Drug Treatment Attenuates Retinal Ganglion Cell Death by Inhibiting Collapsin Response Mediator Protein 2 Phosphorylation in Mouse Models of Normal Tension Glaucoma.

Normal tension glaucoma (NTG) is a progressive neurodegenerative disease in glaucoma families. Typical glaucoma develops because of increased intraocular pressure (IOP), whereas NTG develops despite normal IOP. As a subtype of open-angle glaucoma, NTG is characterized by retinal ganglion cell (RGC) degeneration, gradual loss of axons, and injury to the optic nerve. The relationship between glutamate excitotoxicity and oxidative stress has elicited great interest in NTG studies. We recently reported that suppressing collapsin response mediator protein 2 (CRMP2) phosphorylation in S522A CRMP2 mutant (CRMP2 KIKI) mice inhibited RGC death in NTG mouse models. This study evaluated the impact of the natural compounds huperzine A (HupA) and naringenin (NAR), which have therapeutic effects against glutamate excitotoxicity and oxidative stress, on inhibiting CMRP2 phosphorylation in mice intravitreally injected with N-methyl-D-aspartate (NMDA) and GLAST mutant mice. Results of the study demonstrated that HupA and NAR significantly reduced RGC degeneration and thinning of the inner retinal layer, and inhibited the elevated CRMP2 phosphorylation. These treatments protected against glutamate excitotoxicity and suppressed oxidative stress, which could provide insight into developing new effective therapeutic strategies for NTG.

Biocompatible aggregation-induced emission active polyphosphate-manganese nanosheets with glutamine synthetase-like activity in excitotoxic nerve cells.

Glutamine synthetase (GS) is vital in maintaining ammonia and glutamate (Glu) homeostasis in living organisms. However, the natural enzyme relies on adenosine triphosphate (ATP) to activate Glu, resulting in impaired GS function during ATP-deficient neurotoxic events. To date, no reports demonstrate using artificial nanostructures to mimic GS function. In this study, we synthesize aggregation-induced emission active polyP-Mn nanosheets (STPE-PMNSs) based on end-labeled polyphosphate (polyP), exhibiting remarkable GS-like activity independent of ATP presence. Further investigation reveals polyP in STPE-PMNSs serves as phosphate source to activate Glu at low ATP levels. This self-feeding mechanism offers a significant advantage in regulating Glu homeostasis at reduced ATP levels in nerve cells during excitotoxic conditions. STPE-PMNSs can effectively promote the conversion of Glu to glutamine (Gln) in excitatory neurotoxic human neuroblastoma cells (SH-SY5Y) and alleviate Glu-induced neurotoxicity. Additionally, the fluorescence signal of nanosheets enables precise monitoring of the subcellular distribution of STPE-PMNSs. More importantly, the intracellular fluorescence signal is enhanced in a conversion-responsive manner, allowing real-time tracking of reaction progression. This study presents a self-sustaining strategy to address GS functional impairment caused by ATP deficiency in nerve cells during neurotoxic events. Furthermore, it offers a fresh perspective on the potential biological applications of polyP-based nanostructures.

KYNA Ameliorates Glutamate Toxicity of HAND by Enhancing Glutamate Uptake in A2 Astrocytes.

Reactive astrocytes are key players in HIV-associated neurocognitive disorders (HAND), and different types of reactive astrocytes play opposing roles in the neuropathologic progression of HAND. A recent study by our group found that gp120 mediates A1 astrocytes (neurotoxicity), which secrete proinflammatory factors and promote HAND disease progression. Here, by comparing the expression of A2 astrocyte (neuroprotective) markers in the brains of gp120 tgm mice and gp120+/α7nAChR-/- mice, we found that inhibition of alpha 7 nicotinic acetylcholine receptor (α7nAChR) promotes A2 astrocyte generation. Notably, kynurenine acid (KYNA) is an antagonist of α7nAChR, and is able to promote the formation of A2 astrocytes, the secretion of neurotrophic factors, and the enhancement of glutamate uptake through blocking the activation of α7nAChR/NF-κB signaling. In addition, learning, memory and mood disorders were significantly improved in gp120 tgm mice by intraperitoneal injection of kynurenine (KYN) and probenecid (PROB). Meanwhile, the number of A2 astrocytes in the mouse brain was significantly increased and glutamate toxicity was reduced. Taken together, KYNA was able to promote A2 astrocyte production and neurotrophic factor secretion, reduce glutamate toxicity, and ameliorate gp120-induced neuropathological deficits. These findings contribute to our understanding of the role that reactive astrocytes play in the development of HAND pathology and provide new evidence for the treatment of HAND via the tryptophan pathway.

γ-Oryzanol from Rice Bran Antagonizes Glutamate-Induced Excitotoxicity in an In Vitro Model of Differentiated HT-22 Cells.

The excessive activation of glutamate in the brain is a factor in the development of vascular dementia. γ-Oryzanol is a natural compound that has been shown to enhance brain function, but more research is needed to determine its potential as a treatment for vascular dementia. This study investigated if γ-oryzanol can delay or improve glutamate neurotoxicity in an in vitro model of differentiated HT-22 cells and explored its neuroprotective mechanisms. The differentiated HT-22 cells were treated with 0.1 mmol/L glutamate for 24 h then given γ-oryzanol at appropriate concentrations or memantine (10 µmol/L) for another 24 h. Glutamate produced reactive oxygen species and depleted glutathione in the cells, which reduced their viability. Mitochondrial dysfunction was also observed, including the inhibition of mitochondrial respiratory chain complex I activity, the collapse of mitochondrial transmembrane potential, and the reduction of intracellular ATP levels in the HT-22 cells. Calcium influx triggered by glutamate subsequently activated type II calcium/calmodulin-dependent protein kinase (CaMKII) in the HT-22 cells. The activation of CaMKII-ASK1-JNK MAP kinase cascade, decreased Bcl-2/Bax ratio, and increased Apaf-1-dependent caspase-9 activation were also observed due to glutamate induction, which were associated with increased DNA fragmentation. These events were attenuated when the cells were treated with γ-oryzanol (0.4 mmol/L) or the N-methyl-D-aspartate receptor antagonist memantine. The results suggest that γ-oryzanol has potent neuroprotective properties against glutamate excitotoxicity in differentiated HT-22 cells. Therefore, γ-oryzanol could be a promising candidate for the development of therapies for glutamate excitotoxicity-associated neurodegenerative diseases, including vascular dementia.

Hydrogen sulfide mitigates memory impairments via the restoration of glutamatergic neurons in a mouse model of hemorrhage shock and resuscitation.

Impaired long-term memory, a complication of traumatic stress including hemorrhage shock and resuscitation (HSR), has been reported to be associated with multiple neurodegenerations. The ventral tegmental area (VTA) participates in both learned appetitive and aversive behaviors. In addition to being prospective targets for the therapy of addiction, depression, and other stress-related diseases, VTA glutamatergic neurons are becoming more widely acknowledged as powerful regulators of reward and aversion. This study revealed that HSR exposure induces memory impairments and decreases the activation in glutamatergic neurons, and decreased ß power in the VTA. We also found that optogenetic activation of glutamatergic neurons in the VTA mitigated HSR-induced memory impairments, and restored ß power. Moreover, hydrogen sulfide (H2S), a gasotransmitter with pleiotropic roles, has neuroprotective functions at physiological concentrations. In vivo, H2S administration improved HSR-induced memory deficits, elevated c-fos-positive vesicular glutamate transporters (Vglut2) neurons, increased ß power, and restored the balance of γ-aminobutyric acid (GABA) and glutamate in the VTA. This work suggests that glutamatergic neuron stimulation via optogenetic assay and exogenous H2S may be useful therapeutic approaches for improving memory deficits following HSR.

Interleukin 3 Inhibits Glutamate-Cytotoxicity in Neuroblastoma Cell Line.

Interleukin 3 (IL-3) is a well-known pleiotropic cytokine that regulates the proliferation and differentiation of hematopoietic progenitor cells, triggering classical signaling pathways such as JAK/STAT, Ras/MAPK, and PI3K/Akt to carry out its functions. Interestingly, the IL-3 receptor is also expressed in non-hematopoietic cells, playing a crucial role in cell survival. Our previous research demonstrated the expression of the IL-3 receptor in neuron cells and its protective role in neurodegeneration. Glutamate, a principal neurotransmitter in the central nervous system, can induce cellular stress and lead to neurotoxicity when its extracellular concentrations surpass normal levels. This excessive glutamate presence is frequently observed in various neurological diseases. In this study, we uncover the protective role of IL-3 as an inhibitor of glutamate-induced cell death, analyzing the cytokine's signaling pathways during its protective effect. Specifically, we examined the relevance of JAK/STAT, Ras/MAPK, and PI3 K signaling pathways in the molecular mechanism triggered by IL-3. Our results show that the inhibition of JAK, ERK, and PI3 K signaling pathways, using pharmacological inhibitors, effectively blocked IL-3's protective role against glutamate-induced cell death. Additionally, our findings suggest that Bcl-2 and Bax proteins may be involved in the molecular mechanism triggered by IL-3. Our investigation into IL-3's ability to protect neuronal cells from glutamate-induced damage offers a promising therapeutic avenue with potential clinical implications for several neurological diseases characterized by glutamate neurotoxicity.

The Neuroprotective Effect of Sophocarpine against Glutamate-Induced HT22 Cell Cytotoxicity.

Neuronal cell death and dysfunction of the central nervous system can be caused by oxidative stress, which is associated with the development of neurodegenerative diseases. Sophocarpine, an alkaloid compound derived from Sophora moorcroftiana (Benth.) Baker seeds, has a wide range of medicinal value. This study sought to determine how sophocarpine exerts neuroprotective effects by inhibited oxidative stress and apoptosis in mouse hippocampus neuronal (HT22) cells. 20mM glutamate-induced HT22 cells were used to develop an in vitro model of oxidative stress damage. The Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability. According to the instructions on the kits to detect reactive oxygen species (ROS) levels and oxidative stress indicators. HT22 cells were examined using immunofluorescence and Western Blotting to detect Nuclear Factor Erythroid 2-related Factor 2 (Nrf2) expression. The expression of proteins and messenger RNA (mRNA) for heme oxygenase-1 (HO-1) was examined by Western Blotting and Quantitative real time polymerase chain reaction (qRT-PCR). Mitochondrial membrane potential (MMP) and Cell apoptosis were used by 5, 5', 6, 6'-Tetrachloro-1, 1', 3, 3'-tetraethyl-imidacarbocyanine iodide (JC- 1) kit and Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay kit, respectively. Finally, the expression of pro-apoptotic proteins was detected by Western Blotting. The result demonstrated that sophocarpine (1.25 µM-10 µM) can significantly inhibit glutamate-induced cytotoxicity and ROS generation, improve the activity of antioxidant enzymes. Sophocarpine increased the expression of HO-1 protein and mRNA and the nuclear translocation of Nrf2 to play a cytoprotective role; however, cells were transfected with small interfering RNA targeting HO-1 (si-HO-1) reversed the above effects of sophocarpine. In addition, sophocarpine significantly inhibited glutamate induced mitochondrial depolarization and further inhibited cell apoptosis by reducing the expression level of caspase-related proteins.

Urolithin B protects PC12 cells against glutamate-induced toxicity.

BACKGROUND: The involvement of malfunctioning glutamate systems in various central nervous system (CNS) disorders is widely acknowledged. Urolithin B, known for its neuroprotective and antioxidant properties, has shown potential as a therapeutic agent for these disorders. However, little is known about its protective effects against glutamate-induced toxicity in PC12 cells. Therefore, in this study, for the first time we aimed to investigate the ability of Urolithin B to reduce the cytotoxic effects of glutamate on PC12 cells. METHODS: Different non-toxic concentrations of urolithin B were applied to PC12 cells for 24 h before exposure to glutamate (10 mM). The cells were then analyzed for cell viability, intracellular reactive oxygen species (ROS), cell cycle arrest, apoptosis, and the expression of Bax and Bcl-2 genes. RESULTS: The results of MTT assay showed that glutamate at a concentration of 10 mM and urolithin B at a concentration of 114 µM can reduce PC12 cell viability by 50%. However, urolithin B at non-toxic concentrations of 4 and 8 µM significantly reduced glutamate-induced cytotoxicity (p < 0.01). Interestingly, treatment with glutamate significantly enhanced the intracellular ROS levels and apoptosis rate in PC12 cells, while pre-treatment with non-toxic concentrations of urolithin B significantly reduced these cytotoxic effects. The results also showed that pre-treatment with urolithin B can decrease the Bax (p < 0.05) and increase the Bcl-2 (p < 0.01) gene expression, which was dysregulated by glutamate. CONCLUSIONS: Taken together, urolithin B may play a protective role through reducing oxidative stress and apoptosis against glutamate-induced toxicity in PC12 cells, which merits further investigations.

The Neuroprotective Effects of BMSC-Derived Exosomes against Glutamate-Induced HT22 Cell Cytotoxicity.

Many central nervous system diseases are closely related to nerve damage caused by dysregulation of the endogenous neurotransmitter glutamate. Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) play an important role in improving injury and regeneration functions. However, its mechanism remains unknown. Therefore, the aim of this study is to investigate whether and how BMSC-Exos improve neurotoxicity caused by glutamate and to fill the gap in the literature. In this study, glutamate-treated HT22 cells were first exposed to mouse-derived BMSC-Exos at different concentrations to observe their effects on HT22 apoptosis. Next, we treated glutamate-treated HT22 cells with mouse-derived BMSC-Exos. We then inhibited the PI3K/Akt/mTOR signaling pathways using the PI3K/Akt inhibitor and the mTOR inhibitor, respectively, and observed the protective effect of mouse-derived BMSC-Exos on HT22 cells treated with glutamate. Our results show that BMSC-Exos reduced apoptosis triggered by glutamate stimulation, increased cell vitality, and decreased the levels of proapoptotic proteins while increasing the levels of anti-apoptotic proteins. The protective effect of BMSC-Exos was weakened when PI3K/Akt inhibitor and mTOR inhibitor were added. To sum up, we draw the following conclusions: BMSC-Exos can reduce neuronal apoptosis and apoptosis-related protein expression after glutamate stimulation by regulating the PI3K/Akt/mTOR signaling pathway.

Neuroprotective effects of cordycepin inhibit glutamate-induced apoptosis in hippocampal neurons.

Glutamate is a neurotransmitter that can cause excitatory neurotoxicity when its extracellular concentration is too high, leading to disrupted calcium balance and increased production of reactive oxygen species (ROS). Cordycepin, a nucleoside adenosine derivative, has been shown to protect against excitatory neurotoxicity induced by glutamate. To investigate its potential neuroprotective effects, the present study employed fluorescence detection and spectrophotometry techniques to analyze primary hippocampal-cultured neurons. The results showed that glutamate toxicity reduced hippocampal neuron viability, increased ROS production, and increased intracellular calcium levels. Additionally, glutamate-induced cytotoxicity activated acetylcholinesterase and decreased glutathione levels. However, cordycepin inhibited glutamate-induced cell death, improved cell viability, reduced ROS production, and lowered Ca2+ levels. It also inhibited acetylcholinesterase activation and increased glutathione levels. This study suggests that cordycepin can protect against glutamate-induced neuronal injury in cell models, and this effect was inhibited by adenosine A1 receptor blockers, indicating that its neuroprotective effect is achieved through activation of the adenosine A1 receptor.

Trolox aids coenzyme Q10 in neuroprotection against NMDA induced damage via upregulation of VEGF in rat model of glutamate excitotoxicity.

Glutamate induced damage to retinal ganglion cells (RGCs) requires tight physiological regulation of the N-methyl-D-aspartate (NMDA) receptors. Previously, studies have demonstrated the neuroprotective abilities of antioxidants like coenzyme Q10 (CoQ10) and vitamin E analogs like α-tocopherol against neuropathies resulting from NMDA insult, but have failed to shed light on the effect of CoQ10 and trolox, a hydrophilic analog of vitamin E, on glaucomatous neurodegeneration. In the current study, we wanted to investigate whether the combined effect of trolox with CoQ10 could alleviate NMDA-induced death of retinal cells while also trying to elucidate the underlying mechanism in relation to the yet unexplained role of vascular endothelial growth factor (VEGF) in NMDA-mediated excitotoxicity. After successful NMDA-induced degeneration, we followed it up with the treatment of combination of Trolox and CoQ10. The structural damage by NMDA was repaired significantly and retina retained structural integrity comparable to levels of control in the treatment group of Trolox and CoQ10. Detection of ROS generation after NMDA insult showed that together, Trolox and CoQ10 could significantly bring down the high levels of free radicals while also rescuing mitochondrial membrane potential (MMP). A significant increase in NMDA receptor Grin2A by CoQ10 alone as well as by CoQ10 and trolox was accompanied by a lowered Grin2B receptor expression, suggesting neuroprotective action of Trolox and CoQ10. Subsequently, lowered VEGFR1 and VEGFR2 receptor expression by NMDA treatment also recovered when subjected to combined treatment of Trolox and CoQ10. Western blot analyses also indicated the same whereby Trolox and CoQ10 could increase the diminished levels of phosphorylated VEGFR2. Immunofluorescence studies also indicated a positive correlation between recovered VEGFR2 and NMDAR2A levels and diminished levels of NMDAR2D, confirming the results obtained by RT-PCR analysis. This is the first report in our knowledge that demonstrates the efficacy of trolox in combination with CoQ10 highlighting the importance of maintaining VEGF levels that are lowered in ocular diseases due to NMDA-related toxicities.

Changes in excitatory amino acid transporters in response to remote ischaemic preconditioning and glutamate excitotoxicity.

The successful implementation of remote ischaemic conditioning as a clinical neuroprotective strategy requires a thorough understanding of its basic principles, which can be modified for each patient. The mechanisms of glutamate homeostasis appear to be a key component. In the current study, we focused on the brain-to-blood glutamate shift mediated by glutamate transporters (excitatory amino acid transports [EAATs]) and the effect of remote ischaemic preconditioning (RIPC) as a mediator of ischaemic tolerance. We used model mimicking ischaemia-mediated excitotoxicity (intracerebroventricular administration of glutamate) to avoid the indirect effect of ischaemia-triggered mechanisms. We found quantitative changes in EAAT2 and EAAT3 and altered membrane trafficking of EAAT1 on the cells of the choroid plexus. These changes could underlie the beneficial effects of ischaemic tolerance. There was reduced oxidative stress and increased glutathione level after RIPC treatment. Moreover, we determined the stimulus-specific response on EAATs. While glutamate overdose stimulated EAAT2 and EAAT3 overexpression, RIPC induced membrane trafficking of EAAT1 and EAAT2 rather than a change in their expression. Taken together, mechanisms related to glutamate homeostasis, especially EAAT-mediated transport, represents a powerful tool of ischaemic tolerance and allow a certain amount of flexibility based on the stimulus used.

Design, synthesis, and characterization of novel system xC- transport inhibitors: inhibition of microglial glutamate release and neurotoxicity.

Neuroinflammation appears to involve some degree of excitotoxicity promulgated by microglia, which release glutamate via the system xC- (SxC-) cystine-glutamate antiporter. With the aim of mitigating this source of neuronal stress and toxicity, we have developed a panel of inhibitors of the SxC- antiporter. The compounds were based on L-tyrosine, as elements of its structure align with those of glutamate, a primary physiological substrate of the SxC- antiporter. In addition to 3,5-dibromotyrosine, ten compounds were synthesized via amidation of that parent molecule with a selection of acyl halides. These agents were tested for the ability to inhibit release of glutamate from microglia activated with lipopolysaccharide (LPS), an activity exhibited by eight of the compounds. To confirm that the compounds were inhibitors of SxC-, two of them were further tested for the ability to inhibit cystine uptake. Finally, these agents were shown to protect primary cortical neurons from the toxicity exhibited by activated microglia. These agents may hold promise in reducing the neurodegenerative effects of neuroinflammation in conditions, such as encephalitis, traumatic brain injury, stroke, or neurodegenerative diseases.

Comparative Study of the Protective and Neurotrophic Effects of Neuronal and Glial Progenitor Cells-Derived Conditioned Media in a Model of Glutamate Toxicity In Vitro.

Cell therapy represents a promising approach to the treatment of neurological diseases, offering potential benefits not only by cell replacement but also through paracrine secretory activities. However, this approach includes a number of limiting factors, primarily related to safety. The use of conditioned stem cell media can serve as an equivalent to cell therapy while avoiding its disadvantages. The present study was a comparative investigation of the antioxidant, neuroprotective and neurotrophic effects of conditioned media obtained from neuronal and glial progenitor cells (NPC-CM and GPC-CM) on the PC12 cell line in vitro. Neuronal and glial progenitor cells were obtained from iPSCs by directed differentiation using small molecules. GPC-CM reduced apoptosis, ROS levels and increased viability, expressions of the antioxidant response genes HMOX1 and NFE2L2 in a model of glutamate-induced oxidative stress. The neurotrophic effect was evidenced by a change in the morphology of pheochromocytoma cells to a neuron-like phenotype. Moreover, neurite outgrowth, expression of GAP43, TUBB3, MAP2, SYN1 genes and increased levels of the corresponding MAP2 and TUBB3 proteins. Treatment with NPC-CM showed moderate antiapoptotic effects and improved cell viability. This study demonstrated the potential application of CM in the field of regenerative medicine.

L-glutamic acid-g-poly hydroxyethyl methacrylate nanoparticles: acute and sub-acute toxicity and biodistribution potential in mice.

The aim of this safety study in mice was to determine in vivo toxicity and biodistribution potential of a single and multiple doses of L-glutamic acid-g-p(HEMA) polymeric nanoparticles as a drug delivery system. The single dose did not cause any lethal effect, and its acute oral LD50 was >2.000 mg/kg body weight (bw). Multiple doses (25, 50, or 100 mg/kg bw) given over 28 days resulted in no significant differences in body and relative organ weights compared to control. These results are supported by biochemical and histological findings. Moreover, nanoparticle exposure did not result in statistically significant differences in micronucleus counts in bone marrow cells compared to control. Nanoparticle distribution was time-dependent, and they reached the organs and even bone marrow by hour 6, as established by ex vivo imaging with the IVIS® spectrum imaging system. In conclusion, L-glutamic acid-g-p(HEMA) polymeric nanoparticles appear biocompatible and have a potential use as a drug delivery system.

BANF1 promotes glutamate-induced apoptosis of HT-22 hippocampal neurons.

BACKGROUND: Glutamate exposure was fatal to HT-22 neuronal cells that derived from mouse hippocampus. This is often used as a model for hippocampus neurodegeneration in vitro. The targets relevant to glutamate-induced neuronal toxicity is not fully understood. In this study, we aimed to identify crucial factors associated with glutamate-induced cytotoxicity in HT-22 cells. METHODS: HT-22 cells were treated with 7.5 mM glutamate for 24 h and isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis conducted to identify the differentially expressed proteins. Differential proteins were subjected to Gene Ontology analyses. Upregulation of barrier to autointegration factor (BANF1/BANF1) protein was confirmed by RT-qPCR and western blotting. Cell viability was measured by CKK-8 and MTT assays. Cell apoptosis rates and intracellular reactive oxygen species (ROS) levels were detected using flow cytometry. RESULTS: A total of 5811 proteins were quantified by iTRAQ, 50 of which were recognized as significantly differential proteins (fold change ≥ 1.5 and P ≤ 0.05); 26 proteins were up-regulated and 24 were down-regulated after exposure to glutamate. GO enrichment analysis showed that the apoptotic signaling pathway was involved in cell death induced by glutamate. BANF1 expression level was markedly increased in HT-22 cells after glutamate treatment. Further, knockdown of BANF1 alleviated glutamate-mediated cell death with lower ROS levels. CONCLUSIONS: In conclusion, we successfully filtered out differential proteins relevant to glutamate-mediated cytotoxicity. BANF1 upregulation promoted glutamate-induced apoptosis of HT-22 cells by enhancing ROS generation.

Moschus ameliorates glutamate-induced cellular damage by regulating autophagy and apoptosis pathway.

Alzheimer's disease (AD), a neurodegenerative disorder, causes short-term memory and cognition declines. It is estimated that one in three elderly people die from AD or other dementias. Chinese herbal medicine as a potential drug for treating AD has gained growing interest from many researchers. Moschus, a rare and valuable traditional Chinese animal medicine, was originally documented in Shennong Ben Cao Jing and recognized for its properties of reviving consciousness/resuscitation. Additionally, Moschus has the efficacy of "regulation of menstruation with blood activation, relief of swelling and pain" and is used for treating unconsciousness, stroke, coma, and cerebrovascular diseases. However, it is uncertain whether Moschus has any protective effect on AD patients. We explored whether Moschus could protect glutamate (Glu)-induced PC12 cells from cellular injury and preliminarily explored their related action mechanisms. The chemical compounds of Moschus were analyzed and identified by GC-MS. The Glu-induced differentiated PC12 cell model was thought to be the common AD cellular model. The study aims to preliminarily investigate the intervention effect of Moschus on Glu-induced PC12 cell damage as well as their related action mechanisms. Cell viability, lactate dehydrogenase (LDH), mitochondrial reactive oxygen species, mitochondrial membrane potential (MMP), cell apoptosis, autophagic vacuoles, autolysosomes or autophagosomes, proteins related to apoptosis, and the proteins related to autophagy were examined and analyzed. Seventeen active compounds of the Moschus sample were identified based on GC-MS analysis. In comparison to the control group, Glu stimulation increased cell viability loss, LDH release, mitochondrial damage, loss of MMP, apoptosis rate, and the number of cells containing autophagic vacuoles, and autolysosomes or autophagosomes, while these results were decreased after the pretreatment with Moschus and 3-methyladenine (3-MA). Furthermore, Glu stimulation significantly increased cleaved caspase-3, Beclin1, and LC3II protein expression, and reduced B-cell lymphoma 2/BAX ratio and p62 protein expression, but these results were reversed after pretreatment of Moschus and 3-MA. Moschus has protective activity in Glu-induced PC12 cell injury, and the potential mechanism might involve the regulation of autophagy and apoptosis. Our study may promote research on Moschus in the field of neurodegenerative diseases, and Moschus may be considered as a potential therapeutic agent for AD.

Neuroprotective Potential of L-Glutamate Transporters in Human Induced Pluripotent Stem Cell-Derived Neural Cells against Excitotoxicity.

Human induced pluripotent stem cell (hiPSC)-derived neural cells have started to be used in safety/toxicity tests at the preclinical stage of drug development. As previously reported, hiPSC-derived neurons exhibit greater tolerance to excitotoxicity than those of primary cultures of rodent neurons; however, the underlying mechanisms remain unknown. We here investigated the functions of L-glutamate (L-Glu) transporters, the most important machinery to maintain low extracellular L-Glu concentrations, in hiPSC-derived neural cells. We also clarified the contribution of respective L-Glu transporter subtypes. At 63 days in vitro (DIV), we detected neuronal circuit functions in hiPSC-derived neural cells by a microelectrode array system (MEA). At 63 DIV, exposure to 100 µM L-Glu for 24 h did not affect the viability of neural cells. 100 µM L-Glu in the medium decreased to almost 0 µM in 60 min. Pharmacological inhibition of excitatory amino acid transporter 1 (EAAT1) and EAAT2 suppressed almost 100% of L-Glu decrease. In the presence of this inhibitor, 100 µM L-Glu dramatically decreased cell viability. These results suggest that in hiPSC-derived neural cells, EAAT1 and EAAT2 are the predominant L-Glu transporters, and their uptake potentials are the reasons for the tolerance of hiPSC-derived neurons to excitotoxicity.

Protective Effects of 3'-Epilutein and 3'-Oxolutein against Glutamate-Induced Neuronal Damage.

Dietary lutein can be naturally metabolized to 3'-epilutein and 3'-oxolutein in the human body. The epimerization of lutein can happen in acidic pH, and through cooking, 3'-epilutein can be the product of the direct oxidation of lutein in the retina, which is also present in human serum. The 3'-oxolutein is the main oxidation product of lutein. Thus, the allylic oxidation of dietary lutein can result in the formation of 3'-oxolutein, which may undergo reduction either to revert to dietary lutein or epimerize to form 3'-epilutein. We focused on the effects of 3'-epilutein and 3'-oxolutein itself and on glutamate-induced neurotoxicity on SH-SY5Y human neuroblastoma cells to identify the possible alterations in oxidative stress, inflammation, antioxidant capacity, and iron metabolism that affect neurological function. ROS measurements were performed in the differently treated cells. The inflammatory state of cells was followed by TNFα, IL-6, and IL-8 cytokine ELISA measurements. The antioxidant status of the cells was determined by the total antioxidant capacity kit assay. The alterations of genes related to ferroptosis and lipid peroxidation were followed by gene expression measurements; then, thiol measurements were performed. Lutein metabolites 3'-epilutein and 3'-oxolutein differently modulated the effect of glutamate on ROS, inflammation, ferroptosis-related iron metabolism, and lipid peroxidation in SH-SY5Y cells. Our results revealed the antioxidant and anti-inflammatory features of 3'-epilutein and 3'-oxolutein as possible protective agents against glutamate-induced oxidative stress in SH-SY5Y cells, with greater efficacy in the case of 3'-epilutein.

Novel Probucol Analogue, 4,4'-Diselanediylbis (2,6-Di-tert-Butylphenol), Prevents Oxidative Glutamate Neurotoxicity In Vitro and Confers Neuroprotection in a Rodent Model of Ischemic Stroke.

Oxidative glutamate toxicity is regarded as one of the injurious mechanisms associated with ischemic stroke, which represents a major health problem and requires improved pharmacological treatments. We designed and synthesized two new probucol analogues [2,6-di-tert-butyl-4-selenocyanatophenol (C1) and 4,4'-diselanediylbis (2,6-di-tert-butylphenol) (C2)] and investigated their effects against glutamate-induced neuronal oxidative toxicity in vitro in cultured HT22 cells, compared with their parental compound (probucol). In addition, C2, which exhibited the lowest toxicity, was investigated in an in vivo rodent model of ischemic stroke. Glutamate caused concentration- and time-dependent cytotoxicity in HT22 neuronal cells, which was preceded by increased levels of oxidants and depletion of the antioxidant glutathione. The analogues (C1 and C2), but not probucol, significantly decreased the levels of oxidants (including mitochondrial superoxide anion and lipid reactive oxygen species (ROS)) and protected against glutamate-induced cytotoxicity. In the in vivo model of ischemic stroke, which was based on central injections of the vasoconstrictor agent endothelin-1 (800 pmol/site), C2 (20 or 50 mg/kg/day, intraperitoneally, for 4 consecutive days after stroke) displayed significant beneficial effects against ischemic injury in vivo, improving rats' motor-related behavioral skills and decreasing stroke-related striatal gliosis. This is the first study to design, synthesize, and present a probucol analogue (C2) with in vivo beneficial effects against ischemic stroke. This novel compound, which was able to mitigate glutamate-induced oxidative toxicity in vitro, represents a promising neuroprotective drug.