Search Results (150)

To investigate how dysregulated transient receptor potential canonical channels (TRPCs) are associated with Alzheimer’s disease (AD), we challenged primary neurons with amyloid-β (Aβ). Both the naturally secreted or synthetic Aβ oligomers (AβOs) induced long-lasting increased TRPC3 and downregulated the TRPC6 expression in mature excitatory neurons (CaMKIIα-high) via a Ca²⁺-dependent calcineurin-coupled NFAT transcriptionally and calpain-mediated protein degradation, respectively. The TRPC3 expression was also found to be upregulated in pyramidal neurons of human AD brains. The selective downregulation of the Trpc6 gene induced synaptotoxicity, while no significant effect was observed from the Trpc3-targeting siRNA, suggesting potentially differential roles of TRPC3 and 6 in modulating the synaptic morphology and functions. Electrophysiological recordings of mouse hippocampal slices overexpressing TRPC3 revealed increased neuronal hyperactivity upon the TRPC3 channel activation by its agonist. Furthermore, the AβO-mediated synaptotoxicity appeared to be positively correlated with the degrees of the induced dendritic Ca²⁺ flux in neurons, which was completely prevented by the co-treatment with two pyrazole-based TRPC3-selective antagonists Pyr3 or Pyr10. Taken together, our findings suggest that the aberrantly upregulated TRPC3 is another ion channel critically contributing to the process of AβO-induced Ca²⁺ overload, neuronal hyperexcitation, and synaptotoxicity, thus representing a potential therapeutic target of AD. Full article

Background/Objectives: Kv2 channels have important conducting and nonconducting functions and are regulated by their co-assembly with ‘silent’ Kv subunits, including Kv9.1. Kv9.1 is co-expressed with Kv2 channels in sensory neurons, and a common allele that changes Ile489 to Val in human Kv9.1 is associated with pain hypersensitivity in patients. The mechanism responsible for this association remains unknown, but we hypothesise that these two variants differ in their regulation of Kv2.1 properties, and this is what we set out to test. Methods: Expression was carried out using HEK293 cells, OHeLa cells, and primary cultures of hippocampal neurons, and the biophysical and trafficking properties of homomeric and heteromeric channels were assessed by confocal fluorescence microscopy and patch clamp analysis. Results: Both Kv9.1Ile and Kv9.1Val were retained within the endoplasmic reticulum when expressed individually, but when co-expressed with Kv2.1, they co-localised with Kv2.1 within the surface clusters. Both variants reduced the surface expression of Kv2.1 channels and the size of channel clusters, with Kv9.1Val producing a greater reduction in surface expression in both the HeLa cells and neurons. They both caused a similar hyperpolarising shift in the voltage dependence of channel activation and inactivation. Concatamers of Kv2.1 and Kv9.1 suggested that both 3:1 and 2:2 ratios of Kv2.1 to Kv9.1 were permitted, although 2:2 resulted in lower surface expression and function. Conclusions: The Ile489Val substitution in Kv9.1 does not disrupt its ability to co-assemble with Kv2 channels, nor its effects on the voltage-dependence of channel gating, but it did produce a greater reduction in the Kv2.1 surface expression, suggesting that this underlies its association with pain hypersensitivity. Full article

Terahertz (THz) waves, a novel type of radiation with quantum and electronic properties, have attracted increasing attention for their effects on the nervous system. Spatial working memory, a critical component of higher cognitive function, is coordinated by brain regions such as the infralimbic cortex (IL) region of the medial prefrontal cortex and the ventral cornu ammonis 1 (vCA1) of hippocampus. However, the regulatory effects of THz waves on spatial working memory and the underlying mechanisms remain unclear. In this study, the effects of 0.152 THz waves on glutamatergic neuronal activity and spatial working memory and the related mechanisms were investigated in cell, brain slice, and mouse models. Cellular experiments revealed that THz waves exposure for 60 min significantly increased the intrinsic excitability of primary hippocampal neurons, enhanced glutamatergic neuron activity, and upregulated the expression of molecules involved in glutamate metabolism. In brain slice experiments, THz waves markedly elevated neuronal activity, promoted synaptic plasticity, and increased glutamatergic synaptic transmission within the IL and vCA1 regions. Molecular dynamics simulations found that THz waves could inhibit the ion transport function of glutamate receptors. Moreover, Y-maze tests demonstrated that mice exposed to THz waves exhibited significantly improved spatial working memory. Multiomics analyses indicated that THz waves could induce changes in chromatin accessibility and increase the proportion of excitatory neurons. These findings suggested that exposure to 0.152 THz waves increased glutamatergic neuronal activity, promoted synaptic plasticity, and improved spatial working memory, potentially through modifications in chromatin accessibility and excitatory neuron proportions. Full article

Learning and memory formation rely on synaptic plasticity, the process that changes synaptic strength in response to neuronal activity. In the tripartite synapse concept, molecular signals that affect synapse strength and morphology originate not only from the pre- and post-synaptic neuronal terminals but also from astrocytic processes ensheathing many synapses. Despite significant progress made in understanding astrocytic contribution to synaptic plasticity, only a few astrocytic plasticity-related proteins have been identified so far. In this study, we present evidence indicating the role of astrocyte-secreted Lipocalin-2 (Lcn2) in neuronal plasticity. We show that Lcn2 expression is induced in hippocampal astrocytes in a kainate-evoked aberrant plasticity model. Next, we demonstrate that chemically induced long-term potentiation (cLTP) similarly increases Lcn2 expression in astrocytes of neuronal–glial co-cultures, and that glutamate causes the immediate release of Lcn2 from these cultures. Additionally, through experiments in primary astrocytic cultures, we reveal that Lcn2 release is triggered by calcium signaling, and we demonstrate that a brief treatment of neuronal–glial co-cultures with Lcn2 alters the morphology of dendritic spines. Based on these findings, we propose Lcn2 as an activity-dependent molecule released by astrocytes that influences dendritic spine morphology. Full article

Traumatic brain injury (TBI) is one of the primary causes of mortality and disability, with arterial blood pressure being an important factor in the clinical management of TBI. Spontaneously hypertensive rats (SHRs), widely used as a model of essential hypertension and vascular dementia, demonstrate dysfunction of the hypothalamic–pituitary–adrenal axis, which may contribute to glucocorticoid-mediated hippocampal damage. The aim of this study was to assess acute post-TBI seizures, delayed mortality, and hippocampal pathology in SHRs and normotensive Sprague Dawley rats (SDRs). Male adult SDRs and SHRs were subjected to lateral fluid-percussion injury. Immediate seizures were video recorded, corticosterone (CS) was measured in blood plasma throughout the study, and hippocampal morphology assessed 3 months post-TBI. Acute and remote survival rates were significantly higher in the SHRs compared to the SDRs (overall mortality 0% and 58%, respectively). Immediate seizure duration predicted acute but not remote mortality. TBI did not affect blood CS in the SHRs, while the CS level was transiently elevated in the SDRs, predicting remote mortality. Neuronal cell loss in the polymorph layer of ipsilateral dentate gyrus was found in both the SDRs and SHRs, while thinning of hippocampal pyramidal and granular cell layers were strain- and area-specific. No remote effects of TBI on the density of astrocytes or microglia were revealed. SHRs possess a unique resilience to TBI as compared with normotensive SDRs. SHRs show shorter immediate seizures and reduced CS response to the injury, suggesting the development of long-term adaptative mechanisms associated with chronic hypertension. Though remote post-traumatic hippocampal damage in ipsilateral dentate gyrus is obvious in both SHRs and SDRs, the data imply that physiological adaptations to high blood pressure in SHRs may be protective, preventing TBI-induced mortality but not hippocampal neurodegeneration. Understanding the mechanisms of resilience to TBI may also help improve clinical recommendations for patients with hypertension. Limitation: since more than a half of the SDRs with prolonged immediate seizures or elevated CS 3 days after TBI have died, survivorship bias might hamper correct interpretation of the data. Full article

Artemisinin is a sesquiterpene lactone derived from the plant Artemisia annua L., renowned for its antimalarial activity. Based on this compound, various derivatives and analogues have been obtained that exhibit diverse biological activities, including clinically approved drugs. Recently, increasing evidence has highlighted the neuroprotective potential of artemisinin. In this study, we evaluated the effects of artemisinin on the viability of neuronal-like cells, including primary hippocampal neuronal cultures. Artemisinin exhibited a stimulating effect on SH-SY5Y and HEK-293 cells and enhanced the survival of primary neurons at low concentrations (1 µM). In contrast, artemisinin derivatives, such as dihydroartemisinin, anhydrodihydroartemisinin, and artemisitene, did not display similar stimulatory activity, suggesting that the intact lactone ring is crucial for this property. Furthermore, artemisinin demonstrated a protective effect against endoplasmic reticulum (ER) stress induced by the proteasome inhibitor MG132 in SH-SY5Y cells. However, it did not exhibit protective activity against oxidative stress induced by sodium arsenite. Additionally, artemisinin effectively inhibited the aggregation of mutated TDP-43 protein in transfected SH-SY5Y cells. These findings suggest that artemisinin exerts neuroprotective effects by targeting key molecular pathways associated with neurodegeneration, offering potential therapeutic insights for related conditions. Full article

RAC3 encodes a small GTPase of the Rho family that plays a critical role in actin cytoskeleton remodeling and intracellular signaling regulation. Pathogenic variants in RAC3, all of which reported thus far affect conserved residues within its functional domains, have been linked to neurodevelopmental disorders characterized by diverse phenotypic features, including structural brain anomalies and facial dysmorphism (NEDBAF). Recently, a novel de novo RAC3 variant (NM_005052.3): c.196C>T, p.R66W was identified in a prenatal case with fetal akinesia deformation sequence (a spectrum of conditions that interfere with the fetus’s ability to move), and complex brain malformations featuring corpus callosum agenesis, diencephalosynapsis, kinked brainstem, and vermian hypoplasia. To investigate the mechanisms underlying the association between RAC3 deficiency and this unique, distinct clinical phenotype, we explored the pathophysiological significance of the p.R66W variant in brain development. Biochemical assays revealed a modest enhancement in intrinsic GDP/GTP exchange activity and an inhibitory effect on GTP hydrolysis. Transient expression studies in COS7 cells demonstrated that RAC3-R66W interacts with the downstream effectors PAK1, MLK2, and N-WASP but fails to activate SRF-, AP1-, and NFkB-mediated transcription. Additionally, overexpression of RAC3-R66W significantly impaired differentiation in primary cultured hippocampal neurons. Acute expression of RAC3-R66W in vivo by in utero electroporation resulted in impairments in cortical neuron migration and axonal elongation during corticogenesis. Collectively, these findings suggest that the p.R66W variant may function as an activated version in specific signaling pathways, leading to a distinctive and severe prenatal phenotype through variant-specific mechanisms. Full article

Foeniculum vulgare Mill, commonly known as fennel, is an aromatic herb traditionally used for culinary and medicinal purposes, with potential therapeutic effects on neurological disorders. However, limited research has focused on its neurotrophic impact, particularly on neuronal maturation and synaptic development. This study investigates the neurotrophic effects of F. vulgare ethanol extracts (FVSE) on the maturation of rat primary hippocampal neurons. Results show that FVSE and its prominent component, anethole, significantly promote neurite outgrowth in a dose-dependent manner. Optimal axonal and dendritic growth occurred at concentrations of 40 µg/mL FVSE and 20 µM anethole, respectively, without causing cytotoxicity, underscoring the safety of FVSE for neuronal health. Additionally, FVSE enhances the formation of synapses, essential for neuronal communication. Network pharmacology analysis revealed that FVSE components influence critical neurotrophic pathways, including PI3K-AKT and Alzheimer’s disease pathways. Specifically, FVSE modulates key proteins, including tropomyosin receptor kinase (Trk), glycogen synthase kinase 3 (GSK3β^ser9), phosphatidylinositol 3-kinase (PI3K), and extracellular signal-regulated protein kinase (Erk1/2). Anethole was found to play a key role in regulating these pathways, which was confirmed by immunocytochemistry experiments demonstrating its effect on promoting neuronal growth and synaptic development. In conclusion, this study highlights the neurotrophic properties of FVSE, with anethole emerging as a critical bioactive compound. These findings provide valuable insights into the therapeutic potential of fennel in treating neurological disorders, offering a basis for future research into interventions promoting neuronal growth and survival. Full article

Optogenetics is a combination of optical and genetic technologies used to activate or, conversely, inhibit specific cells in living tissues. The possibilities of using optogenetics approaches for the treatment of epilepsy, Parkinson’s and Alzheimer’s disease (AD) are being actively researched. In recent years, it has become clear that one of the most important players in the development of AD is astrocytes. Astrocytes affect amyloid clearance, participate in the development of neuroinflammation, and regulate the functioning of neural networks. We used an adeno-associated virus carrying the glial fibrillary acidic protein (GFAP) promoter driving the optogenetic channelrhodopsin-2 (ChR2) gene to transduce astrocytes in primary mouse hippocampal cultures. We recorded the bioelectrical activity of neural networks from day 14 to day 21 of cultivation using multielectrode arrays. A single optogenetic stimulation of astrocytes at 14 day of cultivation (DIV14) did not cause significant changes in neural network bioelectrical activity. Chronic optogenetic stimulation from DIV14 to DIV21 exerts a stimulatory effect on the bioelectrical activity of primary hippocampal cultures (the proportion of spikes included in network bursts significantly increased since DIV19). Moreover, chronic optogenetic stimulation over seven days partially preserved the activity and functional architecture of neuronal network in amyloidosis modeling. These results suggest that the selective optogenetic activation of astrocytes may represent a promising novel therapeutic strategy for combating AD. Full article

In this study, the distinct patterns of glial response and neurodegeneration within the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus were examined in 5XFAD mice at 6 and 12 months of age. The primary feature of this transgenic mouse model is the rapid onset of amyloid pathology. We employed quantitative assessments via immunohistochemistry, incorporating double staining techniques, followed by observation with light microscopy and subsequent digital analysis of microscopic images. We identified significantly increased Aβ deposition in these three hippocampal regions at 6 and 12 months of transgenic mice. Moreover, the CA1 and CA3 regions showed higher vulnerability, with signs of reactive astrogliosis such as increased astrocyte density and elevated GFAP expression. Additionally, we observed a significant rise in microglia density, along with elevated inflammatory markers (TNFα) in these hippocampal regions. These findings highlight a non-uniform glial and neuronal response to Aβ plaque deposition within the hippocampal regions of 5xFAD mice, potentially contributing to the neurodegenerative and memory deficit characteristics of Alzheimer’s disease in this model. Full article

The insulin-regulated aminopeptidase (IRAP; oxytocinase) is part of the M1 aminopeptidase family and is highly expressed in many tissues, including the neocortex and hippocampus of the brain. IRAP is involved in various physiological functions and has been identified as a receptor for the endogenous hexapeptide Angiotensin IV (Ang IV). The binding of Ang IV inhibits the enzymatic activity of IRAP and has been proven to enhance learning and memory in animal models. The macrocyclic compound 9 (C9) is a potent synthetic IRAP inhibitor developed from the previously reported inhibitor HA08. In this study, we have examined compound C9 and its effects on cognitive markers drebrin, microtubule-associated protein 2 (MAP2), and glial fibrillary acidic protein (GFAP) in primary hippocampal and cortical cultures. Cells from Sprague Dawley rats were cultured for 14 days before treatment with C9 for 4 consecutive days. The cells were analysed for protein expression of drebrin, MAP2, GFAP, glucose transporter type 4 (GLUT4), vesicular glutamate transporter 1 (vGluT1), and synapsin I using immunocytochemistry. The gene expression of related proteins was determined using qPCR, and viability assays were performed to evaluate toxicity. The results showed that protein expression of drebrin and MAP2 was increased, and the corresponding mRNA levels were decreased after treatment with C9 in the hippocampal cultures. The ratio of MAP2-positive neurons and GFAP-positive astrocytes was altered and there were no toxic effects observed. In conclusion, the IRAP inhibitor compound C9 enhances the expression of the pro-cognitive markers drebrin and MAP2, which further confirms IRAP as a relevant pharmaceutical target and C9 as a promising candidate for further investigation. Full article

Complement C5a protein has been shown to play a major role in tissue regeneration through interaction with its receptor (C5aR) on target cells. Expression of this receptor has been reported in the nervous system which, upon injury, has no treatment to restore the lost functions. This work aimed at investigating the Complement C5a effect on axonal growth after axotomy in vitro. Primary hippocampal neurons were isolated from embryonic Wistar rats. Cell expression of C5aR mRNA was verified by RT-PCR while its membrane expression, localization, and phosphorylation were investigated by immunofluorescence. Then, the effects of C5a on injured axonal growth were investigated using a 3D-printed microfluidic device. Immunofluorescence demonstrated that the primary cultures contained only mature neurons (93%) and astrocytes (7%), but no oligodendrocytes or immature neurons. Immunofluorescence revealed a co-localization of NF-L and C5aR only in the mature neurons where C5a induced the phosphorylation of its receptor. C5a application on injured axons in the microfluidic devices significantly increased both the axonal growth speed and length. Our findings highlight a new role of C5a in regeneration demonstrating an enhancement of axonal growth after axotomy. This may provide a future therapeutic tool in the treatment of central nervous system injury. Full article

Background: The peptidyl-prolyl isomerase (PIN1) plays a vital role in cellular processes, including intracellular signaling and apoptosis. While oxidative stress is considered one of the primary mechanisms of pathogenesis in brain ischemic injury, the precise function of PIN1 in this disease remains to be elucidated. Objective: We constructed a cell-permeable PEP-1–PIN1 fusion protein and investigated PIN1’s function in HT-22 hippocampal cells as well as in a brain ischemic injury gerbil model. Methods: Transduction of PEP-1–PIN1 into HT-22 cells and signaling pathways were determined by Western blot analysis. Intracellular reactive oxygen species (ROS) production and DNA damage was confirmed by DCF-DA and TUNEL staining. Cell viability was determined by MTT assay. Protective effects of PEP-1-PIN1 against ischemic injury were examined using immunohistochemistry. Results: PEP-1–PIN1, when transduced into HT-22 hippocampal cells, inhibited cell death in H₂O₂-treated cells and markedly reduced DNA fragmentation and ROS production. This fusion protein also reduced phosphorylation of mitogen-activated protein kinase (MAPK) and modulated expression levels of apoptosis-signaling proteins in HT-22 cells. Furthermore, PEP-1–PIN1 was distributed in gerbil hippocampus neuronal cells after passing through the blood–brain barrier (BBB) and significantly protected against neuronal cell death and also decreased activation of microglia and astrocytes in an ischemic injury gerbil model. Conclusions: These results indicate that PEP-1–PIN1 can inhibit ischemic brain injury by reducing cellular ROS levels and regulating MAPK and apoptosis-signaling pathways, suggesting that PIN1 plays a protective role in H₂O₂-treated HT-22 cells and ischemic injury gerbil model. Full article

The branched architecture of neuronal dendrites is a key factor in how neurons form ordered networks and discoveries continue to be made identifying proteins and protein–protein interactions that direct or execute the branching and extension of dendrites. Our prior work showed that the molecular scaffold Pdlim5 and delta-catenin, in conjunction, are two proteins that help regulate the branching and elongation of dendrites in cultured hippocampal neurons and do so through a phosphorylation-dependent mechanism triggered by upstream glutamate signaling. In this report we have focused on Pdlim5’s multiple scaffolding domains and how each contributes to dendrite branching. The three identified regions within Pdlim5 are the PDZ, DUF, and a trio of LIM domains; however, unresolved is the intra-molecular conformation of Pdlim5 as well as which domains are essential to regulate dendritic branching. We address Pdlim5’s structure and function by examining the role of each of the domains individually and using deletion mutants in the context of the full-length protein. Results using primary hippocampal neurons reveal that the Pdlim5 DUF domain plays a dominant role in increasing dendritic branching. Neither the PDZ domain nor the LIM domains alone support increased branching. The central role of the DUF domain was confirmed using deletion mutants in the context of full-length Pdlim5. Guided by molecular modeling, additional domain mapping studies showed that the C-terminal LIM domain forms a stable interaction with the N-terminal PDZ domain, and we identified key amino acid residues at the interface of each domain that are needed for this interaction. We posit that the central DUF domain of Pdlim5 may be subject to modulation in the context of the full-length protein by the intra-molecular interaction between the N-terminal PDZ and C-terminal LIM domains. Overall, our studies reveal a novel mechanism for the regulation of Pdlim5’s function in the regulation of neuronal branching and highlight the critical role of the DUF domain in mediating these effects. Full article

Perinatal asphyxia (PA) and hypoxic-ischemic encephalopathy can result in severe, long-lasting neurological deficits. In vitro models, such as oxygen–glucose deprivation (OGD), are used experimentally to investigate neuronal response to metabolic stress. However, multiple variables can affect the severity level of OGD/PA and may confound any measured treatment effect. Oxytocin (OXT) has emerged as a potential neuroprotective agent against the deleterious effects of PA. Previous studies have demonstrated OXT’s potential to enhance neuronal survival in immature hippocampal cultures exposed to OGD, possibly by modulating gamma-aminobutyric acid-A receptor activity. Moreover, OXT’s precise impact on developing hippocampal neurons under different severities of OGD/PA remains uncertain. In this study, we investigated the effects of OXT (0.1 µM and 1 µM) on 7-day-old primary rat hippocampal cultures subjected to 2 h OGD/sham normoxic conditions. Cell culture viability was determined using the resazurin assay. Our results indicate that the efficacy of 1 µM OXT treatment varied according to the severity of the OGD-induced lesion, exhibiting a protective effect (p = 0.022) only when cellular viability dropped below 49.41% in non-treated OGD cultures compared to normoxic ones. Furthermore, administration of 0.1 µM OXT did not yield significant effects, irrespective of lesion severity (p > 0.05). These findings suggest that 1 µM OXT treatment during OGD confers neuroprotection exclusively in severe lesions in hippocampal neurons after 7 days in vitro. Further research is warranted to elucidate the mechanisms involved in OXT-mediated neuroprotection. Full article