David Kremer

The complex world of oligodendroglial differentiation inhibitors.

Myelination is a central nervous system (CNS) process wherein oligodendrocyte‐axon interactions lead to the establishment of myelin sheaths that stabilize, protect, and electrically insulate axons. In inflammatory demyelinating diseases such as multiple sclerosis (MS), the degeneration and eventual loss of functional myelin sheaths slows and blocks saltatory conduction in axons, which results in clinical impairment. However, remyelination can occur, and lesions can be partially repaired, resulting in clinical remission. The recruitment and activation of resident oligodendrocyte precursor cells (OPCs) play a critical role in the repair process because these cells have the capacity to differentiate into functional myelinating cells. Mature oligodendrocytes, however, are thought to have lost the capacity to develop new myelin sheaths and frequently undergo programmed cell death in MS. The endogenous capacity to generate new oligodendrocytes in MS is limited, and this is predominantly due to the presence of inhibitory components that block OPC differentiation and maturation. Here, we present an overview of recently identified negative regulators of oligodendroglial differentiation and their potential relevance for CNS repair in MS. Because currently available immunomodulatory drugs for MS mainly target inflammatory cascades outside the brain and fail to repair existing lesions, achieving more efficient lesion repair constitutes an important goal for future MS therapies. Ann Neurol 2011;69:602–618

Human endogenous retrovirus type W envelope protein inhibits oligodendroglial precursor cell differentiation.

Differentiation of oligodendroglial precursor cells is crucial for central nervous system remyelination and is influenced by both extrinsic and intrinsic factors. Recent studies showed that human endogenous retrovirus type W (HERV‐W) contributes significantly to brain damage. In particular, its envelope protein ENV can mediate injury to specific cell types of the brain and immune system. Here, we investigated whether ENV protein affects oligodendroglial differentiation.

Activation of CXCR7 receptor promotes oligodendroglial cell maturation.

Differentiation of oligodendroglial precursor cells is crucial for central nervous system (re)myelination and is influenced by multiple extrinsic and intrinsic factors. Chemokines, a group of small proteins, are highly conserved among mammals and have been implicated in a variety of biological processes during development, tissue homeostasis, and repair. We investigated whether the chemokine CXCL12 influences oligodendrocytes and what cellular differentiation/maturation processes are controlled by this molecule.

p57kip2 is dynamically regulated in experimental autoimmune encephalomyelitis and interferes with oligodendroglial maturation

The mechanisms preventing efficient remyelination in the adult mammalian central nervous system after demyelinating inflammatory diseases, such as multiple sclerosis, are largely unknown. Partial remyelination occurs in early disease stages, but repair capacity diminishes over time and with disease progression. We describe a potent candidate for the negative regulation of oligodendroglial differentiation that may underlie failure to remyelinate. The p57kip2 gene is dynamically regulated in the spinal cord during MOG-induced experimental autoimmune encephalomyelitis. Transient down-regulation indicated that it is a negative regulator of post-mitotic oligodendroglial differentiation. We then applied short hairpin RNA-mediated gene suppression to cultured oligodendroglial precursor cells and demonstrated that down-regulation of p57kip2 accelerates morphological maturation and promotes myelin expression. We also provide evidence that p57kip2 interacts with LIMK-1, implying that p57kip2 affects cytoskeletal dynamics during oligodendroglial maturation. These data suggest that sustained down-regulation of p57kip2 is important for oligodendroglial maturation and open perspectives for future therapeutic approaches to overcome the endogenous remyelination blockade in multiple sclerosis.

Promoting remyelination in multiple sclerosis: Current drugs and future prospects

Myelin destruction due to inflammatory oligodendrocyte cell damage or death in conjunction with axonal degeneration are among the major histopathological hallmarks of multiple sclerosis (MS). The majority of available immunomodulatory medications for MS are approved for relapsing–remitting (RR) MS, for which they reduce relapse rate, MRI measures of inflammation, and the accumulation of disability. These medications are, however, of little benefit during progressive MS where axonal degeneration following demyelination outweighs inflammation. This has sparked great interest in the development of new remyelination therapies aimed at reversing the neurodegenerative damage observed in this disease. Remyelination as a result of oligodendrocyte production from oligodendrocyte precursor cells (OPCs) is considered a promising potential target for the treatment of all stages of MS. In this review we present an overview of a) approved medications (some of them FDA-and EMA-approved for other diseases) with a proposed role in regeneration, b) regenerative treatments under investigation in clinical trials, and c) promising future therapeutic approaches aiming specifically at facilitating endogenous repair.

Pushing Forward: Remyelination as the New Frontier in CNS Diseases

The evolutionary acquisition of myelin sheaths around large caliber axons in the central nervous system (CNS) represented a milestone in the development of vertebrate higher brain function. Myelin ensheathment of axons enabled saltatory conduction and thus accelerated information processing. However, a number of CNS diseases harm or destroy myelin and oligodendrocytes (myelin-producing cells), ultimately resulting in demyelination. In the adult CNS, new oligodendrocytes can be generated from a quiescent pool of precursor cells, which - upon differentiation - can replace lost myelin sheaths. The efficiency of this spontaneous regeneration is limited, which leads to incomplete remyelination and residual clinical symptoms. Here, we discuss CNS pathologies characterized by white matter degeneration and regeneration and highlight drugs that could potentially serve as remyelination therapies.

The neutralizing antibody GNbAC1 abrogates HERV-W envelope protein-mediated oligodendroglial maturation blockade

Background: The envelope protein (ENV) of the human endogenous retrovirus type W is implicated in inflammatory reactions in multiple sclerosis (MS) but also interferes with oligodendroglial maturation. A neutralizing antibody GNbAC1 has been developed and successfully been tested in clinical trials. Objectives and methods: We stimulated primary oligodendroglial cells with ENV upon preincubation with GNbAC1 and assessed for nitrosative stress and myelin expression. Results: Neutralization of ENV by GNbAC1 reduces its ability to induce stress reactions resulting in a rescue of myelin expression. Conclusions: Beyond immune cell modulation, this monoclonal antibody may therefore help to overcome the oligodendroglial differentiation blockade in MS.

Deciphering the Oligodendrogenic Program of Neural Progenitors: Cell Intrinsic and Extrinsic Regulators

In the developing and adult CNS, neural stem/progenitor cells (NSPCs) and oligodendroglial progenitor cells (OPCs) follow an oligodendrogenic process with the aim of myelinating axons. This process is to a high degree regulated by an oligodendrogenic program (OPr) composed of intrinsic and extrinsic factors that modulate the different steps required for NSPCs to differentiate into myelinating oligodendrocytes. Even though NSPCs and OPCs are present in the diseased CNS and have the capacity to generate oligodendrocytes, sparse remyelination of axons constitutes a major constraint in therapies toward multiple sclerosis (MS) and spinal cord injury (SCI). Lack of pro-oligodendrogenic factors and presence of anti-oligodendrogenic activities are thought to be the main reasons for this limitation. Thus, molecular and cellular strategies aiming at remyelination and at targeting such pro- and anti-oligodendrogenic mechanisms are currently under investigation. The present review summarizes the current knowledge on the OPr; it implements our own findings on mesenchymal stem cell-derived pro-oligodendroglial factors and on the role of p57/kip2 in oligodendroglial differentiation. Moreover, it describes molecular and cellular approaches for the development of future therapies toward remyelination.

Lidocaine metabolites inhibit glycine transporter 1: a novel mechanism for the analgesic action of systemic lidocaine?

Background: Lidocaine exerts antinociceptive effects when applied systemically. The mechanisms are not fully understood but glycinergic mechanisms might be involved. The synaptic glycine concentration is controlled by glycine transporters. Whereas neurons express two types of glycine transporters, astrocytes specifically express glycine transporter 1 (GlyT1). This study focuses on effects of lidocaine and its major metabolites on GlyT1 function. Methods: The effects of lidocaine and its metabolites monoethylglycinexylidide (MEGX), glycinexylidide, and N-ethylglycine on GlyT1 function were investigated in uptake experiments with [14C]-labeled glycine in primary rat astrocytes. Furthermore, the effect of lidocaine and its metabolites on glycine-induced currents were investigated in GlyT1-expressing Xenopus laevis oocytes. Results: Lidocaine reduced glycine uptake only at toxic concentrations. The metabolites MEGX, glycinexylidide, and N-ethylglycine, however, significantly reduced glycine uptake (P < 0.05). Inhibition of glycine uptake by a combination of lidocaine with its metabolites at a clinically relevant concentration was diminished with increasing extracellular glycine concentrations. Detailed analysis revealed that MEGX inhibits GlyT1 function (P < 0.05), whereas N-ethylglycine was identified as an alternative GlyT1 substrate (EC50 = 55 &mgr;M). Conclusions: Although lidocaine does not function directly on GlyT1, its metabolites MEGX and glycinexylidide were shown to inhibit GlyT1-mediated glycine uptake by at least two different mechanisms. Whereas glycinexylidide was demonstrated to be an alternative GlyT1 substrate, MEGX was shown to inhibit GlyT1 activity in both primary astrocytes and in GlyT1-expressing Xenopus laevis oocytes at clinically relevant concentrations. These findings provide new insights into the possible mechanisms for the antinociceptive effect of systemic lidocaine.

Midazolam activates the intrinsic pathway of apoptosis independent of benzodiazepine and death receptor signaling.

Background and Objectives: Midazolam has neurotoxic properties when administered neuraxially in vivo. Furthermore, midazolam induces neurodegeneration in neonatal animal models in combination with other general anesthetics. Therefore, this study focuses on the mechanism of neurotoxicity by midazolam in neuronal and nonneuronal cells. The study aims to evaluate the apoptotic pathway and to investigate the protective effects of the benzodiazepine antagonist flumazenil and the caspase inhibitor N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)-methylketone. Methods: The apoptosis-inducing effect of preservative-free midazolam on human lymphoma and neuroblastoma cell lines was evaluated using flow cytometric analysis of early apoptotic stages (annexin V/7AAD) and caspase 3 activation. B-cell lymphoma (Bcl2) protein overexpressing and caspase 9-deficient lymphoma cells were used to determine the role of the mitochondrial (intrinsic) pathway. Caspase 8-deficient and Fas-associated protein with death domain (FADD)-deficient cells were used to evaluate the death receptor (extrinsic) pathway. The protective effects of flumazenil and the caspase inhibitor N-(2-quinolyl)valyl-aspartyl-(2,6-difluorophenoxy)-methylketone were investigated in neuroblastoma cells and primary rat neurons using metabolic activity assays (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) and immunofluorescence microscopy. Results: Midazolam induced apoptosis in all investigated cell types in a concentration-dependent manner, indicated by flow cytometry. Bcl2-overexpression and caspase 9 deficiency protected against toxicity, whereas caspase 8 or FADD deficiency had no effect. Pancaspase inhibition had a strong protective effect, whereas flumazenil did not inhibit midazolam-induced apoptosis. Conclusions: Midazolam induces apoptosis via activation of the mitochondrial pathway in a concentration-dependent manner. The mechanism of midazolam toxicity switches from caspase-dependent apoptosis to necrosis with increasing concentrations. The induction of apoptosis and necrosis by midazolam is presumably unrelated to GABAA receptor pathway signaling.