Expression Of Glutamate Transporter Proteins Research Articles

Acute Liver Toxicity Modifies Protein Expression of Glutamate Transporters in Liver and Cerebellar Tissue.

Glutamate is the main excitatory amino acid acting at the level of pre and postsynaptic neurons, as well as in glial cells. It is involved in the coordinated modulation of energy metabolism, glutamine synthesis, and ammonia detoxification. The relationship between the functional status of liver and brain has been known for many years. The most widely recognized aspect of this relation is the brain dysfunction caused by acute liver injury that manifests a wide spectrum of neurologic and psychiatric abnormalities. Inflammation, circulating neurotoxins, and impaired neurotransmission have been reported in this pathophysiology. In the present contribution, we report the effect of a hepatotoxic compound like CCl4 on the expression of key proteins involved in glutamate uptake and metabolism as glutamate transporters and glutamine synthetase in mice liver, brain, and cerebellum. Our findings highlight a differential expression pattern of glutamate transporters in cerebellum. A significant Purkinje cells loss, in parallel to an up-regulation of glutamine synthetase, and astrogliosis in the brain have also been noticed. In the intoxicated liver, glutamate transporter 1 expression is up-regulated, in contrast to glutamine synthetase which is reduced in a time-dependent manner. Taken together our results demonstrate that the exposure to an acute CCl4 insult, leads to the disruption of glutamate transporters expression in the liver-brain axis and therefore a severe alteration in glutamate-mediated neurotransmission might be present in the central nervous system.

Siponimod (BAF312) Activates Nrf2 While Hampering NFκB in Human Astrocytes, and Protects From Astrocyte-Induced Neurodegeneration

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS) with heterogeneous pathophysiology. In its progressive course oligodendrocyte and neuroaxonal damage is sustained by compartmentalized inflammation due to glial dysregulation. Siponimod (BAF312), a modulator of two sphingosine-1-phosphate (S1P) receptors (S1P1 and S1P5) is the first oral treatment specifically approved for active secondary progressive MS. To address potential direct effects of BAF312 on glial function and glia-neuron interaction, we set up a series of in vitro functional assays with astrocytes generated from human fibroblasts. These cells displayed the typical morphology and markers of astroglia, and were susceptible to the action of inflammatory mediators and BAF312, because expressing receptors for IL1, IL17, and S1P (namely S1P1 and S1P3). Targeting of S1P signaling by BAF312 inhibited NFκB translocation evoked by inflammatory cytokines, indicating a direct anti-inflammatory activity of the drug on the human astrocyte. Further, while glia cells exposed to IL1 or IL17 downregulated protein expression of glutamate transporters, BAF312-treated astrocytes maintained high levels of GLAST and GLT1 regardless of the presence of inflammatory mediators. Interestingly, despite potential glial susceptibility to S1P signaling via S1P3, which is not targeted by BAF312, NFκB translocation and downregulation of glutamate transporters in response to S1P were inhibited at similar levels by BAF312 and FTY720, another S1P signaling modulator targeting also S1P3. Accordingly, specific inhibition of S1P1 via NIBR-0213 blocked S1P-evoked NFκB translocation, demonstrating that modulation of S1P1 is sufficient to dampen signaling via other S1P receptors. Considering that NFκB-dependent responses are regulated by Nrf2, we measured activation of this critical transcription factor for anti-oxidant reactions, and observed that BAF312 rapidly induced nuclear translocation of Nrf2, but this effect was attenuated in the presence of an inflammatory milieu. Finally, in vitro experiments with spinal neurons exposed to astrocyte-conditioned media showed that modulation of S1P or cytokine signaling in astrocytes via BAF312 prevented neurons from astrocyte-induced degeneration. Overall, these experiments on human astrocytes suggest that during neuroinflammation targeting of S1P1 via BAF312 may modulate key astrocyte functions and thereby attain neuroprotection indirectly.

Anxiolytic activity of paraoxon is associated with alterations in rat brain glutamatergic system

Exposure to organophosphate (OP) compounds leads to behavioral alterations. To determine whether paraoxon has effects on anxiety, anxiety-like behaviors were assessed in paraoxon-exposed rats. Protein expression of glutamate transporters has also been measured in hippocampus and prefrontal cortex. Three doses of paraoxon (0.3, 0.7, or 1 mg/kg) or corn oil (vehicle) were intraperitoneally injected to adult male rats. At 14 or 28 days after exposure, behavioral tests were done using elevated plus-maze (EPM) or open field tests. Thereafter, animals were sacrificed and both hippocampi and prefrontal cortices were extracted for cholinesterase assay and western blotting. Animals treated with convulsive doses of paraoxon (0.7 and 1 mg/kg) showed an increase in percentage of time spent in open arms and percentage of open arm entries in the EPM. In the open field test, an increase in the time spent in central area was observed in rats treated with the same doses of paraoxon. These effects of paraoxon were independent of any changes in locomotor activity. There was an increase in both astrocytic glutamate transporter proteins (GLAST and GLT-1) in the hippocampus of animals treated with 0.7 and 1 mg/kg of paraoxon. In the prefrontal cortex, protein levels of the GLAST and GLT-1 increased in 0.7 and decreased in 1 mg/kg groups. Only a significant decrease in EAAC1 protein was observed in the prefrontal cortex at 14 days following exposure to 1 mg/kg of paraoxon. Collectively, this study showed that exposure to convulsive doses of paraoxon induced anxiolytic-like behaviors in both behavioral tests. This effect may be attributed to alterations of glutamate transporter proteins in the rat hippocampus and prefrontal cortex.

Alterations in mRNA and protein expression of glutamate transporters in rat hippocampus after paraoxon exposure

Organophosphates affect brain function through a variety of mechanisms beyond their shared role as cholinesterase inhibitors. The aim of the current study was to investigate the changes in the expression of glial (GLAST and GLT-1) and neuronal (EAAC1) glutamate transporters at mRNA and protein levels in paraoxon-treated rat hippocampus. Adult male Wistar rats were intraperitoneally treated with either vehicle (corn oil) or one of three dosages of paraoxon (0.3, 0.7 or 1mg/kg). After 4 or 18h, both hippocampi of each rat were collected to detect mRNA and protein expression of glutamate transporters using the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blotting, respectively. Animals treated with 0.3mg/kg paraoxon showed no difference in mRNA and protein levels of the glutamate transporters when compared with control group. At 4h after exposure with 0.7 and 1mg/kg paraoxon, the expression of GLAST and GLT-1 increased at mRNA and protein levels and remained elevated after 18h. No difference in the expression of EAAC1 at mRNA and protein levels was observed in any paraoxon-treated groups compared with the control group. This study showed an increased expression of glial (GLAST and GLT-1), but not neuronal (EAAC1) glutamate transporters, in adult rat hippocampus following administration of convulsive dosages of paraoxon. These suggest a protective and compensatory adaptation for effective uptake of glutamate in hippocampus induced by paraoxon and thus attenuating seizure activity.

Resveratrol Prevents Retinal Dysfunction by Regulating Glutamate Transporters, Glutamine Synthetase Expression and Activity in Diabetic Retina.

This study investigated the effects of resveratrol (RSV) on retinal functions, glutamate transporters (GLAST) and glutamine synthetase (GS) expression in diabetic rats retina, and on glutamate uptake, GS activity, GLAST and GS expression in high glucose-cultured Müller cells. The electroretinogram was used to evaluate retinal functions. Müller cells cultures were prepared from 5- to 7-day-old Sprague-Dawley rats. The expression of GLAST and GS was examined by qRT-PCR, ELISA and western-blotting. Glutamate uptake was measured as (3)H-glutamate contents of the lysates. GS activity was assessed by a spectrophotometric assay. 1- to 7-month RSV administrations (5 and 10mg/kg/day) significantly alleviated hyperglycemia and weight loss in diabetic rats. RSV administrations also significantly attenuated diabetes-induced decreases in amplitude of a-wave in rod response, decreases in amplitude of a-, and b-wave in cone and rod response and decreases in amplitude of OP2 in oscillatory potentials. 1- to 7-month RSV treatments also significantly inhibited diabetes-induced delay in OP2 implicit times in scotopic 3.0 OPS test. The down-regulated mRNA and protein expression of GLAST and GS in diabetic rats retina was prevented by RSV administrations. In high glucose-treated cultures, Müller cells' glutamate uptake, GS activity, GLAST and GS expression were decreased significantly compared with normal control cultures. RSV (10, 20, and 30mmol/l) significantly inhibited the HG-induced decreases in glutamate uptake, GS activity, GLAST and GS expression (at least P<0.05). These beneficial results suggest that RSV may be considered as a therapeutic option to prevent from diabetic retinopathy.

Intrathecal Ultra-Low Dose Naloxone Enhances the Antinociceptive Effect of Morphine by Enhancing the Reuptake of Excitatory Amino Acids from the Synaptic Cleft in the Spinal Cord of Partial Sciatic Nerve–Transected Rats

In this study, we examined the effects of ultra-low dose naloxone on the antinociceptive effect of morphine and on spinal cord dorsal horn glutamate transporter expression in rats with neuropathic pain. Neuropathic pain was induced in male Wistar rats by partial transection of the left sciatic nerve and an intrathecal catheter was implanted for drug administration; in some rats, an intrathecal microdialysis probe for cerebrospinal fluid (CSF) dialysate collection was also implanted. Nociception was assessed using the plantar test, a Hargreaves radiant heat apparatus, and by the von Frey test, using a dynamic plantar anesthesiometer. Glutamate transporter protein expression in the left spinal cord dorsal horn was examined by Western blotting and immunohistochemistry. Levels of the excitatory amino acids (EAAs) glutamate and aspartate in the CSF dialysate were measured using high-performance liquid chromatography. Reduced astrocyte expression of glutamate transporters (GLT-1 and GLAST levels were 55% and 53%, respectively, of that in sham-operated rats) in laminae I and II of the spinal cord dorsal horn ipsilateral to the partial sciatic nerve transection (PST), and hyperalgesia and allodynia in the PST hindlimb were observed. High-dose naloxone (15 μg) attenuated the antihyperalgesia and antiallodynia effects of the morphine (10 μg). In contrast, ultra-low dose (15 ng) naloxone enhanced the antinociceptive effect of morphine (10 μg), with an increase in the paw withdrawal threshold to thermal stimulus (from 19% to 35%) and to tactile stimulus (from 33% to 55%) compared with morphine treatment alone, and this was associated with restoration of GLAST and GLT-1 expression to control levels (102% and 114%, respectively) in the astrocytes of laminae I and II in the spinal cord dorsal horn ipsilateral to the PST hindlimb and a decrease in EAA levels in the CSF dialysate (glutamate: 10.0 μM; aspartate: 1.1 μM). Ultra-low dose naloxone enhanced the antinociceptive effect of morphine in PST rats, possibly by restoration of GLAST and GLT-1 expression in astrocytes, which inhibited the accumulation of EAAs in the synapses, resulting in a neuroprotective effect.

Expression of PACAP and glutamate transporter proteins in satellite oligodendrocytes of the human CNS

White matter oligodendrocytes have been shown to actively regulate extracellular glutamate levels in the CNS. Such function has yet not been examined in satellite oligodendrocytes of gray matter. Similar to those in white matter, satellite oligodendrocytes are involved in myelination. In addition, they modulate the activity of surrounding neurons. This study examined whether satellite oligodendrocytes express PACAP and glutamate transporter proteins and whether this expression is influenced by global ischemia. We demonstrated expression of PACAP27 and PACAP38 in a major fraction of satellite oligodendrocytes in normal neocortex and hippocampus of human brain tissues obtained post-mortem. All three glutamate transporters EAAT1, EAAT2 and EAAT3 were expressed in satellite oligodendrocytes from these tissues. Thus, satellite oligodendrocytes may participate in the perineuronal glutamate homeostasis. Following transient global ischemia, the total number of satellite oligodendrocytes expressing PACAP or glutamate transporter proteins was significantly decreased in cerebral neocortex and hippocampus. However, alterations of PACAP and glutamate transporter protein expression were region and time specific. In satellite oligodendrocytes of CA1 an early strong reduction of PACAP and glutamate transporter expression was observed. This contrasted with late reduction of PACAP27, PACAP38 and glutamate transporters EAAT1, EAAT2 and EAAT3 in satellite oligodendrocytes of neocortex. Further studies should clarify whether these alterations in protein expression are primary or secondary to neuronal cell death.

Altered discharges of spinal wide dynamic range neurons and down-regulation of glutamate transporter expression in rats with paclitaxel-induced hyperalgesia

Changes in the signaling of wide dynamic range neurons and the expression of glutamate transporters in the lumbar spinal dorsal horn of rats with Taxol-induced hyperalgesia are detailed in this report. Deep spinal lamina neurons have significantly increased spontaneous activity and after-discharges to noxious mechanical stimuli, increased responses to both skin heating and cooling, and increased after-discharges and abnormal windup to transcutaneous electrical stimuli. The expression of glutamate transporter proteins in the dorsal horn is decreased at the time point corresponding to the physiological changes. These results suggest a state of increased excitability develops in spinal pain-signaling neurons as a consequence of decreased glutamate clearance. These changes in dorsal horn neurobiology likely in turn contribute to the hyper-responsiveness to sensory stimuli seen in animals treated with Taxol and may play a role in the pain seen in cancer patients receiving Taxol.

Early expression of glutamate transporter proteins in ramified microglia after controlled cortical impact injury in the rat.

Traumatic brain injury is followed by increased extracellular glutamate concentration. Uptake of glutamate is mainly mediated by the glial glutamate transporters GLAST and GLT-1. Extent and distribution of GLAST and GLT-1 were studied in a rat model of controlled cortical impact injury (CCII). Western Blot analysis revealed lowest levels of GLAST and GLT-1 with a decrease by 40%-54% and 42%-49% between 24 and 72 h posttrauma. By 8 h after CCII, CSF glutamate levels were increased (10.5 microM vs. 2.56 microM in controls; P < 0.001), reaching maximum values by 48 h. A significant increase in de novo GLAST and GLT-1 expressing ramified microglia was observed within 4 h, reached a stable level by 48 h, and remained high up to 72 h after CCII. Furthermore, ramified microglia de novo expressed the neuronal glutamate transporter EAAC1 after CCII. Following CCII, GLAST/GLT-1 and GFAP coexpressing astrocytes were immediately reduced, reaching minimum levels within 8 h. This reduction of expression could be either due to protein downregulation or loss of astrocytes. At 72 h, a marked population of GLAST- and GLT-1-positive reactive astrocytes appeared. These results support the hypothesis that reduced astrocytic GLAST and GLT-1 protein levels following CCII contribute to evolving secondary injury. Microglia are capable of de novo expressing glutamate transporter proteins, indicating that the expression of glial and neuronal glutamate transporters is not restricted to a specific glial or neuronal lineage. Ramified microglia may play an important compensatory role in the early regulation of extracellular glutamate after CCII.

The expression of the glial glutamate transporter protein EAAT2 in motor neuron disease: an immunohistochemical study.

Emerging evidence suggests that a disturbance of the glutamate neurotransmitter system may be a contributory factor to motor neuron injury in motor neuron disease. Previous autoradiographic and immunoblotting studies have suggested that there may be reduced expression of glutamate transporter proteins in pathologically affected areas of the CNS in motor neuron disease. This study further explores the possible alteration in expression of the excitatory amino acid transporter protein EAAT2 in MND, by examining the protein expression in situ, in frozen sections, using immunohistochemistry. The aim of the study was to compare the distribution and density of EAAT2 in the motor cortex and spinal cord of MND cases (n = 16) compared with neurologically normal controls (n = 12), matched for relevant parameters. A novel, previously characterized, monoclonal antibody to EAAT2 was employed. EAAT2 immunoreactivity in motor neuron disease and control cases was compared using relative optical density measurements generated by computerized image analysis. In the motor cortex, EAAT2 immunoreactivity was laminated comprising a superficial intense band (corresponding to layers 1 and 2); a paler middle band (layer 3 and part of 5) and a more intense deep layer (layers 5 and 6). In the spinal cord, the ventral horn showed strong immunoreactivity with dense perisomatic staining around motor neuron cell bodies, the substantia gelatinosa showed moderate diffuse staining and the intermediate spinal laminae showed weak staining. This general pattern of immunoreactivity was preserved in the motor neuron disease cases. However, in the motor neuron disease cases compared with controls, the optical density values for EAAT2 immunoreactivity were significantly reduced in all grey matter regions of the lumbar spinal cord (P < 0.001) and were increased in the middle laminae of the motor cortex (P < 0.05). This study indicates that glutamate transporter pathology in motor neuron disease may be a more complex phenomenon than previously recognized.