Oligodendrocytes elaborate an extensive network of multibranched processes and flat membranous sheets. Microtubules (MT) participate in the elaboration and stabilization of myelin-forming processes and are essential for cellular sorting processes. Microtubule-associated proteins (MAPs) are involved in the regulation and stabilization of the dynamic MT network. It has been shown previously that oligodendrocytes express the MAP tau, a phosphoprotein most abundant in neurons of the CNS. In this article, we demonstrate for the first time that oligodendrocytes contain all six tau isoforms, and that tau mRNA and protein expression is developmentally regulated. Immunoblot analysis reveals that tau protein is more abundant, and mature isoforms are more prominent at later stages of development. During the first week of culture maturation, a marked decrease in phosphorylation is observable. Using an RT-PCR approach, we can show that oligodendrocytes express small amounts of exon 3 containing isoforms and that during culture maturation, tau mRNA splice products with 3 MT-binding domains (3R) decrease and mRNA with 4 MT-binding domains (4R) increase. In situ hybridization study demonstrates that tau mRNA is present in precursor cells and in mature oligodendrocytes. Tau mRNA is actively transported into the cellular processes, is specifically present in the primary and some of the secondary processes, enriched at the turning and branching points and the growing tips, and often appears as small patches. Hence, localized tau translation at specific sites in the cellular extensions might contribute to the regulation of MT stability during process formation, early axonal contact establishment, and myelination.
Although protein phosphorylation has been characterized more extensively, modulation of the acetylation state of signaling molecules is now being recognized as a key means of signal transduction. The enzymes responsible for mediating these changes include histone acetyl transferases and histone deacetylases (HDACs). Members of the HDAC family of enzymes have been identified as potential therapeutic targets for diseases ranging from cancer to ischemia and neurodegeneration. We initiated a project to conduct comprehensive gene expression mapping of the 11 HDAC isoforms (HDAC1-11) (classes I, II, and IV) throughout the rat brain using high-resolution in situ hybridization (ISH) and imaging technology. Internal and external data bases were employed to identify the appropriate rat sequence information for probe selection. In addition, immunohistochemistry was performed on these samples to separately examine HDAC expression in neurons, astrocytes, oligodendrocytes, and endothelial cells in the CNS. This double-labeling approach enabled the identification of specific cell types in which the individual HDACs were expressed. The signals obtained by ISH were compared to radiolabeled standards and thereby enabled semiquantitative analysis of individual HDAC isoforms and defined relative levels of gene expression in >50 brain regions. This project produced an extensive atlas of 11 HDAC isoforms throughout the rat brain, including cell type localization, providing a valuable resource for examining the roles of specific HDACs in the brain and the development of future modulators of HDAC activity.
In glaucoma surgery, fibrotic processes occur, leading to impairment of liquid outflow. Activated fibroblasts are responsible for postoperative scarring. The transforming growth factor-β (TGF-β) pathway plays a key role in fibroblast function, differentiation and proliferation. The aim of this study was the characterization of the fibrotic potential of two subtypes of primary human ocular fibroblasts and the attempt to inhibit fibrotic processes specifically, without impairing cell viability. For fibrosis inhibition we focused on the small molecule pirfenidone, which has been shown to prevent pulmonary fibrosis by the decrease of the expression of TGF-β1, TGF-β2 and TGF-β3 cytokines. For in vitro examinations, isolated human primary fibroblasts from Tenon capsule and human intraconal orbital fat tissues were used. These fibroblast subpopulations were analyzed in terms of the expression of matrix components responsible for postoperative scarring. We concentrated on the expression of collagen I, III, VI and fibronectin. Additionally, we analyzed the expression of α-smooth muscle actin, which serves as a marker for fibrosis and indicates transformation of fibroblasts into myofibroblasts. Gene expression was analyzed by rtPCR and synthesized proteins were examined by immunofluorescence and Western blot methods. Proliferation of fibroblasts under different culture conditions was assessed using BrdU assay. TGF-β1 induced a significant increase of cell proliferation in both cell types. Also the expression of some fibrotic markers was elevated. In contrast, pirfenidone decreased cell proliferation and matrix synthesis in both fibroblast subpopulations. Pirfenidone slightly attenuated TGF-β1 induced expression of fibronectin and α-smooth muscle actin in fibroblast cultures, without impairing cell viability. To summarize, manipulation of the TGF-β signaling pathway by pirfenidone represents a specific antifibrotic approach with no toxic side effects in two human orbital fibroblast subtypes. We presume that pirfenidone is a promising candidate for the treatment of fibrosis following glaucoma surgery.
Proteasomal dysfunction has been implicated in neurodegenerative diseases, and molecular chaperones may provide a first line of defence against protein aggregate formation. We have shown before that oligodendrocytes respond to proteasomal inhibition by the onset of apoptotic cell death, whereas astrocytes have a higher capability to cope with stressful conditions that might be causally related to their high constitutive level of HSP25. This study was undertaken to investigate the effects of the proteasomal inhibitor MG-132 on aggregate formation in astrocytes, and to test if HSP25 exerts a protective means. Our data show that upon proteasomal inhibition aggresomes are formed in astrocytes that contain the small HSPs, HSP25 and alpha B-crystallin, and ubiquitinated proteins. HSP expression is induced and HSP25, alpha B-crystallin and ubiquitinated proteins are translocated from the soluble to the detergent-insoluble fraction. Simultaneously, the cytoskeletal organization is disturbed, microfilaments are fragmented, GFAP intermediate filaments and microtubules surround the aggresome, and mitochondria are assembled in these structures. Mitochondria membrane potential, however, stays intact. Aggresome formation and apoptotic cell death do not correlate. After the removal of MG-132, the observed effects are reversible. MG-132 promotes the formation of small oligomers of HSP25, which have been connected to the protection of the microfilament system. Downregulation of HSP25 by siRNA approach causes actin filament breakdown in control cells in the absence of stress stimuli, and sensitizes astrocytes against stress induced by proteasomal inhibition. Hence, HSP25 enables astrocytes to prevent irreversible damage and to recover after removal of the proteasomal inhibitor MG-132.
Oligodendrocytes, the myelin-forming cells of the CNS, are specifically sensitive to oxidative stress and respond by the onset of programmed cell death (PCD). To further unravel the molecular events underlying their enhanced susceptibility, we have investigated whether mitochondrial damage occurs during oxidative stress-induced PCD in cultured rat brain oligodendrocytes. Mitochondria are considered as a central control point of apoptosis, and mitochondrial dysfunction has been linked to neurodegenerative disease. Upon a number of stimuli through the release of cytochrome c, they coordinate caspase activation, causing morphological and biochemical changes associated with PCD. Oxidative stress was exerted by the application of hydrogen peroxide. The data show that hydrogen peroxide-induced apoptosis in oligodendrocytes involves mitochondrial damage and cytochrome c release and is accompanied by the activation of the death-related caspases 3 and 9. Concomitantly, the activation and nuclear translocation of extracellular signal regulated kinases ERK1,2 are observed, which have been implicated to participate in the regulation of cell death and survival. DNA fragmentation could not be attenuated by the ERK1,2 inhibitor PD 98059, indicating that the ERK1,2- pathway in oligodendrocytes may be involved in the initial survival response after exposure to stressful stimuli.
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