Expression Of Glutamate Transporters Research Articles

Effects of Hydrocodone Overdose and Ceftriaxone on Astrocytic Glutamate Transporters and Glutamate Receptors, and Associated Signaling in Nucleus Accumbens as well as Locomotor Activity in C57/BL Mice.

Chronic opioid treatments dysregulate the glutamatergic system, inducing a hyperglutamatergic state in mesocorticolimbic brain regions. This study investigated the effects of exposure to hydrocodone overdose on locomotor activity, expression of target proteins related to the glutamatergic system, signaling kinases, and neuroinflammatory factors in the nucleus accumbens. The locomotor activity of mice was measured using the Comprehensive Laboratory Animal Monitoring System (CLAMS). CLAMS data showed that exposure to hydrocodone overdose increased locomotion activity in mice. This study tested ceftriaxone, known to upregulate major glutamate transporter 1 (GLT-1), in mice exposed to an overdose of hydrocodone. Thus, ceftriaxone normalized hydrocodone-induced hyperlocomotion activity in mice. Furthermore, exposure to hydrocodone overdose downregulated GLT-1, cystine/glutamate antiporter (xCT), and extracellular signal-regulated kinase activity (p-ERK/ERK) expression in the nucleus accumbens. However, exposure to an overdose of hydrocodone increased metabotropic glutamate receptor 5 (mGluR5), neuronal nitric oxide synthase activity (p-nNOS/nNOS), and receptor for advanced glycation end products (RAGE) expression in the nucleus accumbens. Importantly, ceftriaxone treatment attenuated hydrocodone-induced upregulation of mGluR5, p-nNOS/nNOS, and RAGE, as well as hydrocodone-induced downregulation of GLT-1, xCT, and p-ERK/ERK expression. These data demonstrated that exposure to hydrocodone overdose can cause dysregulation of the glutamatergic system, neuroinflammation, hyperlocomotion activity, and the potential therapeutic role of ceftriaxone in attenuating these effects.

Salidroside derivative SHPL-49 attenuates glutamate excitotoxicity in acute ischemic stroke via promoting NR2A-CAMKⅡα-Akt /CREB pathway

BackgroundIschemic stroke is a significant cause of death and disability with a limited treatment time window. The reduction of early glutamate excitotoxicity using neuroprotective agents targeting N-methyl-D-aspartic acid (NMDA) receptors have attracted recent research attention. SHPL-49, a structurally modified derivative of salidroside, was synthesized by our team. Previous studies have confirmed the neuroprotective efficacy of SHPL-49 in rats with ischemic stroke. However, the underlying mechanisms need to be clarified. MethodsWe conducted in vivo experiments using the permanent middle cerebral artery occlusion rat model to investigate the role of SHPL-49 in glutamate release at different time points and treatment durations. Glutamate transporters and receptor proteins and neural survival proteins in the brain were also examined at the same time points. In vitro, primary neurons and the coculture system of primary neurons–astrocytes were subjected to oxygen–glucose deprivation and glutamate injury. Proteomics and parallel reaction monitoring analyses were performed to identify potential therapeutic targets of SHPL-49, which were further confirmed through in vitro experiments on the inhibition and mutation of the target. ResultsSHPL-49 significantly reduced glutamate release caused by hypoxia–ischemia. One therapeutic pathway of SHPL-49 was promoting the expression of glutamate transporter-1 to increase glutamate reuptake and further reduce the occurrence of subsequent neurotoxicity. In addition, we explored the therapeutic targets of SHPL-49 and its regulatory effects on glutamate receptors for the first time. SHPL-49 enhanced neuroprotection by activating the NMDA subunit NR2A, which upregulated the cyclic-AMP response binding protein (CREB) neural survival pathway and Akt phosphorylation. Since calcium/calmodulin-dependent kinase IIα (CaMKIIα) is necessary for synaptic transmission of NMDA receptors, we explored the interaction between CaMKIIα and SHPL-49, which protected CaMKIIα from hypoxia–ischemia-induced autophosphorylation damage. ConclusionOverall, SHPL-49 enhanced neuronal survival and attenuated acute ischemic stroke by promoting the NR2A–CAMKⅡα–Akt/CREB pathway. Our study provides the first evidence demonstrating that the neuroprotective effect of SHPL-49 is achieved by promoting the NR2A subunit to extend the treatment time window, making it a promising drug for ischemic stroke.

Neuroprotection Role of Vitamin C by Upregulating Glutamate Transporter-1 in Auditory Cortex of Noise-Induced Tinnitus Animal Model.

Vitamin C (Vc) plays a pivotal role in a series of pathological processes, such as tumors, immune diseases, and neurological disorders. However, its therapeutic potential for tinnitus management remains unclear. In this study, we find that Vc relieves tinnitus in noise-exposed rats. In the 7-day therapy groups, spontaneous firing rate (SFR) increases from 1.17 ± 0.10 Hz to 1.77 ± 0.15 Hz after noise exposure. Vc effectively reduces the elevated SFR to 0.99 ± 0.07 and 0.55 ± 0.05 Hz at different doses. The glutamate level in auditory cortex of noise-exposed rats (3.78 ± 0.42 μM) increases relative to that in the control group (1.34 ± 0.22 μM). High doses of Vc (500 mg/kg/day) effectively reduce the elevated glutamate levels (1.49 ± 0.28 μM). Mechanistic studies show that the expression of glutamate transporter 1 (GLT-1) is impaired following noise exposure and that Vc treatment effectively restores GLT-1 expression in the auditory cortex. Meanwhile, the GLT-1 inhibitor, dl-threo-beta-benzyloxyaspartic acid (dl-TBOA), invalidates the protection role of Vc. Our finding shows that Vc substantially enhances glutamate clearance by upregulating GLT-1 and consequently alleviates noise-induced tinnitus. This study provides valuable insight into a novel biological target for the development of therapeutic interventions that may prevent the onset of tinnitus.

High-frequency repetitive transcranial magnetic stimulation improves depressive-like behaviors in CUMS-induced rats by modulating astrocyte GLT-1 to reduce glutamate toxicity

Impaired glutamate recycling plays an important role in the pathophysiology of depression, and it has been demonstrated that glutamate transporter-1 (GLT-1) on astrocytes is involved in glutamate uptake. Studies have shown that repetitive transcranial magnetic stimulation (rTMS) is effective in treating depression, however, the exact mechanism of rTMS treatment remains unclear. Here, we used a chronic unpredictable mild stress (CUMS) protocol to induce depression-like behaviors in rats followed by rTMS treatment. Behavioral assessment was primarily through SPT, FST, OFT and body weight. Histological analysis focused on GFAP and GLT-1 expression, synaptic plasticity, apoptosis and PI3K/Akt/CREB pathway-related proteins. The results showed that rTMS treatment increased sucrose preference, improved locomotor activity, shortened immobility time as well as increased body weight. And rTMS intervention reversed the elevated glutamate concentration in the hippocampus of CUMS rats using an ELISA kit. Moreover, rTMS ameliorated the reduction in GFAP and GLT-1 expression, alleviated the decrease in BDNF, PSD95 and synapsin-1 expression, also reversed the expression levels of BAX and Bcl2 in the hippocampus of CUMS-induced rats. Moreover, rTMS also increased the protein phosphorylation level of PI3K/Akt/CREB pathway. These results suggest that rTMS treatment ameliorates depression-like behaviors in the rat model by reversing the reduction of GLT-1 on astrocytes and reducing glutamate accumulation in the synaptic cleft, which in turn ameliorates synaptic plasticity damage and neuronal apoptosis. The regulation of GLT-1 by rTMS may be through the PI3K/Akt/CREB pathway.

PFOS Elicits Cytotoxicity in Neuron Through Astrocyte-Derived CaMKII-DLG1 Signaling In Vitro Rat Hippocampal Model.

Both epidemiological investigation and animal experiments demonstrated that pre-/postnatal exposure to perfluorooctane sulfonic acid (PFOS) could induce neurodevelopmental disorders. Previous studies showed that astrocyte was involved in PFOS-induced neurotoxicity, while little information is available. In the present study, the role of astrocyte-derived calmodulin-dependent protein kinase II (CaMKII)-phosphorylated discs large homolog 1 (DLG1) signaling in PFOS eliciting cytotoxicity in neuron was explored with primary cultured hippocampal astrocyte and neuron. The application of PFOS showed a decreased cell viability, synapse length and glutamate transporter 1 (GLT-1) expression, but an increased CaMKII, DLG1 and cyclic AMP response element binding protein (CREB) expression in primary cultured astrocyte. With 2-(2-hydroxyethylamino)-6-aminohexylcarbamic acid tert-butyl ester-9-isopropylpurine (CK59), the CaMKII inhibitor, the disturbed cell viability and molecules induced by PFOS could be alleviated (CREB expression was excluded) in astrocytes. The cytotoxic effect of neuron exposed to astrocyte conditional medium collected from PFOS (PFOS-ACM) pretreated with CK59 was also decreased. These results indicated that PFOS mediated GLT-1 expression through astrocyte-derived CaMKII-DLG signaling, which might be associated with injuries on neurons. The present study gave an insight in further exploration of mechanism in PFOS-induced neurotoxicity.

Sulbactam protects neurons against double neurotoxicity of amyloid beta and glutamate load by upregulating glial glutamate transporter 1

Amyloid beta (Abeta) synergistically enhances excitotoxicity of glutamate load by impairing glutamate transporter 1 (GLT1) expression and function, which exacerbates the development of Alzheimer’s disease (AD). Our previous studies suggested that sulbactam can upregulate the expression levels and capacity of GLT1. Therefore, this study aims to investigate whether sulbactam improves neuronal tolerance against neurotoxicity of Abeta and glutamate load by up-regulating GLT1 in primary neuron-astrocyte co-cultures. Early postnatal P0–P1 Wistar rat pups’ cortices were collected for primary neuron–astrocyte cultures. Hoechst–propidium iodide (HO–PI) stain and lactate dehydrogenase (LDH) assays were used to analyze neuronal death. Cell counting kit 8 (CCK8) was applied to determine cell viability. Immunofluorescence staining and western blotting were used to assess protein expressions including GLT1, B-cell lymphoma 2 (BCL2), BCL2 associated X (BAX), and cleaved caspase 3 (CCP3). Under the double effect of Abeta and glutamate load, more neurons were lost than that induced by Abeta or glutamate alone, shown as decreased cell viability, increased LDH concentration in the cultural medium, HO–PI positive stains, high CCP3 expression, and high BAX/BCL2 ratio resulting from increased BAX and decreased BCL2 expressions. Notably, pre-incubation with sulbactam significantly attenuated the neuronal loss and activation of apoptosis induced by both Abeta and glutamate in a dose-dependent manner. Simultaneously, both astrocytic and neuronal GLT1 expressions were upregulated after sulbactam incubation. Taken together, it could be concluded that sulbactam protected neurons against double neurotoxicity of Abeta and glutamate load by upregulating GLT1 expression. The conclusion provides evidence for potential intervention using sulbactam in AD research.

Activating astrocytic α2A adrenoceptors in hippocampus reduces glutamate toxicity to attenuate sepsis-associated encephalopathy in mice

BackgroundGlutamate metabolism disorder is an important mechanism of sepsis-associated encephalopathy (SAE). Astrocytes regulate glutamate metabolism. In septic mice, α2A adrenoceptor (α2A-AR) activation in the central nervous system provides neuroprotection. α2A-ARs are expressed abundantly in hippocampal astrocytes. This study was performed to determine whether hippocampal astrocytic α2A-AR activation confers neuroprotection against SAE and whether this protective effect is astrocyte specific and achieved by the modulation of glutamate metabolism. MethodsMale C57BL/6 mice with and without α2A-AR knockdown were subjected to cecal ligation and puncture (CLP). They were treated with intrahippocampal guanfacine (an α2A-AR agonist) or intraperitoneal dexmedetomidine in the presence or absence of dihydrokainic acid [DHK; a glutamate transporter 1 (GLT-1) antagonist] and/or UCPH-101 [a glutamate/aspartate transporter (GLAST) antagonist]. Hippocampal tissue was collected for the measurement of astrocyte reactivity, GLT-1 and GLAST expression, and glutamate receptor subunit 2B (GluN2B) phosphorylation. In vivo real-time extracellular glutamate concentrations in the hippocampus were measured by ultra-performance liquid chromatography tandem mass spectrometry combined with microdialysis, and in vivo real-time hippocampal glutamatergic neuron excitability was assessed by calcium imaging. The mice were subjected to the Barnes maze and fear conditioning tests to assess their learning and memory. Golgi staining was performed to assess changes in the hippocampal synaptic structure. In vitro, primary astrocytes with and without α2A-AR knockdown were stimulated with lipopolysaccharide (LPS) and treated with guanfacine or dexmedetomidine in the presence or absence of 8-bromo- cyclic adenosine monophosphate (8-Br-cAMP, a cAMP analog). LPS-treated primary and BV2 microglia were also treated with guanfacine or dexmedetomidine. Astrocyte reactivity, PKA catalytic subunit, GLT-1 an GLAST expression were determined in primary astrocytes. Interleukin-1β, interleukin-6 and tumor necrosis factor-alpha in the medium of microglia culture were measured. ResultsCLP induced synaptic injury, impaired neurocognitive function, increased astrocyte reactivity and reduced GLT-1 and GLAST expression in the hippocampus of mice. The extracellular glutamate concentration, phosphorylation of GluN2B at Tyr-1472 and glutamatergic neuron excitability in the hippocampus were increased in the hippocampus of septic mice. Intraperitoneal dexmedetomidine or intrahippocampal guanfacine administration attenuated these effects. Hippocampal astrocytes expressed abundant α2A-ARs; expression was also detected in neurons but not microglia. Specific knockdown of α2A-ARs in hippocampal astrocytes and simultaneous intrahippocampal DHK and UCPH-101 administration blocked the neuroprotective effects of dexmedetomidine and guanfacine. Intrahippocampal administration of DHK or UCPH-101 alone had no such effect. In vitro, guanfacine or dexmedetomidine inhibited astrocyte reactivity, reduced PKA catalytic subunit expression, and increased GLT-1 and GLAST expression in primary astrocytes but not in primary astrocytes that received α2A-AR knockdown or were treated with 8-Br-cAMP. Guanfacine or dexmedetomidine inhibited microglial reactivity in BV2 but not primary microglia. ConclusionsOur results suggest that neurocognitive protection against SAE after hippocampal α2A-AR activation is astrocyte specific. This protection may involve the inhibition of astrocyte reactivity and alleviation of glutamate neurotoxicity, thereby reducing synaptic injury. The cAMP/protein kinase A (PKA) signaling pathway is a potential cellular mechanism by which activating α2A-AR modulates astrocytic function.

A preliminary in vitro study on Adult Neural Stem cells from 3xTg mice

AbstractBackgroundIn AD, abnormalities in brain wiring and variations of the brain energetic metabolism are considered a key pathological framework of cognitive decline (Heininger, 2000). Neural stem cells (NSCs) can contribute to brain regeneration and, their transplantation improves the behaviour of neurodegenerative mice models (De Gioia et.al., 2021) although, the molecular mechanisms involved are still unidentified. Our preliminary study aims to explore the potential differences between NSCs‐derived neurons from 6‐months‐old healthy and AD mice in terms of neuronal network morphology. Furthermore, the study wants to provide a tool to investigate whether cell energy metabolism is modified, considering that bioenergetic failure can influence the regenerative potential of adult AD brain, an issue that is still ill defined.MethodNSCs were isolated from subventricular zone of healthy (H) and 3xTg (AD) mice. NSCs selection was based on their ability to proliferate and to form 3D floating neurospheres. Isolated cells were amplified and characterised for their expression of stem‐related markers. Subsequently, neuron and astrocyte differentiation protocols were set up. Morphological analyses were performed on H and AD NSC‐derived neurons and the expressions of glutamate transporters and sodium/calcium exchangers (NCXs) were investigated.ResultH and AD NSCs were strongly positive for Nestin and Sox2 stem markers and a faint expression of NeuN and Gfap, markers related to neuron and astrocyte mature phenotype respectively, was detected. These data confirmed the purity of the cell cultures as well as the reliability of the protocols. Morphological investigations highlighted a different distribution of the neurite network between NSCs‐derived neurons isolated from H and AD mice. Furthermore, H and AD mature cells expressed glutamate transporters essential not only for cellular transmission but also for energy production by their interplay with NCXs.ConclusionPreliminary data suggest that the pathological features of adult neurons from AD brain can be traced back to “stem level” and continue during differentiation. The different morphological organization of neurite network observed in NSCs‐derive AD neurons could be involved in the alteration of the impulse transmission and plasticity. Further studies will be necessary to better clarify the consequence of dysregulated glutamate transporters as well the role of astrocytes.

Overexpression of astrocytic glutamate transporters alleviates hyperexcitability in mouse model of Alzheimer’s Disease

AbstractBackgroundAstrocytes are the main cells responsible for clearing glutamate from the synapse; their glutamate transporters quickly and efficiently clear glutamate within the synaptic cleft, preventing neuronal excitotoxicity. In mouse models and human cases of Alzheimer’s Disease (AD), the astrocyte‐specific glutamate transporter (Glt1 in rodents and EAAT2 in humans) is decreased. Our goal is to study the effects of decreased glutamate transporters on synaptic measures of function and plasticity.MethodUsing a 5XFAD mouse model of amyloid pathology, we utilized an adeno‐associated virus (AAV) to over‐express Glt1 in the hippocampus. Electrophysiological field recordings examined the role of astrocyte‐specific glutamate transporters in synaptic plasticity and hyperexcitability. Animals received injections of AAV‐Luciferase (control) in one hippocampus, and AAV‐Glt1a in the contralateral hippocampus. Field recordings were performed to assess excitatory postsynaptic potentials in the hippocampal CA1 stratum radiatum elicited by stimulation of CA3 axons.ResultCompared to control (AAV‐Luciferase), AAV‐Glt1 significantly increased the expression of glutamate transporters in hippocampal tissue. We then compared measures of neuronal hyperexcitability, synaptic strength, and long‐term synaptic potentiation. Results indicate that there is more hyperexcitability in control tissues compared to AAV‐Glt injections, as evident by decreased population spike thresholds.ConclusionOthers have reported hippocampal hyperexcitability may lead to memory impairment; in human cases of AD it is thought to encourage epileptic episodes. By using our AAV‐directed technique to increase glutamate transporters, and thus reduce network hyperexcitability, we will be able to examine how reactive astrocytes and their loss of glutamate transporters contribute to memory or cognition loss. Ongoing studies will do exactly this‐ use a mouse model of AD with and without AAV Glt1 overexpression to determine changes in behavioral cognitive outcome measures that may be attributed to hyperexcitability in neuronal networks.

Aging-Related Changes in Expression and Function of Glutamate Transporters in Rat Spinal Cord Astrocytes

Astrocytes make up the predominant cell population among glial cells in the mammalian brain, and they play a vital role in ensuring its optimal functioning. They promote neuronal health and survival and protect neurons from glutamate-induced excitotoxicity. In the spinal cord’s dorsal horn (DH) and ventral horn (VH) regions, astrocytes serve crucial roles. Notably, VH motor neurons exhibit a heightened sensitivity to glutamate-induced damage. It is posited that this selective sensitivity could be related to their localized presence within the VH, where astrocytes possess a distinct set of mechanisms for managing glutamate. As organisms age, the risk of damage from glutamate increases, indicating a potential decline in the efficiency of astrocytic glutamate regulation. Our research involved an analysis of astrocytic structure, glutamate transporter levels, and glutamate uptake capabilities within the DH and VH through immunohistochemical methods, protein analysis via Western blot, and patch-clamp studies in electrophysiology. The investigations revealed a decrease in both the number and coverage of astroglia in the spinal cord, more so within the VH as aging progressed. Notably, levels of the excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2) also decreased with age, particularly within the VH. Patch-clamp analyses of astrocytes from both spinal regions confirmed a significant reduction in glutamate uptake activity as age advanced, indicating an age-related impairment in glutamate processing. The findings indicate aging leads to distinct changes in DH and VH astrocytes, impairing their glutamate management abilities, which could contribute significantly to the development of late-onset neurodegenerative conditions.

Marrow Mesenchymal Stem Cell-Derived Exosomes Upregulate Astrocytic Glutamate Transporter-1 Expression via miR-124/mTOR Pathway against Oxygen-Glucose Deprivation/Reperfusion Injury.

Experimental investigations have reported the efficacy of marrow mesenchymal stem cell-derived exosomes (MSC-Exos) for the treatment of ischemic stroke. The therapeutic mechanism, however, is still unknown. The purpose of the study is to show whether MSC-Exos increases astrocytic glutamate transporter-1 (GLT-1) expression in response to ischemic stroke and to investigate further mechanisms. An in vitro ischemia model (oxygen-glucose deprivation/reperfusion, OGD/R) was used. MSC-Exos was identified by Western blot (WB) and transmission electron microscopy (TEM). To further investigate the mechanism, MSC-Exos, miR-124 inhibitor, and mimics, and a mTOR pathway inhibitor (rapamycin, Rap) were used. The interaction between GLT-1 and miR-124 was analyzed by luciferase reporter assay. The GLT-1 RNA expression and miR-124 was assessed by quantitative real-time polymerase chain reaction (qRTPCR). The protein expressions of GLT-1, S6, and pS6 were detected by WB. Results demonstrated that MSC-Exos successfully inhibited the decrease of GLT-1 and miR-124 expression and the increase of pS6 expression in astrocytes after OGD/R. miR-124 inhibitor suppressed the effect of MSC-Exos on GLT-1 upregulation after OGD/R. Rapamycin notably decreased pS6 expression with significantly higher GLT-1 expression in astrocytes injured by OGD/R. Luciferase activity of the reporter harboring the wild-type or mutant GLT-1 3'UTR was not inhibited by miR-124 mimics. Further results showed that the inhibiting effect of MSC-Exos on pS6 expression and promoting effect of MSC-Exos on GLT-1 expression could be reversed by miR-124 inhibitor after OGD/R; meanwhile, the above conditions could be reversed again by rapamycin. Results show that miR-124 and the mTOR pathway are involved in regulation of MSC-Exos on GLT-1 expression in astrocytes injured by OGD/R. miR-124 does not directly target GLT-1. MSC-Exos upregulates GLT-1 expression via the miR-124/mTOR pathway in astrocytes injured by OGD/R.

Effects of Chronic Hydrocodone Exposure and Ceftriaxone on the Expression of Astrocytic Glutamate Transporters in Mesocorticolimbic Brain Regions of C57/BL Mice.

Exposure to opioids can lead to the alteration of several neurotransmitters. Among these neurotransmitters, glutamate is thought to be involved in opioid dependence. Glutamate neurotransmission is mainly regulated by astrocytic glutamate transporters such as glutamate transporter 1 (GLT-1) and cystine/glutamate antiporter (xCT). Our laboratory has shown that exposure to lower doses of hydrocodone reduced the expression of xCT in the nucleus accumbens (NAc) and the hippocampus. In the present study, we investigated the effects of chronic exposure to hydrocodone, and tested ceftriaxone as a GLT-1 upregulator in mesocorticolimbic brain regions such as the NAc, the amygdala (AMY), and the dorsomedial prefrontal cortex (dmPFC). Eight-week-old male mice were divided into three groups: (1) the saline vehicle control group; (2) the hydrocodone group; and (3) the hydrocodone + ceftriaxone group. Mice were injected with hydrocodone (10 mg/kg, i.p.) or saline for 14 days. On day seven, the hydrocodone/ceftriaxone group was injected with ceftriaxone (200 mg/kg, i.p.) for last seven days. Chronic exposure to hydrocodone reduced the expression of GLT-1, xCT, protein kinase B (AKT), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal Kinase (JNK) in NAc, AMY, and dmPFC. However, hydrocodone exposure increased the expression of G-protein-coupled metabotropic glutamate receptors (mGluR5) in the NAc, AMY, and dmPFC. Importantly, ceftriaxone treatment normalized the expression of mGluR5, GLT-1, and xCT in all these brain regions, except for xCT in the AMY. Importantly, ceftriaxone treatment attenuated hydrocodone-induced downregulation of signaling pathways such as AKT, ERK, and JNK expression in the NAc, AMY, and dmPFC. These findings demonstrate that ceftriaxone has potential therapeutic effects in reversing hydrocodone-induced downregulation of GLT-1 and xCT in selected reward brain regions, and this might be mediated through the downstream kinase signaling pathways such as AKT, ERK, and JNK.

THU495 Glutamate Metabolic Enzymes GLUD1/2 And GPX4 Are Associated with Tumor Size In Black Women With Invasive Lobular Breast Cancer

Abstract Disclosure: T. Young: None. T.C. Abalum: None. N. Hodgins: None. S. Persaud: None. A.I. Fernandez: None. A.O. Olukoya: None. A.M. Rozeboom: None. R.B. Riggins: None. Background and Objective: Invasive Lobular Breast Cancer (ILC) is a special histologic type of breast cancer with unique genetic and biological features. ILC may be less dependent on glucose metabolism than the more common histologic type, invasive ductal breast cancer (IDC), instead relying more on lipid and amino acid metabolism. Our lab has previously published that expression of metabotropic glutamate receptors is increased in more rapidly growing and therapy-resistant cell line models of ILC. We hypothesized that increased expression of glutamate transporters and regulatory enzymes is associated with poor prognostic features. Methods and Analysis: We used multispectral immunohistochemical staining to measure the expression of four functionally related proteins: CD98, GLUD1/2, GPX4, and SLC7A11 in a cohort of 72 women with ILC (23.5% Black, 64.7% White, 11.8% Other). The cohort was in a two-fold redundant tissue microarray (TMA) format comprised of primary tumors collected by the Georgetown University Histopathology and Tissue Shared Resource. SLC7A11 and CD98 form a heterodimer that exports glutamate from, and imports cystine to, the cell. GLUD1/2 are two isoenzymes that convert glutamate into α-ketoglutarate to replenish the TCA cycle, or recycle ammonia to support amino acid synthesis. GPX4 is an enzyme that protects cells from lipid peroxidation-induced death. We compared expression of these proteins with clinical, pathological, and demographic variables (overall survival, hormone receptor expression, tumor size, race, age) using t test/ANOVA, Kaplan-Meier survival analysis, and linear regression. Results: In the 68 evaluable cases, the percentage of ILC tumor cells stained positive for GLUD1/2 and GPX4 was significantly higher than for CD98 or SLC7A11 (P<0.001), so further analyses focused on these. Women with a combination of high (above median) GLUD1/2+ (>49%) and GPX4+ (>54%) expression had a trend toward decreased survival (Hazard Ratio, HR 3.53, P=0.159). GPX4, GLUD1/2, and estrogen receptor (ER) expression were associated with tumor size in the entire cohort (R2 = 0.124, P = 0.041), but this relationship was markedly stronger in Black women (R2 = 0.487, P = 0.040). In univariate analyses, GLUD1/2 (R2 = 0.43, P<0.01) and GPX4 (R2 = 0.27, P<0.05) expression were each associated with tumor size in Black women, but not in the entire cohort. GPX4 expression and tumor size were both significantly higher (P<0.05) in Black women over 60 years of age, compared to those under 60. Conclusion: GLUD1/2 and GPX4 expression may be prognostic in ILC, as our results show that tumor size increases with higher expression of these proteins (especially among Black women). Limitations of this study include small sample size and lack of an independent cohort for validation. Future studies will include analysis of these proteins in a TMA comprised of tissue from women with IDC. Presentation: Thursday, June 15, 2023

Analysis of cnidarian Gcm suggests a neuronal origin of glial EAAT1 function

In bilaterian central nervous systems, coordination of neurotransmission by glial cells enables highly sophisticated neural functions. The diversity of transcription factors (TFs) involved in gliogenesis suggests multiple evolutionary origins of various glial cell types of bilaterians. Many of these TFs including the glial cells missing (Gcm) are also present in genomes of Cnidaria, the closest outgroup to Bilateria, but their function remains to be elucidated. In this study, we analyzed the function of Gcm, a multifunctional TF involved in development of glial and non-glial cell types, in the sea anemone, Nematostella vectensis. siRNA-mediated knockdown of Nematostella Gcm altered expression of cell adhesion proteins, glutamate and GABA transporters, ion channels, metabolic enzymes, and zinc finger and Ets-related TFs. NvGcm and mRNAs of downstream genes are expressed in broad neural cell clusters. However, immunostaining of a NvGcm target protein, the glutamate transporter, NvEAAT1, visualized a novel class of cells with flat cell bodies and no clear processes. Together with the finding of unique morphological features of NvEAAT1-functioning cells, these data suggest that extracellular glutamate metabolism, one of major glial functions, is deployed downstream of Gcm in specific neural cell types in Cnidaria.

Liver Metabolomics and Inflammatory Profiles in Mouse Model of Fentanyl Overdose Treated with Beta-Lactams.

Fentanyl is a highly potent opioid analgesic that is approved medically to treat acute and chronic pain. There is a high potential for overdose-induced organ toxicities, including liver toxicity, and this might be due to the increase of recreational use of opioids. Several preclinical studies have demonstrated the efficacy of beta-lactams in modulating the expression of glutamate transporter-1 (GLT-1) in different body organs, including the liver. The upregulation of GLT-1 by beta-lactams is associated with the attenuation of hyperglutamatergic state, which is a characteristic feature of opioid use disorders. A novel experimental beta-lactam compound with no antimicrobial properties, MC-100093, has been developed to attenuate dysregulation of glutamate transport, in part by normalizing GLT-1 expression. A previous study showed that MC-100093 modulated hepatic GLT-1 expression with subsequent attenuation of alcohol-increased fat droplet content in the liver. In this study, we investigated the effects of fentanyl overdose on liver metabolites, and determined the effects of MC-100093 and ceftriaxone in the liver of a fentanyl overdose mouse model. Liver samples from control, fentanyl overdose, and fentanyl overdose ceftriaxone- or MC-100093-treated mice were analyzed for metabolomics using gas chromatography-mass spectrometry. Heatmap analysis revealed that both MC-100093 and ceftriaxone attenuated the effects of fentanyl overdose on several metabolites, and MC-100093 showed superior effects. Statistical analysis showed that MC-100093 reversed the effects of fentanyl overdose in some metabolites. Moreover, enrichment analysis revealed that the altered metabolites were strongly linked to the glucose-alanine cycle, the Warburg effect, gluconeogenesis, glutamate metabolism, lactose degradation, and ketone body metabolism. The changes in liver metabolites induced by fentanyl overdose were associated with liver inflammation, an effect attenuated with ceftriaxone pre-treatments. Ceftriaxone normalized fentanyl-overdose-induced changes in liver interleukin-6 and cytochrome CYP3A11 (mouse homolog of human CYP3A4) expression. Our data indicate that fentanyl overdose impaired liver metabolites, and MC-100093 restored certain metabolites.

Proteomic Analysis of the Amygdala Reveals Dynamic Changes in Glutamate Transporter-1 During Progression of Complete Freund’s Adjuvant-Induced Pain Aversion

Pain sufferer usually show an aversion to the environment associated with pain, identified as pain aversion. The amygdala, an almond-shaped limbic structure in the medial temporal lobe, exerts a critical effect on emotion and pain formation. However, studies on inflammatory pain-induced aversion are still relatively limited, and the available evidence is not enough to clarify its inherent mechanisms. Proteomics is a high-throughput, comprehensive, and objective study method that compares the similarities and differences of protein expression under different conditions to screen potential targets. The current study aimed to identify potential pivotal proteins in the amygdala of rats after complete Freund’s adjuvant (CFA)-induced pain aversion via proteomics analysis. Immunohistochemistry was performed to confirm the expression of glutamate transporter-1 (GLT-1) in the amygdala during different periods of pain aversion. Thirteen proteins were found to be different between the day 2 and day 15 groups. Among the 13 differentially expressed proteins, Q8R64 denotes GLT-1, which utilises synaptic glutamate to remain optimal extracellular glutamic levels, thereby preventing accumulation in the synaptic cleft and consequent excitotoxicity. The variation in GLT-1 expression was correlated with the variation tendency of pain aversion, which implies a potential link between the modulation of pain aversion and the excitability of glutamatergic neurons. This study demonstrated that exposure to inflammatory pain results in aversion induced from pain, leading to extensive biological changes in the amygdala.

Chronic Inhalation of Pod-based e-cigarette Aerosols on Inflammatory Biomarkers in the Central Nervous and Peripheral Systems

Electronic cigarettes (e-cigs) use has been dramatically increased recently, especially among youths. In this study, we evaluated the effect of one- and three-months continuous exposure to e-cig vapor (JUUL pods), containing high nicotine concentration, on the expression of glutamate receptors and transporters and mainly the associated neuroinflammatory markers in drug reward brain regions such as nucleus accumbens (NAc) core (NAc-core), NAc shell (NAc-shell) and hippocampus (HIP) in female C57BL/6 mice. We revealed that three months’ exposure to mint or mango flavored-JUUL (containing 5% nicotine, 59 mg/ml) induced upregulation in metabotropic glutamate receptor 1 (mGluR1) and postsynaptic density protein 95 (phosphorylated and total PSD95) expression, and downregulation of mGluR5 and glutamate transporter 1 (GLT-1) in the NAc-shell. Three months’ exposure to JUUL induced upregulation of mGluR5 and GLT-1 expression in the HIPP. We further revealed that mice exposed to JUUL Mango and JUUL Mint for one month had significantly increased TNF-α expression in the NAc-core and NAc-shell, with elevation in IL-1β as well. NAc-shell also showed significantly increased levels of IL-6 and HMGB-1 for both flavors. There were significantly increased levels of TNF-α in the NAc-core and NAc-shell for both Mint- and Mango-exposed JUUL mice for three months. NAc-shell also showed significantly increased levels of IL-1β, IL-6, HMGB-1, and RAGE after three months of JULL exposure, while the hippocampus showed significantly decreased levels of HMGB-1 for both flavors. These findings demonstrated that exposure to e-cig vapor containing high nicotine concentrations induced differential effects on glutamatergic system as well as induction of proinflammatory markers in the brain.

Aspirin Effect on Tobacco Smoke Withdrawal-Induced Anxiety In Female Rats

Chronic exposure to cigarette smoke produces neuroinflammation and long-term changes in neurotransmitter systems, especially glutamatergic systems. We examined the effects of cigarette smoke on astroglial glutamate transporters as well as NF-κB expression in mesocorticolimbic brain regions including the prefrontal cortex (PFC) and the nucleus accumbens (NAc). The behavioral consequences of cigarette smoke exposure on withdrawal-induced anxiety-like behavior were assessed using open field (OF) and light/dark (LD) tests. Sprague-Dawley rats were randomly assigned to 5 experimental groups: a control group exposed only to standard room air, a cigarette smoke exposed group treated with saline vehicle, two cigarette smoke exposed groups treated with aspirin (15mg/kg and 30mg/kg, respectively). Lastly, a group treated only with aspirin (30 mg/kg). Cigarette smoke exposure was performed for 2hr/day, 5days/week, for 31days. Behavioral tests were conducted weekly, 24hrs after cigarette smoke exposure, during acute withdrawal. At the end of week 4, rats were given either saline or aspirin 45 min before cigarette exposure for 11 days. Cigarette smoke increased withdrawal-induced anxiety, and 30 mg/kg aspirin attenuated this effect. Cigarette smoke exposure increased the relative mRNA and the protein level for nuclear factor ĸB (NFĸB) in the PFC and the NAc, and aspirin treatment reversed this effect. In addition, cigarette smoke decreased the relative mRNA and the protein levels of glutamate transporter 1 (GLT-1) and the cystine-glutamate transporter (xCT) in the PFC and the NAc, while aspirin treatment normalized their expression. Thus, cigarette smoke caused neuroinflammation, alterations in relative mRNA glutamate transporter expression, and increased anxiety-like behavior, and these effects were attenuated by aspirin treatment.

Vesicular glutamate transporter 2 expression in the ventral tegmental area of outbred male rats following exposure to nicotine and alcohol

BackgroundInitiation of use/co-use of nicotine and alcohol, commonly occurring in an episodic manner during adolescence, can imprint vulnerability to the developing brain and lead to addiction. The ventral tegmental area (VTA) is a key heterogeneous region of the mesocorticolimbic circuit involved in the binge-drinking and intoxication step of the addiction circuit. Higher human post-mortem VTA expression of vesicular glutamate transporter 2 (VGLUT2), a marker of the glutamatergic phenotype also expressed in dopaminergic [Tyrosine Hydroxylase (Th)-positive] neurons, has been associated with chronic nicotine use and co-use with alcohol. MethodsThe present study aimed to map and characterize the Vglut2- and Th-expressing neurons in the VTA of adolescent male rats exposed or not to prolonged (six-weeks) episodic (three consecutive days/week) nicotine and/or alcohol administration. Nicotine (0.35 mg/kg free base) was injected subcutaneously, whereas alcohol (2 g/kg 20%) was administrated via gavage. Vglut2 and Th mRNA was assessed in the anterior and posterior VTA by use of in situ hybridization. ResultsThe profile of neurons varied with substance-exposure among VTA subregions. Th-only expressing neurons were more abundant in the posterior VTA of the group exposed to nicotine-only, compared to controls. The same neurons were, on the contrary, less present in the anterior VTA of animals exposed to alcohol-only, who also displayed a higher number of Vglut2-expressing neurons in the lateral anterior VTA. ConclusionsVTA Vglut2- and Th-only neurons seem differentially involved in the effects of adolescent episodic nicotine and alcohol exposure in the anterior and posterior VTA.

Glioprotective effects of resveratrol in hypothalamic astrocyte cultures obtained from interferon receptor knockout (IFNα/βR-/-) mice.

Astrocytes play essential roles in the central nervous system (CNS), such as the regulation of glutamate metabolism, antioxidant defenses, and inflammatory/immune responses. Moreover, hypothalamic astrocytes seem to be crucial in the modulation of inflammatory processes, including those related to type I interferon signaling. In this regard, the polyphenol resveratrol has emerged as an important glioprotective molecule to regulate astrocyte functions. Therefore, this study aimed to investigate the immunomodulatory and protective effects of resveratrol in hypothalamic astrocyte cultures obtained from mouse depleted of type I interferon receptors (INF-α/β-/-), a condition that can impair immune and inflammatory functions. Resveratrol upregulated glutamate transporter and glutamine synthetase gene expression, as well as modulated the release of wide range of cytokines and genes involved in the control of inflammatory response, besides the expression of adenosine receptors, which display immunomodulatory functions. Resveratrol also increased genes associated with redox balance, mitochondrial processes, and trophic factors signaling. The putative genes associated with glioprotective effects of resveratrol, including nuclear factor erythroid derived 2 like 2 (Nrf2), heme oxygenase 1 (HO-1), sirtuin 1 (SIRT1), and phosphoinositide 3-kinase (PI3K)/Akt, were further upregulated by resveratrol. Thus, our data show that resveratrol was able to modulate key genes associated with glial functionality and inflammatory response in astrocyte cultures derived from IFNα/βR-/- mice. These data are in agreement with previous results, reinforcing its glioprotective effects even in hypothalamic astrocytes with altered inflammatory and immune signaling. Finally, this polyphenol can prepare astrocytes to better respond to injuries, including those associated with neuroimmunology defects.