Downregulation of FTO in the hippocampus is associated with mental disorders induced by fear stress during pregnancy.

Mental disorders (MD), such as anxiety, depression, and cognitive impairment, are very common during pregnancy and predispose to adverse pregnancy outcomes; however, the underlying mechanisms are still under intense investigation. Although the most common RNA modification in epigenetics, N6-methyladenosine (m6A) has been widely studied, its role in MD has not been investigated. Here, we observed that fat mass and obesity-associated protein (FTO) are downregulated in the hippocampus of pregnant rats with MD induced by fear stress and demonstrated that FTO participates in and regulates MD induced by fear stress. In addition, we identified four genes with anomalous modifications and expression (double aberrant genes) that were directly regulated by FTO, namely Angpt2, Fgf10, Rpl21, and Adcy7. Furthermore, we found that these genes might induce MD by regulating the PI3K/Akt and Rap1 signaling pathways. It appears that FTO-mediated m6A modification is a key regulatory mechanism in MD caused by fear stress during pregnancy.

Specific contribution of Reelin expressed by Cajal-Retzius cells or GABAergic interneurons to cortical lamination.

The extracellular protein Reelin, expressed by Cajal-Retzius (CR) cells at early stages of cortical development and at late stages by GABAergic interneurons, regulates radial migration and the "inside-out" pattern of positioning. Current models of Reelin functions in corticogenesis focus on early CR cell-derived Reelin in layer I. However, developmental disorders linked to Reelin deficits, such as schizophrenia and autism, are related to GABAergic interneuron-derived Reelin, although its role in migration has not been established. Here we selectively inactivated the Reln gene in CR cells or GABAergic interneurons. We show that CR cells have a major role in the inside-out order of migration, while CR and GABAergic cells sequentially cooperate to prevent invasion of cortical neurons into layer I. Furthermore, GABAergic cell-derived Reelin compensates some features of the reeler phenotype and is needed for the fine tuning of the layer-specific distribution of cortical neurons. In the hippocampus, the inactivation of Reelin in CR cells causes dramatic alterations in the dentate gyrus and mild defects in the hippocampus proper. These findings lead to a model in which both CR and GABAergic cell-derived Reelin cooperate to build the inside-out order of corticogenesis, which might provide a better understanding of the mechanisms involved in the pathogenesis of neuropsychiatric disorders linked to abnormal migration and Reelin deficits.

Targeting acetyl-CoA metabolism attenuates the formation of fear memories through reduced activity-dependent histone acetylation.

Histone acetylation is a key component in the consolidation of long-term fear memories. Histone acetylation is fueled by acetyl-coenzyme A (acetyl-CoA), and recently, nuclear-localized metabolic enzymes that produce this metabolite have emerged as direct and local regulators of chromatin. In particular, acetyl-CoA synthetase 2 (ACSS2) mediates histone acetylation in the mouse hippocampus. However, whether ACSS2 regulates long-term fear memory remains to be determined. Here, we show that Acss2 knockout is well tolerated in mice, yet the Acss2-null mouse exhibits reduced acquisition of long-term fear memory. Loss of Acss2 leads to reductions in both histone acetylation and expression of critical learning and memory-related genes in the dorsal hippocampus, specifically following fear conditioning. Furthermore, systemic administration of blood-brain barrier-permeable Acss2 inhibitors during the consolidation window reduces fear-memory formation in mice and rats and reduces anxiety in a predator-scent stress paradigm. Our findings suggest that nuclear acetyl-CoA metabolism via ACSS2 plays a critical, previously unappreciated, role in the formation of fear memories.

Effects of methylphenidate after a long period of discontinuation include changes in exploratory behavior and increases brain activities of Na+,K+-ATPase and acetylcholinesterase.

Methylphenidate (MPH) has been widely misused by children and adolescents who do not meet all diagnostic criteria for attention-deficit/hyperactivity disorder. Since it is not yet known whether MPH can be administered in childhood without consequences in adulthood, in the present study we proposed to investigate the effects of chronic early treatment with MPH after a long period of discontinuation. Wistar male rats were injected with MPH (2 mg/kg, intraperitoneally) or saline solution once daily from 15th to 44th day of life. Two months after the last MPH administration, we evaluated the animal's performances on a battery of behavior tests. We also tested Na+,K+-ATPase and acetylcholinesterase activities in prefrontal cortex and hippocampus, which may be associated with behavior. Rats treated with MPH during peri-adolescence show changes in exploratory behavior in adulthood in the open field but not in the elevated plus maze and light-dark transition tests. MPH-treated rats showed a lower latency to find the platform in the training phase, as well as a better performance in the test phase in the Morris water maze test. No differences were observed in the object recognition index and working memory. Acetylcholinesterase was increased in prefrontal cortex and hippocampus, while Na+,K+-ATPase was increased only in hippocampus. These findings provide additional evidence that early-life exposure to MPH can have complex effects in adulthood and new basis for understanding the behavioral and neurochemical consequences associated with chronic use of MPH during the development of central nervous system.

Structure-Activity Relationship in the Leucettine Family of Kinase Inhibitors.

The protein kinase DYRK1A is involved in Alzheimer's disease, Down syndrome, diabetes, viral infections, and leukemia. Leucettines, a family of 2-aminoimidazolin-4-ones derived from the marine sponge alkaloid Leucettamine B, have been developed as pharmacological inhibitors of DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). We report here on the synthesis and structure-activity relationship (SAR) of 68 Leucettines. Leucettines were tested on 11 purified kinases and in 5 cellular assays: (1) CLK1 pre-mRNA splicing, (2) Threonine-212-Tau phosphorylation, (3) glutamate-induced cell death, (4) autophagy and (5) antagonism of ligand-activated cannabinoid receptor CB1. The Leucettine SAR observed for DYRK1A is essentially identical for CLK1, CLK4, DYRK1B, and DYRK2. DYRK3 and CLK3 are less sensitive to Leucettines. In contrast, the cellular SAR highlights correlations between inhibition of specific kinase targets and some but not all cellular effects. Leucettines deserve further development as potential therapeutics against various diseases on the basis of their molecular targets and cellular effects.

Bisphenol-A exposure leads to neurotoxicity through upregulating the expression of histone deacetylase 2 in vivo and in vitro.

Bisphenol-A (BPA), an environmental endocrine disruptor, is toxic to the central nervous system. Although recent studies have shown BPA-induced neurotoxicity, it is far from clear what precisely epigenetic mechanisms are involved in BPA-induced cognitive deficits. In this study, pheochromocytoma (PC12) cells were treated with BPA at 1 µM for 36 h in vitro. In vivo, C57BL/6 mice were administered to BPA at a dose of 1 mg/kg/day for 10 weeks. The results showed that 1 µM BPA exposure for 36 h impaired neurite outgrowth of PC12 cells through decreasing the primary and secondary branches. Besides, BPA exposure decreased the level of Ac-H3K9 (histone H3 Lys9 acetylation) by upregulating the expression of HDAC2 (histone deacetylases 2) in PC12 cells. Furthermore, treatment of both TSA (Trichostatin A, inhibitor of the histone deacetylase) and shHDAC2 plasmid (HDAC2 knockdown construct) resulted in amelioration neurite outgrowth deficits induced by BPA. In addition, it was shown that repression of HDAC2 could markedly rescue the spine density impairment in the hippocampus and prevent the cognitive impairment caused by BPA exposure in mice. Collectively, HDAC2 plays an essential role in BPA-induced neurotoxicity, which provides a potential molecular target for medical intervention.

Hippocampal MSK1 regulates the behavioral and biological responses of mice to chronic social defeat stress: Involving of the BDNF-CREB signaling and neurogenesis.

Depression is one of the most common psychiatric diseases in the 21st century, while its pathogenesis is not yet fully understood. Currently, besides to the monoaminergic system, the brain-derived neurotrophic factor (BDNF)-cAMP response element-binding protein (CREB) signaling is one of the most attractive signaling pathways for treating depression. Mitogen and stress-activated kinase (MSK) 1 and 2 are nuclear proteins activated downstream of the ERK1/2 or p38 MAPK pathways, and it has been demonstrated that MSKs are involved in the BDNF-CREB signaling. Here we assumed that MSKs may play a role in depression, and various methods including the chronic social defeat stress (CSDS) model of depression, western blotting, immunofluorescence and virus-mediated gene transfer were used together. It was found that CSDS fully enhanced the expression of both phosphorylated MSK1 and total MSK1 in the hippocampus but not the medial prefrontal cortex (mPFC). CSDS did not influence the expression of phosphorylated MSK2 and total MSK2 in the two brain regions. Genetic over-expression of hippocampal MSK1 fully prevented not only the CSDS-induced depressive-like behaviors but also the CSDS-induced dysfunction in the hippocampal BDNF-CREB signaling and neurogenesis in mice, while genetic knockdown of hippocampal MSK1 aggravated the CSDS-induced depressive-like symptomatology in mice. Our results collectively suggest that although CSDS evidently enhances the activity of hippocampal MSK1, it is not a contributor to the CSDS-induced dysfunction in the brain but a defensive feedback regulator which protects against CSDS. Therefore, hippocampal MSK1 participates in the pathogenesis of depression and is a feasible and potential antidepressant target.

Target enzymes in oxaliplatin-induced peripheral neuropathy in Swiss mice: A new acetylcholinesterase inhibitor as therapeutic strategy.

In the present study it was hypothesized that 5-((4-methoxyphenyl)thio)benzo[c][1,2,5] thiodiazole (MTDZ), a new acetylcholinesterase inhibitor, exerts antinociceptive action and reduces the oxaliplatin (OXA)-induced peripheral neuropathy and its comorbidities (anxiety and cognitive deficits). Indeed, the acute antinociceptive activity of MTDZ (1 and 10 mg/kg; per oral route) was observed for the first time in male Swiss mice in formalin and hot plate tests and on mechanical withdrawal threshold induced by Complete Freund's Adjuvant (CFA). To evaluate the MTDZ effect on OXA-induced peripheral neuropathy and its comorbidities, male and female Swiss mice received OXA (10 mg/kg) or vehicle intraperitoneally, on days 0 and 2 of the experimental protocol. Oral administration of MTDZ (1 mg/kg) or vehicle was performed on days 2-14. OXA caused cognitive impairment, anxious-like behaviour, mechanical and thermal hypersensitivity in animals, with females more susceptible to thermal sensitivity. MTDZ reversed the hypersensitivity, cognitive impairment and anxious-like behaviour induced by OXA. Here, the negative correlation between the paw withdrawal threshold caused by OXA and acetylcholinesterase (AChE) activity was demonstrated in the cortex, hippocampus, and spinal cord. OXA inhibited the activity of total ATPase, Na+ K+ - ATPase, Ca2+ - ATPase and altered Mg2+ - ATPase in the cortex, hippocampus, and spinal cord. OXA exposure increased reactive species (RS) levels and superoxide dismutase (SOD) activity in the cortex, hippocampus, and spinal cord. MTDZ modulated ion pumps and reduced the oxidative stress induced by OXA. In conclusion, MTDZ is an antinociceptive molecule promising to treat OXA-induced neurotoxicity since it reduced nociceptive and anxious-like behaviours, and cognitive deficit in male and female mice.

Oral glutamine supplementation increases seizure severity in a rodent model of mesial temporal lobe epilepsy.

Background: Glutamine synthetase (GS) is the only enzyme known to synthesize significant amounts of glutamine in mammals, and loss of GS in the hippocampus has been implicated in the pathophysiology of medication refractory mesial temporal lobe epilepsy (MTLE). Moreover, loss-of-function mutations of the GS gene causes severe epileptic encephalopathy, and supplementation with glutamine has been shown to normalize EEG and possibly improve the outcome in these patients. Here we examined whether oral glutamine supplementation is an effective treatment for MTLE by assessing the frequency and severity of seizures after supplementation in a translationally relevant model of the disease.Methods: Male Sprague Dawley rats (380-400 g) were allowed to drink unlimited amounts of glutamine in water (3.6% w/v; n = 8) or pure water (n = 8) for several weeks. Ten days after the start of glutamine supplementation, GS was chronically inhibited in the hippocampus to induce MTLE. Continuous video-intracranial EEG was collected for 21 days to determine the frequency and severity of seizures.Results: While there was no change in seizure frequency between the groups, the proportion of convulsive seizures was significantly higher in glutamine treated animals during the first three days of GS inhibition.Conclusion: The results suggest that oral glutamine supplementation transiently increases seizure severity in the initial stages of an epilepsy model, indicating a potential role of the amino acid in seizure propagation and epileptogenesis.

Chronic Intestinal Inflammation Suppresses Brain Activity by Inducing Neuroinflammation in Mice.

Chronic gut inflammation such as inflammatory bowel disease is believed to be associated with neurodegenerative diseases in humans. However, the direct evidence for and the underlying mechanism of this brain-gut interaction remain elusive. In this study, manganese-enhanced magnetic resonance imaging was used to assess functional brain activity from awake and freely moving mice with chronic colitis. Manganese ion uptake (indicative of Ca2+ influx into neuronal cells) and accumulation were reduced in the hippocampus of chronic colitis mice compared with control mice. Long-term memory declined and neuroinflammatory signals, including IL-1ß production and activation of caspase-1, caspase-11, and gasdermin, were induced. High-mobility group box 1 (HMGB1) levels were elevated both in the serum and in the hippocampus; however, lipopolysaccharide (LPS) levels remained at low levels without significant changes in these samples. The blood-brain barrier permeability was increased in chronic colitis mice. In the presence of LPS, HMGB1 treatment induced the activation of caspase-11 and gasdermin in the mouse microglial cell line SIM-A9. These findings suggest that HMGB1 released from the inflamed intestine may move to the brain through the blood circulatory system; in conjunction with a low level of endogenous LPS, elevated HMGB1 can subsequently activate caspase-mediated inflammatory responses in the brain.

Conserved and divergent features of neuronal CaMKII holoenzyme structure, function, and high-order assembly.

Neuronal CaMKII holoenzymes (α and ß isoforms) enable molecular signal computation underlying learning and memory but also mediate excitotoxic neuronal death. Here, we provide a comparative analysis of these signaling devices, using single-particle electron microscopy (EM) in combination with biochemical and live-cell imaging studies. In the basal state, both isoforms assemble mainly as 12-mers (but also 14-mers and even 16-mers for the ß isoform). CaMKIIα and ß isoforms adopt an ensemble of extended activatable states (with average radius of 12.6 versus 16.8 nm, respectively), characterized by multiple transient intra- and inter-holoenzyme interactions associated with distinct functional properties. The extended state of CaMKIIß allows direct resolution of intra-holoenzyme kinase domain dimers. These dimers could enable cooperative activation by calmodulin, which is observed for both isoforms. High-order CaMKII clustering mediated by inter-holoenzyme kinase domain dimerization is reduced for the ß isoform for both basal and excitotoxicity-induced clusters, both in vitro and in neurons.

Synaptotagmin 7 is targeted to the axonal plasma membrane through γ-secretase processing to promote synaptic vesicle docking in mouse hippocampal neurons.

Synaptotagmin 7 (SYT7) has emerged as a key regulator of presynaptic function, but its localization and precise role in the synaptic vesicle cycle remain the subject of debate. Here, we used iGluSnFR to optically interrogate glutamate release, at the single-bouton level, in SYT7KO-dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired-pulse facilitation, and synaptic vesicle replenishment and found that SYT7 contributes to each of these processes to different degrees. 'Zap-and-freeze' electron microscopy revealed that a loss of SYT7 diminishes docking of synaptic vesicles after a stimulus and inhibits the recovery of depleted synaptic vesicles after a stimulus train. SYT7 supports these functions from the axonal plasma membrane, where its localization and stability require both γ-secretase-mediated cleavage and palmitoylation. In summary, SYT7 is a peripheral membrane protein that controls multiple modes of synaptic vesicle (SV) exocytosis and plasticity, in part, through enhancing activity-dependent docking of SVs.

Legumain knockout improved cognitive impairment via reducing neuroinflammation in right unilateral common carotid artery occlusion mice.

AIMS: Chronic cerebral hypoperfusion (CCH) is a state of chronic cerebral blood flow reduction, and it is the main cause of cognitive impairment and neurodegenerative diseases. The abnormal upregulation of legumain, a lysosomal cysteine protease, trigger synaptic plasticity impairment and neuroinflammation, which are involved in the underlying pathophysiology of CCH. At present, few studies have reported the role of legumain in cognitive impairment caused by CCH. In our study, we aimed to investigate the involvement of legumain knockout in cognitive function and neuroinflammation in a CCH mouse model. MAIN METHODS: In this study, right unilateral common carotid artery occlusion (rUCCAO) was used to simulate the pathological state of cerebral ischemic injury. Various behavioural tests were executed to assess cognitive performance. In vivo electrophysiological recordings were used to measure synaptic functions. Western blotting, Golgi staining, haematoxylin/eosin staining, and immunofluorescence assays were conducted to examine pathological changes and molecular mechanisms. KEY FINDINGS: The data showed that the level of legumain was significantly increased in the hippocampus of mice subjected to rUCCAO. Legumain knockout significantly improved cognitive function and synaptic plasticity induced by rUCCAO, suggesting that legumain knockout-regulation effectively protected against CCH-induced behavioural dysfunctions. Moreover, legumain knockout suppressed rUCCAO-induced microglial activation, reduced the abnormal expression of inflammatory cytokines and the inflammasome complex, and impeded the activation of P65 and pyroptosis. SIGNIFICANCE: These findings suggest that legumain is an effective regulator of CCH, and may be an ideal target for the development of cerebral ischemia treatments in the future.

Quercetin stimulates the non-amyloidogenic pathway via activation of ADAM10 and ADAM17 gene expression in aluminum chloride-induced Alzheimer's disease rat model.

AIMS: Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder characterized by declined cognitive functions in the elderly. Quercetin (Q) is a potent flavonol that has neuroprotective effects on AD derangements. The present study aimed to evaluate the α-secretase stimulatory function of Q through activation of ADAM10 and ADAM17 gene expression in the aluminum chloride (AlCl3)-induced AD rat model. MAIN METHODS: After induction of AD in rats by oral administration of AlCl3 (50 mg/kg) for 28 days, the Q doses (25 and 50 mg/kg) were orally administered for 28 days. Rats performed the behavioral assessments during the last week of the treatment period. Hippocampi were harvested for assessments of the neurochemical and histopathological examinations and gene expression analysis. KEY FINDINGS: Administration of Q to AlCl3-induced AD rat model attenuated behavioral deficits, improved cholinergic and dopaminergic dysfunctions, and diminished insoluble amyloid ß (Aß) plaques aggregation in the hippocampus. These ameliorative effects of Q were associated with down-regulation of APP, BACE1, APH1, and PSEN1 and up-regulation of ADAM10 and ADAM17 gene expression levels in the hippocampus. SIGNIFICANCE: The present study suggests that Q might attenuate neurotransmission impairment, Aß aggregation in the hippocampus, and behavioral deficits in the AlCl3-induce AD rat model via up-regulating ADAM 10 and ADAM 17 (α-secretase) gene expression, leading to the inhibition of the amyloidogenic pathway. In support of the present finding, we suggest that ADAM10 and ADAM17 activation might be potential drug targets for AD to counteract the Aß aggregation and cognitive deterioration.

Super-resolution study of PIAS SUMO E3-ligases in hippocampal and cortical neurons.

The SUMOylation machinery is a regulator of neuronal activity and synaptic plasticity. It is composed of SUMO isoforms and specialized enzymes named E1, E2 and E3 SUMO ligases. Recent studies have highlighted how SUMO isoforms and E2 enzymes localize with synaptic markers to support previous functional studies but less information is available on E3 ligases. PIAS proteins - belonging to the protein inhibitor of activated STAT (PIAS) SUMO E3-ligase family - are the best-characterized SUMO E3-ligases and have been linked to the formation of spatial memory in rodents. Whether however they exert their function co-localizing with synaptic markers is still unclear. In this study, we applied for the first time structured illumination microscopy (SIM) to PIAS ligases to investigate the co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal and cortical murine neurons. The results indicate partial co-localization of PIAS1 and PIAS3 with synaptic markers in hippocampal neurons and much rarer occurrence in cortical neurons. This is in line with previous super-resolution reports describing the co-localization with synaptic markers of other components of the SUMOylation machinery.

Mitochondrial Metabolism behind Region-Specific Resistance to Ischemia-Reperfusion Injury in Gerbil Hippocampus. Role of PKCßII and Phosphate-Activated Glutaminase.

Ischemic episodes are a leading cause of death worldwide with limited therapeutic interventions. The current study explored mitochondrial phosphate-activated glutaminase (GLS1) activity modulation by PKCßII through GC-MS untargeted metabolomics approach. Mitochondria were used to elucidate the endogenous resistance of hippocampal CA2-4 and dentate gyrus (DG) to transient ischemia and reperfusion in a model of ischemic episode in gerbils. In the present investigation, male gerbils were subjected to bilateral carotids occlusion for 5 min followed by reperfusion (IR). Gerbils were randomly divided into three groups as vehicle-treated sham control, vehicle-treated IR and PKCßII specific inhibitor peptide ßIIV5-3-treated IR. Vehicle or ßIIV5-3 (3 mg/kg, i.v.) were administered at the moment of reperfusion. The gerbils hippocampal tissue were isolated at various time of reperfusion and cell lysates or mitochondria were isolated from CA1 and CA2-4,DG hippocampal regions. Recombinant proteins PKCßII and GLS1 were used in in vitro phosphorylation reaction and organotypic hippocampal cultures (OHC) transiently exposed to NMDA (25 µM) to evaluate the inhibition of GLS1 on neuronal viability. PKCßII co-precipitates with GAC (GLS1 isoform) in CA2-4,DG mitochondria and phosphorylates GLS1 in vitro. Cell death was dose dependently increased when GLS1 was inhibited by BPTA while inhibition of mitochondrial pyruvate carrier (MPC) attenuated cell death in NMDA-challenged OHC. Fumarate and malate were increased after IR 1h in CA2-4,DG and this was reversed by ßIIV5-3 what correlated with GLS1 activity increases and earlier showed elevation of neuronal death (Krupska et al., 2017). The present study illustrates that CA2-4,DG resistance to ischemic episode at least partially rely on glutamine and glutamate utilization in mitochondria as a source of carbon to tricarboxylic acid cycle. This phenomenon depends on modulation of GLS1 activity by PKCßII and remodeling of MPC: all these do not occur in ischemia-vulnerable CA1.

Tadalafil and bergapten mitigate streptozotocin-induced sporadic Alzheimer's disease in mice via modulating neuroinflammation, PI3K/Akt, Wnt/ß-catenin, AMPK/mTOR signaling pathways.

Sporadic Alzheimer's disease (SAD) is a slowly progressive neurodegenerative disorder. This study aimed to investigate neuroprotective potential of tadalafil (TAD) and bergapten (BG) in SAD-induced cognitive impairment in mice. SAD was induced by single injection of streptozotocin (STZ; 3 mg/kg, ICV). STZ resulted in AD-like pathologies including Aß deposition, tau aggregation, impaired insulin and Wnt/ß-catenin signaling, as well as autophagic dysfunction and neuroinflammation. Administration of TAD or BG at doses of 20 and 25 mg/kg, respectively, for 21 consecutive days attenuated STZ-induced hippocampal insult, preserved neuronal integrity, and improved cognitive function in the Morris water maze and object recognition tests paralleled by reduction in Aß expression by 79 and 89% and tau hyperphosphorylation by 60 and 61%, respectively. TAD and BG also enhanced protein expression of pAkt, pGSK-3ß, beclin-1 and methylated protein phosphatase 2A (PP2A) and gene expression of cyclin D1, while raised BDNF immunoreactivity. Furthermore, TAD and BG boosted hippocampal levels of cGMP, PKG, Wnt3a, and AMPK and reduced expression of ß-catenin and mTOR by 74% and 51%, respectively. TAD and BG also halted neuroinflammation by reducing IL-23 and IL-27 levels, as well as protein expression of NF-κB by 62% & 61%, respectively. In conclusion, this study offers novel insights on the neuroprotective effects of TAD or BG in the management of SAD as evidenced by improved cognitive function and histological architecture. This could be attributed to modulation of the crosstalk among PI3K/Akt/GSK-3ß, PP2A, mTOR/autophagy, cGMP/PKG, and Wnt/ß-catenin signaling cascades and mitigation of neuroinflammation.

Acetylation of calmodulin regulates synaptic plasticity and fear learning.

Synaptic plasticity is critical for brain function, including learning and memory. It is regulated by gene transcription and protein synthesis as well as posttranslational modifications at synapses. Although protein acetylation has been shown to be involved in the regulation of synaptic plasticity, this was mainly for histone protein acetylation. To investigate whether acetylation of nonhistone proteins is important for synaptic plasticity, we analyzed mouse brain acetylome and found that calmodulin (CaM), a ubiquitous Ca2+ sensor, was acetylated on three lysine residues, which were conserved across species. NMDA receptor-dependent long-term potentiation (LTP) is considered the most compelling form of synaptic plasticity. During LTP induction, activation of NMDA receptor triggers Ca2+ influx, and the Ca2+ binds with CaM and activates calcium/calmodulin-dependent protein kinase IIα (CaMKIIα), which is essential for LTP induction. By using home-generated and site-specific antibodies against acetylated CaM, we show that CaM acetylation is upregulated by neural activities in an NMDA receptor-dependent manner. Moreover, mutation of acetyllysines in CaM1 proteins disrupts synaptic plasticity and fear learning in a mouse model. We further demonstrate that acetylation of CaM reduces the binding free energy and increases the binding affinity toward CaMKIIα, a protein kinase pivotal to synaptic plasticity and learning. Taken together, our results demonstrate importance of CaM acetylation in regulating synaptic plasticity and learning.

BOTOX® counteracts the innate anxiety-related behaviours in correlation with increased activities of key antioxidant enzymes in the hippocampus of ageing experimental mice.

Cholinergic crisis and oxidative stress in the hippocampus of the brain have been known to induce anxiety disorders upon ageing. BOTOX® is a widely used therapeutic form of botulinum neurotoxin that acts by inhibiting the release of acetylcholine (ACh) from the nerve terminals at the neuromuscular junction. BOTOX® can migrate from the muscle to the brain through retrograde axonal transport and modulate neuroplasticity. While a mild dose of BOTOX® has been used to manage various neurological deficits and psychiatric complications including depression, the efficacy and experimental evidence for its anxiolytic effects and antioxidant properties remain limited. In this study, we have investigated the effect of BOTOX® on the innate anxiety-like behaviours in ageing mice upon exposure to different behavioural paradigms like open field test, elevated plus maze and light-dark box test, and estimated the enzymatic activities of key antioxidants in the hippocampus. Results revealed that animals injected with a mild intramuscular dosage of BOTOX® showed reduced level of innate anxiety-related symptoms and increased activities of hippocampal antioxidant enzymes compared to the control group. This study strongly supports that BOTOX® could be implemented to prevent or treat anxiety and hippocampal oxidative stress resulting from ageing, emotional and mood disorders.

Apocynin Prevents Anxiety-Like Behavior and Histone Deacetylases Overexpression Induced by Sub-Chronic Stress in Mice.

Anxiety disorders are common mental health diseases affecting up to 7% of people around the world. Stress is considered one of the major environmental risk factors to promote anxiety disorders through mechanisms involving epigenetic changes. Moreover, alteration in redox balance and increased reactive oxygen species (ROS) production have been detected in anxiety patients and in stressed-animal models of anxiety. Here we tested if the administration of apocynin, a natural origin antioxidant, may prevent the anxiety-like phenotype and reduction of histone acetylation induced by a subchronic forced swimming stress (FSS) paradigm. We found that apocynin prevented the enhanced latency time in the novelty-suppressed feeding test, and the production of malondialdehyde induced by FSS. Moreover, apocynin was able to block the upregulation of p47phox, a key subunit of the NADPH oxidase complex. Finally, apocynin prevented the rise of hippocampal Hdac1, Hdac4 and Hdac5, and the reduction of histone-3 acetylation levels promoted by FSS exposure. In conclusion, our results provide evidence that apocynin reduces the deleterious effect of stress and suggests that oxidative stress may regulate epigenetic mechanisms.