Search Results (63)

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

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

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

Alzheimer’s disease (AD) is the main cause of dementia worldwide. Given that learning and memory are impaired in this pathology, NMDA receptors (NMDARs) appear as key players in the onset and progression of the disease. NMDARs are glutamate receptors, mainly located at the post-synapse, which regulate voltage-dependent influx of calcium into the neurons. They are heterotetramers, and there are different subunits that can be part of the receptors, which are usually composed of two obligatory GluN1 subunits plus either two NR2A or two NR2B subunits. NR2A are mostly located at the synapse, and their activation is involved in the expression of pro-survival genes. Conversely, NR2B are mainly extrasynaptic, and their activation has been related to cell death and neurodegeneration. Thus, activation of NR2A and/or inactivation of NR2B-containing NMDARS has been proposed as a therapeutic strategy to treat AD. Here, we wanted to investigate the main differences between both subunits signalling in neuronal primary cultures of the cortex and hippocampus. It has been observed that Aβ induces a significant increase in calcium release and also in MAPK phosphorylation signalling in NR2B-containing NMDAR in cortical and hippocampal neurons. However, while NR2A-containing NMDAR decreases neuronal death and favours cell viability after Aβ treatment, NR2B-containing NMDAR shows higher levels of cytotoxicity and low levels of neuronal survival. Finally, it has been detected that NMDAR has no effect on pTau axonal transport. The present results demonstrate a different role between GluNA and GluNB subunits in neurodegenerative diseases such as Alzheimer’s. Full article

Microglia and astrocytes are essential in sustaining physiological networks in the central nervous system, with their ability to remodel the extracellular matrix, being pivotal for synapse plasticity. Recent findings have challenged the traditional view of homogenous glial populations in the brain, uncovering morphological, functional, and molecular heterogeneity among glial cells. This diversity has significant implications for both physiological and pathological brain states. In the present study, we mechanically induced a Schaffer collateral lesion (SCL) in mouse entorhino–hippocampal slice cultures to investigate glial behavior, i.e., microglia and astrocytes, under metalloproteinases (MMPs) modulation in the lesioned area, CA3, and the denervated region, CA1. We observed distinct response patterns in the microglia and astrocytes 3 days after the lesion. Notably, GFAP-expressing astrocytes showed no immediate changes post-SCL. Microglia responses varied depending on their anatomical location, underscoring the complexity of the hippocampal neuroglial network post-injury. The MMPs inhibitor GM6001 did not affect microglial reactions in CA3, while increasing the number of Iba1-expressing cells in CA1, leading to a withdrawal of their primary branches. These findings highlight the importance of understanding glial regionalization following neural injury and MMPs modulation and pave the way for further research into glia-targeted therapeutic strategies for neurodegenerative disorders. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is characterized by amyloid-beta (Aβ) plaques and tau neurofibrillary tangles (NFT). Modelling aspects of AD is challenging due to its complex multifactorial etiology and pathology. The present study aims to establish a cost-effective and rapid method to model the two primary pathologies in organotypic brain slices. Coronal hippocampal brain slices (150 µm) were generated from postnatal (day 8–10) C57BL6 wild-type mice and cultured for 9 weeks. Collagen hydrogels containing either an empty load or a mixture of human Aβ42 and P301S aggregated tau were applied to the slices. The media was further supplemented with various intracellular pathway modulators or heavy metals to augment the appearance of Aβ plaques and tau NFTs, as assessed by immunohistochemistry. Immunoreactivity for Aβ and tau was significantly increased in the ventral areas in slices with a mixture of human Aβ42 and P301S aggregated tau compared to slices with empty hydrogels. Aβ plaque- and tau NFT-like pathologies could be induced independently in slices. Heavy metals (aluminum, lead, cadmium) potently augmented Aβ plaque-like pathology, which developed intracellularly prior to cell death. Intracellular pathway modulators (scopolamine, wortmannin, MHY1485) significantly boosted tau NFT-like pathologies. A combination of nanomolar concentrations of scopolamine, wortmannin, MHY1485, lead, and cadmium in the media strongly increased Aβ plaque- and tau NFT-like immunoreactivity in ventral areas compared to the slices with non-supplemented media. The results highlight that we could harness the potential of the collagen hydrogel-based spreading of human Aβ42 and P301S aggregated tau, along with pharmacological manipulation, to produce pathologies relevant to AD. The results offer a novel ex vivo organotypic slice model to investigate AD pathologies with potential applications for screening drugs or therapies in the future. Full article

The primary neuronal and astrocyte culture described here is from the stress-hyperreactive Wistar Kyoto (WKY) More Immobile (WMI) rat with premature aging-related memory deficit, and its nearly isogenic control, the Less Immobile (WLI) strain. Primary WMI hippocampal neurons and cortical astrocytes are significantly more sensitive to oxidative stress (OS) generated by administration of H₂O₂ compared to WLI cells as measured by the trypan blue cell viability assay. Intrinsic genetic vulnerability is also suggested by the decreased gene expression in WMI neurons of catalase (Cat), and in WMI cortical astrocytes of insulin-like growth factor 2 (Igf2), synuclein gamma (Sncg) and glutathione peroxidase 2 (Gpx2) compared to WLI. The expressions of several mitochondrial genes are dramatically increased in response to H₂O₂ treatment in WLI, but not in WMI cortical astrocytes. We propose that the vulnerability of WMI neurons to OS is due to the genetic differences between the WLI and WMI. Furthermore, the upregulation of mitochondrial genes may be a compensatory response to the generation of free radicals by OS in the WLIs, and this mechanism is disturbed in the WMIs. Thus, this pilot study suggests intrinsic vulnerabilities in the WMI hippocampal neurons and cortical astrocytes, and affirm the efficacy of this bimodal in vitro screening system for finding novel drug targets to prevent oxidative damage in illnesses. Full article

High levels of alcohol intake alter brain gene expression and can produce long-lasting effects. FK506-binding protein 51 (FKBP51) encoded by Fkbp5 is a physical and cellular stress response gene and has been associated with alcohol consumption and withdrawal severity. Fkbp5 has been previously linked to neurite outgrowth and hippocampal morphology, sex differences in stress response, and epigenetic modification. Presently, primary cultured Fkbp5 KO and WT mouse neurons were examined for neurite outgrowth and mitochondrial signal with and without alcohol. We found neurite specification differences between KO and WT; particularly, mesh-like morphology was observed after alcohol treatment and confirmed higher MitoTracker signal in cultured neurons of Fkbp5 KO compared to WT at both naive and alcohol-treated conditions. Brain regions that express FKBP51 protein were identified, and hippocampus was confirmed to possess a high level of expression. RNA-seq profiling was performed using the hippocampus of naïve or alcohol-injected (2 mg EtOH/Kg) male and female Fkbp5 KO and WT mice. Differentially expressed genes (DEGs) were identified between Fkbp5 KO and WT at baseline and following alcohol treatment, with female comparisons possessing a higher number of DEGs than male comparisons. Pathway analysis suggested that genes affecting calcium signaling, lipid metabolism, and axon guidance were differentially expressed at naïve condition between KO and WT. Alcohol treatment significantly affected pathways and enzymes involved in biosynthesis (Keto, serine, and glycine) and signaling (dopamine and insulin receptor), and neuroprotective role. Functions related to cell morphology, cell-to-cell signaling, lipid metabolism, injury response, and post-translational modification were significantly altered due to alcohol. In summary, Fkbp5 plays a critical role in the response to acute alcohol treatment by altering metabolism and signaling-related genes. Full article

S100B is a calcium-binding protein produced and secreted by astrocytes in response to various extracellular stimuli. C6 glioma cells are a lineage commonly employed for astroglial studies due to the expression of astrocyte specific markers and behavior. However, in high-glucose medium, C6 S100B secretion increases, in contrast to the trend in primary astrocyte cultures. Additionally, S100B secretion decreases due to fluorocitrate (FC), a Krebs cycle inhibitor, highlighting a connection between S100B and metabolism. Herein, we investigate the impact of FC on S100B secretion in primary astrocyte cultures, acute hippocampal slices and C6 glioma cells, as well as lactate mediation. Our results demonstrated that C6 responded similarly to astrocytes in various parameters, despite the decrease in S100B secretion, which was inversely observed in astrocytes and slices. Furthermore, FC inversely altered extracellular lactate in both models, suggesting a role for lactate in S100B secretion. This was reinforced by a decrease in S100B secretion in hippocampal slices treated with lactate and its agonist, but not in C6 cells, despite HCAR1 expression. Our findings indicate that extracellular lactate mediates the decrease in S100B secretion in astrocytes exposed to FC. They also emphasize the differences in C6 glioma cells regarding energetic metabolism. The proposed mechanism via HCAR1 provides further compelling evidence of the relationship between S100B and glucose metabolism. Full article