RhoA and lysophosphatidic acid are involved in the actin cytoskeleton reorganization of astrocytes exposed to ethanol

Astroglial cells play an important role in maintaining neuronal function in the adult and in the developing nervous system. Ethanol exposure induces profound alterations in the astrogliogenesis process, affecting important cell functions, including intracellular protein trafficking. Because the actin cytoskeleton plays a crucial role in intracellular protein transport, the aim of the present study was to analyze the effects of ethanol on actin cytoskeleton organization and the involvement of the RhoA signaling pathway in these effects. We show that RhoA and lysophosphatidic acid (LPA), an upstream activator of RhoA, stimulate the formation of stress fibers and focal adhesion in cortical astrocytes in primary culture. Exposure of cultured astrocytes to different concentrations of ethanol profoundly disorganizes the actin cytoskeleton, leading to the formation of actin rings at the cell periphery and decreasing the content of focal adhesion proteins. Furthermore, LPA treatment or RhoA transfection revert the ethanol-induced actin alterations in astrocytes, whereas transfection with an inactive mutant of RhoA is unable to revert the actin ring organization. In addition, inhibition of endogenous RhoA by C3 exoenzyme effectively blocks ethanol-induced actin ring formation. These results suggest that the effects of alcohol on actin cytoskeleton organization are mediated by the RhoA signaling pathway. Disruptions in actin organization may impair important astrocyte functions, participating in ethanol-induced astroglial and brain damage during development.     

Oligodendrocyte progenitor migration in response to injury of glial monolayers requires the polysialic neural cell-adhesion molecule

Injury to the nervous system results in reactive astrogliosis that is a critical determinant of neuronal regeneration. To analyze glial responses to mechanical injury and the role of the polysialic neural cell adhesion molecule (PSA-NCAM) in this process, we established primary glia cultures from newborn rat cerebral cortex. Scratching a confluent monolayer of primary glial cells resulted in two major events: rapid migration of oligodendrocyte progenitor-like (O-2A) cells into the wounded area and development of polarized morphology of type 1 astrocytes at the wound edge. Migrating O-2A progenitors had a bipolar morphology and exhibited A2B5 and O4 immunolabeling. Once these cells were established inside the wounded area, they lost A2B5 immunoreactivity and differentiated into glial fibrillary acidic protein-positive astrocytes. Migrating O-2A cells expressed PSA-NCAM, but type 1 astrocytes at the wound edge did not. Treatment of wounded cultures with Endo-N, which specifically removes PSA from the surface of cells, resulted in a significant decrease in O-2A cell migration into the wounded area and completely blocked the wound closure. Video time-lapse analysis showed that, in the presence of Endo-N, O-2A cells remained motile and migrated short distances but did not move away from the monolayer. These results demonstrate that O-2A progenitors contribute to reactive astrogliosis in culture and that PSA-NCAM is involved in this process by regulating cell migration.     

Mechanical trauma induces rapid astroglial activation of ERK/MAP kinase: Evidence for a paracrine signal

Astrogliosis is a prominent and ubiquitous reaction of astrocytes to many forms of CNS injury, often implicated in the poor regenerative capacity of the adult mammalian CNS. Transmembrane signals that rapidly trigger and maintain astroglial responses to injury are largely undefined. Several candidate inducers of astrogliosis, including growth factors and neuropeptides, act via the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. We previously observed chronically activated ERK/MAPK in human reactive astrocytes. To investigate mechanisms of pathway activation in a defined in vitro model, primary cultured astroglial monolayers were subjected to focal mechanical injury. Within 2-10 min, ERK/MAPK was activated, but only in cells near the wound edge. By 30 min, the entire monolayer showed activation, which persisted for 4 to 8 h. ERK/MAPK activation was specifically blocked by application of the MEK inhibitors, PD98059 and U0126. Cell-cell contact was not necessary for intercellular spread of ERK/MAPK activation, and ERK/MAPK-stimulating activity was found in the injury-conditioned medium. The activating factor was shown to have a native size of 50-100 kD and did not signal through the classical EGF receptor. Injury-induced signaling to ERK/MAPK required Ras, as demonstrated by specific blockade after transient transfection with a dominant negative Ha-RasN17 construct. Finally, we demonstrated that focal lesioning of adult rat cortex induces a rapid activation and spreading of astroglial ERK/MAPK, suggesting that similar mechanisms may operate in astroglial activation following acute brain injury.     

Demyelination and axonal dystrophy in alpha A-crystallin transgenic mice

Homozygous mice transgenic for alphaA-crystallin, one of the structural eye lens proteins, developed hindlimb paralysis after 8 weeks of age. To unravel the pathogenesis of this unexpected finding and the possible role of alphaA-crystallin in this pathological process, mice were subjected to a histopathological and immunohistochemical investigation. Immunohistochemistry showed large deposits of alphaA-crystallin in the astrocytes of the spinal cord, and in the Schwann cells of dorsal roots and sciatic nerves. Additionally, microscopy showed dystrophic axons in the spinal cord and digestion chambers as a sign of ongoing demyelination in dorsal roots and sciatic nerves. Apart from a few areas with slight alphaA-crystallin-immunopositive structures, the brain was normal. Because the alphaA-crystallin protein expression appeared in specific cells of the nervous system (astrocytes and Schwann cells), the most plausible explanation for the paralysis is a disturbance of cell function caused by the excessive intracytoplasmic accumulation of the alphaA-crystallin protein. This is followed by a sequence of secondary changes (demyelination, axonal dystrophy) and finally arthrosis. In conclusion, alphaA-crystallin transgenic mice develop a peripheral and central neuropathy primarily affecting spinal cord areas at the dorsal side, dorsal root and sciatic nerve.     

Parkinsonism, dementia and vertical gaze palsy in a Guamanian with atypical neuroglial degeneration

A 58-year-old Chamorro female patient, who died in 1993, was examined clinicopathologically. At the age of 51, she suffered from hemiparkinsonism, then bradykinesia, rigidity without tremor, and dementia. Extrapyramidal symptoms developed, and at the age of 57, vertical gaze palsy was noted. The clinical diagnosis was parkinsonism-dementia complex (PDC) with vertical gaze palsy. The brain showed atrophy in the frontal and temporal lobes, and the atrophy was accentuated in the dentate gyrus, Ammon’s horn and parahippocampal gyrus. The basal ganglia, thalamus and midbrain were moderately atrophic. The substantia nigra and locus ceruleus were completely depigmented. Numerous neurofibrillary tangles (NFTs) were seen in the subiculum and amygdaloid nucleus. Many NFTs were evident in the parahippocampal gyrus, lateral occipitotemporal gyrus, insula, Sommer sector, basal nucleus of Meynert, lateral nucleus of the thalamus, subthalamic nucleus and brain stem, and several were observed in the globus pallidus and hypothalamus. The Sommer sector, substantia nigra, locus ceruleus and basal nucleus of Meynert showed severe loss of neurons, and a moderate loss of neurons was exhibited by the globus pallidus. These findings were apparently consistent with those associated with PDC. However, in this patient, severe neuronal loss was seen in the subthalamic nucleus and lateral nucleus of the thalamus, and grumose degeneration, which has not previously been reported in PDC, was seen in the dentate nucleus. In addition, many tufted astrocytes, which have been reported to occur in progressive supranuclear palsy (PSP) and postencephalitic parkinsonism, but scarcely observed in PDC, were present. Furthermore, astrocytic plaques, which have been considered as a specific finding of corticobasal degeneration (CBD), were observed in the cerebral cortex. On the other hand, granular hazy astrocytic inclusions, previously reported to occur in PDC, were not seen. Chromatolytic neurons were not observed. The question thus arises as to whether it is appropriate to consider this patient as having suffered from a combination of PDC, PSP and CBD. From the view points of absence of granular hazy astrocytic inclusions and chromatolytic neurons, and of tufted astrocytes in the neostriatum, it is conceivable that this patient is a case of a new disease entity. Received: 11 January 1999 / Revised, accepted: 6 April 1999     

局灶性脑缺血/再灌注时大鼠脑皮质GFAP及c-fos的表达变化

目的 研究大鼠局灶性脑缺血／再灌注时皮质区星形胶质细胞胶质纤维酸性蛋白（GFAP）与神经元c-fos的表达变化。方法70只大鼠，随机分成缺血组及假手术组，缺血组再根据再灌注时间的不同，分别分为再灌注2、4、6、12、24、48、72h7个亚组，应用免疫组织化学单标记法、蛋白印迹法观察缺血皮质区各时间点GFAP和c-fos表达，用免疫组织化学双重染色法显示2组再灌注4、24、48hGFAP和c-fos表达的相互关系。结果大鼠脑缺血2h再灌注2h神经元c-fos的表达增加，4h达高峰，随之下降，2组间各时间点均有差异（P〈0．05）；于再灌注4h星形胶质细胞被激活、GFAP表达增加，随时间延长而逐渐升高，于48h达高峰，随后下降，2组间6、12、24、48、72h5个时间点具有差异（P〈0．05）；且在同一部位，c-fos阳性神经元周围伴有激活的星形胶质细胞表达GFAP。结论脑缺血／再灌注时皮质区星形胶质细胞及神经元均被激活，并伴有GFAP、c-fos的不同的时程规律表达变化。     

载脂蛋白D在两型星形胶质细胞和少突胶质细胞中的表达

目的: 通过对载脂蛋白D(ApoD)在分化成熟的两型星形胶质细胞和少突胶质细胞中表达的检测,探讨ApoD等脂代谢相关基因对神经髓鞘生成的调控机制.方法: 用激光共聚焦免疫荧光标记技术检测ApoD在分化成熟的两型星形胶质细胞和少突胶质细胞中的表达情况.结果: 激光共聚焦显微镜检测显示在O-2A谱系来源的成熟2型星形胶质细胞和少突胶质细胞中ApoD标记为阳性,而在T1A谱系来源的成熟1型星形胶质细胞中则未检测到ApoD的表达,从而在蛋白质水平验证本室研究两型星形胶质细胞基因表达谱差异基因芯片结果中ApoD mRNA在T2A中高表达,而在T1A中低表达的现象.结论: ApoD等脂代谢相关基因可能在O-2A谱系发生和分化过程中起重要作用,O-2A谱系与脑内脂质代谢以及神经髓鞘发生内在机制密切相关.     

CS2对原代星形胶质细胞的毒性研究

为探讨ＣＳ２的神经毒性机制及体外评价方法,我们研究了ＣＳ２对原代星形胶质细胞的毒性,采用常规培养方法,观察ＣＳ２染毒（０,１０,１００,１０００,１００００μｍｏｌ／Ｌ对星形胶质细胞脱落、细胞形态学、及Ｎａ＋－Ｋ＋－ＡＴＰ酶活性的影响。结果表明：ＣＳ２染毒可使细胞脱落数增加,且与接触剂量和接触时间有关;细胞形态学明显异常,电镜下观察表现为髓样小体的形成及空泡样改变;Ｎａ＋－Ｋ＋－ＡＴＰ酶活性下降。提示ＣＳ２对于星形胶质细胞有明显的毒性,此酶活性的下降可作为ＣＳ２毒性的一种标志物     

大鼠脑组织微注射红藻氨酸与γ—刀照射后GFAP反应变化

目的：分别以红藻氨酸（ＫＡ）微注射和γ－刀照射大鼠单侧尾状核,比较两者星形胶质细胞（ＡＳ）的适应性变化。方法：用抗胶质纤维酸性蛋白（ＧＦＡＰ）的ＡＢＣ法检测在受损伤３ｈ至３０ｄ的不同时期ＡＳ的变化,结果：２种损伤方式都可引起靶区ＧＦＡＰ阳性反应的细胞数量的增加,并且都可见到两种类型的阳性细胞,即胞体小、突起细的纤维型和胞体大、突起粗的肥大型,但二者在ＡＳ的反应顺序上有不同,在ＫＡ微注射组,２４ｈ内     

Local uptake of (14)C-labeled acetate and butyrate in rat brain in vivo during spreading cortical depression.

Spreading depression severely disrupts ion homeostasis, causes sensory neglect and motor impairment, and is associated with stroke and migraine. Glucose utilization (CMR(glc)) and lactate production rise during spreading depression, but the metabolic changes in different brain cell types are unknown. Uptake of (14)C-labeled compounds known to be preferentially metabolized by the glial tricarboxylic acid cycle was, therefore, examined during unilateral KCl-induced spreading cortical depression in conscious, normoxic rats. [(14)C]Metabolites derived from [(14)C]butyrate in K+ -treated tissue rose 21% compared to that of untreated contralateral control cortex, whereas incorporation of H(14)CO(3) into metabolites in K+ -treated tissue was reduced to 86% of control. Autoradiographic analysis showed that laminar labeling of cerebral cortex by both (14)C-labeled acetate and butyrate was elevated heterogeneously throughout cortex by an average of 23%; the increase was greatest (approximately 40%) in tissue adjacent to the K+ application site. Local uptake of acetate, butyrate, and deoxyglucose showed similar patterns, and monocarboxylic acid uptake was highest in the structures in which apparent loss of labeled metabolites of [6-(14)C]glucose was greatest. Enhancement of net uptake of acetate and butyrate in cerebral cortex during spreading depression is tentatively ascribed to increased astrocyte metabolism.