Enhanced GLT-1 mediated glutamate uptake and migration of primary astrocytes directed by fibronectin-coated electrospun poly-l-lactic acid fibers

Bioengineered fiber substrates are increasingly studied as a means to promote regeneration and remodeling in the injured central nervous system (CNS). Previous reports largely focused on the ability of oriented scaffolds to bridge injured regions and direct outgrowth of axonal projections. In the present work, we explored the effects of electrospun microfibers on the migration and physiological properties of brain astroglial cells. Primary rat astrocytes were cultured on either fibronectin-coated poly-l-lactic acid (PLLA) films, fibronectin-coated randomly oriented PLLA electrospun fibers, or fibronectin-coated aligned PLLA electrospun fibers. Aligned PLLA fibers strongly altered astrocytic morphology, orienting cell processes, actin microfilaments, and microtubules along the length of the fibers. On aligned fibers, astrocytes also significantly increased their migration rates in the direction of fiber orientation. We further investigated if fiber topography modifies astrocytic neuroprotective properties, namely glutamate and glutamine transport and metabolism. This was done by quantifying changes in mRNA expression (qRT-PCR) and protein levels (Western blotting) for a battery of relevant biomolecules. Interestingly, we found that cells grown on random and/or aligned fibers increased the expression levels of two glutamate transporters, GLAST and GLT-1, and an important metabolic enzyme, glutamine synthetase, as compared to the fibronectin-coated films. Functional assays revealed increases in glutamate transport rates due to GLT-1 mediated uptake, which was largely determined by the dihydrokainate-sensitive GLT-1. Overall, this study suggests that aligned PLLA fibers can promote directed astrocytic migration, and, of most importance, our in vitro results indicate for the first time that electrospun PLLA fibers can positively modify neuroprotective properties of glial cells by increasing rates of glutamate uptake.     

Neuronal stimulation leading to upregulation of glutamate transporter-1 (GLT-1) in rat microglia in vitro

As previously reported, activated microglia facilitate the expression of a glial cell-type glutamate transporter, glutamate transporter-1 (GLT-1; EAAT2), around injured motoneurons in axotomized rat facial nucleus. This phenomenon suggests that the motoneurons stimulate microglia to enhance the levels of GLT-1. In the present study, we investigated the effects of neuronal stimulus on the uptake of glutamate (Glu) by microglia and on the expression of GLT-1 protein in microglia in vitro. A 14C-Glu uptake experiment revealed that microglia enhance uptake of Glu by stimulation with neuronal conditioned medium (NCM). The NCM-stimulated uptake was significantly suppressed in the presence of dihydrokinate (a specific GLT-1 inhibitor), suggesting that GLT-1 is a major glutamate transporter for the uptake. Furthermore, immunoblotting analysis revealed that the amounts of GLT-1, but not another glial cell-type glutamate transporter glutamate-aspartate transporter (GLAST: EAAT1), increased significantly in microglia by treatment with NCM. Altogether, neuronal stimulus was found to promote the uptake of Glu in microglia, probably due to the increased levels of GLT-1.     

Differential expression of two glutamate transporters,GLAST and GLT-1, in an experimental rat model of glaucoma

Glutamate is the major excitatory neurotransmitter of the mammalian retina, and excessive glutamate has been implicated in the pathogenesis of glaucoma. It is well known that glutamate transport, mainly via GLAST and GLT-1, is cardinal mechanism for maintaining glutamate homeostasis in normal and pathological conditions, including ischemia in the brain. In an effort to understand the role of glutamate and the glutamate regulation system of the retina in the pathogenesis of glaucoma, we examined changes in the expression of two glutamate transporters, GLAST and GLT-1, by Western blot analysis and immunocytochemistry in a rat glaucoma model. GLT-1 was expressed in cone photoreceptors and some cone bipolar cells and the levels of expression were significantly increased in the cauterized eyes throughout the entire experimental period. In contrast, GLAST expression, which occurred in Müller cells, the main retinal glial cells, remained stable during the experimental period. These results suggest that GLT-1 may be a prerequisite for the maintenance of glutamate homeostasis in the retina undergoing glaucoma.     

Downregulation of glutamate transporters is associated with elevation in extracellular glutamate concentration following rat microsphere embolism

Sodium-dependent glutamate transporters expressed in astroglial cells and neurons are essential for clearance of extracellular glutamate. In the present study, we found elevation of extracellular glutamate concentration associated with concomitant downregulation of glutamate transporters following rat microsphere embolism (ME). A marked increase in extracellular glutamate in the rat striatum was observed by microdialysis immediately after ME induction, and glutamate remained elevated at least 12 h after ischemia. Concomitantly, impairment of high KCl (146 mM)-induced glutamate release was observed in the striatum 12 h after ME. Consistent with the persistent increase in extracellular glutamate, expression of the glutamate transporters EAAC1 and GLT-1 significantly decreased 6 h after insult without a change in GLAST levels. GLT-1 expression was restored to basal levels within 48 h, whereas EAAC1 expression remained decreased up to at least 72 h after ME. Restoration of GLT-1 was associated with increased expression of the astroglial marker GFAP, whereas markedly reduced EACC1 levels were correlated with reduced levels of the neuronal marker MAP2, likely due to loss of vulnerable neurons. Taken together, downregulation of glutamate transporters after ME is associated with dysregulation of basal glutamate concentrations and KCl-induced glutamate release in the brain.     

Distribution of the glutamate transporters GLT-1 (SLC1A2) and GLAST (SLC1A3) in peripheral organs

The glutamate transporters GLT-1 and GLAST are widely expressed in astrocytes in the brain where they fulfill important functions during glutamatergic neurotransmission. The present study examines their distribution in peripheral organs using in situ hybridization (ISH) and immunocytochemistry. GLAST was found to be more widely distributed than GLT-1. GLAST was expressed primarily in epithelial cells, cells of the macrophage-lineage, lymphocytes, fat cells, interstitial cells, and salivary gland acini. GLT-1 was primarily expressed in glandular tissue, including mammary gland, lacrimal gland, and ducts and acini in salivary glands, but also by perivenous hepatocytes and follicular dendritic cells in spleen and lymph nodes. The findings demonstrate that, although expressed by the same cells in the brain, these two glutamate transporters have different distribution patterns in peripheral tissues and that they fulfill glutamate transport functions apart from glutamatergic neurotransmission in these areas.     

Up-regulation of spinal glutamate transporters contributes to anti-hypersensitive effects of valproate in rats after peripheral nerve injury

Valproate produces analgesia in animals and humans, however, its mechanisms of action are yet unknown. The present study examined effects of repeated administration of valproate on behavioral hypersensitivity and expression of glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in the spinal dorsal horn in rats after L5-L6 spinal nerve ligation (SNL). SNL significantly reduced mechanical withdrawal threshold and expression of GLT-1 and GLAST in the spinal dorsal horn. Repeated oral administration of valproate reduced hypersensitivity, restored down-regulated expression of GLT-1 and GLAST in the spinal dorsal horn, and enhanced analgesia from the glutamate transporter activator riluzole. This analgesia from valproate was blocked by the selective GLT-1 blocker dihydrokainic acid (DHK). These data suggest that valproate restores down-regulated expression of glutamate transporters in the spinal cord to presumably reduce glutamate signaling and to reduce hypersensitivity after nerve injury, and that combination of valproate with riluzole produces enhanced analgesia which relies on the spinal glutamate transporters.     

Contribution of glutamate transporter GLT-1 to removal of synaptically released glutamate at climbing fiber-Purkinje cell synapses

Rapid removal of synaptically released glutamate from the extracellular space ensures a high signal-to-noise ratio in excitatory neurotransmission. In the cerebellum, glial glutamate transporters, GLAST and GLT-1, are co-localized in the processes of Bergmann glia wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, the decay kinetics of climbing fiber-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(−/−) mice are not different from those in wild-type (WT) mice. This raises a possibility that GLT-1 plays a significant role in clearing glutamate at CF-PC synapses despite its smaller amount of expression. Here, we studied the functions of GLT-1 and GLAST in the clearance of glutamate using GLAST(−/−) mice and GLT-1(−/−) mice. In the presence of cyclothiazide (CTZ) that attenuates the desensitization of AMPA receptors, the decay time constant of CF-EPSCs (τ_w) in GLT-1(−/−) mice was slower than that in WT mice. However, the degree of this prolongation of τ_w was less prominent compared to that in GLAST(−/−) mice. The values of τ_w in GLT-1(−/−) mice and GLAST(−/−) mice were comparable to those estimated in WT mice in the presence of a potent blocker of glial glutamate transporters (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA) at 10 and 100 nM, which reduced the amplitudes of glutamate transporter currents elicited by CF stimulation in Bergmann glia to ∼81 and ∼28%, respectively. We conclude that GLT-1 plays a minor role compared to GLAST in clearing synaptically released glutamate at CF-PC synapses.     

Spinal glial glutamate transporters downregulate in rats with taxol-induced hyperalgesia

Changes in the expression of glial glutamate transporters (GLAST and GLT-1) were examined in the spinal cord of rats with chemotherapy (taxol)-induced mechanical hyperalgesia. Immunohistochemical studies show that the expression of both GLAST and GLT-1 in the L4-L5 spinal dorsal horn is decreased by 24% (P<0.001) and 23% (P<0.001), respectively, in rats with taxol-induced hyperalgesia as compared with those in control rats. These changes were further confirmed using an enzyme-linked immunosorbent assay that confirmed downregulation of GLAST by 36% (P<0.05) and GLT-1 by 18% (P<0.05) in the L4-L5 spinal cord of taxol-treated rats. These data indicate that downregulation of glutamate transporters may contribute to the development of hyperalgesia induced by taxol and suggest that glutamate transporters may be a new target for treatment of pain.     

Propentofylline-induced astrocyte modulation leads to alterations in glial glutamate promoter activation following spinal nerve transection

We have previously shown that the atypical methylxanthine, propentofylline, reduces mechanical allodynia after peripheral nerve transection in a rodent model of neuropathy. In the present study, we sought to determine whether propentofylline-induced glial modulation alters spinal glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in vivo, which may contribute to reduced behavioral hypersensitivity after nerve injury. In order to specifically examine the expression of the spinal glutamate transporters, a novel line of double transgenic GLT-1-enhanced green fluorescent protein (eGFP)/GLAST-Discosoma Red (DsRed) promoter mice was used. Adult mice received propentofylline (10 mg/kg) or saline via i.p. injection starting 1 h prior to L5-spinal nerve transection and then daily for 12 days. Mice receiving saline exhibited punctate expression of both eGFP (GLT-1 promoter activation) and DsRed (GLAST promoter activation) in the dorsal horn of the spinal cord, which was decreased ipsilateral to nerve injury on day 12. Propentofylline administration reinstated promoter activation on the injured side as evidenced by an equal number of eGFP (GLT-1) and DsRed (GLAST) puncta in both dorsal horns. As demonstrated in previous studies, propentofylline induced a concomitant reversal of L5 spinal nerve transection-induced expression of glial fibrillary acidic protein (GFAP). The ability of propentofylline to alter glial glutamate transporters highlights the importance of controlling aberrant glial activation in neuropathic pain and suggests one possible mechanism for the anti-allodynic action of this drug.     

Spinal upregulation of glutamate transporter GLT-1 by ceftriaxone: therapeutic efficacy in a range of experimental nervous system disorders

Glutamate neurotransmission is highly regulated, largely by glutamate transporters. In the spinal cord, the glutamate transporter GLT-1 is primarily responsible for glutamate clearance. Downregulation of GLT-1 can occur in activated astrocytes, and is associated with increased extracellular glutamate and neuroexcitation. Among other conditions, astrocyte activation occurs following repeated opioids and in models of chronic pain. If GLT-1 downregulation occurs in these states, GLT-1 could be a pharmacological target for improving opioid efficacy and controlling chronic pain. The present studies explored whether daily intrathecal treatment of rats with ceftriaxone, a β-lactam antibiotic that upregulates GLT-1 expression, could prevent development of hyperalgesia and allodynia following repeated morphine, reverse pain arising from central or peripheral neuropathy, and reduce glial activation in these models. Ceftriaxone pre-treatment attenuated the development of hyperalgesia and allodynia in response to repeated morphine, and prevented associated astrocyte activation. In a model of multiple sclerosis (experimental autoimmune encephalomyelitis; EAE), ceftriaxone reversed tactile allodynia and halted the progression of motor weakness and paralysis. Similarly, ceftriaxone reversed tactile allodynia induced by chronic constriction nerve injury (CCI). EAE and CCI each significantly reduced the expression of membrane-bound, dimerized GLT-1 protein in lumbar spinal cord, an effect normalized by ceftriaxone. Lastly, ceftriaxone normalized CCI- and EAE-induced astrocyte activation in lumbar spinal cord. Together, these data indicate that increasing spinal GLT-1 expression attenuates opioid-induced paradoxical pain, alleviates neuropathic pain, and suppresses associated glial activation. GLT-1 therefore may be a therapeutic target that could improve available treatment options for patients with chronic pain.     

Functional role for redox in the epileptogenesis: molecular regulation of glutamate in the hippocampus of FeCl<Subscript>3</Subscript>-induced limbic epilepsy model

We used western blotting to measure the quantity of glutamate and γ-aminobutyric acid (GABA) transporters proteins within hippocampal tissue obtained from rats who had undergone epileptogenesis. Chronic seizures were induced by amygdalar injection of FeCl₃. We found that the glial glutamate transporters GLAST and GLT-1 were down-regulated at 60 days after initiation of chronic and recurrent seizures. However, the neuronal glutamate transporter EAAC-1 and the GABA transporter GAT-3 were increased. We performed in vivo microdialysis in freely moving animals to estimate in vivo redox state. We found that the hippocampal tissues were oxidized, resulting in even further impairment of glutamate transport. Our data show that epileptogenesis in rats resulting in chronic and recurrent seizures is associated with collapse of glutamate regulation caused by both the molecular down-regulation of glial glutamate transporters combined with the functional failure due to oxidation.     

Molecular regulation of glutamate and GABA transporter proteins by valproic acid in rat hippocampus during epileptogenesis

Epileptiform discharges and behavioral seizures may be the consequences of the presence of either excessive excitation associated with the neurotransmitter glutamate or from inadequate inhibitory effects associated with gamma-aminobutyric acid (GABA). Synaptic effects of these neurotransmitters are terminated by the action of transporter proteins that remove these amino acids from the synaptic cleft. The glial transporters glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), and the neuronal transporter excitatory amino acids carrier-1 (EAAC-1) limit excitation initiated by synaptic release of glutamate. Transporter proteins GABA transporter-1 (GAT-1) and GABA transporter-3 (GAT-3) remove GABA from synaptic regions. To assess the molecular effects of the antiepileptic drug valproate, albino rats with chronic, spontaneous, recurrent seizures induced by amygdalar injection of FeCl3 were treated for 14 days with either valproic acid or with saline as an injection control. Regions of the hippocampus were assayed for glutamate and GABA transporters by western blot. While epileptogenesis is thought to correlate with the downregulation of GLAST and upregulation of EAAC-1, valproate caused an increase in the quantity of GLAST protein measured in the hippocampus. Valproate treatment decreased GLT-1 in both control and experimental animals in both hippocampi. EAAC-1 was unchanged by valproate treatment. GABA transporters GAT-1 and GAT-3 in the hippocampus were upregulated by FeCl3 injection into the amygdala. However, valproate caused the downregulation of these GABA transporters in both control and experimental animals. Altered molecular regulation of glutamate appears to be critical in the development of sustained, spontaneous limbic seizures. Our data suggest that valproate may have unique mechanisms of action; specifically, it may affect the removal of glutamate by upregulating GLAST and decreasing GABA transport, which could result in increased tissue concentrations of GABA.     

Functional changes in astrocytes by human T-lymphotropic virus type-1 T-lymphocytes

The human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of a chronic progressive myelopathy (TSP/HAM) in which lesions of the central nervous system (CNS) are associated with infiltration of HTLV-1-infected T-cells. In a model that mimics the interaction between glial and T-cells, we show that transient contact with T-lymphocytes chronically infected with HTLV-1 induce profound metabolic alterations in astrocytes. Within the first week post-contact, an overall activation of astrocyte metabolism was observed as assessed by enhanced uptake of glutamate and glucose, and lactate release. In contrast, longer examination showed a reduced astrocytic accumulation of glutamate. The time course of the change in glutamate uptake was in fact biphasic. Previous observations indicated that HTLV-1 protein Tax-1 was involved in this delayed decrease, via the induction of TNF-alpha. The expression of the glial glutamate transporters, GLAST and GLT-1 decreased in parallel. These decreases in glutamate uptake and transporters' expression were associated with an imbalance in the expression of the catabolic enzymes of glutamate, GS and GDH, presumably due to Tax-1. Given the fact that impairment of glutamate management in astrocytes is able to compromise the functional integrity of neurons and oligodendrocytes, our results altogether give new insights into the physiopathology of TSP/HAM.     

成年大鼠侧脑室下区星形胶质细胞的分离培养及纯化研究

成年哺乳动物侧脑室下区星形胶质细胞(AST)的神经干细胞特性研究是当前神经科学领域的热点,为了进一步了解AST生物学特性,体外培养是探讨AST细胞学特性的有力手段,而怎样通过体外培养得到高纯度的成年AST是亟待解决的问题。本研究以2.5月的成年大鼠侧脑室下区为实验材料进行AST培养,并对传统的培养方法进行改良。首先在AST培养基中增加B27添加剂,结合成年AST体外生长特性,采用低浓度血清(2.5%～5%)和血清浓度调整法对培养的AST进行纯化。然后采用GFAP免疫细胞化学染色方法对培养至14d的细胞进行鉴定及纯度检测,采用流式细胞仪检测B27添加剂对AST细胞周期的影响。结果显示:B27添加剂能有效促进体外培养的成年AST的生长和增殖,采用此方法培养的成年AST纯度达90%以上;B27添加剂与低浓度血清的组合以及根据细胞特性及时调整血清浓度是一种有效的纯化成年AST方法。本方法的建立可望为AST的神经干细胞特性研究提供理想的细胞模型。     

The ionic stoichiometry of the GLAST glutamate transporter in salamander retinal glia

Maintaining a low extracellular glutamate concentration in the central nervous system is important for terminating synaptic transmission and preventing excitotoxic cell death. The stoichiometry of the most abundant glutamate transporter, GLT-1, predicts that a very low glutamate concentration, ∼2 n m , should be reached in the absence of glutamate release, yet microdialysis measurements give a value of ∼1 μ m . If other glutamate transporters had a different stoichiometry, the predicted minimum glutamate concentration could be higher, for example if those transporters were driven by the cotransport of 2 Na⁺ (rather than of 3 Na⁺ as for GLT-1). Here we investigated the ionic stoichiometry of the glutamate transporter GLAST, which is the major glutamate transporter expressed in the retina and cerebellum, is expressed in other adult brain areas at a lower level than GLT-1, and is present throughout the brain early in development when expression of GLT-1 is low. Glutamate transport by GLAST was found to be driven, as for GLT-1, by the cotransport of 3 Na⁺ and 1 H⁺ and the counter-transport of 1 K⁺, suggesting that the minimum extracellular glutamate concentration should be similar during development and in the adult brain. A less powerful accumulation of glutamate by GLAST than by GLT-1 cannot be used to explain the high glutamate concentration measured by microdialysis.     

bFGF和EGF对胚胎神经干细胞分化为神经元及神经胶质细胞的影响

目的：观察碱性成纤维生长因子（bFGF）和表皮生长因子（EGF）对体外培养胚胎神经管神经干细胞生长和分化的影响。方法：从孕12天大鼠胚胎神经管分离神经干细胞，进行原代培养，分为bFGF组、EGF组、bFGF＋EGF组及对照组：培养过程中观察干细胞的生长，培养2小时做nestin染色鉴定神经干细胞，培养第5天用免疫组化方法检测培养细胞神经元特异烯醇化酶（NSE）和胶质纤维酸性蛋白（GFAP）的表达，以观察神经干细胞分化为神经元及神经胶质细胞的状况。结果：取材细胞大部分为nestin免疫阳性细胞；各实验组均可促进培养细胞的生长和分 化。免疫组化中，EGF使神经干细胞增殖成团，增加GFAP的表达（P＜0．01）；bFGF能明显增加NSE及GFAP的表达（P＜0．01）；两种因子联合应用，神经元和神经胶质细胞均比对照组增多（P＜0．01）。结论：EGF和bFGF两类生长因子均能促进胚胎神经干细胞的生长，在分化方面，EGF倾向于诱导干细胞增并向着胶质细胞分化，bFGF则诱导干细胞分 成更多的神经元。     

GLAST1b, the exon-9 skipping form of the glutamate-aspartate transporter EAAT1 is a sensitive marker of neuronal dysfunction in the hypoxic brain

In normal brain, we previously demonstrated that the exon-9 skipping form of glutamate-aspartate transporter (GLAST; which we refer to as GLAST1b) is expressed by small populations of neurons that appear to be sick or dying and suggested that these cells were subject to inappropriate local glutamate-mediated excitation. To test this hypothesis we examined the expression of GLAST1b in the hypoxic pig brain. In this model glial glutamate transporters such as GLAST and glutamate transporter 1 (GLT-1) are down-regulated in susceptible regions, leading to regional loss of glutamate homeostasis and thus to brain damage. We demonstrate by immunohistochemistry that in those brain regions where astroglial glutamate transporters are lost, GLAST1b expression is induced in populations of neurons and to a lesser extent in some astrocytes. These neurons were also immunolabeled by antibodies against the carboxyl-terminal region of GLAST but did not label with antibodies directed against the amino-terminal region. Our Western blotting data indicate that GLAST1b expressed by neurons lacks the normal GLAST amino-terminal region and may be further cleaved to a smaller approximately 30-kDa fragment. We propose that GLAST1b represents a novel and sensitive marker for the detection of neurons at risk of dying in response to hypoxic and other excitotoxic insults and may have wider applicability in experimental and clinical contexts.     

Role of IL-6 in spinal cord injury in a mouse model

In recent years, various studies have been conducted toward the goal of achieving regeneration of the central nervous system using neural stem cells. However, various complex factors are involved in the regulation of neural stem cell differentiation, and many unresolved questions remain. It has been reported that after spinal cord injury, the intrinsic neural stem cells do not differentiate into neurons but, rather, into astrocytes, resulting in the formation of glial scars. Based on reports that the expression of interleukin (IL)-6 and the IL-6 receptor (IL-6R) is sharply increased in the acute stages after spinal cord injury and that IL-6 may serve as a factor strongly inducing the differentiation of neural stem cells into astrocytes, we examined the effects of an antibody to IL-6R in cases of spinal cord injury and found that the antibody suppressed secondary injury (caused by inflammatory reactions) and glial scar formation, facilitating functional recovery. This article presents the data from this investigation and discusses the relationship between IL-6 signals and spinal cord injury.