L-theanine alleviates myocardial ischemia/reperfusion injury by suppressing oxidative stress and apoptosis through activation of the JAK2/STAT3 pathway in mice.

BACKGROUND: L-theanine is a unique non-protein amino acid in tea that is widely used as a safe food additive. We investigated the cardioprotective effects and mechanisms of L-theanine in myocardial ischemia-reperfusion injury (MIRI). METHODS: The cardioprotective effects and mechanisms of L-theanine and the role of Janus Kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling were investigated in MIRI mice using measures of cardiac function, oxidative stress, and apoptosis. RESULTS: Administration of L-theanine (10 mg/kg, once daily) suppressed the MIRI-induced increase in infarct size and serum creatine kinase and lactate dehydrogenase levels, as well as MIRI-induced cardiac apoptosis, as evidenced by an increase in Bcl-2 expression and a decrease in Bax/caspase-3 expression. Administration of L-theanine also decreased the levels of parameters reflecting oxidative stress, such as dihydroethidium, malondialdehyde, and nitric oxide, and increased the levels of parameters reflecting anti-oxidation, such as total antioxidant capacity (T-AOC), glutathione (GSH), and superoxide dismutase (SOD) in ischemic heart tissue. Further analysis showed that L-theanine administration suppressed the MIRI-induced decrease of phospho-JAK2 and phospho-STAT3 in ischemic heart tissue. Inhibition of JAK2 by AG490 (5 mg/kg, once daily) abolished the cardioprotective effect of L-theanine, suggesting that the JAK2/STAT3 signaling pathway may play an essential role in mediating the anti-I/R effect of L-theanine. CONCLUSIONS: L-theanine administration suppresses cellular apoptosis and oxidative stress in part via the JAK2/STAT3 signaling pathway, thereby attenuating MIRI-induced cardiac injury. L-theanine could be developed as a potential drug to alleviate cardiac damage in MIRI.

Experimentally evolved Staphylococcus aureus shows increased survival in the presence of Pseudomonas aeruginosa by acquiring mutations in the amino acid transporter, GltT.

When cultured together under standard laboratory conditions Pseudomonas aeruginosa has been shown to be an effective inhibitor of Staphylococcus aureus. However, P. aeruginosa and S. aureus are commonly observed in coinfections of individuals with cystic fibrosis (CF) and in chronic wounds. Previous work from our group revealed that S. aureus isolates from CF infections are able to persist in the presence of P. aeruginosa strain PAO1 with a range of tolerances with some isolates being eliminated entirely and others maintaining large populations. In this study, we designed a serial transfer, evolution experiment to identify mutations that allow S. aureus to survive in the presence of P. aeruginosa. Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa PAO1. After eight coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT, that were unique to evolved P. aeruginosa-tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompeted wild-type S. aureus when glutamate was absent from chemically defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two are cultured together.

L-theanine abates oxidative stress and mitochondrial dysfunction in myocardial ischemia-reperfusion injury by positively regulating the antioxidant response.

L-theanine (L-THE), a non-protein amino acid isolated from Camelia sinensis, has antioxidant properties that could prevent oxidative damage and mitochondrial dysfunction generated by myocardial ischemia and reperfusion (I/R) injury. The present study aimed to identify the effects of pretreatment with L-THE in rat hearts undergoing I/R. Wistar rats received vehicle or 250 mg/Kg L-THE intragastrically for 10 days. On day 11, hearts were removed under anesthesia and exposed to I/R injury in the Langendorff system. Measurement of left ventricular developed pressure and heart rate ex vivo demonstrates that L-THE prevents I/R-induced loss of cardiac function. Consequently, the infarct size of hearts subjected to I/R was significantly decreased when L-THE was administered. L-THE also mitigated I/R-induced oxidative injury in cardiac tissue by decreasing reactive oxygen species and malondialdehyde levels, while increasing the activity of antioxidant enzymes, SOD and CAT. Additionally, L-THE prevents oxidative phosphorylation breakdown and loss of inner mitochondrial membrane potential caused by I/R, restoring oxygen consumption levels, increasing respiratory control and phosphorylation efficiency, as well as buffering calcium overload. Finally, L-THE modifies the expression of genes involved in the antioxidant response through the overexpression of SOD1, SOD2 and CAT; as well as the transcriptional factors PPARα and Nrf2 in hearts undergoing I/R. In conclusion, L-THE confers cardioprotection against I/R injury by preventing oxidative stress, protecting mitochondrial function, and promoting overexpression of antioxidant genes. More studies are needed to place L-THE at the forefront of cardiovascular research and recommend its therapeutic use.

A Pharmacological Evaluation of the Analgesic Effect and Hippocampal Protein Modulation of the Ketamine Metabolite (2R,6R)-Hydroxynorketamine in Murine Pain Models.

BACKGROUND: The ketamine metabolite (2R,6R)-hydroxynorketamine ([2R,6R]-HNK) has analgesic efficacy in murine models of acute, neuropathic, and chronic pain. The purpose of this study was to evaluate the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) dependence of (2R,6R)-HNK analgesia and protein changes in the hippocampus in murine pain models administered (2R,6R)-HNK or saline. METHODS: All mice were CD-1 IGS outbred mice. Male and female mice underwent plantar incision (PI) (n = 60), spared nerve injury (SNI) (n = 64), or tibial fracture (TF) (n = 40) surgery on the left hind limb. Mechanical allodynia was assessed using calibrated von Frey filaments. Mice were randomized to receive saline, naloxone, or the brain-penetrating AMPA blocker (1,2,3,4-Tetrahydro-6-nitro-2,3-dioxobenzo [f]quinoxaline-7-sulfonamide [NBQX]) before (2R,6R)-HNK 10 mg/kg, and this was repeated for 3 consecutive days. The area under the paw withdrawal threshold by time curve for days 0 to 3 (AUC 0-3d ) was calculated using trapezoidal integration. The AUC 0-3d was converted to percent antiallodynic effect using the baseline and pretreatment values as 0% and 100%. In separate experiments, a single dose of (2R,6R)-HNK 10 mg/kg or saline was administered to naive mice (n = 20) and 2 doses to PI (n = 40), SNI injury (n = 40), or TF (n = 40) mice. Naive mice were tested for ambulation, rearing, and motor strength. Immunoblot studies of the right hippocampal tissue were performed to evaluate the ratios of glutamate ionotropic receptor (AMPA) type subunit 1 (GluA1), glutamate ionotropic receptor (AMPA) type subunit 2 (GluA2), phosphorylated voltage-gated potassium channel 2.1 (p-Kv2.1), phosphorylated-calcium/calmodulin-dependent protein kinase II (p-CaMKII), brain-derived neurotrophic factor (BDNF), phosphorylated protein kinase B (p-AKT), phosphorylated extracellular signal-regulated kinase (p-ERK), CXC chemokine receptor 4 (CXCR4), phosphorylated eukaryotic translation initiation factor 2 subunit 1 (p-EIF2SI), and phosphorylated eukaryotic translation initiation factor 4E (p-EIF4E) to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RESULTS: No model-specific gender difference in antiallodynic responses before (2R,6R)-HNK administration was observed. The antiallodynic AUC 0-3d of (2R,6R)-HNK was decreased by NBQX but not with pretreatment with naloxone or saline. The adjusted mean (95% confidence interval [CI]) antiallodynic effect of (2R,6R)-HNK in the PI, SNI, and TF models was 40.7% (34.1%-47.3%), 55.1% (48.7%-61.5%), and 54.7% (46.5%-63.0%), greater in the SNI, difference 14.3% (95% CI, 3.1-25.6; P = .007) and TF, difference 13.9% (95% CI, 1.9-26.0; P = .019) compared to the PI model. No effect of (2R,6R)-HNK on ambulation, rearing, or motor coordination was observed. Administration of (2R,6R)-HNK was associated with increased GluA1, GluA2, p-Kv2.1, and p-CaMKII and decreased BDNF ratios in the hippocampus, with model-specific variations in proteins involved in other pain pathways. CONCLUSIONS: (2R,6R)-HNK analgesia is AMPA-dependent, and (2R,6R)-HNK affected glutamate, potassium, calcium, and BDNF pathways in the hippocampus. At 10 mg/kg, (2R,6R)-HNK demonstrated a greater antiallodynic effect in models of chronic compared with acute pain. Protein analysis in the hippocampus suggests that AMPA-dependent alterations in BDNF-TrkB and Kv2.1 pathways may be involved in the antiallodynic effect of (2R,6R)-HNK.

The Response of Growth and Transcriptome Profiles of Tea Grey Blight Disease Pathogen Pestalotiopsis theae to the Variation of Exogenous L-Theanine.

Tea grey blight disease is one of the most destructive diseases that infects tea and is caused by the pathogen Pestalotiopsis theae (Sawada) Steyaert. L-theanine is a unique non-protein amino acid of the tea plant. Different concentrations of L-theanine exhibit significant inhibitory effects on the growth and sporulation ability of the pathogen causing tea grey blight disease. To understand the effect mechanism of L-theanine on P. theae, transcriptome profiling was performed on the pathogenic mycelium treated with three different concentrations of L-theanine: no L-theanine treatment (TH0), 20 mg/mL theanine treatment (TH2), and 40 mg/mL theanine treatment (TH4). The colony growths were significantly lower in the treatment with L-theanine than those without L-theanine. The strain cultured with a high concentration of L-theanine produced no spores or only a few spores. In total, 2344, 3263, and 1158 differentially expressed genes (DEGs) were detected by RNA-sequencing in the three comparisons, Th2 vs. Th0, Th4 vs. Th0, and Th4 vs. Th2, respectively. All DEGs were categorized into 24 distinct clusters. According to GO analysis, low concentrations of L-theanine primarily affected molecular functions, while high concentrations of L-theanine predominantly affected biological processes including external encapsulating structure organization, cell wall organization or biogenesis, and cellular amino acid metabolic process. Based on KEGG, the DEGs of Th2 vs. Th0 were primarily involved in pentose and glucuronate interconversions, histidine metabolism, and tryptophan metabolism. The DEGs of Th4 vs. Th0 were mainly involved in starch and sucrose metabolism, amino sugar, and nucleotide sugar metabolism. This study indicated that L-theanine has a significant impact on the growth and sporulation of the pathogen of tea grey blight disease and mainly affects amino acid metabolism, carbohydrate metabolism, and cellular structure-related biosynthesis processes of pathogenic fungi. This work provides insights into the direct control effect of L-theanine on pathogenic growth and also reveals the molecular mechanisms of inhibition of L-theanine to P. theae.

L-Theanine Prolongs the Lifespan by Activating Multiple Molecular Pathways in Ultraviolet C-Exposed Caenorhabditis elegans.

L-theanine, a unique non-protein amino acid, is an important bioactive component of green tea. Previous studies have shown that L-theanine has many potent health benefits, such as anti-anxiety effects, regulation of the immune response, relaxing neural tension, and reducing oxidative damage. However, little is known concerning whether L-theanine can improve the clearance of mitochondrial DNA (mtDNA) damage in organisms. Here, we reported that L-theanine treatment increased ATP production and improved mitochondrial morphology to extend the lifespan of UVC-exposed nematodes. Mechanistic investigations showed that L-theanine treatment enhanced the removal of mtDNA damage and extended lifespan by activating autophagy, mitophagy, mitochondrial dynamics, and mitochondrial unfolded protein response (UPRmt) in UVC-exposed nematodes. In addition, L-theanine treatment also upregulated the expression of genes related to mitochondrial energy metabolism in UVC-exposed nematodes. Our study provides a theoretical basis for the possibility that tea drinking may prevent mitochondrial-related diseases.

Δ3-tubulin impairs mitotic spindle morphology and increases nuclear size in pancreatic cancer cells.

Cancer cell proliferation is affected by post-translational modifications of tubulin. Especially, overexpression or depletion of enzymes for modifications on the tubulin C-terminal region perturbs dynamic instability of the spindle body. Those modifications include processing of C-terminal amino acids of α-tubulin; detyrosination, and a removal of penultimate glutamic acid (Δ2). We previously found a further removal of the third last glutamic acid, which generates so-called Δ3-tubulin. The effects of Δ3-tubulin on spindle integrities and cell proliferation remain to be elucidated. In this study, we investigated the impacts of forced expression of Δ3-tubulin on the structure of spindle bodies and cell division in a pancreatic cancer cell line, PANC-1. Overexpression of HA-tagged Δ3-tubulin impaired the morphology and orientation of spindle bodies during cell division in PANC-1 cells. In particular, spindle bending was most significantly increased. Expression of EGFP-tagged Δ3-tubulin driven by the endogenous promoter of human TUBA1B also deformed and misoriented spindle bodies. Spindle bending and condensation defects were significantly observed by EGFP-Δ3-tubulin expression. Furthermore, EGFP-Δ3-tubulin expression increased the nuclear size in a dose-dependent manner of EGFP-Δ3-tubulin expression. The expression of EGFP-Δ3-tubulin tended to slow down cell proliferation. Taken together, our results demonstrate that Δ3-tubulin affects the spindle integrity and cell division.

Caution may be required in using l-theanine in diabetes mellitus: A study on the rats.

BACKGROUND: The study aimed to identify the effects of l-theanine on kidney and heart tissues in diabetic rats. 24 male rats included in the study were divided into 4 groups (n = 6/group): SHAM, LTEA, DM and DM + LTEA. For 28 days, drinking water was given to SHAM and DM, and LTEA (200 mg/kg/day) to LTEA and DM + LTEA groups, intragastrically. DM was induced by 120 mg/kg nicotinamide (NA) + 60 mg/kg streptozotocin (STZ). The levels of cystatin C (CysC) and angiotensin-converting enzyme 2 (ACE2) were determined by ELISA kits, homocysteine, electrolytes and iron by an autoanalyzer, the ratio of oxidized/total reduced glutathione (GSSG/TGSH) by assay kits. The tissues were histopathologically analyzed. RESULTS: LTEA alleviated histopathological degenerations. However, it decreased significantly serum iron and homocysteine levels (p < 0.05). CONCLUSION: LTEA did not exhibit significant protective effects on kidney and heart tissues; it may have affected the homocysteine and iron metabolisms in diabetics.

Synergistic photoactivation of VTA-catecholaminergic and BLA-glutamatergic projections induces long-term potentiation in the insular cortex.

The presentation of novel stimuli induces a reliable dopamine release in the insular cortex (IC) from the ventral tegmental area (VTA). The novel stimuli could be associated with motivational and emotional signals induced by cortical glutamate release from the basolateral amygdala (BLA). Dopamine and glutamate are essential for acquiring and maintaining behavioral tasks, including visual and taste recognition memories. In this study, we hypothesize that the simultaneous activation of dopaminergic and glutamatergic projections to the neocortex can underlie synaptic plasticity. High-frequency stimulation of the BLA-IC circuit has demonstrated a reliable long-term potentiation (LTP), a widely acknowledged synaptic plasticity that underlies memory consolidation. Therefore, the concurrent optogenetic stimulation of the insula's glutamatergic and dopaminergic terminal fibers would induce reliable LTP. Our results confirmed that combined photostimulation of the VTA and BLA projections to the IC induces a slow-onset LTP. We also found that optogenetically-induced LTP in the IC relies on both glutamatergic NMDA receptors and dopaminergic D1/D5 receptors, suggesting that the combined effects of these neurotransmitters can trigger synaptic plasticity in the neocortex. Overall, our findings provide compelling evidence supporting the essential role of both dopaminergic and glutamatergic projections in modulating synaptic plasticity within the IC. Furthermore, our results suggest that the synergistic actions of these projections have a pivotal influence on the formation of motivational memories.

Inhibition of astroglial hemichannels ameliorates infrasonic noise induced short-term learning and memory impairment.

As a kind of environmental noise, infrasonic noise has negative effects on various human organs. To date, research has shown that infrasound impairs cognitive function, especially the ability for learning and memory. Previously, we demonstrated that impaired learning and memory induced by infrasound was closely related with glia activation; however, the underlying mechanisms remain unclear. Connexin 43 hemichannels (Cx43 HCs), which are mainly expressed in hippocampal astrocytes, are activated under pathological conditions, lending support to the hypothesis that Cx43 HCs might function in the impaired learning and memory induced by infrasound. This study revealed that that blocking hippocampal Cx43 HCs or downregulating hippocampal Cx43 expression significantly alleviated impaired learning and memory induced by infrasound. We also observed that infrasound exposure led to the abundant release of glutamate and ATP through Cx43 HCs. In addition, the abundant release of glutamate and ATP depended on proinflammatory cytokines. Our finds suggested that the enhanced release of ATP and glutamate by astroglial Cx43 HCs may be involved in the learning and memory deficits caused by infrasound exposure.

Dose-response effect of L-theanine on psychomotor speed, sustained attention, and inhibitory control: a double-blind, placebo-controlled, crossover study.

BACKGROUND: L-theanine is a non-protein-forming amino acid found in tea. Previous research shows high doses (100-400 mg) of L-theanine enhances attention, mainly by reducing mind wandering and distracter processing. We hypothesized that these indirect mechanisms could significantly improve the performance of low-level attentional tasks, whereas the relative contribution could be less in complex attentional tasks that require active, higher-order processing of target stimuli. METHODS: To test this hypothesis, we conducted a double-blind, placebo-controlled, counterbalanced, four-way crossover study in 32 healthy young adults, where we compared the effects of three doses of L-theanine (100, 200 and 400 mg) with a placebo (distilled water), administered before and 50 min after dosing, on three attentional tasks from the Cambridge Neuropsychological Test Automated Battery [viz. Reaction Time (RTI)-visuomotor speed, Rapid Visual Information Processing (RVP)-sustained attention, and Stop Signal Task (SST)-inhibitory control]. Results were analyzed in dose × time repeated measures ANOVA models, with subsequent pairwise comparisons. RESULTS: Active doses significantly improved reaction times in the RTI (100-200 mg) and RVP (200-400 mg) tasks from baseline (p < 0.05), but once controlled for the change-from-baseline caused by placebo, only the RTI simple reaction times showed significant improvements, following 100 mg (Δ = 16.3 ms, p = 0.009) and 200 mg (Δ = 16.9 ms, p = 0.009) of L-theanine. CONCLUSIONS: Consistent with our hypothesis, these findings suggest that L-theanine significantly improves attention in simple visuomotor tasks, but not in more complex sustained attention tasks, or executive control tasks that require top-down inhibition of pre-active responses.

Comparative Investigation of Expression of Glutamatergic and GABAergic Genes in the Rat Hippocampus after Focal Brain Ischemia and Central LPS Administration.

Among the responses in the early stages of stroke, activation of neurodegenerative and proinflammatory processes in the hippocampus is of key importance for the development of negative post-ischemic functional consequences. However, it remains unclear, what genes are involved in these processes. The aim of this work was a comparative study of the expression of genes encoding glutamate and GABA transporters and receptors, as well as inflammation markers in the hippocampus one day after two types of middle cerebral artery occlusion (according to Koizumi et al. method, MCAO-MK, and Longa et al. method, MCAO-ML), and direct pro-inflammatory activation by central administration of bacterial lipopolysaccharide (LPS). Differences and similarities in the effects of these challenges on gene expression were observed. Expression of a larger number of genes associated with activation of apoptosis and neuroinflammation, glutamate reception, and markers of the GABAergic system changed after the MCAO-ML and LPS administration than after the MCAO-MK. Compared with the MCAO-ML, the MCAO-MK and LPS challenges caused changes in the expression of more genes involved in glutamate transport. The most pronounced difference between the responses to different challenges was the changes in expression of calmodulin and calmodulin-dependent kinases genes observed after MCAO, especially MCAO-ML, but not after LPS. The revealed specific features of the hippocampal gene responses to the two types of ischemia and a pro-inflammatory stimulus could contribute to further understanding of the molecular mechanisms underlying diversity of the post-stroke consequences both in the model studies and in the clinic.

Optofluidic control of rodent learning using cloaked caged glutamate.

Glutamate is the major excitatory neurotransmitter in the brain, and photochemical release of glutamate (or uncaging) is a chemical technique widely used by biologists to interrogate its physiology. A basic prerequisite of these optical probes is bio-inertness before photolysis. However, all caged glutamates are known to have strong antagonism toward receptors of γ-aminobutyric acid, the major inhibitory transmitter. We have developed a caged glutamate probe that is inert toward these receptors at concentrations that are effective for photolysis with violet light. Pharmacological tests in vitro revealed that attachment of a fifth-generation (G5) dendrimer (i.e., cloaking) to the widely used 4-methoxy-7-nitro-indolinyl(MNI)-Glu probe prevented such off-target effects while not changing the photochemical properties of MNI-Glu significantly. G5-MNI-Glu was used with optofluidic delivery to stimulate dopamine neurons of the ventral tegmental area of freely moving mice in a conditioned place-preference protocol so as to mediate Pavlovian conditioning.

L-theanine combination therapy with fluvoxamine in moderate-to-severe obsessive-compulsive disorder: A placebo-controlled, double-blind, randomized trial.

AIM: The main aim of this study was to investigate the additional effects of L-theanine, an amino acid in tea and an analog of glutamate with neuroprotective and anti-depressant properties, on obsessive-compulsive disorder (OCD) symptoms in combination with fluvoxamine. METHODS: Patients from either sex aged between 18 and 60 years diagnosed with OCD, based on the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), who had a Yale-Brown Obsessive Compulsive Scale (Y-BOCS) score of more than 21 were enrolled in a double-blinded, parallel-group, placebo-controlled, clinical trial of 10 weeks to receive either L-theanine (100 mg twice daily) and fluvoxamine (100 mg daily initially followed by 200 mg daily after week 5) or placebo and fluvoxamine. The primary outcome of interest in this study was the Y-BOCS total score decrease from baseline. RESULTS: From a total of 95 evaluated patients, 50 completed our study; 30 were randomly assigned to each group. Multivariate analysis (ANOVA) showed a significant effect of time × $$ \times $$ treatment for L-theanine in obsession subscale (F = 5.51, P = 0.008) of the Y-BOCS score but not in the total and compulsion scores. Our results showed significantly more improvement in obsession subscale scores in L-theanine compared to placebo group (P = 0.007, Cohen's d = 0.82). Also, total Y-BOCS scores were lower in L-theanine compared to placebo group at week 5 (P = 0.039, Cohen's d = 0.60) and 10 (P = 0.008, Cohen's d = 0.80). However, there was no significant between-group differences in compulsion subscale scores. Complete response was also more frequent in the L-theanine group (P = 0.0001). CONCLUSION: Findings in this study suggest L-theanine as a relatively safe and effective adjuvant therapy for moderate to severe OCD.

The important role of glial transmitters released by astrocytes in Alzheimer's disease: A perspective from dynamical modeling.

This paper aims to establish a coupling model of neuronal populations and astrocytes and, on this basis, explore the possible mechanism of electroencephalography (EEG) slowing in Alzheimer's disease (AD) from the viewpoint of dynamical modeling. First and foremost, excitatory and inhibitory time constants are shown to induce the early symptoms of AD. The corresponding dynamic nature is mainly due to changes in the amplitude and frequency of the oscillatory behavior. However, there are also a few cases that can be attributed to the change of the oscillation mode caused by the limit cycle bifurcation and birhythmicity. Then, an improved neural mass model influenced by astrocytes is proposed, considering the important effects of glutamate and adenosine triphosphate (ATP) released by astrocytes on the synaptic transmission process reported in experiments. The results show that a dysfunctional astrocyte disrupts the physiological state, causing three typical EEG slowing phenomena reported clinically: the decreased dominant frequency, the decreased rhythmic activity in the α band, and the increased rhythmic activity in the δ+θ band. In addition, astrocytes may control AD when the effect of ATP on synaptic connections is greater than that of glutamate. The control rate depends on the ratio of the effect of glutamate on excitatory and inhibitory synaptic connections. These modeling results can not only reproduce some experimental and clinical results, but, more importantly, may offer a prediction of some underlying phenomena, helping to inspire the disease mechanisms and therapeutic methods of targeting astrocytes.

Theanine, a Tea-Leaf-Specific Amino Acid, Alleviates Stress through Modulation of Npas4 Expression in Group-Housed Older Mice.

Group rearing is a common housing condition, but group-housed older mice show increased adrenal hypertrophy, a marker of stress. However, the ingestion of theanine, an amino acid unique to tea leaves, suppressed stress. We aimed to elucidate the mechanism of theanine's stress-reducing effects using group-reared older mice. The expression of repressor element 1 silencing transcription factor (REST), which represses excitability-related genes, was increased in the hippocampus of group-reared older mice, whereas the expression of neuronal PAS domain protein 4 (Npas4), which is involved in the regulation of excitation and inhibition in the brain, was lower in the hippocampus of older group-reared mice than in same-aged two-to-a-house mice. That is, the expression patterns of REST and Npas4 were found to be just inversely correlated. On the other hand, the expression levels of the glucocorticoid receptor and DNA methyltransferase, which suppress Npas4 transcription, were higher in the older group-housed mice. In mice fed theanine, the stress response was reduced and Npas4 expression tended to be increased. These results suggest that Npas4 expression was suppressed by the increased expression of REST and Npas4 downregulators in the group-fed older mice, but that theanine avoids the decrease in Npas4 expression by suppressing the expression of Npas4 transcriptional repressors.

Estrogen modulation of cortical spreading depression.

BACKGROUND AND AIMS: Cortical spreading depression (CSD), a transient neuronal and glial depolarization that propagates slowly across the cerebral cortex, is the putative electrophysiological event underlying migraine aura and a headache trigger. Migraine is three times more prevalent in women than men, linked to circulating female hormones. High estrogen levels or estrogen withdrawal may be a migraine trigger for many women. We, therefore, aimed to examine whether sex, gonadectomy, and female hormone supplementation and withdrawal affect the susceptibility to CSD. METHODS: To determine CSD susceptibility, we recorded the frequency of CSDs triggered during 2-h topical KCl application in intact or gonadectomized female and male rats, without or with estradiol or progesterone supplementation via daily intraperitoneal injections. Estrogen or progesterone treatment followed by withdrawal was studied in a separate cohort. To take the first step towards identifying potential mechanisms, we studied glutamate and GABAA receptor binding using autoradiography. RESULTS: The CSD frequency in intact female rats was higher than intact male and ovariectomized rats. We did not detect a change in CSD frequency during different stages of the estrous cycle in intact females. Daily estrogen injections for three weeks did not change CSD frequency. However, one-week estrogen withdrawal after two weeks of treatment significantly increased CSD frequency compared with the vehicle group in gonadectomized females. The same protocol of estrogen treatment and withdrawal was ineffective in gonadectomized males. In contrast to estrogen, daily progesterone injections for three weeks elevated CSD susceptibility, and one-week withdrawal after two weeks of treatment partially normalized this effect. Autoradiography did not reveal significant changes in glutamate or GABAA receptor binding density after estrogen treatment and withdrawal. CONCLUSIONS: These data suggest that females are more susceptible to CSD, and sexual dimorphism is abrogated by gonadectomy. Moreover, estrogen withdrawal after prolonged daily treatment enhances CSD susceptibility. These findings may have implications for estrogen-withdrawal migraine, although the latter tends to be without aura.

l-Theanine Prevents Long-Term Affective and Cognitive Side Effects of Adolescent Δ-9-Tetrahydrocannabinol Exposure and Blocks Associated Molecular and Neuronal Abnormalities in the Mesocorticolimbic Circuitry.

Chronic adolescent exposure to Δ-9-tetrahydrocannabinol (THC) is linked to elevated neuropsychiatric risk and induces neuronal, molecular and behavioral abnormalities resembling neuropsychiatric endophenotypes. Previous evidence has revealed that the mesocorticolimbic circuitry, including the prefrontal cortex (PFC) and mesolimbic dopamine (DA) pathway are particularly susceptible to THC-induced pathologic alterations, including dysregulation of DAergic activity states, loss of PFC GABAergic inhibitory control and affective and cognitive abnormalities. There are currently limited pharmacological intervention strategies capable of preventing THC-induced neuropathological adaptations. l-Theanine is an amino acid analog of l-glutamate and l-glutamine derived from various plant sources, including green tea leaves. l-Theanine has previously been shown to modulate levels of GABA, DA, and glutamate in various neural regions and to possess neuroprotective properties. Using a preclinical model of adolescent THC exposure in male rats, we report that l-theanine pretreatment before adolescent THC exposure is capable of preventing long-term, THC-induced dysregulation of both PFC and VTA DAergic activity states, a neuroprotective effect that persists into adulthood. In addition, pretreatment with l-theanine blocked THC-induced downregulation of local GSK-3 (glycogen synthase kinase 3) and Akt signaling pathways directly in the PFC, two biomarkers previously associated with cannabis-related psychiatric risk and subcortical DAergic dysregulation. Finally, l-theanine powerfully blocked the development of both affective and cognitive abnormalities commonly associated with adolescent THC exposure, further demonstrating functional and long-term neuroprotective effects of l-theanine in the mesocorticolimbic system.SIGNIFICANCE STATEMENT With the increasing trend of cannabis legalization and consumption during adolescence, it is essential to expand knowledge on the potential effects of adolescent cannabis exposure on brain development and identify potential pharmacological strategies to minimize Δ-9-tetrahydrocannabinol (THC)-induced neuropathology. Previous evidence demonstrates that adolescent THC exposure induces long-lasting affective and cognitive abnormalities, mesocorticolimbic dysregulation, and schizophrenia-like molecular biomarkers that persist into adulthood. We demonstrate for the first time that l-theanine, an amino acid analog of l-glutamate and l-glutamine, is capable of preventing long-term THC side effects. l-Theanine prevented the development of THC-induced behavioral aberrations, blocked cortical downregulation of local GSK-3 (glycogen synthase kinase 3) and Akt signaling pathways, and normalized dysregulation of both PFC and VTA DAergic activity, demonstrating powerful and functional neuroprotective effects against THC-induced developmental neuropathology.

Pridopidine rescues BDNF/TrkB trafficking dynamics and synapse homeostasis in a Huntington disease brain-on-a-chip model.

Huntington disease (HD) is a neurodegenerative disorder caused by polyglutamine-encoding CAG repeat expansion in the huntingtin (HTT) gene. HTT is involved in the axonal transport of vesicles containing brain-derived neurotrophic factor (BDNF). In HD, diminished BDNF transport leads to reduced BDNF delivery to the striatum, contributing to striatal and cortical neuronal death. Pridopidine is a selective and potent sigma-1 receptor (S1R) agonist currently in clinical development for HD. The S1R is located at the endoplasmic reticulum (ER)-mitochondria interface, where it regulates key cellular pathways commonly impaired in neurodegenerative diseases. We used a microfluidic device that reconstitutes the corticostriatal network, allowing the investigation of presynaptic dynamics, synaptic morphology and transmission, and postsynaptic signaling. Culturing primary neurons from the HD mouse model HdhCAG140/+ provides a "disease-on-a-chip" platform ideal for investigating pathogenic mechanisms and drug activity. Pridopidine rescued the trafficking of BDNF and TrkB resulting in an increased neurotrophin signaling at the synapse. This increased the capacity of HD neurons to release glutamate and restored homeostasis at the corticostriatal synapse. These data suggest that pridopidine enhances the availability of corticostriatal BDNF via S1R activation, leading to neuroprotective effects.

Chemogenetic activation of noradrenergic A5 neurons increases blood pressure and visceral sympathetic activity in adult rats.

In mammals, the pontine noradrenergic system influences nearly every aspect of central nervous system function. A subpopulation of pontine noradrenergic neurons, called A5, are thought to be important in the cardiovascular response to physical stressors, yet their function is poorly defined. We hypothesized that activation of A5 neurons drives a sympathetically mediated increase in blood pressure (BP). To test this hypothesis, we conducted a comprehensive assessment of the cardiovascular effects of chemogenetic stimulation of A5 neurons in male and female adult rats using intersectional genetic and anatomical targeting approaches. Chemogenetic stimulation of A5 neurons in freely behaving rats elevated BP by 15 mmHg and increased cardiac baroreflex sensitivity with a negligible effect on resting HR. Importantly, A5 stimulation had no detectable effect on locomotor activity, metabolic rate, or respiration. Under anesthesia, stimulation of A5 neurons produced a marked elevation in visceral sympathetic nerve activity (SNA) and no change in skeletal muscle SNA, showing that A5 neurons preferentially stimulate visceral SNA. Interestingly, projection mapping indicates that A5 neurons target sympathetic preganglionic neurons throughout the spinal cord and parasympathetic preganglionic neurons throughout in the brainstem, as well as the nucleus of the solitary tract, and ventrolateral medulla. Moreover, in situ hybridization and immunohistochemistry indicate that a subpopulation of A5 neurons coreleases glutamate and monoamines. Collectively, this study suggests A5 neurons are a central modulator of autonomic function with a potentially important role in sympathetically driven redistribution of blood flow from the visceral circulation to critical organs and skeletal muscle.