Excitotoxic Brain Injury Suppresses Striatal High-Affinity Glutamate Uptake in Perinatal Rats

In immature rodent brain, the glutamate receptor agonist N -methyl-D-aspartate (NMDA) is a potent neurotoxin. In postnatal day (PND)-7 rats, intrastriatal injection of 25 nmol of NMDA results in extensive ipsilateral forebrain injury. In this study, we examined alterations in high-affinity [ 3 H]glutamate uptake (HAGU) in NMDA-lesioned striatum. HAGU was assayed in synaptosomes, prepared from lesioned striatum, the corresponding contralateral striatum, or unlesioned controls. Twenty-four hours after NMDA injection (25 nmol), HAGU declined 44 ± 8% in lesioned tissue, compared with the contralateral striatum (mean ± SEM, n = 6 assays, p < 0.006, paired t test). Doses of 5–25 nmol of NMDA resulted in increasing suppression of HAGU (5 nmol, n = 3; 12.5 nmol, n = 3; and 25 nmol, n = 5 assays; p < 0.01, regression analysis). The temporal evolution of HAGU suppression was biphasic. There was an early transient suppression of HAGU (−28 ± 4% at 1 h; p < 0.03, analysis of variance, comparing changes at 0.5, 1, 2, and 3 h after lesioning); 1 or 5 days postinjury there was sustained loss of HAGU (at 5 days, −56 ± 11%, n = 3, p < 0.03, paired t test, lesioned versus contralateral striata). Treatment with the noncompetitive NMDA antagonist MK-801 (1 mg/kg i.p.) attenuated both the early and subsequent irreversible suppression of HAGU (1 h postlesion −28 ± 4%, n = 6 assays versus −12.6 ± 5% with MK-801, n = 4, p = 0.005; 24 h postlesion, −44 ± 8%, n = 5, versus +2.4 ± 6%, n = 3 with MK-801, p = 0.01, Wilcoxon ranked sum tests). In immature brain excitotoxic lesions produce an acute reversible suppression of HAGU, and a delayed long-lasting reduction in HAGU secondary to brain injury. These data suggest that accumulation of endogenous glutamate, as a consequence of the acute disruption of HAGU, could contribute to the pathogenesis of excitotoxic neuronal injury.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65556/1/j.1471-4159.1991.tb02011.x.pd

Perinatal Hypoxia-Ischemia Disrupts Striatal High-Affinity [ 3 H]Glutamate Uptake into Synaptosomes

: We examined the impact of hypoxia-ischemia on high-affinity [ 3 H]glutamate uptake into a synaptosomal fraction prepared from immature rat corpus striatum. In 7-day-old pups the right carotid artery was ligated, and pups were exposed to 8% oxygen for 0, 0.5, 1, or 2.5 h, and allowed to recover for up to 24 h before they were killed. High-affinity glutamate uptakes in striatal synaptosomes derived from tissue ipsilateral and contralateral to ligation were compared. After 1 h of hypoxia plus ischemia, high-affinity glutamate uptake in the striatum was reduced by 54 ± 13% compared with values from the opposite (nonischemic) side of the brain (p < 0.01, t test versus ligates not exposed to hypoxia). There were similar declines after 2.5 h of hypoxiaischemia. Activity remained low after a 1 h recovery period in room air, but after 24 h of recovery, high-affinity glutamate uptake was equal bilaterally. Kinetic analysis revealed that loss of activity could be attributed primarily to a 40% reduction in the number of uptake sites. Hypoxia alone had no effect on high-affinity glutamate uptake although it reduced synaptosomal uptake of [ 3 H]3,4-dihydroxyphenyl-ethylamine. Addition of 1 mg/ml of bovine serum albumin to the incubation medium preferentia'ly stimulated high-affinity glutamate uptake in hypoxic-ischemic brain compared with its effects in normal tissue. These studies demonstrate that hypoxia-ischemia reversibly inhibits high-affinity glutamate uptake and this occurs earlier than the time required to produce neuronal damage in the model.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66361/1/j.1471-4159.1986.tb00803.x.pd

β-Amyloid 25-35 Peptide Reduces the Expression of Glutamine Transporter SAT1 in Cultured Cortical Neurons

β-Amyloid (Aβ) peptides may cause malfunction and death of neurons in Alzheimer’s disease. We investigated the effect of Aβ on key transporters of amino acid neurotransmission in cells cultured from rat cerebral cortex. The cultures were treated with Aβ(25-35) at 3 and 10 μM for 12 and 24 h followed by quantitative analysis of immunofluorescence intensity. In mixed neuronal–glial cell cultures (from P1 rats), Aβ reduced the concentration of system A glutamine transporter 1 (SAT1), by up to 50% expressed relative to the neuronal marker microtubule-associated protein 2 (MAP2) in the same cell. No significant effects were detected on vesicular glutamate transporters VGLUT1 or VGLUT2 in neurons, or on glial system N glutamine transporter 1 (SN1). In neuronal cell cultures (from E18 rats), Aβ(25-35) did not reduce SAT1 immunoreactivity, suggesting that the observed effect depends on the presence of astroglia. The results indicate that Aβ may impair neuronal function and transmitter synthesis, and perhaps reduce excitotoxicity, through a reduction in neuronal glutamine uptake

Neurotrophic Effects of a Cyanine Dye via the PI3K-Akt Pathway: Attenuation of Motor Discoordination and Neurodegeneration in an Ataxic Animal Model

BACKGROUND: Neurotrophic factors may be future therapeutic agents for neurodegenerative disease. In the screening of biologically active molecules for neurotrophic potency, we found that a photosensitizing cyanine dye, NK-4, had remarkable neurotrophic activities and was a potent radical scavenger. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we evaluated the effect of NK-4 on the protection of neurons against oxidative damage and investigated the associated intracellular signaling pathways. Subsequently, we evaluated the effect of NK-4 in an animal model of neurodegeneration. In vitro, NK-4 showed dose-dependent protection of PC12 cells from toxicity induced by oxidative stress caused by hydrogen peroxide (H(2)O(2)) or 6-hydroxydopamine (6-OHDA). Comparison of extracellular signal-regulated kinase signaling pathways between treatment with NK-4 and nerve growth factor (NGF) using K252a, an inhibitor of the NGF receptor TrkA, revealed that NK-4 activity occurs independently of NGF receptors. LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocked the protective effect of NK-4, and NK-4 caused activation of Akt/protein kinase B, a downstream effector of PI3K. These results suggest that the neuroprotective effects of NK-4 are mediated by the PI3K-Akt signaling pathway. NK-4 treatment also attenuated stress-induced activation of SAPK/JNK, which suggests that NK-4 activates a survival signaling pathway and inhibits stress-activated apoptotic pathways independently of the TrkA receptor in neuronal cells. In vivo, administration of NK-4 improved motor coordination in genetic ataxic hamsters, as assessed by rota-rod testing. Histological analysis showed that cerebellar atrophy was significantly attenuated by NK-4 treatment. Notably, the Purkinje cell count in the treated group was threefold higher than that in the vehicle group. CONCLUSIONS/SIGNIFICANCE: These results suggest that NK-4 is a potential agent for therapy for neurodegenerative disorders based on the activation of survival signaling pathways

Cardiac Glycosides Ouabain and Digoxin Interfere with the Regulation of Glutamate Transporter GLAST in Astrocytes Cultured from Neonatal Rat Brain

Glutamate transport (GluT) in brain is mediated chiefly by two transporters GLT and GLAST, both driven by ionic gradients generated by (Na+, K+)-dependent ATPase (Na+/K+-ATPase). GLAST is located in astrocytes and its function is regulated by translocations from cytoplasm to plasma membrane in the presence of GluT substrates. The phenomenon is blocked by a naturally occurring toxin rottlerin. We have recently suggested that rottlerin acts by inhibiting Na+/K+-ATPase. We now report that Na+/K+-ATPase inhibitors digoxin and ouabain also blocked the redistribution of GLAST in cultured astrocytes, however, neither of the compounds caused detectable inhibition of ATPase activity in cell-free astrocyte homogenates (rottlerin inhibited app. 80% of Pi production from ATP in the astrocyte homogenates, IC50 = 25 μM). Therefore, while we may not have established a direct link between GLAST regulation and Na+/K+-ATPase activity we have shown that both ouabain and digoxin can interfere with GluT transport and therefore should be considered potentially neurotoxic

Purinergic Receptor Stimulation Reduces Cytotoxic Edema and Brain Infarcts in Mouse Induced by Photothrombosis by Energizing Glial Mitochondria

Treatments to improve the neurological outcome of edema and cerebral ischemic stroke are severely limited. Here, we present the first in vivo single cell images of cortical mouse astrocytes documenting the impact of single vessel photothrombosis on cytotoxic edema and cerebral infarcts. The volume of astrocytes expressing green fluorescent protein (GFP) increased by over 600% within 3 hours of ischemia. The subsequent growth of cerebral infarcts was easily followed as the loss of GFP fluorescence as astrocytes lysed. Cytotoxic edema and the magnitude of ischemic lesions were significantly reduced by treatment with the purinergic ligand 2-methylthioladenosine 5′ diphosphate (2-MeSADP), an agonist with high specificity for the purinergic receptor type 1 isoform (P2Y1R). At 24 hours, cytotoxic edema in astrocytes was still apparent at the penumbra and preceded the cell lysis that defined the infarct. Delayed 2MeSADP treatment, 24 hours after the initial thrombosis, also significantly reduced cytotoxic edema and the continued growth of the brain infarction. Pharmacological and genetic evidence are presented indicating that 2MeSADP protection is mediated by enhanced astrocyte mitochondrial metabolism via increased inositol trisphosphate (IP3)-dependent Ca2+ release. We suggest that mitochondria play a critical role in astrocyte energy metabolism in the penumbra of ischemic lesions, where low ATP levels are widely accepted to be responsible for cytotoxic edema. Enhancement of this energy source could have similar protective benefits for a wide range of brain injuries

Synaptic proximity enables NMDAR signalling to promote brain metastasis.

Metastasis-the disseminated growth of tumours in distant organs-underlies cancer mortality. Breast-to-brain metastasis (B2BM) is a common and disruptive form of cancer and is prevalent in the aggressive basal-like subtype, but is also found at varying frequencies in all cancer subtypes. Previous studies revealed parameters of breast cancer metastasis to the brain, but its preference for this site remains an enigma. Here we show that B2BM cells co-opt a neuronal signalling pathway that was recently implicated in invasive tumour growth, involving activation by glutamate ligands of N-methyl-D-aspartate receptors (NMDARs), which is key in model systems for metastatic colonization of the brain and is associated with poor prognosis. Whereas NMDAR activation is autocrine in some primary tumour types, human and mouse B2BM cells express receptors but secrete insufficient glutamate to induce signalling, which is instead achieved by the formation of pseudo-tripartite synapses between cancer cells and glutamatergic neurons, presenting a rationale for brain metastasis.This work was principally supported by grants from the Swiss National Science Foundation and the European Research Council, and by a gift from the Biltema Foundation that was administered by the ISREC Foundation, Lausanne, Switzerland

Two Distinct Modes of Hypoosmotic Medium-Induced Release of Excitatory Amino Acids and Taurine in the Rat Brain In Vivo

A variety of physiological and pathological factors induce cellular swelling in the brain. Changes in cell volume activate several types of ion channels, which mediate the release of inorganic and organic osmolytes and allow for compensatory cell volume decrease. Volume-regulated anion channels (VRAC) are thought to be responsible for the release of some of organic osmolytes, including the excitatory neurotransmitters glutamate and aspartate. In the present study, we compared the in vivo properties of the swelling-activated release of glutamate, aspartate, and another major brain osmolyte taurine. Cell swelling was induced by perfusion of hypoosmotic (low [NaCl]) medium via a microdialysis probe placed in the rat cortex. The hypoosmotic medium produced several-fold increases in the extracellular levels of glutamate, aspartate and taurine. However, the release of the excitatory amino acids differed from the release of taurine in several respects including: (i) kinetic properties, (ii) sensitivity to isoosmotic changes in [NaCl], and (iii) sensitivity to hydrogen peroxide, which is known to modulate VRAC. Consistent with the involvement of VRAC, hypoosmotic medium-induced release of the excitatory amino acids was inhibited by the anion channel blocker DNDS, but not by the glutamate transporter inhibitor TBOA or Cd2+, which inhibits exocytosis. In order to elucidate the mechanisms contributing to taurine release, we studied its release properties in cultured astrocytes and cortical synaptosomes. Similarities between the results obtained in vivo and in synaptosomes suggest that the swelling-activated release of taurine in vivo may be of neuronal origin. Taken together, our findings indicate that different transport mechanisms and/or distinct cellular sources mediate hypoosmotic medium-induced release of the excitatory amino acids and taurine in vivo