Glutamate Transporter Research Articles

Anakinra Promotes M2 Microglia Activation during the Latent Phase of the Lithium-Pilocarpine Model of Temporal Lobe Epilepsy

Astrocytes and microglia and their polarization are thought to contribute to the progression of epilepsy. One of the processes affecting polarization is neuroinflammation, which plays an important role in epileptogenesis. However, the specific mechanisms of its involvement in shifting the pro- and anti-inflammatory reactivation of astro- and microglia have not been clarified. In this study, we examined the effect of 7-day interleukin-1 receptor antagonist (anakinra) administration on glial cell polarization during the latent phase of the lithium-pilocarpine model in 7-week-old male Wistar rats. In temporal cortex, dorsal and ventral hippocampus the mRNA expression levels of the following genes were analyzed: (i) markers of astroglia (S100b) and microglia (Aif1) activation, (ii) astrocytic proteins involved in glutamate transport and metabolism (Slc1a3, Glul, Gja1), (iii) pro-inflammatory pathway interleukin-1β (Nlrp3, Il1b, Il1rn) and transforming growth factor β1 (Tgfb1), (iv) markers of astroglia polarization (Lcn2, S100a10, Gbp2, Ptx3), and (v) microglia polarization (Nos2 and Arg1). The mRNA expression levels of S100b and Aif1 were significantly increased, and anakinra administration did not reduce their overexpression. This indicates reactivation of astroglia and microglia regardless of the anakinra administered. The expression of Slc1a3, Glul, and Gja1 genes increased in the hippocampus; anakinra administration did not affect their hyperexpression, but promoted increased expression of Gja1 in the temporal cortex. The mRNA production of Lcn2, S100a10, Gbp2, Ptx3, Nlrp3, Il1b, Il1rn and Tgfb1 increased in all structures. Administration of anakinra reduced the gene expression of Il1b. Among the markers of microglia polarization, downregulation of Arg1 expression in the dorsal hippocampus and Nos2 expression in the temporal cortex was detected. Anakinra administration enhanced the decrease in Nos2 expression and restored the level of Arg1 expression to control values. Thus, anakinra administration did not affect the intensity of glial cell reactivation, but improved M2 reactivation of microglia.

Postnatal Neuronal Nogo-A Knockdown Decreased the Message of Glutamatergic Synaptic Proteins.

It is well known that oligodendrocyte-associated Nogo-A protein is an important regulator of axonal outgrowth and an important inhibitor of functional recovery and anatomical plasticity after central nervous system (CNS) injury. Abundant studies of oligodendrocyte-associated Nogo-A function in the uninjured rodent have suggested a role in neuronal development and synaptic function. On the other hand, the roles of neuron-associated (i.e., neuronal) Nogo-A have not been fully investigated. We have previously shown that neuronal Nogo-A influence dendritic spine density and morphology in pyramidal neurons of the intact neocortex. To further examine the role of neuronal Nogo-A in this synaptic population, we designed an RNAi directed against Nogo-A, delivered to the developing rat sensorimotor cortex using a neurotropic viral vector adeno-associated virus (AAV) 2/8. We examined the transduced neocortex for molecules important for synaptic plasticity, including N-Methyl-D-Aspartate (NMDA) receptor subunits GRIN2A; glutamate receptor subunit epsilon-1 (NR2A), and GRIN2B; glutamate receptor subunit epsilon-2 (NR2B), as well as postsynaptic density-95 (PSD-95). Furthermore, we also determined the density of excitatory synapses by examining the presynaptic protein vesicular glutamate transporter 1 (vGLut1) as a marker for potential excitatory synapses. Our results showed that neuronal Nogo-A knockdown in postnatal pyramidal neurons of the sensorimotor cortex led to a significant decrease in NMDA receptor subunits NR2A and NR2B messenger RNA when examined as adults. However, there was no difference in PSD-95 expression in comparison to controls. In addition, the decrease in the number of vGlut1(+) puncta on branches of apical dendrites of pyramidal neurons indicated the loss of synapses that have a strong influence on direct current entering the dendrite. Taken together, these results indicate that neuronal Nogo-A may regulate synaptic plasticity by modulating the components of excitatory synapses. This finding represents a novel role in excitatory synaptic formation for neuronal Nogo-A in developing neurons of the uninjured CNS.

AKR1C1 interacts with STAT3 to increase intracellular glutathione and confers resistance to oxaliplatin in colorectal cancer

Oxaliplatin (OXA), a platinum-based chemotherapeutic agent, remains a mainstay in first-line treatments for advanced colorectal cancer (CRC). However, the eventual development of OXA resistance represents a significant clinical challenge. In the present study, we demonstrate that the aldo-keto reductase 1C1 (AKR1C1) is overexpressed in CRC cells upon acquisition of OXA resistance, evident in OXA-resistant CRC cell lines. We employed genetic silencing and pharmacological inhibition strategies to establish that suppression of AKR1C1 restores OXA sensitivity. Mechanistically, AKR1C1 interacts with and activates the transcription factor STAT3, which upregulates the glutamate transporter EAAT3, thereby elevating intracellular glutathione levels and conferring OXA resistance. Alantolactone, a potent natural product inhibitor of AKR1C1, effectively reverses this chemoresistance, restricting the growth of OXA-resistant CRC cells both in vitro and in vivo. Our findings uncover a critical AKR1C1-dependent mechanism behind OXA resistance and propose a promising combinatorial therapeutic strategy to overcome this resistance in CRC.

Exploring the role of spinal astrocytes in the onset of hyperalgesic priming signals in acid-induced chronic muscle pain.

Hyperalgesic priming, a form of pain plasticity initiated by initial injury, leads to heightened sensitivity to subsequent noxious stimuli, contributing to chronic pain development in animals. While astrocytes play active roles in modulating synaptic transmission in various pain models, their specific involvement in hyperalgesic priming remains elusive. Here, we show that spinal astrocytes are essential for hyperalgesic priming formation in a mouse model of acid-induced muscle pain. We observed spinal astrocyte activation 4 h after initial acid injection, and inhibition of this activation prevented chronic pain development upon subsequent acid injection. Chemogenetic activation of spinal astrocytes mimicked the first acid-induced hyperalgesic priming. We also demonstrated that spinal phosphorylated extracellular regulated kinase (pERK)-positive neurons were mainly vesicular glutamate transporter-2 positive (Vglut2+) neurons after the first acid injection, and inhibition of spinal pERK prevented astrocyte activation. Furthermore, pharmacological inhibition of astrocytic glutamate transporters glutamate transporter-1 and glutamate-aspartate transporter abolished the hyperalgesic priming. Collectively, our results suggest that pERK activation in Vglut2+ neurons activate astrocytes through astrocytic glutamate transporters. This process eventually establishes hyperalgesic priming through spinal D-serine. We conclude that spinal astrocytes play a crucial role in the transition from acute to chronic pain.

Troriluzole rescues glutamatergic deficits, amyloid and tau pathology, and synaptic and memory impairments in 3xTg-AD mice.

Alzheimer's disease (AD) is a neurodegenerative condition in which clinical symptoms are highly correlated with the loss of glutamatergic synapses. While later stages of AD are associated with markedly decreased glutamate levels due to neuronal loss, in the early stages, pathological accumulation of glutamate and hyperactivity contribute to AD pathology and cognitive dysfunction. There is increasing awareness that presynaptic dysfunction, particularly synaptic vesicle (SV) alterations, play a key role in mediating this early-stage hyperactivity. In the current study, we sought to determine whether the 3xTg mouse model of AD that exhibits both beta-amyloid (Aβ) and tau-related pathology would exhibit similar presynaptic changes as previously observed in amyloid or tau models separately. Hippocampal cultures from 3xTg mice were used to determine whether presynaptic vesicular glutamate transporters (VGlut) and glutamate are increased at the synaptic level while controlling for postsynaptic activity. We observed that 3xTg hippocampal cultures exhibited increased VGlut1 associated with an increase in glutamate release, similar to prior observations in cultures from tau mouse models. However, the SV pool size was also increased in 3xTg cultures, an effect not previously observed in tau mouse models but observed in Aβ models, suggesting the changes in pool size may be due to Aβ and not tau. Second, we sought to determine whether treatment with troriluzole, a novel 3rd generation tripeptide prodrug of the glutamate modulator riluzole, could reduce VGlut1 and glutamate release to restore cognitive deficits in 8-month-old 3xTg mice. Treatment with troriluzole reduced VGlut1 expression, decreased basal and evoked glutamate release, and restored cognitive deficits in 3xTg mice. Together, these findings suggest presynaptic alterations are early events in AD that represent potential targets for therapeutic intervention, and these results support the promise of glutamate-modulating drugs such as troriluzole in Alzheimer's disease.

Glutamate uptake is transiently compromised in the perilesional cortex following controlled cortical impact.

Glutamate, the primary excitatory neurotransmitter in the CNS, is regulated by the excitatory amino acid transporters (EAATs) GLT-1 and GLAST. Following traumatic brain injury (TBI), extracellular glutamate levels increase, contributing to excitotoxicity, circuit dysfunction, and morbidity. Increased neuronal glutamate release and compromised astrocyte-mediated uptake contribute to elevated glutamate, but the mechanistic and spatiotemporal underpinnings of these changes are not well established. Using the controlled cortical impact (CCI) model of TBI and iGluSnFR glutamate imaging, we quantified extracellular glutamate dynamics after injury. Three days post-injury, glutamate release was increased, and glutamate uptake and GLT-1 expression were reduced. 7- and 14-days post-injury, glutamate dynamics were comparable between sham and CCI animals. Changes in peak glutamate response were unique to specific cortical layers and proximity to injury. This was likely driven by increases in glutamate release, which was spatially heterogenous, rather than reduced uptake, which was spatially uniform. The astrocyte K + channel, Kir4.1, regulates activity-dependent slowing of glutamate uptake. Surprisingly, Kir4.1 was unchanged after CCI and accordingly, activity-dependent slowing of glutamate uptake was unaltered. This dynamic glutamate dysregulation after TBI underscores a brief period in which disrupted glutamate uptake may contribute to dysfunction and highlights a potential therapeutic window to restore glutamate homeostasis.

Photobiomodulation with near-infrared laser for tinnitus management: preliminary animal experiments and randomized clinical trials.

This study investigates the effectiveness of photobiomodulation therapy (PBMT) in treating chronic high-frequency tinnitus with the TINI device, a near-infrared (830nm) laser. The study includes preliminary animal experiments with 28 mice and a randomized controlled trial with 56 participants to examine the functional and molecular changes in the auditory system that PBMT may cause. The animal model used sodium salicylate to induce tinnitus, followed by PBMT, which showed promising reductions in the behavioral evidence of tinnitus and a reversal of tinnitus-associated upregulation of vesicular glutamate transporters 2 expression in the ipsilateral dorsal cochlear nucleus (p < 0.05). In the clinical trial, participants with chronic high-frequency tinnitus received trans-tympanic application of the TINI device. The results did not show a significant difference in tinnitus score at the final time point when compared to the sham group. However, questionnaires revealed significant improvements in tinnitus symptoms and psychological outcomes following treatment with the TINI device compared to before treatment (p < 0.05). These findings suggest that while PBMT has potential benefits, its clinical effectiveness may be unclear due to its complex nature and interaction with other conditions. Further research is required to optimize treatment parameters and gain a complete understanding of the therapeutic potential of PBMT in managing tinnitus.

LT-102, an AMPA receptor potentiator, alleviates depression-like behavior and synaptic plasticity impairments in prefrontal cortex induced by sleep deprivation

BackgroundSleep loss is closely related to the onset and development of depression, and the mechanisms involved may include impaired synaptic plasticity. Considering the important role of glutamate α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) in synaptic plasticity as well as depression, we introduce LT-102, a novel AMPARs potentiator, to evaluate the potential of LT-102 in treating sleep deprivation-induced depression-like behaviors. MethodsWe conducted a comprehensive behavioral assessment to evaluate the effects of LT-102 on depression-like symptoms in male C57BL/6J mice. This assessment included the open field test to measure general locomotor activity and anxiety-like behavior, the forced swimming test and tail suspension test to assess despair behaviors indicative of depressive states, and the sucrose preference test to quantify anhedonia, a core symptom of depression. Furthermore, to explore the impact of LT-102 on synaptic plasticity, we utilized a combination of Western blot analysis to detect protein expression levels, Golgi-Cox staining to visualize neuronal morphology, and immunofluorescence to examine the localization of synaptic proteins. Additionally, we utilized primary cortical neurons to delineate the signaling pathway modulated by LT-102. ResultsTreatment with LT-102 significantly reduced depression-like behaviors associated with sleep deprivation. Quantitative Western blot (WB) analysis revealed a significant increase in GluA1 phosphorylation in the prefrontal cortex (PFC), triggering the Ca2+/calmodulin-dependent protein kinase II/cAMP response element-binding protein/brain-derived neurotrophic factor (CaMKII/CREB/BDNF) and forkhead box protein P2/postsynaptic density protein 95 (FoxP2/PSD95) signaling pathways. Immunofluorescence imaging confirmed that LT-102 treatment increased spine density and co-labeling of PSD95 and vesicular glutamate transporter 1 (VGLUT1) in the PFC, reversing the reductions typically observed following sleep deprivation. Golgi staining further validated these results, showing a substantial increase in neuronal dendritic spine density in sleep-deprived mice treated with LT-102. Mechanistically, application of LT-102 to primary cortical neurons, resulted in elevated levels of phosphorylated AKT (p-AKT) and phosphorylated glycogen synthase kinase-3 beta (p-GSK3β), key downstream molecules in the BDNF signaling pathway, which in turn upregulated FoxP2 and PSD95 expression. LimitationsIn our study, we chose to exclusively use male mice to eliminate potential influences of the estrous cycle on behavior and physiology. As there is no widely accepted positive drug control for sleep deprivation studies, we did not include one in our research. ConclusionOur results suggest that LT-102 is a promising therapeutic agent for counteracting depression-like behaviors and synaptic plasticity deficits induced by sleep deprivation, primarily through the activation of CaMKII/CREB/BDNF and AKT/GSK3β/FoxP2/PSD95 signaling pathways.

Ovariectomy exacerbates the disturbance of excitation- inhibition balance in the brain of APP/PS-1/tau mice.

The prevalence of Alzheimer's disease (AD) is significantly gender-differentiated, with the number of female AD patients far exceeding that of males, accounting for two-thirds of the total prevalence. Although postmenopausal AD mice have been shown to have more prominent pathologic features and memory impairments than normal AD mice, the relevant molecular mechanisms leading to these outcomes have not been well elucidated. In the present study, we used the disturbance of excitation-inhibition balance in the postmenopausal brain as an entry point to explore the link between estrogen deficiency, disorders of the glutamatergic-GABAergic nervous system, and memory impairment. Wild-type (WT) mice and APP/PS1/tau (3 × Tg-AD) mice (10 months old) were randomly divided into four groups: WT+Sham group, WT+OVX group, 3 × Tg-AD+Sham group and 3 × Tg-AD+OVX group. Ovariectomy (OVX) was performed in the WT+OVX group and the 3 × Tg-AD+OVX group, and sham surgery was performed in the WT+Sham group and the 3 × Tg-AD+Sham group. The learning and memory ability and the anxiety and depression-like behavior changes of mice were evaluated by behavioral experiments, and the association between estrogen-estrogen receptors pathway and glutamatergic/GABAergic nervous system and female AD was evaluated by neurochemical experiments. In WT and 3 × Tg-AD mice, OVX resulted in impaired learning and memory abilities and anxiety and depression-like behaviors; reduced estrogen levels and downregulated the expression of estrogen receptors; upregulated the expression of amyloid-β, amyloid precursor protein, presenilin 1, and p-tau; upregulated the expression of Bcl-2-associated X protein and downregulated the expression of B-cell lymphoma-2, promoting cell apoptosis; reduced the number of neuronal dendrites and downregulated the expression of postsynaptic density protein-95; more importantly, OVX increased brain glutamate levels but downregulated the expression of N-methyl-D-aspartate receptor-2B, excitatory amino acid transporter 1, excitatory amino acid transporter 2, γ-aminobutyric acid receptor-A and γ-aminobutyric acid receptor-B. Our results suggested that OVX-induced estrogen-estrogen receptors pathway disruption caused learning and memory impairment and anxiety and depression-like behaviors, upregulated the expression of AD pathological markers, promoted apoptosis, destroyed neuronal structure, and most importantly, caused glutamatergic/GABAergic nervous system disorders.

The cell-permeant antioxidant D-thiol ester D-cysteine ethyl ester overcomes physical dependence to morphine in male Sprague Dawley rats.

The ability of morphine to decrease cysteine transport into neurons by inhibition of excitatory amino acid transporter 3 (EAA3) may be a key molecular mechanism underlying the acquisition of physical and psychological dependence to morphine. This study examined whether co-administration of the cell-penetrant antioxidant D-thiol ester, D-cysteine ethyl ester (D-CYSee), with morphine, would diminish the development of physical dependence to morphine in male Sprague Dawley rats. Systemic administration of the opioid receptor antagonist, naloxone (NLX), elicited pronounced withdrawal signs (e.g., wet-dog shakes, jumps, rears, circling) in rats that received a subcutaneous depot of morphine (150mg/kg, SC) for 36h and continuous intravenous infusion of vehicle (20μL/h, IV). The NLX-precipitated withdrawal signs were reduced in rats that received an infusion of D-CYSee, but not D-cysteine, (both at 20.8μmol/kg/h, IV) for the full 36h. NLX elicited pronounced withdrawal signs in rats treated for 48h with morphine (150mg/kg, SC), plus continuous infusion of vehicle (20μL/h, IV) that began at the 36h timepoint of morphine treatment. The NLX-precipitated withdrawal signs were reduced in rats that received a 12h infusion of D-CYSee, but not D-cysteine, (both at 20.8μmol/kg/h, IV) that began at the 36h timepoint of morphine treatment. These findings suggest that D-CYSee may attenuate the development of physical dependence to morphine and reverse established dependence to the opioid in male Sprague Dawley rats. Alternatively, D-CYSee may simply suppress the processes responsible for NLX-precipitated withdrawal. Nonetheless, D-CYSee and analogues may be novel therapeutics for the treatment of opioid use disorders.

Exogenous leptin alleviates glutamate-excitotoxic injury caused by cerebral ischemia–reperfusion in mice by affecting the expression of glutamate transporters

Ischemic stroke is characterized by high morbidity and mortality and a lack of effective therapeutic interventions. Leptin plays an important role in regulating oxidative stress, angiogenesis, hematopoiesis, etc. Although recent studies have found a neuroprotective effect of leptin, little is known about its role in cerebral ischemia. This study explores the possible roles and potential preventative mechanisms of leptin in cerebral ischemia–reperfusion injury (CIRI). An in vivo middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model was used to replicate the CIRI model, low (0.5 mg/kg), medium (1 mg/kg) and high (2 mg/kg) concentrations of leptin were injected intraperitoneally immediately after inserting the embolic line. After 1.5 h of ischemia and 24 h of reperfusion, we examined the neural function of the mice, collected brain tissue for histological examination, and screened for the optimal concentrations of leptin intervention. On this basis, we observed the changes of cortical apoptosis injury, intracellular calcium fluorescence intensity and astrocyte glial fibrillary acidic protein (GFAP) expression and morphological changes. In addition, we also tested the expression of transporters and metabolism-related enzymes (VGLUT-1, VGLUT-2, GLAST, GLT-1, GS, ATP1α1), the expression of inflammatory factors and the content of glutamate (Glu). Compared with the I/R group, we found that leptin improved neurological deficits, reduced the area of infarcts, maintained the normal morphology of astrocytes (AST), downregulated the expression of VGLUT-1, and upregulates the expression of GLT-1 and GLAST, thereby reducing the content of Glu in the synaptic cleft. Our studies suggest that leptin may have a neuroprotective effect by decreasing the excitotoxicity of glutamate.

Astrocytic glutamate transport is essential for the memory-enhancing effects of 17β-estradiol in ovariectomized mice

Infusion of 17β-estradiol (E2) into the dorsal hippocampus (DH) of ovariectomized (OVX) mice enhances memory consolidation, an effect that depends on rapid phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Astrocytic glutamate transporter 1 (GLT-1) modulates neurotransmission via glutamate uptake from the synaptic cleft. However, little is known about the contribution of DH astrocytes, and astrocytic glutamate transport, to the memory-enhancing effects of E2. This study was designed to test whether DH astrocytes contribute to estrogenic modulation of memory consolidation by determining the extent to which DH GLT-1 is necessary for E2 to enhance memory in object recognition and object placement tasks and trigger rapid phosphorylation events in DH astrocytes. OVX female mice were bilaterally cannulated into the DH or the DH and dorsal third ventricle (ICV). Post-training DH infusion of the GLT-1 inhibitor dihydrokainic acid (DHK) dose-dependently impaired memory consolidation in both tasks. Moreover, the memory-enhancing effects of ICV-infused E2 in each task were blocked by DH DHK infusion. E2 increased p42 ERK and Akt phosphorylation in DH astrocytes, and these effects were blocked by DHK. Results suggest the necessity of DH GLT-1 activity for object and spatial memory consolidation, and for E2 to enhance consolidation of these memories and to rapidly activate cell signaling in DH astrocytes. Findings indicate that astrocytic function in the DH of OVX females is necessary for memory formation and is regulated by E2, and suggest an essential role for DH astrocytic GLT-1 activity in the memory-enhancing effects of E2.

Neuroprotective Effects of VGLUT1 Inhibition in HT22 Cells Overexpressing VGLUT1 Under Oxygen Glucose Deprivation Conditions

Glutamate (Glu) is a major excitatory neurotransmitter in the brain, essential for synaptic plasticity, neuronal activity, and memory formation. However, its dysregulation leads to excitotoxicity, implicated in neurodegenerative diseases and brain ischemia. Vesicular glutamate transporters (VGLUTs) regulate Glu loading into synaptic vesicles, crucial for maintaining optimal extracellular Glu levels. This study investigates the neuroprotective effects of VGLUT1 inhibition in HT22 cells overexpressing VGLUT1 under oxygen glucose deprivation (OGD) conditions. HT22 cells, a hippocampal neuron model, were transduced with lentiviral vectors to overexpress VGLUT1. Cells were subjected to OGD, with pre-incubation of Chicago Sky Blue 6B (CSB6B), an unspecific VGLUT inhibitor. Cell viability, lactate dehydrogenase (LDH) release, mitochondrial membrane potential, and hypoxia-related protein markers (PARP1, AIF, NLRP3) were assessed. Results indicated that VGLUT1 overexpression increased vulnerability to OGD, evidenced by higher LDH release and reduced cell viability. CSB6B treatment improved cell viability and reduced LDH release in OGD conditions, particularly at 0.1 μM and 1.0 μM concentrations. Moreover, CSB6B preserved mitochondrial membrane potential and decreased levels of PARP1, AIF, and NLRP3 proteins, suggesting neuroprotective effects through mitigating excitotoxicity. This study demonstrates that VGLUT1 inhibition could be a promising therapeutic strategy for ischemic brain injury, warranting further investigation into selective VGLUT1 inhibitors.

Dietary supplementation of microencapsulated botanicals and organic acids enhances the expression and function of intestine epithelial digestive enzymes and nutrient transporters in broiler chickens

Organic acids and botanicals have shown protective effects on gut barrier and against inflammation in broilers. However, their effects on intestinal digestive enzymes and nutrients transporters expression and functions have not been fully studied. The objective of this study was to understand how a microencapsulated blend of botanicals and organic acids affected intestinal enzyme activities and nutrient transporters expression and functions in broilers. A total of 288 birds were assigned to a commercial control diet or diet supplemented with 500 g/MT (metric ton) of the microencapsulated additive. Growth performance was recorded weekly. At d 21 and d 42, jejunum and ileum were isolated for enzyme (maltase, sucrase, and aminopeptidase) and transporter (SGLT1, GLUT2, GLUT1, EAAT3, B0AT1, and PepT1) analyses. Jejunum specific nutrients (glucose, alanine, and glutamate) transport activities were evaluated by Ussing chamber. Protein expression of nutrient transporters in small intestine were measured in mucosa and brush-border membrane (BBM) samples by western blot. Intestinal gene expression of the transporters was determined by RT-PCR. Statistical analysis was performed using Student's t-test comparing the supplemented diet to the control. The feed efficiency was significantly improved through the study period in the supplemented group (P ≤ 0.05). Significant changes of intestinal histology were shown in both jejunum (P ≤ 0.10) and ileum (P ≤ 0.05) after 21 d of treatment. At d21, jejunal maltase activity was upregulated (P ≤ 0.10). The Ussing chamber transport of glucose and alanine was increased, which was in line with increased gene expression (GLUT2, GLUT1, EAAT3, and B0AT1) (P ≤ 0.10 and P ≤ 0.05, respectively) and BBMV protein levels (B0AT1, P < 0.10). At d21, ileal sucrase and maltase activities were upregulated (P ≤ 0.05). Increased expressions of GLUT1, EAAT3, and B0AT1 were observed in both mRNA and protein levels (P ≤ 0.05). Similar pattern of changes was also shown at d42 of age. Our results suggest that feeding microencapsulated additives improves intestinal nutrient digestion and transporter expression and function in broilers, thereby enhancing feed efficiency.

β-Hydroxybutyrate enhances astrocyte glutamate uptake through EAAT1 expression regulation

β-Hydroxybutyrate (BHB) has been reported to exert neuroprotective functions and is considered a promising treatment for neurodegenerative diseases such as Parkinson's and Alzheimer's. Numerous studies have revealed BHB's multifaceted roles, including anti-senescence, anti-oxidative, and anti-inflammatory activities. However, the underlying mechanisms warrant further investigation. Astrocytes, the most abundant glial cells in the central nervous system, play a pivotal role in the development and progression of neurodegenerative diseases. While BHB is known to alter neuronal metabolism and function, its effects on astrocytes remain poorly understood. In this study, we conducted transcriptome sequencing analysis to identify differentially expressed genes induced by BHB in astrocytes and found that the gene Solute carrier family 1 member 3 (Slc1a3), encoding the glutamate transporter EAAT1, was significantly upregulated by BHB treatment. Cellular and animal-based experiments confirmed an increase in EAAT1 protein expression in primary astrocytes and the hippocampus of mice treated with BHB. This upregulation may be due to the activation of the Ca2+/CAMKII pathway by BHB. Furthermore, BHB improved astrocytes' glutamate uptake and partially restored neuronal viability impaired by glutamate-induced excitotoxicity when astrocytes were functionalized. Our results suggest that BHB may alleviate neuronal damage caused by excessive glutamate by enhancing the glutamate absorption and uptake capacity of astrocytes. This study proposes a novel mechanism for the neuroprotective effects of BHB and reinforces its beneficial impact on the central nervous system (CNS).

Neuroinflammation and Neurometabolomic Profiling in Fentanyl Overdose Mouse Model Treated with Novel β-Lactam, MC-100093, and Ceftriaxone.

Opioid-related deaths are attributed to overdoses, and fentanyl overdose has been on the rise in many parts of the world, including the USA. Glutamate transporter 1 (GLT-1) has been identified as a therapeutic target in several preclinical models of substance use disorders, and β-lactams effectively enhance its expression and function. In the current study, we characterized the metabolomic profile of the nucleus accumbens (NAc) in fentanyl-overdose mouse models, and we evaluated the protective effects of the functional enhancement of GLT-1 using β-lactams, ceftriaxone, and MC-100093. BALB/c mice were divided into four groups: control, fentanyl, fentanyl/ceftriaxone, and fentanyl/MC-100093. While the control group was intraperitoneally (i.p.) injected with normal saline simultaneously with other groups, all fentanyl groups were i.p. injected with 1 mg/kg of fentanyl as an overdose after habituation with four repetitive non-consecutive moderate doses (0.05 mg/kg) of fentanyl for a period of seven days. MC-100093 (50 mg/kg) and ceftriaxone (200 mg/kg) were i.p. injected from days 5 to 9. Gas chromatography-mass spectrometry (GC-MS) was used for metabolomics, and Western blotting was performed to determine the expression of target proteins. Y-maze spontaneous alternation performance and the open field activity monitoring system were used to measure behavioral manifestations. Fentanyl overdose altered the abundance of about 30 metabolites, reduced the expression of GLT-1, and induced the expression of inflammatory mediators IL-6 and TLR-4 in the NAc. MC-100093 and ceftriaxone attenuated the effects of fentanyl-induced downregulation of GLT-1 and upregulation of IL-6; however, only ceftriaxone attenuated fentanyl-induced upregulation of TRL4 expression. Both of the β-lactams attenuated the effects of fentanyl overdose on locomotor activities but did not induce significant changes in the overall metabolomic profile. Our findings revealed that the exposure to a high dose of fentanyl causes alterations in key metabolic pathways in the NAc. Pretreatment with ceftriaxone and MC-100093 normalized fentanyl-induced downregulation of GLT-1 expression with subsequent attenuation of neuroinflammation as well as the hyperactivity, indicating that β-lactams may be promising drugs for treating fentanyl use disorder.

Neuroprotective effects of L-Dopa-modified zinc oxide nanoparticles on the rat model of 6-OHDA-ınduced Parkinson’s disease

Parkinson’s disease (PD) is a chronic neurodegenerative case. As the disease progresses, the response time to doses of levodopa (L-Dopa) becomes shorter and the effects of the drug are severely limited by some undesirable side effects such as the ‘on–off’ phenomenon. In several diseases, including Parkinson’s, nanoparticles can deliver antioxidant compounds that reduce oxidative stress. This study evaluates and compares the neuroprotective effects of L-Dopa-modified zinc nanoparticles (ZnNPs) in the 6-hydroxydopamine (6-OHDA)-induced PD rat model. For this purpose, the synthesis of NPs was carried out. Scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectrophotometer were used for characterization. The rats were randomized into 9 experimental groups: control, lesion group (6-OHDA), 6-OHDA + 5 mg/kg L-Dopa, 6-OHDA + 10 mg/kg L-Dopa, 6-OHDA + 20 mg/kg L-Dopa, 6-OHDA + 20 mg/kg ZnNPs, 6-OHDA + 40 mg/kg ZnNPs, 6-OHDA + 30 mg/kg ZnNPs + L-Dopa, and 6-OHDA + 60 mg/kg ZnNPs + L-Dopa. Behavioral tests were performed on all groups 14 days after treatment. Phosphatase and tensin homolog, Excitatory amino acid transporter 1/2, and Glutamine synthetase gene analyses were performed on brain samples taken immediately after the tests. In addition, histological and immunohistochemical methods were used to determine the general structure and properties of the tissues. We obtained important findings that L-Dopa-modified ZnNPs increased the activity of glutamate transporters. Our experiment showed that glutamate increases neuronal cell vitality and improves behavioral performance. Therefore, L-Dopa-modified ZnNPs can be used to prevent neurotoxicity. According to what we found, results show that L-Dopa-modified ZnNPs will lend to the effective avoidance and therapy of PD.

YTHDF1 regulates GID8-mediated glutamine metabolism to promote colorectal cancer progression in m6A-dependent manner

Dysregulation of epigenetics is a hallmark of cancer development, and YTHDF1 stands out as a crucial epigenetic regulator with the highest DNA copy number variation among all N6-methyladenosine (m6A) regulators in colorectal cancer (CRC) patients. Here, we aimed to investigate the specific contribution of YTHDF1 overexpression to CRC progression and its consequences. Through multiple bioinformatic analyses of human cancer databases and clinical CRC samples, we identified GID8/Twa1 as a crucial downstream target of YTHDF1. YTHDF1 manipulates GID8 translation efficiency in an m6A-dependent manner, and high expression of GID8 is associated with more aggressive tumor progression and poor overall survival. Mechanistically, GID8 is intimately associated with glutamine metabolic demands by maintaining active glutamine uptake and metabolism through the regulation of excitatory amino acid transporter 1 (SLC1A3) and glutaminase (GLS), thereby facilitating the malignant progression of CRC. Inhibition of GID8 attenuated CRC proliferation and metastasis both in vitro and in vivo. In summary, we identified a previously unknown target pertaining to glutamine uptake and metabolism in tumor cells, underscoring the potential of GID8 in the treatment of CRC.

Albiflorin Decreases Glutamate Release from Rat Cerebral Cortex Nerve Terminals (Synaptosomes) through Depressing P/Q-Type Calcium Channels and Protein Kinase A Activity.

The purpose of this study was to investigate whether and how albiflorin, a natural monoterpene glycoside, affects the release of glutamate, one of the most important neurotransmitters involved in neurotoxicity, from cerebrocortical nerve terminals (synaptosomes) in rats. The results showed that albiflorin reduced 4-aminopyridine (4-AP)-elicited glutamate release from synaptosomes, which was abrogated in the absence of extracellular Ca2+ or in the presence of the vesicular glutamate transporter inhibitor or a P/Q-type Ca2+ channel inhibitor, indicating a mechanism of action involving Ca2+-dependent depression of vesicular exocytotic glutamate release. Albiflorin failed to alter the increase in the fluorescence intensity of 3,3-diethylthiacarbocyanine iodide (DiSC3(5)), a membrane-potential-sensitive dye. In addition, the suppression of protein kinase A (PKA) abolished the effect of albiflorin on glutamate release. Albiflorin also reduced the phosphorylation of PKA and synaptosomal-associated protein of 25 kDa (SNAP-25) and synapsin I at PKA-specific residues, which correlated with decreased available synaptic vesicles. The results of transmission electron microscopy (TEM) also observed that albiflorin reduces the release competence of synaptic vesicles evoked by 4-AP in synaptosomes. In conclusion, by studying synaptosomally released glutamate, we suggested that albiflorin reduces vesicular exocytotic glutamate release by decreasing extracellular Ca2+ entry via P/Q-type Ca2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation.

Structural basis of the obligatory exchange mode of human neutral amino acid transporter ASCT2

ASCT2 is an obligate exchanger of neutral amino acids, contributing to cellular amino acid homeostasis. ASCT2 belongs to the same family (SLC1) as Excitatory Amino Acid Transporters (EAATs) that concentrate glutamate in the cytosol. The mechanism that makes ASCT2 an exchanger rather than a concentrator remains enigmatic. Here, we employ cryo-electron microscopy and molecular dynamics simulations to elucidate the structural basis of the exchange mechanism of ASCT2. We establish that ASCT2 binds three Na+ ions per transported substrate and visits a state that likely acts as checkpoint in preventing Na+ ion leakage, both features shared with EAATs. However, in contrast to EAATs, ASCT2 retains one Na+ ion even under Na+-depleted conditions. We demonstrate that ASCT2 cannot undergo the structural transition in TM7 that is essential for the concentrative transport cycle of EAATs. This structural rigidity and the high-affinity Na+ binding site effectively confine ASCT2 to an exchange mode.