Myelination of mouse cerebellar explants by rat cultured oligodendrocytes

It has been shown that transplanted central nervous system tissue containing oligodendrocytes will myelinate neuronal processes in vitro and in situ. In this study we propose to show that cultured rat oligodendrocytes have the capacity to myelinate mouse cerebellar neuronal processes in vitro. Cultured rat oligodendrocytes were transplanted to cytosine arabinoside-treated mouse cerebellar explant cultures, then observed for myelination. Ultrastructural examination showed myelin and myelin-like figures in co-cultures. Control cytosine arabinoside-treated cultures and cultured oligodendroglia were without compact myelin.     

ABCA2蛋白在大鼠面神经组织内雪旺细胞成熟过程中的表达

目的本研究通过检测面神经发育过程中ATP结合盒式转运蛋A2(ABCA2)的表达方式,探讨ABCA2蛋白在周围神经系统髓鞘形成过程中的作用。方法应用多重免疫荧光方法检测发育过程中面神经组织内ABCA2和特异雪旺细胞标志抗体的表达。结果 ABCA2蛋白由出生后第8d在成髓鞘雪旺细胞特异抗体-锌指端转录因子Krox20和S100阳性的细胞中开始少量表达;在出生后第14d,近50%髓鞘化的该类细胞中可检测到ABCA2蛋白;在成熟期,所有该类细胞中都可检测到ABCA2蛋白。结论 ABCA2在生后雪旺细胞的表达随髓鞘化的发育成熟而增加。     

The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development

The nonreceptor tyrosine phosphatase Shp2 (PTPN11) has been implicated in tyrosine kinase, cytokine, and integrin receptor signaling. We show here that conditional mutation of Shp2 in neural crest cells and in myelinating Schwann cells resulted in deficits in glial development that are remarkably similar to those observed in mice mutant for Neuregulin-1 (Nrg1) or the Nrg1 receptors, ErbB2 and ErbB3. In cultured Shp2 mutant Schwann cells, Nrg1-evoked cellular responses like proliferation and migration were virtually abolished, and Nrg1-dependent intracellular signaling was altered. Pharmacological inhibition of Src family kinases mimicked all cellular and biochemical effects of the Shp2 mutation, implicating Src as a primary Shp2 target during Nrg1 signaling. Together, our genetic and biochemical analyses demonstrate that Shp2 is an essential component in the transduction of Nrg1/ErbB signals.     

Minocycline alleviates hypoxic-ischemic injury to developing oligodendrocytes in the neonatal rat brain

The role of minocycline in preventing white matter injury, in particular the injury to developing oligodendrocytes was examined in a neonatal rat model of hypoxia-ischemia. Hypoxia-ischemia was achieved through bilateral carotid artery occlusion followed by exposure to hypoxia (8% oxygen) for 15 min in postnatal day 4 Sprague-Dawley rats. A sham operation was performed in control rats. Minocycline (45 mg/kg) or normal phosphate-buffered saline was administered intraperitoneally 12 h before and immediately after bilateral carotid artery occlusion+hypoxia and then every 24 h for 3 days. Nissl staining revealed pyknotic cells in the white matter area of the rat brain 1 and 5 days after hypoxia-ischemia. Hypoxia-ischemia insult also resulted in apoptotic oligodendrocyte cell death, loss of O4+ and O1+ oligodendrocyte immunoreactivity, and hypomyelination as indicated by decreased myelin basic protein immunostaining and by loss of mature oligodendrocytes in the rat brain. Minocycline significantly attenuated hypoxia-ischemia-induced brain injury. The protective effect of minocycline was associated with suppression of hypoxia-ischemia-induced microglial activation as indicated by the decreased number of activated microglia, which were also interleukin-1beta and inducible nitric oxide synthase expressing cells. The protective effect of minocycline was also linked with reduction in hypoxia-ischemia-induced oxidative and nitrosative stress as indicated by 4-hydroxynonenal and nitrotyrosine positive oligodendrocytes, respectively. The reduction in hypoxia-ischemia-induced oxidative stress was also evidenced by the decreases in the content of 8-isoprostane in the minocycline-treated hypoxia-ischemia rat brain as compared with that in the vehicle-treated hypoxia-ischemia rat brain. The overall results suggest that reduction in microglial activation may protect developing oligodendrocytes in the neonatal brain from hypoxia-ischemia injury.     

Shift of adenosine kinase expression from neurons to astrocytes during postnatal development suggests dual functionality of the enzyme

Adenosine is a potent modulator of excitatory neurotransmission, especially in seizure-prone regions such as the hippocampal formation. In adult brain ambient levels of adenosine are controlled by adenosine kinase (ADK), the major adenosine-metabolizing enzyme, expressed most strongly in astrocytes. Since ontogeny of the adenosine system is largely unknown, we investigated ADK expression and cellular localization during postnatal development of the mouse brain, using immunofluorescence staining with cell-type specific markers. At early postnatal stages ADK immunoreactivity was prominent in neurons, notably in cerebral cortex and hippocampus. Thereafter, as seen best in hippocampus, ADK gradually disappeared from neurons and appeared in newly developed nestin- and glial fibrillary acidic protein (GFAP)-positive astrocytes. Furthermore, the region-specific downregulation of neuronal ADK coincided with the onset of myelination, as visualized by myelin basic protein staining. After postnatal day 14 (P14), the transition from neuronal to astrocytic ADK expression was complete, except in a subset of neurons that retained ADK until adulthood in specific regions, such as striatum. Moreover, neuronal progenitors in the adult dentate gyrus lacked ADK. Finally, recordings of excitatory field potentials in acute slice preparations revealed a reduced adenosinergic inhibition in P14 hippocampus compared with adult. These findings suggest distinct roles for adenosine in the developing and adult brain. First, ADK expression in young neurons may provide a salvage pathway to utilize adenosine in nucleic acid synthesis, thus supporting differentiation and plasticity and influencing myelination; and second, adult ADK expression in astrocytes may offer a mechanism to regulate adenosine levels as a function of metabolic needs and synaptic activity, thus contributing to the differential resistance of young and adult animals to seizures.     

Oligodendrocyte responses to buprenorphine uncover novel and opposing roles of μ-opioid- and nociceptin/orphanin FQ receptors in cell development: implications for drug addiction treatment during pregnancy

Although the classical function of myelin is the facilitation of saltatory conduction, this membrane and the oligodendrocytes, the cells that make myelin in the central nervous system (CNS), are now recognized as important regulators of plasticity and remodeling in the developing brain. As such, oligodendrocyte maturation and myelination are among the most vulnerable processes along CNS development. We have shown previously that rat brain myelination is significantly altered by buprenorphine, an opioid analogue currently used in clinical trials for managing pregnant opioid addicts. Perinatal exposure to low levels of this drug induced accelerated and increased expression of myelin basic proteins (MBPs), cellular and myelin components that are markers of mature oligodendrocytes. In contrast, supra-therapeutic drug doses delayed MBP brain expression and resulted in a decreased number of myelinated axons. We have now found that this biphasic-dose response to buprenorphine can be attributed to the participation of both the μ-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOP receptor) in the oligodendrocytes. This is particularly intriguing because the NOP receptor/nociceptin system has been primarily linked to behavior and pain regulation, but a role in CNS development or myelination has not been described before. Our findings suggest that balance between signaling mediated by (a) MOR activation and (b) a novel, yet unidentified pathway that includes the NOP receptor, plays a crucial role in the timing of oligodendrocyte maturation and myelin synthesis. Moreover, exposure to opioids could disrupt the normal interplay between these two systems altering the developmental pattern of brain myelination.     

Role of transmembrane semaphorin Sema6A in oligodendrocyte differentiation and myelination

Myelination is regulated by extracellular proteins, which control interactions between oligodendrocytes and axons. Semaphorins are repulsive axon guidance molecules, which control the migration of oligodendrocyte precursors during normal development and possibly in demyelinating diseases. We show here that the transmembrane semaphorin 6A (Sema6A) is highly expressed by myelinating oligodendrocytes in the postnatal mouse brain. In adult mice, Sema6A expression is upregulated in demyelinating lesions in cuprizone‐treated mice. The analysis of the optic nerve and anterior commissure of Sema6A‐deficient mice revealed a marked delay of oligodendrocyte differentiation. Accordingly, the development of the nodes of Ranvier is also transiently delayed. We also observed an arrest in the in vitro differentiation of purified oligodendrocytes lacking Sema6A, with a reduction of the expression level of Myelin Basic Protein. Their morphology is also abnormal, with less complex and ramified processes than wild‐type oligodendrocytes. In myelinating co‐cultures of dorsal root ganglion neurons and purified oligodendrocytes we found that myelination is perturbed in absence of Sema6A. These results suggest that Sema6A might have a role in myelination by controlling oligodendrocyte differentiation. © 2012 Wiley Periodicals, Inc     

Abnormal Schwann cell/axon interactions in the Trembler-J mouse

The Trembler-J ( Tr ^J ) mouse has a point mutation in the gene coding for peripheral myelin protein 22 (PMP22). Disturbances in PMP22 are associated with abnormal myelination in a range of inherited peripheral neuropathies both in mice and humans. PMP22 is produced mainly by Schwann cells in the peripheral nervous system where it is localised to compact myelin. The function of PMP22 is unclear but its low abundance (∼5% of total myelin protein) means that it is unlikely to play a structural role. Its inclusion in a recently discovered family of proteins suggests a function in cell proliferation/differentiation and possibly in adhesion. Nerves from Tr ^J and the allelic Trembler ( Tr ) mouse are characterised by abnormally thin myelin for the size of the axon and an increased number of Schwann cells. We report ultrastructural evidence of abnormal Schwann cell-axon interactions. Schwann cell nuclei have been found adjacent to the nodes of Ranvier whereas in normal animals they are located near the centre of the internodes. In some fibres the terminal myelin loops faced outwards into the extracellular space instead of turning inwards and terminating on the axon. In severely affected nerves many axons were only partially surrounded by Schwann cell cytoplasm. All these features suggest a failure of Schwann cell–axon recognition or interaction. In addition to abnormalities related to abnormal myelination there was significant axonal loss in the dorsal roots.     

Acute inhalation of combustion smoke triggers neuroinflammation and persistent anxiety-like behavior in the mouse

Context: Acute inhalation of combustion smoke triggers neurologic sequelae in survivors. Due to the challenges posed by heterogeneity of smoke exposures in humans, mechanistic links between acute smoke inhalation and neuropathologic sequelae have not been systematically investigated.

Methods: Here, using mouse model of acute inhalation of combustion smoke, we studied longitudinal neurobehavioral manifestations of smoke exposures and molecular/cellular changes in the mouse brain.

Results: Immunohistochemical analyses at eight months post-smoke, revealed hippocampal astrogliosis and microgliosis accompanied by reduced myelination. Elevated expression of proinflammatory cytokines was also detected. Longitudinal testing in different neurobehavioral paradigms in the course of post-smoke recovery, revealed lasting anxiety-like behavior. The examined paradigms included the open field exploration/anxiety testing at two, four and six months post-smoke, which detected decreases in total distance traveled and time spent in the central arena in the smoke-exposed compared to sham-control mice, suggestive of dampened exploratory activity and increased anxiety-like behavior. In agreement with reduced open field activity, cued fear conditioning test revealed increased freezing in response to conditioned auditory stimulus in mice after acute smoke inhalation. Similarly, elevated plus maze testing demonstrated lesser presence in open arms of the maze, consistent with anxiety-like behavior, for the post-smoke exposure mice.

Conclusions: Taken together, our data demonstrate for the first time persistent neurobehavioral manifestations of acute inhalation of combustion smoke and provide new insights into long-term progression of events initiated by disrupted brain oxygenation that might contribute to lasting adverse sequelae in survivors of smoke inhalation injuries.     

Transport of proteolipid protein to the plasma membrane does not depend on glycosphingolipid cotransport in oligodendrocyte cultures

The possibility that transport of proteolipid protein (PLP) from its site of synthesis to the plasma membrane is dependent on cotransport with (sulfo)galacto-cerebrosides was investigated in primary cultured oligodendrocytes and Chinese hamster ovary (CHO) cells expressing PLP. Sulfation was inhibited by growing oligodendrocytes in the presence of a competitive inhibitor of this process, sodium chlorate. Under these circumstances, sulfatide synthesis was inhibited by 85%. Nevertheless, PLP was still delivered to the plasma membrane in quantitative amounts. Furthermore, when PLP was expressed in CHO cells, which normally synthesize very low amounts of galactosyl ceramide (GalCer) and no sulfatide, PLP was transported to the plasma membrane. Moreover, in CHO cells coexpressing PLP and ceramide galactosyl transferase, PLP cell surface labeling was unaltered. Noting that it has been demonstrated that proteins destined for the apical surface of epithelial cells colocalize with glycolipid-enriched microdomains, we isolated detergent-insoluble membrane complexes from cultured oligodendrocytes. We found, however, that most of the PLP is present in the detergent-soluble fraction and, furthermore, that PLP could not be chased into or out of the insoluble fraction. Taken together, these data make it very likely that in oligodendrocytes PLP transport takes place irrespective of the presence of glycosphingolipids GalCer and sulfatide. J. Neurosci. Res. 51:371–381, 1998. © 1998 Wiley-Liss, Inc.