up, but instead plays a fundamental role in facilitating the reorganization of neuronal circuits and triggering repair. Insufficient clearance by microglia, prevalent in several neurodegenerative diseases and declining with ageing, is associated with an inadequate regenerative response. Thus, understanding the mechanism and functional significance of microglial-mediated clearance of tissue debris following injury may open up exciting new therapeutic avenues.

Abnormally phosphorylated tau is associated with neuronal and axonal loss in experimental autoimmune encephalomyelitis and multiple sclerosis.

The pathological correlate of clinical disability and progression in multiple sclerosis is neuronal and axonal loss; however, the underlying mechanisms are unknown. Abnormal phosphorylation of tau is a common feature of some neurodegenerative disorders, such as Alzheimer's disease. We investigated the presence of tau hyperphosphorylation and its relationship with neuronal and axonal loss in chronic experimental autoimmune encephalomyelitis (CEAE) and in brain samples from patients with secondary progressive multiple sclerosis. We report the novel finding of abnormal tau phosphorylation in CEAE. We further show that accumulation of insoluble tau is associated with both neuronal and axonal loss that correlates with progression from relapsing-remitting to chronic stages of EAE. Significantly, analysis of secondary progressive multiple sclerosis brain tissue also revealed abnormally phosphorylated tau and the formation of insoluble tau. Together, these observations provide the first evidence implicating abnormal tau in the neurodegenerative phase of tissue injury in experimental and human demyelinating disease.

Remyelination in experimental models of toxin-induced demyelination.

Remyelination is the regenerative process by which demyelinated axons are reinvested with new myelin sheaths. It is associated with functional recovery and maintenance of axonal health. It occurs as a spontaneous regenerative response following demyelination in a range of pathologies including traumatic injury as well as primary demyelinating disease such as multiple sclerosis (MS). Experimental models of demyelination based on the use of toxins, while not attempting to accurately mimic a disease with complex etiology and pathogenesis such as MS, have nevertheless proven extremely useful for studying the biology of remyelination. In this chapter, we review the main toxin models of demyelination, drawing attention to their differences and how they can be used to study different aspects of remyelination. We also describe the optimal use of these models, highlighting potential pitfalls in interpretation, and how remyelination can be unequivocally recognized. Finally, we discuss the role of toxin models alongside viral and immune-mediated models of demyelination.

Congenital unilateral absence of the corticospinal tract in a Siamese cat.

A Siamese kitten presented with mild gait dysfunction associated with periodic circling. Pathologic investigation revealed unilateral (right-sided) absence of the corticospinal (pyramidal) tract throughout its normal course. Although an infectious cause cannot be completely ruled out a genetic etiology was suspected.

[Promoting remyelination in multiple sclerosis by endogenous adult neural stem/precursor cells]. / Promouvoir la remyélinisation dans le cadre de la sclérose en plaque à l'aide des cellules neurales/précurseurs et/ou souches endogènes adultes.

Although the treatment of multiple sclerosis has made significant strides in the last decade, successful translation from laboratory to clinical medicine of neuronal repair remains a therapeutic challenge. Nevertheless, advances in the biology of stem and precursor cells, particularly in relation to myelin damage, make this a realistic proposition during the next decade. Replacing lost myelin (remyelination) is currently thought to be an important clinical objective because of the role it might play in slowing or preventing axonal degeneration. Stem/precursor cell-based strategies for enhancing remyelination can be divided into those in which cell are transplanted into a patients (exogenous or cell therapies) and those in which the patients own stem/precursor cells are mobilised to more efficiently engage in healing areas of demyelination (endogenous or pharmacological therapies). While the two approaches tend to be regarded separately they are not mutually exclusive. This article focuses on the endogenous approach and reviews the nature and nomenclature of the stem and precursor cells present within the adult CNS that engage in remyelination and that are therefore potential targets for pharmacological manipulation.

Schwann cell precursors: a favourable cell for myelin repair in the Central Nervous System.

Cell transplant therapies are currently under active consideration for a number of degenerative diseases. In the immune-mediated demyelinating-neurodegenerative disease multiple sclerosis (MS), only the myelin sheaths of the CNS are lost, while Schwann cell myelin of the PNS remains normal. This, and the finding that Schwann cells can myelinate CNS axons, has focussed interest on Schwann cell transplants to repair myelin in MS. However, the experimental use of these cells for myelin repair in animal models has revealed a number of problems relating to the incompatibility between peripheral glial cells and the CNS glial environment. Here, we have tested whether these difficulties can be avoided by using an earlier stage of the Schwann cell lineage, the Schwann cell precursor (SCP). For direct comparison of these two cell types, we implanted Schwann cells from post-natal rat nerves and SCPs from embryo day 14 (E14) rat nerves into the CNS under various experimental conditions. Examination 1 and 2 months later showed that in the presence of naked CNS axons, both types of cell form myelin that antigenically and ultrastructurally resembles that formed by Schwann cells in peripheral nerves. In terms of every other parameter we studied, however, the cells in these two implants behaved remarkably differently. As expected from previous work, Schwann cell implants survive poorly unless the cells find axons to myelinate, the cells do not migrate significantly from the implantation site, fail to integrate with host oligodendrocytes and astrocytes, and form little myelin when challenged with astrocyte-rich environment in the retina. Following SCP implantation, on the other hand, the cells survive well, migrate through normal CNS tissue, interface smoothly and intimately with host glial cells and myelinate extensively among the astrocytes of the retina. Furthermore, when implanted at a distance from a demyelinated lesion, SCPs but not Schwann cells migrate through normal CNS tissue to reach the lesion and generate new myelin. These features of SCP implants are all likely to be helpful attributes for a myelin repair cell. Since these cells also form Schwann cell myelin that is arguably likely to be resistant to MS pathology, they share some of the main advantages of Schwann cells without suffering from the disadvantages that render Schwann cells less than ideal candidates for transplantation into MS lesions.

Clinical canine spinal cord injury provides an opportunity to examine the issues in translating laboratory techniques into practical therapy.

STUDY DESIGN: Review. OBJECTIVES: To highlight the value of investigating the effects of putative therapeutic interventions in clinical spinal cord injury (SCI) in domestic dogs. SETTING: England, UK. METHODS: Many experimental interventions in laboratory rodents have been shown to ameliorate the functional deficits caused by SCI; the challenge now is to determine whether they can be translated into useful clinical techniques. Important differences between clinical SCI in human patients and that in laboratory rodents are in the size of the spinal cord and heterogeneity of injury severity. A further key issue is whether the statistical difference in outcome in the laboratory will translate into a useful difference in clinical outcome. Here, we stress the value of investigating the effects of putative therapies in clinical SCI in domestic dogs. The causes of injury, ability to categorise the severity and methods available to measure outcome are very similar between canine and human patients. Furthermore, postmortem tissue more rapidly becomes available from dogs because of their short lifespan than from human patients. RESULTS: The role that investigation of canine SCI might play is illustrated by our preliminary trials on intraspinal transplantation of olfactory glial cells for severe SCI. CONCLUSIONS: This canine translational model provides a means of 'filtering' putative treatments before human application.

Nasal and frontal sinus mucosa of the adult dog contain numerous olfactory sensory neurons and ensheathing glia.

Olfactory glial cells have been the focus of much recent research interest because of their possible future use as cellular transplants in repair of spinal cord injury. Although olfactory glial cells can be collected from the olfactory bulb for in vitro culture, alternative sites would be preferable for safer surgical access. This study was designed to investigate the distribution of olfactory sensory neurons and olfactory glial cells within the canine peripheral olfactory system. Using immunohistochemistry and electron microscopy on perfused tissue we demonstrate that olfactory sensory neurons are found in both the caudal nasal and the frontal sinus epithelia. Olfactory ensheathing glia were found in the mucosa at both these sites implying that surgical access for harvesting cells for transplantation would be straightforward.

The responses of oligodendrocyte precursor cells, astrocytes and microglia to a cortical stab injury, in the brain.

The cortical stab injury has been widely used for biochemical analysis of molecular changes following CNS injury. However, the cellular responses to this injury have not been accurately quantified. In order to provide a baseline for biochemical studies and future experiments on the manipulation of the CNS injury response we have undertaken a quantitative analysis of this injury. The proliferative and reactive responses of oligodendrocyte precursor cells, astrocytes and microglia were measured, using antibodies to NG2, glial fibrillary acidic protein (GFAP) and the cd11-b clone OX-42 to characterise these cell types at 2, 4, 7 and 14 days post-injury. Oligodendrocyte precursors and microglia proliferated rapidly during the first week, mostly within 0.3 mm of the lesion. Of the dividing cells over 60% were oligodendrocyte precursor cells with microglia making up the balance of the dividing cells. Minimal numbers of astrocytes divided in response to the lesion. Large cells with one or two short processes that were both NG2 and OX-42 positive were identified very close to the lesion at 2 and 4 days post-lesion but not thereafter. They are likely to be blood-derived cells that express NG2 or have ingested it. NG2 immunohistochemistry and platelet-derived growth factor alpha receptor (PDGFalpha-R) in situ hybridisation on neighbouring sections was performed. In the lesioned area only 12% of NG2 positive (+ive) cells were PDGFalpha-R +ive (a ratio of 1:8 for PDGFalpha-R +ive cells: NG2 +ive cells) compared with 33% in the unlesioned cortex and an almost 100% overlap in the spinal cord.

Systemic progesterone administration results in a partial reversal of the age-associated decline in CNS remyelination following toxin-induced demyelination in male rats.

In order to establish the effects of systemically administered progesterone on central nervous system (CNS) remyelination, a toxin-induced model of CNS demyelination was used in which the rate of remyelination is age-dependent. The rapid remyelination in young adult rats allowed an assessment of potential adverse effects of progesterone while the slow remyelination in older adult rats allowed an assessment of its potentially beneficial effects. There was no significant difference in the rate of remyelination between young control and treated animals. However, a modest but significant increase in the extent of oligodendrocyte remyelination in response to progesterone (and a comparable significant decrease in the proportion of axons that remained demyelinated) was observed in older rats 5 weeks after lesion induction. We also found a significant increase in the proportion of Schwann cell remyelinated axons between 3 and 5 weeks after lesion induction that was not apparent in the control animals. These results indicate that progesterone does not inhibit CNS remyelination and that it has a positive modulating effect on oligodendrocyte remyelination in circumstances where it is occurring sub-optimally.

Steroid hormones and neurosteroids in normal and pathological aging of the nervous system.

Without medical progress, dementing diseases such as Alzheimer's disease will become one of the main causes of disability. Preventing or delaying them has thus become a real challenge for biomedical research. Steroids offer interesting therapeutical opportunities for promoting successful aging because of their pleiotropic effects in the nervous system: they regulate main neurotransmitter systems, promote the viability of neurons, play an important role in myelination and influence cognitive processes, in particular learning and memory. Preclinical research has provided evidence that the normally aging nervous system maintains some capacity for regeneration and that age-dependent changes in the nervous system and cognitive dysfunctions can be reversed to some extent by the administration of steroids. The aging nervous system also remains sensitive to the neuroprotective effects of steroids. In contrast to the large number of studies documenting beneficial effects of steroids on the nervous system in young and aged animals, the results from hormone replacement studies in the elderly are so far not conclusive. There is also little information concerning changes of steroid levels in the aging human brain. As steroids present in nervous tissues originate from the endocrine glands (steroid hormones) and from local synthesis (neurosteroids), changes in blood levels of steroids with age do not necessarily reflect changes in their brain levels. There is indeed strong evidence that neurosteroids are also synthesized in human brain and peripheral nerves. The development of a very sensitive and precise method for the analysis of steroids by gas chromatography/mass spectrometry (GC/MS) offers new possibilities for the study of neurosteroids. The concentrations of a range of neurosteroids have recently been measured in various brain regions of aged Alzheimer's disease patients and aged non-demented controls by GC/MS, providing reference values. In Alzheimer's patients, there was a general trend toward lower levels of neurosteroids in different brain regions, and neurosteroid levels were negatively correlated with two biochemical markers of Alzheimer's disease, the phosphorylated tau protein and the beta-amyloid peptides. The metabolism of dehydroepiandrosterone has also been analyzed for the first time in the aging brain from Alzheimer patients and non-demented controls. The conversion of dehydroepiandrosterone to Delta5-androstene-3beta,17beta-diol and to 7alpha-OH-dehydroepiandrosterone occurred in frontal cortex, hippocampus, amygdala, cerebellum and striatum of both Alzheimer's patients and controls. The formation of these metabolites within distinct brain regions negatively correlated with the density of beta-amyloid deposits.

Steroids and the reversal of age-associated changes in myelination and remyelination.

The myelin sheaths that surround all but the smallest diameter axons within the mammalian central nervous system (CNS) must maintain their structural integrity for many years. Like many tissues, however, this function is prone to the effects of ageing, and various structural anomalies become apparent in the aged CNS. Similarly, the regenerative process by which myelin sheaths, lost as a consequence of exposure to a demyelinating insult, are restored (remyelination) is also affected by age. As animals grow older, the efficiency of remyelination progressively declines. In this article, we review both phenomena and describe how both can be partially reversed by steroid hormones and their derivatives.

Cryopreserved cells isolated from the adult canine olfactory bulb are capable of extensive remyelination following transplantation into the adult rat CNS.

Naturally occurring spinal cord injury in dogs provides a potentially powerful intermediate model for testing the efficacy of therapeutic strategies developed in experimental rodent models before phase 1 trials in human patients. A particularly promising strategy involves transplantation of olfactory ensheathing cells (OECs) that both promote axon regeneration and generate new myelin sheaths. As a first step in developing OEC transplantation in the canine intermediate model we describe the isolation, purification, and characterization of OECs from adult dog olfactory bulb. We also show that the canine OEC behaves in a manner similar to its rodent counterpart following transplantation into demyelinating lesions in rat spinal cord and that these properties are retained following cryopreservation.

Increasing local levels of IGF-I mRNA expression using adenoviral vectors does not alter oligodendrocyte remyelination in the CNS of aged rats.

IGF-I, a growth factor that contributes to developmental myelination, shows increased levels of expression within experimental models of remyelination. The pattern of IGF-I mRNA expression changes with the rate of remyelination, with peak levels of expression occurring earlier during rapid remyelination in young adult rats compared to the slower remyelination in old adult rats. In this study we have attempted to accelerate remyelination in old adult rats by using an IGF-expressing adenoviral vector (IGF-I-Ad) to bring forward the timing of peak level of IGF-I expression. Following injection of IGF-I-Ad into focal areas of lysolecithin-induced demyelination in the spinal white matter of old adult rats we created levels of IGF-I mRNA expression at 10 days that were considerably higher than those normally occurring at this time and more similar to those in young animals. However, despite the elevated levels of IGF-I mRNA expression there was no significant change in the extent of oligodendrocyte remyelination compared to saline controls or animals injected with an adenoviral vector expressing LacZ (NT-LacZ-Ad). There was a small increase in Schwann cell remyelination in IGF-I-Ad- and NT-LacZ-Ad-injected animals compared to saline controls. These results indicate that changing the levels of IGF-I directly within demyelinating lesions undergoing remyelination is not sufficient to alter remyelination and that the proremyelinating effects of systemically delivered IGF-I are unlikely to be due to direct effects on the oligodendrocyte lineage.