Search Results (99)

Background: Epigenetic mechanisms and RNA signalling profoundly impact body growth during the early stages of embryonic development. RNA molecules, like microRNAs, play a vital role in early embryonic development, laying the groundwork for future growth and function. miR-124-3p microinjected into mouse fertilised eggs (miR-124-3p*) exhibited a significantly overgrowth phenotype. Behavioural test results showed that miR-124-3p mice were more physically active, as indicated by total distance and movement velocity. However, the molecular mechanism leading to these phenotypic changes mediated by miR-124-3p remains a mystery. This study aimed to investigate the role of epidermal growth factor (EGF) in developing an overgrowth phenotype in miR-124-3p* mice. Results: In this research, we preferred to work with neurospheres (NSs) due to the challenges of handling a single embryo, as NSs exhibit similar features, especially regarding cell growth, differentiation, and capacity for self-renewal. We examined the mRNA expression levels of Sox8, Sox9, Sox10, Doublecortin (Dcx), and Neurod1 genes, which are linked to a tiny phenotype in knockout mice, in total embryos at E7.5 and hippocampal cells isolated from E19.5-day fetus and neurospheres aged 12 and 21 days, which were derived from these hippocampal cells through primary cell culture. These genes are significantly overexpressed in miR-124-3p* NSs, but not in the E7.5 total embryos or the hippocampus of the E19.5 fetus. Conclusions: These findings suggest a possible link between miR-124-3p microinjection and EGF activation, which may be associated with early neurogenesis and neuronal differentiation in embryos. This molecular shift might contribute to the development of mice exhibiting increased physical activity and enlarged body size, although these observations remain correlative and require further validation. Full article

5-HT4 receptors play an important role in the regulation of synaptic plasticity. However, the effect of 5-HT4Rs on neural network activity and intercellular calcium signaling remains enigmatic. Using calcium imaging and original software, we determined the network-level characteristics of calcium dynamics within primary hippocampal cultures. We found that the single activation of 5-HT4 receptors by BIMU8 significantly reduced the correlation of activity within neuron–glial networks of primary cultures, without altering the proportion of active cells or the frequency of calcium events. In contrast, chronic stimulation of 5-HT4Rs promoted greater cell involvement in Ca²⁺ signal generation and increased the frequency of calcium events, while maintaining the connectivity level of the neuron–glial network. Moreover, our immunocytochemical labeling results indicated that chronic stimulation of 5-HT4Rs increased the size of both presynaptic and postsynaptic terminals. The acute blockade of 5-HT4Rs by RS23597-190 exerted a marked inhibitory effect on calcium activity in primary hippocampal cultures. Network connectivity and correlation of calcium activity were disrupted, and the number of functional connections among cells sharply declined. Our study showed that 5-HT4 receptors exhibit diverse effects based on the type and duration of activation, mediating several key functions in regulating neural network calcium activity. Full article

The piperazine derivative N-(2,6-difluorophenyl)-2-(4-phenylpiperazin-1-yl)propanamide (cmp2) has emerged as a potential transient receptor potential cation channel, subfamily C, member 6 (TRPC6) modulator, offering a promising pathway for Alzheimer’s disease (AD) therapy. Our recent findings identify cmp2 as a novel compound with synaptoprotective effects in primary hippocampal cultures and effective blood–brain barrier (BBB) penetration. In vivo studies demonstrate that cmp2 (10 mg/kg, intraperitoneally) restores synaptic plasticity deficits in 5xFAD mice. This study further shows cmp2’s selectivity towards tetrameric TRPC6 channel in silico. Acute administration of cmp2 is non-toxic, with no indications of chronic toxicity, and Ames testing confirms its lack of mutagenicity. Behavioral assays reveal that cmp2 improves cognitive functions in 5xFAD mice, including increased novel object recognition, better passing of the Morris water maze, and improved fear memory, as well as upregulation of motor function in beam walking tests. These findings suggest that cmp2 holds promise as a candidate for AD treatment. 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

Maternal depression negatively affects the neurodevelopment of offspring, but its pharmacological treatment during gestation remains controversial. This study reports the consequences of maternal depression and/or prenatal antidepressant treatment with mirtazapine on offspring early neurodevelopment via an animal model of maternal depression induced by pregestational chronic unpredictable stress (CUS). Offspring from four groups were studied: nonstressed vehicle-treated dams, nonstressed mirtazapine-treated dams, stressed vehicle-treated dams, and stressed mirtazapine-treated dams. The hippocampal excitability of offspring was examined in primary hippocampal cultures established on the first postnatal day, reflecting mostly prenatal development, and in hippocampal slices prepared on postnatal days 11–13, reflecting an early postnatal development. The pregestational CUS modeling of maternal depression moderately suppressed offspring hippocampal excitability in primary cultures but facilitated it in slices. Mirtazapine administered to CUS-exposed dams partly rectified the changes observed in primary cultures of pups from untreated dams and, more prominently, in slices. Mirtazapine itself negatively affected the hippocampal excitability of nonstressed dam offspring in primary culture, and this effect was diminished in slices. Since altered hippocampal neurotransmission might be responsible, at least in part, for the neuropsychopathologies frequently observed in the offspring of depressed mothers, and mirtazapine was able to partly relieve such changes, this treatment may be also beneficial during the prenatal and perinatal periods. 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

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

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

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

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

The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and neurological dysfunctions. The SARS-CoV-2 envelope protein (E protein) is a structural viroporin able to form ion channels in cell membranes, which is critical for viral replication. However, its effects in primary neurons have not been addressed. Here we used fluorescence microscopy and calcium imaging to study SARS-CoV-2 viroporin E localization and the effects on neuron damage and intracellular Ca²⁺ homeostasis in a model of rat hippocampal neurons aged in vitro. We found that the E protein quickly enters hippocampal neurons and colocalizes with the endoplasmic reticulum (ER) in both short-term (6–8 days in vitro, DIV) and long-term (20–22 DIV) cultures resembling young and aged neurons, respectively. Strikingly, E protein treatment induces apoptosis in aged neurons but not in young neurons. The E protein induces variable increases in cytosolic Ca²⁺ concentration in hippocampal neurons. Ca²⁺ responses to the E protein are due to Ca²⁺ release from intracellular stores at the ER. Moreover, E protein-induced Ca²⁺ release is very small in young neurons and increases dramatically in aged neurons, consistent with the enhanced Ca²⁺ store content in aged neurons. We conclude that the SARS-CoV-2 E protein quickly translocates to ER endomembranes of rat hippocampal neurons where it releases Ca²⁺, probably acting like a viroporin, thus producing Ca²⁺ store depletion and neuron apoptosis in aged neurons and likely contributing to neurological damage in COVID-19 patients. Full article

Development and optimisation of bioelectronic monitoring techniques like microelectrode array-based field potential measurement and impedance spectroscopy for the functional, label-free and non-invasive monitoring of in vitro neuronal networks is widely investigated in the field of biosensors. Thus, these techniques were individually used to demonstrate the capabilities of, e.g., detecting compound-induced toxicity in neuronal culture models. In contrast, extended application for investigating the effects of central nervous system infecting viruses are rarely described. In this context, we wanted to analyse the effect of herpesviruses on functional neuronal networks. Therefore, we developed a unique hybrid bioelectronic monitoring platform that allows for performing field potential monitoring and impedance spectroscopy on the same microelectrode. In the first step, a neuronal culture model based on primary hippocampal cells from neonatal rats was established with reproducible and stable synchronised electrophysiological network activity after 21 days of cultivation on microelectrode arrays. For a proof of concept, the pseudorabies model virus PrV Kaplan-ΔgG-GFP was applied and the effect on the neuronal networks was monitored by impedance spectroscopy and field potential measurement for 72 h in a multiparametric mode. Analysis of several bioelectronic parameters revealed a virus concentration-dependent degeneration of the neuronal network within 24–48 h, with a significant early change in electrophysiological activity, subsequently leading to a loss of activity and network synchronicity. In conclusion, we successfully developed a microelectrode array-based hybrid bioelectronic measurement platform for quantitative monitoring of pathologic effects of a herpesvirus on electrophysiological active neuronal networks. Full article