Multiple sclerosis (MS), a chronic neurodegenerative disease driven by damage to the protective myelin sheath, is currently incurable. Today, all clinically available treatments modulate the immune-mediated symptoms of the disease but they fail to stop neurodegeneration in many patients. Remyelination, the regenerative process of myelin repair by oligodendrocytes, which is considered a necessary step to protect demyelinated axons and stop neuronal death, is impaired in MS patients. One of the major obstacles to finding effective remyelinating drugs is the lack of biomimetic drug screening platforms that enable quantification of compounds' potential to stimulate 3D myelination in the physiologically relevant axon-like environment. To address this need, we built a unique myelination drug discovery platform, by expanding our previously developed technology, artificial axons (AAs), which enables 3D-printing of synthetic axon mimics with the geometry and mechanical properties closely resembling those of biological axons. This platform allows for high-throughput phenotypic myelination assay based on quantification of 3D wrapping of myelin membrane around axons in response to compounds. Here, we demonstrate quantification of 3D myelin wrapping by rat oligodendrocytes around the axon mimics in response to a small library of known pro-myelinating compounds. This assay shows pro-myelinating activity for all tested compounds consistent with the published in vitro and in vivo data, demonstrating predictive power of AA platform. We find that stimulation of myelin wrapping by these compounds is dose-dependent, providing a facile means to quantify the compounds' potency and efficacy in promoting myelin wrapping. Further, the ranking of relative efficacy among these compounds differs in this 3D axon-like environment as compared to a traditional oligodendrocyte 2D differentiation assay quantifying area of deposited myelin membrane. Together, we demonstrate that the artificial axons platform and associated phenotypic myelin wrapping assay afford direct evaluation of myelin wrapping by oligodendrocytes in response to soluble compounds in an axon-like environment, providing a predictive tool for the discovery of remyelinating therapies.

The compact myelin sheath is important for axonal function, and its loss can lead to neuronal cell death and irreversible functional deficits. Myelin is vulnerable to a variety of metabolic, toxic, and autoimmune insults. In diseases like multiple sclerosis, there is currently no therapy to stop myelin loss, underscoring the need for neuroprotective and remyelinating therapies. Noninvasive, robust techniques are also needed to confirm the effect of such therapies in animal models. This article describes the generation, characterization, and potential uses for a myelin basic protein-luciferase (MBP-luci) transgenic mouse model, in which the firefly luciferase reporter gene is selectively controlled by the MBP promoter. In vivo bioluminescence imaging can be used to visualize and quantify demyelination and remyelination at the transcriptional level, noninvasively, and in real time. Transgenic mice were assessed in the cuprizone-induced model of demyelination, and luciferase activity highly correlated with demyelination and remyelination events as confirmed by both magnetic resonance imaging and postmortem histological analysis. Furthermore, MBP-luci mice demonstrated enhanced luciferase signal and remyelination in the cuprizone model after treatment with a peroxisome proliferator activated receptor-delta selective agonist and quetiapine. Imaging sensitivity was further enhanced by using CycLuc 1, a luciferase substrate, which has greater blood–brain barrier penetration. We demonstrated the utility of MBP-luci model in tracking myelin changes in real time and supporting target and therapeutic validation efforts.

ABSTRACT Certain murine leukemia viruses (MLVs) are capable of inducing fatal progressive spongiform motor neuron disease in mice that is largely mediated by viral Env glycoprotein expression within central nervous system (CNS) glia. While the etiologic mechanisms and the glial subtypes involved remain unresolved, infection of NG2 glia was recently observed to correlate spatially and temporally with altered neuronal physiology and spongiogenesis. Since one role of NG2 cells is to serve as oligodendrocyte (OL) progenitor cells (OPCs), we examined here whether their infection by neurovirulent (FrCasE) or nonneurovirulent (Fr57E) ecotropic MLVs influenced their viability and/or differentiation. Here, we demonstrate that OPCs, but not OLs, are major CNS targets of both FrCasE and Fr57E. We also show that MLV infection of neural progenitor cells (NPCs) in culture did not affect survival, proliferation, or OPC progenitor marker expression but suppressed certain glial differentiation markers. Assessment of glial differentiation in vivo using transplanted transgenic NPCs showed that, while MLVs did not affect cellular engraftment or survival, they did inhibit OL differentiation, irrespective of MLV neurovirulence. In addition, in chimeric brains, where FrCasE-infected NPC transplants caused neurodegeneration, the transplanted NPCs proliferated. These results suggest that MLV infection is not directly cytotoxic to OPCs but rather acts to interfere with OL differentiation. Since both FrCasE and Fr57E viruses restrict OL differentiation but only FrCasE induces overt neurodegeneration, restriction of OL maturation alone cannot account for neuropathogenesis. Instead neurodegeneration may involve a two-hit scenario where interference with OPC differentiation combined with glial Env-induced neuronal hyperexcitability precipitates disease. IMPORTANCE A variety of human and animal retroviruses are capable of causing central nervous system (CNS) neurodegeneration manifested as motor and cognitive deficits. These retroviruses infect a variety of CNS cell types; however, the specific role each cell type plays in neuropathogenesis remains to be established. The NG2 glia, whose CNS functions are only now emerging, are a newly appreciated viral target in murine leukemia virus (MLV)-induced neurodegeneration. Since one role of NG2 glia is that of oligodendrocyte progenitor cells (OPCs), we investigated here whether their infection by the neurovirulent MLV FrCasE contributed to neurodegeneration by affecting OPC viability and/or development. Our results show that both neurovirulent and nonneurovirulent MLVs interfere with oligodendrocyte differentiation. Thus, NG2 glial infection could contribute to neurodegeneration by preventing myelin formation and/or repair and by suspending OPCs in a state of persistent susceptibility to excitotoxic insult mediated by neurovirulent virus effects on other glial subtypes.

In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin degradation results in loss of axonal function and eventual axonal degeneration. Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to remyelination of denuded axons occurs regularly in early stages of MS but halts as the pathology transitions into progressive MS. Pharmacological potentiation of endogenous OPC maturation and remyelination is now recognized as a promising therapeutic approach for MS. In this study, we analyzed the effects of modulating the Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective inhibitors of ROCK, on the transformation of OPCs into mature, myelinating oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent and human origin, that ROCK inhibition in OPCs results in a significant generation of branches and cell processes in early differentiation stages, followed by accelerated production of myelin protein as an indication of advanced maturation. Furthermore, inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons and remyelination in rat cerebellar tissue explants previously demyelinated with lysolecithin. Our findings indicate that by direct inhibition of this signaling molecule, the OPC differentiation program is activated resulting in morphological and functional cell maturation, myelin formation, and regeneration. Altogether, we show evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the induction of remyelination in demyelinating pathologies.

Methods: Data were collected on CHD risk factors from 80 patient-partner pairs as part of a multi-factorial exploratory prospective study.This study was unusual in collecting data on both patients and partners early in the treatment trajectory just after it had been confirmed that the patient would go on the waiting list for CABG surgery.Results: The patients had significantly reduced their modifiable CHD risk factors by 4 months post-operatively, whilst the partners significantly increased their CHD risk factors.At 4 months follow-up the patients still had an average 1.78 modifiable CHD risk factors and the partners an average of 1.54 risk factors.There were significant differences between the patients and partners for the total number of modifiable CHD risk factors pre-and post-operatively.The pattern that emerged post-CABG indicated a move towards concordance in risk factors between the patients and partners.The patients' greater post-operative total number of CHD risk factors was significantly predicted by their greater total number of pre-operative CHD risk factors and by being female.Similarly, the partners' greater total number of pre-operative modifiable CHD risk factors significantly predicted their higher total number of post-operative CHD risk factors.Conclusions: This study highlights the potential that pre-operative rehabilitation and the use of interventions pre-and post-operatively that target the patient and their partner have for the primary and secondary prevention of CHD. 50Cognitive outcomes in high-risk patients 3 months and 1 year after off-pump versus on-pump coronary artery bypass grafting (CABG)

Much current knowledge of oligodendrocyte biology, the myelin‐forming cells in the central nervous system, comes from cell culture studies mainly from postnatal rat tissue but mouse cells have been much more difficult to produce in large quantities. We have developed a high yield protocol for production of oligodendrocyte precursor cells from mouse embryonic neural progenitors grown as neurospheres. Neurospheres can be maintained and expanded for long periods in culture in the presence of epidermal growth factor (EGF). When floating neurospheres were plated on substrate‐coated dishes in media supplemented with platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), the spheres attached and generated migrating cells that were predominantly oligodendrocyte‐lineage cells. Furthermore, cells in spheres could be shifted to the oligodendrocyte phenotype prior to plating on substrate, by incubation in suspension with PDGF/bFGF. Single cell suspensions plated after dissociation of either EGF‐treated neurospheres or PDGF/bFGF‐treated oligospheres had the bipolar, elongated morphology characteristic of oligodendrocyte precursor cells. mRNA and protein expression analysis of the cells generated by this method confirmed their oligodendrocyte lineage. Oligodendrocyte precursors generated by this method matured in response to ciliary neurotrophic factor treatment, producing cells with multiple processes and myelin‐like membranes. The most important aspect of this protocol is the ability to generate very high numbers of relatively pure mouse oligodendrocyte progenitor cells, which can be easily transfected. These studies open up many kinds of investigations on transgenic and mutant mouse oligodendrocytes, thereby providing a valuable tool to study oligodendrocyte biology and development. © 2008 Wiley‐Liss, Inc.

The group C of Sry-related high-mobility group (HMG) box (Sox) transcription factors has three members in most vertebrates: Sox4, Sox11 and Sox12. Sox4 and Sox11 have key roles in cardiac, neuronal and other major developmental processes, but their molecular roles in many lineages and the roles of Sox12 remain largely unknown. We show here that the three genes are co-expressed at high levels in neuronal and mesenchymal tissues in the developing mouse, and at variable relative levels in many other tissues. The three proteins have conserved remarkable identity through evolution in the HMG box DNA-binding domain and in the C-terminal 33 residues, and we demonstrate that the latter residues constitute their transactivation domain (TAD). Sox11 activates transcription several times more efficiently than Sox4 and up to one order of magnitude more efficiently than Sox12, owing to a more stable alpha-helical structure of its TAD. This domain and acidic domains interfere with DNA binding, Sox11 being most affected and Sox4 least affected. The proteins are nevertheless capable of competing with one another in reporter gene transactivation. We conclude that the three SoxC proteins have conserved overlapping expression patterns and molecular properties, and might therefore act in concert to fulfill essential roles in vivo.

The pro form of neurotrophic growth factor (pro-NGF), purified by chromatography from human Alzheimer's disease (AD)-affected brains (ADhbi-pro-NGF), has been shown to induce apoptotic cell death in neuronal cell cultures through its interaction with the p75 neurotrophin receptor (p75NTR). In the present work, we report that ADhbi-pro-NGF stimulates processing of p75NTR with alpha- and gamma-secretases, yielding a 20-kd intracellular domain (p75(ICD)) that translocates to the nucleus. This process was accompanied by delayed apoptosis. In AD, p75(ICD) was significantly increased in human entorhinal cortex. Although human frontal cortex has been described as showing a higher pro-NGF increase in AD, the increase in the entorhinal cortex paralleled p75NTR processing in its intracellular domain. In addition, pro-NGF isolated from AD-affected brains differed functionally from pro-NGF isolated from comparably aged control brains, with pro-NGF isolated from control brains being unstable and undergoing degradation to NGF when added to cell culture. As p75(ICD) and pro-NGF are both mediators of apoptosis and are both found in increased levels in the cerebral cortex in AD, the present data have implications for understanding neuronal degeneration in AD.

In the CNS, NO is an important physiological messenger involved in the modulation of brain development, synaptic plasticity, neuroendocrine secretion, sensory processing, and cerebral blood flow [Annu. Rev. Physiol. 57 (1995) 683]. These NO actions are largely mediated by cyclic GMP (cGMP) formed by stimulation of soluble guanylyl cyclase (sGC). NO has also been recognized as a neuropathological agent in conditions such as epilepsy, stroke and neurodegenerative disorders. In these conditions, NO may contribute to excitotoxic cell death and neuroinflammatory cell damage [Brain Res. Bull. 41 (1996) 131; Glia 29 (2000) 1]. NO can be formed in every type of CNS parenchymal cell, however, cGMP appears to be formed mainly in neurons and astroglia [Annu. Rev. Physiol. 57 (1995) 683]. There is a large body of information about the regulation of NO formation in brain cells under both normal and pathological conditions but much less is known about the control of cGMP generation, in particular during neuroinflammation when there is a high NO output. Here we briefly review our present knowledge on the regulation of NO-dependent cGMP formation in brain cells under inflammatory conditions.

In astroglial cells beta-amyloid peptides (betaA) induce a reactive phenotype and increase expression of NO synthase. Here we show that treatment of rat brain astrocytes with betaA decreases their capacity to accumulate cyclic GMP (cGMP) in response to NO as a result of a decreased expression of soluble guanylyl cyclase (sGC) at the protein and mRNA levels. Potentiation of betaA-induced NO formation by interferon-gamma did not result in a larger decrease in cGMP formation and inhibition of NO synthase failed to reverse down-regulation of sGC, indicating that NO is not involved. The betaA effect was prevented by the protein synthesis inhibitor cycloheximide. Intracerebral betaA injection also decreased sGC beta1 subunit mRNA levels in adult rat hippocampus and cerebellum. A loss of sGC in reactive astrocytes surrounding beta-amyloid plaques could be a mechanism to prevent excess signalling via cGMP at sites of high NO production.

In rat brain astroglia-enriched cultures long-term treatment with interleukin-1L induces NO release and stimulation of soluble guanylyl cyclase. The cGMP formed is recovered in the extracellular medium but not in the cell monolayer. The interleukin-1L effect is mediated by type I receptor and potentiated by interferon-Q. In cells treated with bacterial endotoxin a larger NO-dependent cGMP accumulation occurs only intracellularly, however a significant cGMP egression is observed when cells are co-treated with interleukin-1L. The nonselective anion transport inhibitors probenecid and verapamil block cGMP efflux, indicating that interleukin-1L stimulates a cGMP transporter. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.