ABSTRACT
Activated microglia can influence the survival of neural cells through the release of cytotoxic factors. Here, we investigated the interaction between Toll-like receptor 4 (TLR4)-activated microglia and oligodendrocytes or their precursor cells (OPC). Primary rat or N9 microglial cells were activated by exposure to TLR4-specifc lipopolysaccharide (LPS), resulting in mitogen-activated protein kinase activation, increased CD68 and inducible nitric oxide synthase expression, and release of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6). Microglial conditioned medium (MGCM) from LPS-activated microglia attenuated primary OPC proliferation without inducing cell death. The microglial-induced inhibition of OPC proliferation was reversed by stimulating group III metabotropic glutamate receptors in microglia with the agonist L-AP4. In contrast to OPC, LPS-activated MGCM enhanced the survival of mature oligodendrocytes. Further investigation suggested that TNF and IL-6 released from TLR4-activated microglia might contribute to the effect of MGCM on OPC proliferation, insofar as TNF depletion of LPS-activated MGCM reduced the inhibition of OPC proliferation, and direct addition of TNF or IL-6 attenuated or increased proliferation, respectively. OPC themselves were also found to express proteins involved in TLR4 signalling, including TLR4, MyD88, and MAL. Although LPS stimulation of OPC did not induce proinflammatory cytokine release or affect their survival, it did trigger JNK phosphorylation, suggesting that TLR4 signalling in these cells is active. These findings suggest that OPC survival may be influenced not only by factors released from endotoxin-activated microglia but also through a direct response to endotoxins. This may have consequences for myelination under conditions in which microglial activation and cerebral infection are both implicated. , Inc.
Subject(s)
Cell Proliferation , Microglia/physiology , Oligodendroglia/physiology , Stem Cells/physiology , Animals , Animals, Newborn , Bromodeoxyuridine/metabolism , Cell Count/methods , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Proliferation/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Culture Media, Conditioned/pharmacology , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Fibroblast Growth Factor 2/pharmacology , Gangliosides/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Ki-67 Antigen/metabolism , Lipopolysaccharides/pharmacology , Microglia/chemistry , Microglia/drug effects , Myelin Basic Protein/metabolism , Neuroblastoma , Phosphorylation , Platelet-Derived Growth Factor/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Stem Cells/drug effects , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolismABSTRACT
Axonal signals are critical in promoting the survival and maturation of oligodendrocytes during myelination, with contact-dependent signals thought to play a key role. However, the exact nature of these signals remains unclear. Neural cell adhesion molecule (NCAM) is expressed by both axons and oligodendrocytes and is ideally localized to transduce signals from the axon. This study sought to investigate the influence of NCAM on premyelinating oligodendrocytes in vitro. Both a soluble molecule comprising the extracellular domain of NCAM and a peptide derived from the fibroblast growth factor receptor (FGFR) binding motif within the first fibronectin domain stimulated a dose-dependent increase in survival of premyelinating oligodendrocytes in vitro. The survival effect was blocked by a mitogen-activated protein kinase (MAPK) inhibitor and an FGFR inhibitor, suggesting that activation of MAPK signalling pathways following interaction with the FGFR is involved in the survival effect of NCAM. Furthermore, NCAM presented in a cellular monolayer induced an increase in radial process outgrowth of oligodendrocyte progenitor cells. These data suggest that NCAM may play a role in axon-oligodendrocyte signalling during myelination, leading to an increase in oligodendrocyte survival and process outgrowth following axonal contact.
Subject(s)
Cell Differentiation/physiology , Myelin Sheath/metabolism , Neural Cell Adhesion Molecules/metabolism , Oligodendroglia/metabolism , Receptors, Fibroblast Growth Factor/metabolism , Stem Cells/metabolism , Amino Acid Motifs/physiology , Animals , Animals, Newborn , Cell Differentiation/drug effects , Cell Shape/drug effects , Cell Shape/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Coculture Techniques , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Mice , Myelin Sheath/drug effects , NIH 3T3 Cells , Neural Cell Adhesion Molecules/chemistry , Neural Cell Adhesion Molecules/pharmacology , Oligodendroglia/drug effects , Peptide Fragments/pharmacology , Protein Structure, Tertiary/physiology , Rats , Rats, Sprague-Dawley , Receptors, Fibroblast Growth Factor/drug effects , Stem Cells/drug effectsABSTRACT
Myelination and its regenerative counterpart remyelination represent one of the most complex cell-cell interactions in the central nervous system (CNS). The biochemical regulation of axon myelination via the proliferation, migration, and differentiation of oligodendrocyte progenitor cells (OPCs) has been characterized extensively. However, most biochemical analysis has been conducted in vitro on OPCs adhered to substrata of stiffness that is orders of magnitude greater than that of the in vivo CNS environment. Little is known of how variation in mechanical properties over the physiological range affects OPC biology. Here, we show that OPCs are mechanosensitive. Cell survival, proliferation, migration, and differentiation capacity in vitro depend on the mechanical stiffness of polymer hydrogel substrata. Most of these properties are optimal at the intermediate values of CNS tissue stiffness. Moreover, many of these properties measured for cells on gels of optimal stiffness differed significantly from those measured on glass or polystyrene. The dependence of OPC differentiation on the mechanical properties of the extracellular environment provides motivation to revisit results obtained on nonphysiological, rigid surfaces. We also find that OPCs stiffen upon differentiation, but that they do not change their compliance in response to substratum stiffness, which is similar to embryonic stem cells, but different from adult stem cells. These results form the basis for further investigations into the mechanobiology of cell function in the CNS and may specifically shed new light on the failure of remyelination in chronic demyelinating diseases such as multiple sclerosis.