The Protective Effects of Inosine Against Chemical Hypoxia on Cultured Rat Oligodendrocytes

Inosine is a purine nucleoside and is considered protective to neural cells including neurons and astrocytes against hypoxic injury. However, whether oligodendrocytes (OLs) could also be protected from hypoxia by inosine is not known. Here we investigated the effects of inosine on primarily cultured rat OLs injured by rotenone-mediated chemical hypoxia, and the mechanisms of the effects using ATP assay, MTT assay, PI-Hoechst staining, TUNEL, and immunocytochemistry. Results showed that rotenone exposure for 24 h caused cell death and impaired viability in both immature and mature OLs, while pretreatment of 10 mM inosine 30 min before rotenone administration significantly reduced cell death and improved the viability of OLs. The same concentration of inosine given 120 min after rotenone exposure also improved viability of injured mature OLs. Immunocytochemistry for nitrotyrosine and cellular ATP content examination indicated that inosine may protect OLs by providing ATP and scavenging peroxynitrite for cells. In addition, immature OLs were more susceptible to hypoxia than mature OLs; and at the similar degree of injury, inosine protected immature and mature OLs differently. Quantitative real-time PCR revealed that expression of adenosine receptors was different between these two stages of OLs. These data suggest that inosine protect OLs from hypoxic injury as an antioxidant and ATP provider, and the protective effects of inosine on OLs vary with cell differentiation, possibly due to the adenosine receptors expression profile. As OLs form myelin in the central nervous system, inosine could be used as a promising drug to treat demyelination-involved disorders.     

Process outgrowth in oligodendrocytes is mediated by CNP, a novel microtubule assembly myelin protein

Oligodendrocytes (OLs) extend arborized processes that are supported by microtubules (MTs) and microfilaments. Little is known about proteins that modulate and interact with the cytoskeleton during myelination. Several lines of evidence suggest a role for 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in mediating process formation in OLs. In this study, we report that tubulin is a major CNP-interacting protein. In vitro, CNP binds preferentially to tubulin heterodimers compared with MTs and induces MT assembly by copolymerizing with tubulin. CNP overexpression induces dramatic morphology changes in both glial and nonglial cells, resulting in MT and F-actin reorganization and formation of branched processes. These morphological effects are attributed to CNP MT assembly activity; branched process formation is either substantially reduced or abolished with the expression of loss-of-function mutants. Accordingly, cultured OLs from CNP-deficient mice extend smaller outgrowths with less arborized processes. We propose that CNP is an important component of the cytoskeletal machinery that directs process outgrowth in OLs.     

The life, death, and replacement of oligodendrocytes in the adult CNS

Oligodendrocytes (OLs) are mature glial cells that myelinate axons in the brain and spinal cord. As such, they are integral to functional and efficient neuronal signaling. The embryonic lineage and postnatal development of OLs have been well-studied and many features of the process have been described, including the origin, migration, proliferation, and differentiation of precursor cells. Less clear is the extent to which OLs and damaged/dysfunctional myelin are replaced following injury to the adult CNS. OLs and their precursors are very vulnerable to conditions common to CNS injury and disease sites, such as inflammation, oxidative stress, and elevated glutamate levels leading to excitotoxicity. Thus, these cells become dysfunctional or die in multiple pathologies, including Alzheimer's disease, spinal cord injury, Parkinson's disease, ischemia, and hypoxia. However, studies of certain conditions to date have detected spontaneous OL replacement. This review will summarize current information on adult OL progenitors, mechanisms that contribute to OL death, the consequences of their loss and the pathological conditions in which spontaneous oligodendrogenesis from endogenous precursors has been observed in the adult CNS.     

The Role of the Oligodendrocyte Lineage in Acute Brain Trauma

An acute brain injury is commonly characterized by an extended cellular damage. The post-injury process of scar formation is largely determined by responses of various local glial cells and blood-derived immune cells. The role of astrocytes and microglia have been frequently reviewed in the traumatic sequelae. Here, we summarize the diverse contributions of oligodendrocytes (OLs) and their precursor cells (OPCs) in acute injuries. OLs at the lesion site are highly sensitive to a damaging insult, provoked by Ca²⁺ overload after hyperexcitation originating from increased levels of transmitters. At the lesion site, differentiating OPCs can replace injured oligodendrocytes to guarantee proper myelination that is instrumental for healthy brain function. In contrast to finally differentiated and non-dividing OLs, OPCs are the most proliferative cells of the brain and their proliferation rate even increases after injury. There exist even evidence that OPCs might also generate some type of astrocyte beside OLs. Thereby, OPCs can contribute to the generation and maintenance of the glial scar. In the future, detailed knowledge of the molecular cues that help to prevent injury-evoked glial cell death and that control differentiation and myelination of the oligodendroglial lineage will be pivotal in developing novel therapeutic approaches.     

Down‐regulation of long non‐coding RNA MEG3 promotes Schwann cell proliferation and migration and repairs sciatic nerve injury in rats

Peripheral nerve injury and regeneration are complex processes and involve multiple molecular and signalling components. However, the involvement of long non‐coding RNA (lncRNA) in this process is not fully clarified. In this study, we evaluated the expression of the lncRNA maternally expressed gene 3 (MEG3) in rats after sciatic nerve transection and explored its potential mechanisms. The expression of lncRNA MEG3 was up‐regulated following sciatic nerve injury and observed in Schwann cells (SCs). The down‐regulation of lncRNA MEG3 in SCs enhanced the proliferation and migration of SCs via the PTEN/PI3K/AKT pathway. The silencing of lncRNA MEG3 promoted the migration of SCs and axon outgrowth in rats after sciatic nerve transection and facilitated rat nerve regeneration and functional recovery. Our findings indicated that lncRNA MEG3 may be involved in nerve injury and injured nerve regeneration in rats with sciatic nerve defects by regulating the proliferation and migration of SCs. This gene may provide a potential therapeutic target for improving peripheral nerve injury.     

p53 Induction by Tumor Necrosis Factor-α and Involvement of p53 in Cell Death of Human Oligodendrocytes

Oligodendrocytes (OLs) and their myelin membranes are the primary targets in the autoimmune disease multiple sclerosis (MS). The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) has been implicated as a mediator of OL cell injury. TNF-alpha is detectable within MS lesions and induces apoptosis of mature human OLs in vitro. One possible mechanism by which TNF-alpha mediates cell death is through the activation of c-jun N-terminal kinase (JNK). We have previously shown that treatment of human OLs with TNF-alpha leads to activation of JNK. Here we provide evidence that p53, a regulator of the cell cycle and apoptosis, is a mediator of TNF-alpha-induced apoptosis of OLs. Although p53 was undetectable by western blot analysis in adult human OLs, its levels increased within 24 h after TNF-alpha treatment (100 ng/ml). The induced p53 was immunolocalized to the nucleus prior to the appearance of significant numbers of apoptotic cells. Overexpression of p53 by adenovirus-mediated gene transfer into human OLs in vitro resulted in marked apoptosis as revealed by in situ cleavage of DNA (TUNEL positive), decreased mitochondrial function, and release of lactate dehydrogenase into the culture medium. These in vitro studies demonstrate that increased p53 levels are associated with apoptosis of human OLs. The findings further implicate p53 as a target for the JNK pathway activated during TNF-alpha-mediated cell death of human adult OLs.     

Masseteric Nerve Injury Increases Expression of Brain-Derived Neurotrophic Factor in Microglia Within the Rat Mesencephalic Trigeminal Tract Nucleus

The distribution of brain-derived neurotrophic factor was examined in the rat mesencephalic trigeminal tract nucleus after transection and crush of the masseteric nerve. In the intact mesencephalic trigeminal tract nucleus, brain-derived neurotrophic factor was detected in small cells with fine processes. These cells and processes were occasionally located adjacent to tyrosine kinase B receptor-immunoreactive sensory neurons. The transection and crush of the masseteric nerve increased expression of brain-derived neurotrophic factor in the nucleus. The number and size of brain-derived neurotrophic factor-immunoreactive cells and processes were dramatically elevated by the nerve injury. As a result, the density of brain-derived neurotrophic factor-immunoreactive profiles in the mesencephalic trigeminal tract nucleus at 7 days after the injury was significantly higher compared with the intact nucleus. Double immunofluorescence method also revealed that brain-derived neurotrophic factor-immunoreactive cells were mostly immunoreactive for OX-42 but not glial fibrillary acidic protein. In addition, the retrograde tracing method demonstrated that brain-derived neurotrophic factor-immunoreactive cells and processes surrounded retrogradely labeled neurons which showed tyrosine kinase B receptor-immunoreactivity. These findings indicate that the nerve injury increases expression of brain-derived neurotrophic factor in microglia within the mesencephalic trigeminal tract nucleus. The glial neurotrophic factor may be associated with axonal regeneration of the injured primary proprioceptor in the trigeminal nervous system.     

Rapid lamellipodia formation in nerve growth factor-stimulated PC12 cells is dependent on Rac and PI3K activity

Neuronal differentiation of PC12 cells is achieved by stimulation with nerve growth factor (NGF) but not by epidermal growth factor (EGF). However, features of differentiation such as neurite outgrowth are observable at the earliest after several hours. Using actin staining of the cells, we show here that NGF stimulation leads to lamellipodia formation within only 3 min at the periphery of the PC12 cells. EGF stimulation or microinjection of differentiation-inducing c-Crk I protein does not cause lamellipodia. The actin reorganization after NGF stimulation is blocked by microinjecting dominant negative Rac protein. The lamellipodia formation is also abolished by inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY 294002 in a concentration-dependent manner. Phase-contrast time-lapse microscopy was used to analyze membrane dynamics in real time and to confirm the induction of lamellipodia by NGF and their inhibition by pretreatment with both wortmannin and LY 294002. The results indicate that NGF, but not EGF, leads to rapid lamellipodia formation in PC12 cells via phosphatidylinositol 3-kinase and the small GTPase Rac, thereby defining a novel role for these factors in early NGF signaling.     

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish ( Carassius auratus ). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5–40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5–40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo . None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.     

Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury

Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of OX42 + microglial cells and glial fibrillary acidic protein + astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages.     

The origin of neointimal cells in the rat carotid artery after balloon angioplasty

At present the issue of a possible role of circulating stem cells and precursors in pathological vascular wall remodeling after angioplasty remains unsolved. Therefore the origin of neointimal cells was examined in the rat carotid artery after balloon angioplasty using morphological and immunocytochemical approaches. It is shown that at the early stages (1-7 days) after vessel injury acute inflammatory response arises in the arterial wall recruiting neutrophils, monocytes, macrophages as well as large amounts of low-differentiated blood-derived cells. At the late stages (10-28 days), at the area of injured intima, a new hyperplastic intima (neointima) is formed, which consists of cells carrying specific smooth muscle markers--alpha-actin and smoothelin. The study on cell proliferative behaviour in the injured vessel wall by bromodeoxyuridine showed that in the process of neointima formation blood-born rather than resident cells are involved. Probably, early smooth muscle and endothelial precursor cells penetrate into injured area with blood stream, where they proliferative and differentiate into mature cells.     

Shoc2/Sur8 Protein Regulates Neurite Outgrowth

The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.     

FLRT3, a cell surface molecule containing LRR repeats and a FNIII domain, promotes neurite outgrowth

The mature peripheral nervous system has the ability to survive and to regenerate its axons following axonal injury. After nerve injury, the distal axonal and myelin segment undergoes dissolution and absorption by the surrounding cellular environment, a process called Wallerian degeneration. Using cDNA microarrays, we isolated FLRT3 as one of the up-regulated genes expressed in the distal segment of the sciatic nerve 7 days after transection relative to those of the intact sciatic nerve. FLRT3 is a putative type I transmembrane protein containing 10 leucine-rich repeats, a fibronectin type III domain, and an intracellular tail. The neurons plated on CHO cells expressing FLRT3 extended significantly longer neurites than those plated on wild-type CHO cells, demonstrating that FLRT3 promotes neurite outgrowth. FLRT3 mRNA was especially abundant in the basal ganglia, the granular layer of cerebellum, and the hippocampus, except the CA1 region in the adult rat brain. Thus, FLRT3 may contribute to regeneration following axonal injury.     

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG(R)) mRNA increased in RGCs 1-6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.     

Morphological changes and expression of protein kinase CK2 β subunit in the microglia after hypoglossal nerve transection

Following hypoglossal nerve transection, the microglia of the rat hypoglossal nucleus expressed protein kinase CK2 β subunit immunoreactivity. CK2 β immunostaining occurred on the operated side from postoperative day 3; on day 5 we observed strong immunoreactivity and the immunopositive microglial cell processes surrounded the injured neurones. Thereafter, the immunoreactivity decreased gradually and on day 10 the immunopositive cells surrounded only a few injured neurones. Electron microscopic observations on the hypoglossal nucleus revealed microglia-neuronal contact within 3 hours of nerve injury, and by day 3 all the injured neurones were in contact with microglial cells. These observations indicated that microglia-neuronal contact occurred earlier than the CK2 β subunit immunoreactivity. CK2 may not be implicated during the initial migration of the microglia to the injured neurones; however, it may enhance the growth and elongation of the microglial cell processes around the injured neurones.     

Molecular cloning of a novel transmembrane protein MOLT expressed by mature oligodendrocytes

A novel oligodendrocyte (OL)-specific cDNA was isolated from brain capillary endothelial cells and characterized. The cDNA encodes a protein of 1099 amino acids that contains a signal peptide and a transmembrane domain. The protein was expressed in mature OLs in vivo and in vitro cell cultures and was thus designated as mature OL transmembrane protein (MOLT). RT-PCR analysis showed that MOLT mRNA was expressed in brain, lung, pancreas, and testis. A polyclonal antibody raised against a part of the mouse MOLT reacted specifically with multipolar OLs possessing radially oriented processes that penetrated into the gray matter. More cells were detected in the white matter, and these had longitudinally oriented processes. In a rat OL lineage culture system, oligodendrocyte precursor cells did not initially produce MOLT mRNA and protein, but when they begun to differentiate into mature OLs, they started expressing MOLT. Consequently, MOLT may function as OLs become mature and may serve as a cell-surface marker for OL differentiation.     

Rho‐associated kinase (ROCK) inhibitor,Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF

Neurite extension and retraction are very important processes in the formation of neuronal networks. A strategy for fostering axonal regrowth/regeneration of injured adult neurons is attractive therapeutically for various diseases such as traumatic brain injury, stroke and Alzheimer's disease. The Rho family of small GTPases, including Rac and Cdc42 have been shown to be involved in promoting neurite outgrowth. On the other hand, activation of RhoA induces collapse of growth cone and retraction of neurites. Rho‐associated kinase (ROCK) an effector molecule of RhoA, is downstream of a number of axonal outgrowth and growth cone collapse inhibition mechanisms. In the present study, we sought to identify the role of ROCK in neurite outgrowth in PC12 cells. Y27632, a specific inhibitor of ROCK, induced a robust increase in neurite outgrowth in these cells within 24–48 h as visualized by phase contrast microscopy. Staining with FITC‐tubulin or phalloidin show extended neurites in PC12 cells treated with Y27632, comparable to that with 100 ng/mL of NGF. Assessment of other biochemical markers of neurite outgrowth such as GAP43, neurofilament and tyrosine hydroxylase phosphorylation further indicates that inhibition of ROCK in PC12 cells causes differentiation of these cells to a neuronal phenotype.     

Endothelial Injury Induces Vascular Smooth Muscle Cell Proliferation in Highly Localized Regions of a Direct Contact Co-culture System

Though previous studies have indicated a relationship between the proliferation of endothelial cells and vascular smooth muscle cells (VSMCs) in co-culture, the results have been contradictory and the signaling mechanism poorly understood. In this transmembrane co-culture study, VSMCs and endothelial cells were grown to confluence on opposite sides of a microporous membrane to mimic the intima/media border of vessels. The endothelial layer was injured, and then cultured for 3 days, resulting in partial re-endothelialization. VSMC proliferation across from the injured/partially recovered endothelial region was significantly higher than across from the de-endothelialized region (a sevenfold increase) and the uninjured region (a threefold increase). ELISA indicated that PDGF, which was undetectable in uninjured co-culture and homotypic controls, increased after injury and the addition of a piperazinyl-quinazoline carboxamide PDGF receptor inhibitor blocked VSMC proliferation across from the injured/partially recovered region. We conclude that co-culture signaling initiated by endothelial cell injury locally stimulates VSMC proliferation and that this signaling could be mediated by PDGF-BB.