A cell culture preparation equipped with stimulating electrodes was used to investigate whether action potential activity can influence myelination of mouse dorsal root ganglia axons by Schwann cells. Myelination was reduced to one-third of normal by low-frequency impulse activity (0.1 Hz), but higher-frequency stimulation (1 Hz) had no effect. The number of Schwann cells and the ultrastructure of compact myelin were not affected. The frequency of stimulation that inhibited myelination decreased expression of the cell adhesion molecule L1, and stimulation under conditions that prevented the reduction in L1 blocked the effects on myelination. This link between myelination and functional activity in the axon at specific frequencies that change axonal expression of L1 could have important consequences for the structural and functional relationship of myelinating axons.
Decreased phosphorylation of neurofilaments in mice lacking myelin-associated glycoprotein (MAG) was shown to be associated with decreased activities of extracellular-signal regulated kinases (ERK1/2) and cyclin-dependent kinase-5 (cdk5). These in vivo changes could be caused directly by the absence of a MAG-mediated signaling pathway or secondary to a general disruption of the Schwann cell-axon junction that prevents signaling by other molecules. Therefore, in vitro experimental paradigms of MAG interaction with neurons were used to determine if MAG directly influences expression and phosphorylation of cytoskeletal proteins and their associated kinases. COS-7 cells stably transfected with MAG or with empty vector were co-cultured with primary dorsal root ganglion (DRG) neurons. Total amounts of the middle molecular weight neurofilament subunit (NF-M), microtubule-associated protein 1B (MAP1B), MAP2, and tau were up-regulated significantly in DRG neurons in the presence of MAG. There was also increased expression of phosphorylated high molecular weight neurofilament subunit (NF-H), NF-M, and MAP1B. Additionally, in similar in vitro paradigms, total and phosphorylated NF-M were increased significantly in PC12 neurons co-cultured with MAG-expressing COS cells or treated with a soluble MAG Fc-chimera. The increased expression of phosphorylated cytoskeletal proteins in the presence of MAG in vitro was associated with increased activities of ERK 1/2 and cdk5. We propose that interaction of MAG with an axonal receptor(s) induces a signal transduction cascade that regulates expression of cytoskeletal proteins and their phosphorylation by these proline-directed protein kinases.
Microtubule-associated protein (MAP) 1B is a high-molecular-weight cytoskeletal protein that is abundant in developing neuronal processes and appears to be necessary for axonal growth. Various biochemical and immunocytochemical results are reported, indicating that a significant fraction of MAP1B is expressed as an integral membrane glycoprotein in vesicles and the plasma membrane of neurons. MAP1B is present in microsomal fractions isolated from developing rat brain and fractionates across a sucrose gradient in a manner similar to synaptophysin, a well-known vesicular and plasma membrane protein. MAP1B is also in axolemma-enriched fractions (AEFs) isolated from myelinated axons of rat brain. MAP1B in AEFs and membrane fractions from cultured dorsal root ganglion neurons (DRGNs) remains membrane-associated following high-salt washes and contains sialic acid. Furthermore, MAP1B in intact DRGNs is readily degraded by extracellular trypsin and is labeled by the cell surface probe sulfosuccinimidobiotin. Immunocytochemical examination of DRGNs shows that MAP1B is concentrated in vesicle-rich varicosities along the length of axons. Myelinated peripheral nerves immunostained for MAP1B show an enrichment at the axonal plasma membrane. These observations demonstrate that some of the MAP1B in developing neurons is an integral plasma membrane glycoprotein. Key Words: Axolemma-Dorsal root ganglion neurons-GlycoproteinMembrane protein-Microtubule-associated proteinNeurons.
The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of the cellular stress response, but the biological functions of the related Nrf3 protein are largely unknown. Here we demonstrate a novel pro-apoptotic function of Nrf3 in mouse and human keratinocytes. In response to UV irradiation, Nrf3-deficient keratinocytes were protected from apoptosis in vitro and in vivo. The protective function was also seen under oxidative or hyperosmotic stress conditions, but not when apoptosis was induced by disruption of cell–matrix interactions. Mechanistically, we show that Nrf3-deficient keratinocytes exhibit stronger cell–cell and cell-matrix adhesion, which correlates with higher cell surface integrin levels and enhanced activation of focal adhesion kinase. Nrf3-deficient cells also formed more and larger focal adhesions and exhibited a higher motility. These results suggest that the strong expression of Nrf3 in basal keratinocytes promotes their elimination in response to DNA damage-inducing agents, thereby preventing accumulation of mutated stem and transit amplifying cells in the epidermis.
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