Search Results (112)

Background: The retina, a light-sensitive tissue of the central nervous system that is located at the posterior part of the eye, is particularly vulnerable to alterations in oxygen levels. In various retinal diseases, such as central retinal vein occlusion, glaucoma, and diabetic retinopathy, hypoxia (a condition of low oxygen levels) is commonly observed. Müller glia, the principal glial cells in the retina, play a crucial role in supporting the metabolic needs of retinal neurons. They are also responsible for sensing oxygen levels and, in response to hypoxia, express Hypoxia-Inducible Factor 1 (HIF-1), a transcription factor that activates signaling pathways related to hypoxia. Methods: In this study, primary rat Müller glial cells were cultured and exposed to a 1% oxygen for 72 h. Following this, immunohistochemical assays were conducted to assess the effects of hypoxia on various parameters, including HIF-1α expression, cell survival, Müller glia-specific markers (CRALBP and GS), gliosis (GFAP expression), apoptosis (caspase-3 expression), cell proliferation (Ki-67 expression), and metabolic stress (indicated by the number of mitochondria per cell). Results: Under hypoxic conditions, a decrease in Müller glial survival and proliferation was observed. Conversely, there was an increase in HIF-1α expression, GFAP expression, caspase-3-positive cells, and the number of mitochondria per cell. However, no significant changes were noted in the expression of the Müller glial markers GS and CRALBP. Conclusions: In conclusion, hypoxia resulted in reduced proliferation and survival of Müller glial cells, primarily due to increased apoptosis and heightened metabolic stress. Full article

The nervous system maintains homeostasis and coordinated behavior through complex neuronal and glial cells. Traditional models, such as primary rodent neurons and human-induced pluripotent stem cell (hIPSC)-derived neurons, have advanced our understanding of neuronal function and neurotoxic damage; however, they are costly and labor-intensive. SH-SY5Y cells, an immortalized human neuroblastoma cell line, provide a more accessible alternative for studying neuronal processes and neurotoxicity. However, their limited capacity to differentiate into specific neuronal phenotypes remains a challenge. To address this limitation, differentiation protocols using neuronal factors and vitamins have been developed, primarily in two-dimensional (2D) cultures, which reduces physiological relevance. Here, we present a novel three-dimensional (3D) SH-SY5Y model incorporating 2D differentiation protocols to generate cholinergic neurons (ChAT+). This model enhances neurotoxicity studies related to pesticides and mycotoxins. Our protocol produces homogeneous spheroids differentiated into cholinergic neurons using serum restriction and specific factors, maintaining viability and circularity for up to 22 days. Differentiation was validated by immunofluorescence and Western blot by Choline acetyltransferase (ChAT) expression. This scalable and reproducible 3D model provides a valuable in vitro tool for neurotoxicological research, improving physiological relevance and enabling the study of cholinergic neuron differentiation and function. Full article

Epigenetic rearrangements can create a favorable environment for the intrinsic plasticity of brain cells, leading to cellular reprogramming into virtually any cell type through the induction of cell-specific transcriptional programs. In this study, we assessed how chromatin remodeling induced by broad-spectrum HDAC inhibitors affects cellular differentiation trajectories in rat primary neuron–glia cultures using a combination of transcriptomics, qPCR, and cytochemistry. We described the epigenetic regulation of transcriptional programs controlled by master transcription factors and neurotrophins in the context of neuronal and glial differentiation and evaluated the expression of representative cell-specific markers. The results obtained suggest that HDAC inhibitors reduce the proliferative potential of cultured cells and induce transcriptomic changes associated with cell differentiation and specialization. Particularly, we revealed a significant upregulation of genes typically expressed in neuromodulatory neurons and the downregulation of genes expressed in glia and inhibitory neurons. Transcriptional changes were accompanied by continuous elevation of histone serotonylation levels in both neurons and glia. Emerging shortly after HDAC inhibition, a complex chromatin remodeling, which includes histone serotonylation, persists over many hours in distinct brain cells. We assume that this sustained epigenetic mechanism likely helps to maintain transcriptional changes associated with cell fate commitment, possibly priming cells for long-term fate conversion. Full article

Arsenic, a potent metalloid contaminant of drinking water, is known for its ability to act as an initiator and modulator of disease in a variety of human tissues. Upon ingestion, arsenic is bio-transformed in the liver into a variety of metabolites, including arsenite. Arsenite permeates the blood–brain barrier (BBB), inducing oxidative stress that can be detrimental to brain neurons. As the primary glial cell at the BBB interface, astrocytes play a pivotal role in detoxifying xenobiotics such as arsenite via the production of the tripeptide antioxidant γ-glutamylcysteine, or glutathione (GSH). In this study, we assessed the mRNA levels of key components of the GSH synthetic pathway in astrocytes exposed to arsenite compared to vehicle controls. These components included xCT [substrate-specific light chain of the substrate importing transporter, system x_c⁻ (Sx_c⁻)], glutamate-cysteine ligase [both catalytic (GCL_C) and modifying (GCL_M) subunits], and glutathione synthetase (GS). Additionally, we analyzed protein levels of some components by Western blotting and evaluated functional activity of Sx_c⁻ using a fluorescence-based cystine uptake assay. Finally, we utilized a luminescence-based glutathione assay to determine the intracellular and extracellular GSH content in arsenite-treated cells. Arsenite significantly increased xCT, GCL_C, GCL_M, and GS mRNA levels, an effect blocked by the transcriptional inhibitor actinomycin D (ActD). A corresponding increase in Sx_c⁻ activity was also observed in the arsenite treatment groups, along with significant increases in GCL_C and GCL_M protein expression. However, no increase in GS protein expression was detected. Finally, arsenite treatment significantly increased extracellular GSH levels, an effect which was also prevented by the inclusion of ActD. Overall, our study provides evidence that arsenite transcriptionally regulates several cellular processes necessary for GSH synthesis in primary cortical astrocyte cultures, thereby contributing to a better understanding of how this environmental toxicant influences antioxidant defenses in the brain. However, these results should be interpreted with caution regarding their applicability to vivo systems. Full article

Primary cultures of neural cells are important key tools for basic and translational neuroscience research. These primary cell cultures are classically generated from the rodent brain hippocampus or cortex and optimized for enrichment in neurons at the expense of glial cells. Importantly, considerable differences exist in neuronal cell populations and in glial cell contribution between different brain regions. Because many basic and translational research projects aim to identify mechanisms underlying brainstem neuronal networks that affect major vital functions, primary cultures representative of cell populations present in the hindbrain are required. However, the preparation of primary cultures of brainstem/hindbrain neurons is scarcely described in the literature, limiting the possibilities for studying the development and physiology of these brain regions in vitro. The present report describes a reliable protocol to dissociate and culture in vitro embryonic mouse fetal hindbrain neurons in a defined culture medium, while control of astrocytes’ expansion was attained by using a chemically defined, serum-free supplement, namely CultureOne™. The neuronal cells maintained according to this protocol differentiate and, by 10 days in vitro, they develop extensive axonal and dendritic branching. Using immunofluorescence, we further characterized the different cell populations and neuronal subtypes. Patch–clamp recordings demonstrate the excitable nature of these neurons, while colocalization of pre- and postsynaptic neuronal markers showed that neurons form mature synapses, suggesting the establishment of functional networks in vitro. The cultures produced by this method show excellent reproducibility and can be used for molecular, biochemical, and physiological analyses, as illustrated here for tamoxifen-induced Cre recombination in genetically-modified neural cells. Full article

Innovations in spinal cord injury (SCI) models are crucial for developing effective therapies. This study introduces a novel in vitro SCI model using cultures of primary mixed spinal cord cells from rat pups, featuring key spinal cord cell types. This model offers distinct advantages in terms of feasibility, reproducibility, and cost-effectiveness, requiring only basic cell culture equipment. Following hyperosmotic stress via sorbitol treatment, the model recapitulated SCI pathophysiological hallmarks, with a 65% reduction in cell viability and gradual cell death over 48 h, making it ideal for evaluating neuroprotective agents. Notably, the human adipose tissue stem cell (hASC) secretome provided significant protection: it preserved metabolic viability, reduced β amyloid precursor protein (β-APP) expression in surviving neurons, and modulated the shift in the astrocytic morphotype. A transcriptomic profile of the effect of the hASC secretome treatment showed significant functional enrichments related to cell proliferation and cycle progression pathways. In addition to supporting the use of the hASC secretome as a therapy for SCI, this study is the first to use sorbitol as a hyperosmolar stressor to recapitulate key aspects of SCI pathophysiology. Thereby, this model can be used as a promising platform for evaluating therapeutic agents targeting neuroprotection and neuroregeneration, offering outputs related to cell death, neuronal stress, and protection, as well as induction of glial reactivity. 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

Alexander disease (AxD) is an intractable neurodegenerative disease caused by mutations in glial fibrillary acidic protein (GFAP), which is predominantly expressed in astrocytes. Thus, AxD is a primary astrocyte disease. However, it remains unclear how GFAP mutations affect astrocytes and cause AxD pathology. Three features are characteristic of AxD astrocytes in vivo: (1) Rosenthal fibers (RFs), the hallmark of AxD; (2) aberrant Ca²⁺ signals (AxCa); and (3) upregulation of disease-associated genes (AxGen). We established a primary culture system for astrocytes from an AxD transgenic mouse model, and used it to analyze the above features of AxD pathogenesis in astrocytes in vitro. We observed the formation of RFs in AxD primary cultures. The abundance of RFs was greater in AxD-transgene-homozygous compared with -hemizygous astrocytes, indicating a gene dosage effect, and this abundance increased with time in culture, indicating a developmental process effect. However, cultured AxD astrocytes did not exhibit changes in either AxCa or AxGen. We therefore conclude that RFs in astrocytes form via a cell-autonomous mechanism, whereas AxCa and AxGen are likely to occur via a non-cell-autonomous mechanism through interactions with other cells, such as neurons, microglia, and vascular cells. Although primary cultured AxD astrocytes are suitable for elucidating the mechanisms of RFs formation and for intervention studies, it should be noted that they cannot reflect the pathophysiology of non-cell-autonomous events in astrocytes. 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

Within the phylum Cnidaria, sea anemones (class Anthozoa) express a rich diversity of ion-channel peptide modulators with biomedical applications, but corollary discoveries from jellyfish (subphylum Medusozoa) are lacking. To bridge this gap, bioactivities of previously unexplored proteinaceous and small molecular weight (~15 kDa to 5 kDa) venom components were assessed in a mouse dorsal root ganglia (DRG) high-content calcium-imaging assay, known as constellation pharmacology. While the addition of crude venom led to nonspecific cell death and Fura-2 signal leakage due to pore-forming activity, purified small molecular weight fractions of venom demonstrated three main, concentration-dependent and reversible effects on defined heterogeneous cell types found in the primary cultures of mouse DRG. These three phenotypic responses are herein referred to as phenotype A, B and C: excitatory amplification (A) or inhibition (B) of KCl-induced calcium signals, and test compound-induced disturbances to baseline calcium levels (C). Most notably, certain Alatina alata venom fractions showed phenotype A effects in all DRG neurons; Physalia physalis and Chironex fleckeri fractions predominantly showed phenotype B effects in small- and medium-diameter neurons. Finally, specific Physalia physalis and Alatina alata venom components induced direct excitatory responses (phenotype C) in glial cells. These findings demonstrate a diversity of neuroactive compounds in jellyfish venom potentially targeting a constellation of ion channels and ligand-gated receptors with broad physiological implications. Full article

Hydrogen sulfide (H₂S), a known inhibitor of the electron transport chain, is endogenously produced in the periphery as well as in the central nervous system, where is mainly generated by glial cells. It affects, as a cellular signaling molecule, many different biochemical processes. In the central nervous system, depending on its concentration, it can be protective or damaging to neurons. In the study, we have demonstrated, in a primary mouse spinal cord cultures, that it is particularly harmful to motor neurons, is produced by glial cells, and is stimulated by inflammation. However, its role on glial cells, especially astrocytes, is still under-investigated. The present study was designed to evaluate the impact of H₂S on astrocytes and their phenotypic heterogeneity, together with the functionality and homeostasis of mitochondria in primary spinal cord cultures. We found that H₂S modulates astrocytes’ morphological changes and their phenotypic transformation, exerts toxic properties by decreasing ATP production and the mitochondrial respiration rate, disturbs mitochondrial depolarization, and alters the energetic metabolism. These results further support the hypothesis that H₂S is a toxic mediator, mainly released by astrocytes, possibly acting as an autocrine factor toward astrocytes, and probably involved in the non-cell autonomous mechanisms leading to motor neuron death. Full article

Neurological symptoms associated with COVID-19, acute and long term, suggest SARS-CoV-2 affects both the peripheral and central nervous systems (PNS/CNS). Although studies have shown olfactory and hematogenous invasion into the CNS, coinciding with neuroinflammation, little attention has been paid to susceptibility of the PNS to infection or to its contribution to CNS invasion. Here we show that sensory and autonomic neurons in the PNS are susceptible to productive infection with SARS-CoV-2 and outline physiological and molecular mechanisms mediating neuroinvasion. Our infection of K18-hACE2 mice, wild-type mice, and golden Syrian hamsters, as well as primary peripheral sensory and autonomic neuronal cultures, show viral RNA, proteins, and infectious virus in PNS neurons, satellite glial cells, and functionally connected CNS tissues. Additionally, we demonstrate, in vitro, that neuropilin-1 facilitates SARS-CoV-2 neuronal entry. SARS-CoV-2 rapidly invades the PNS prior to viremia, establishes a productive infection in peripheral neurons, and results in sensory symptoms often reported by COVID-19 patients. Full article

Background: Transferrin receptor 1 (TfR1), glucose transporter 1 (GLUT1), and CD98hc are candidates for targeted therapy at the blood–brain barrier (BBB). Our objective was to challenge the expression of TfR1, GLUT1, and CD98hc in brain capillaries using the histone deacetylase inhibitor (HDACi) valproic acid (VPA). Methods: Primary mouse brain capillary endothelial cells (BCECs) and brain capillaries isolated from mice injected intraperitoneally with VPA were examined using RT-qPCR and ELISA. Targeting to the BBB was performed by injecting monoclonal anti-TfR1 (Ri7217)-conjugated gold nanoparticles measured using ICP-MS. Results: In BCECs co-cultured with glial cells, Tfrc mRNA expression was significantly higher after 6 h VPA, returning to baseline after 24 h. In vivo Glut1 mRNA expression was significantly higher in males, but not females, receiving VPA, whereas Cd98hc mRNA expression was unaffected by VPA. TfR1 increased significantly in vivo after VPA, whereas GLUT1 and CD98hc were unchanged. The uptake of anti-TfR1-conjugated nanoparticles was unaltered by VPA despite upregulated TfR expression. Conclusions: VPA upregulates TfR1 in brain endothelium in vivo and in vitro. VPA does not increase GLUT1 and CD98hc proteins. The increase in TfR1 does not result in higher anti-TfR1 antibody targetability, suggesting targeting sufficiently occurs with available transferrin receptors without further contribution from accessory VPA-induced TfR1. Full article

Zika virus (ZIKV), a mosquito-borne flavivirus, is prominently associated with microcephaly in babies born to infected mothers as well as Guillain-Barré Syndrome in adults. Each cell type infected by ZIKV—neuronal cells (radial glial cells, neuronal progenitor cells, astrocytes, microglia cells, and glioblastoma stem cells) and non-neuronal cells (primary fibroblasts, epidermal keratinocytes, dendritic cells, monocytes, macrophages, and Sertoli cells)—displays its own characteristic changes to their cell physiology and has various impacts on disease. Here, we provide an in-depth review of the ZIKV life cycle and its cellular targets, and discuss the current knowledge of how infections cause neuropathologies, as well as what approaches researchers are currently taking to further advance such knowledge. A key aspect of ZIKV neuropathogenesis is virus-induced neuronal apoptosis via numerous mechanisms including cell cycle dysregulation, mitochondrial fragmentation, ER stress, and the unfolded protein response. These, in turn, result in the activation of p53-mediated intrinsic cell death pathways. A full spectrum of infection models including stem cells and co-cultures, transwells to simulate blood–tissue barriers, brain-region-specific organoids, and animal models have been developed for ZIKV research. Full article

Limited substrate availability because of the blood–brain barrier (BBB) has made the brain develop specific molecular mechanisms to survive, using lactate synthesized by astrocytes as a source of energy in neurons. To understand if lactate improves cellular viability and susceptibility to glutamate toxicity, primary cortical cells were incubated in glucose- or lactate-containing media and toxic concentrations of glutamate for 24 h. Cell death was determined by immunostaining and lactate dehydrogenase (LDH) release. Mitochondrial membrane potential and nitric oxide (NO) levels were measured using Tetramethylrhodamine, methyl ester (TMRM) and 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate (DAF-FM) live staining, respectively. LDH activity was quantified in single cells in the presence of lactate (LDH substrate) and oxamate (LDH inhibitor). Nuclei of cells were stained with DAPI and neurons with MAP2. Based on the distance between neurons and glial cells, they were classified as linked (<10 µm) and non-linked (>10 µm) neurons. Lactate increased cell death rate and the mean value of endogenous NO levels compared to glucose incubations. Mitochondrial membrane potential was lower in the cells cultured with lactate, but this effect was reversed when glutamate was added to the lactate medium. LDH activity was higher in linked neurons compared to non-linked neurons, supporting the hypothesis of the existence of the lactate shuttle between astrocytes and at least a portion of neurons. In conclusion, glucose or lactate can equally preserve primary cortical neurons, but those neurons having a low level of LDH activity and incubated with lactate cannot cover high energetic demand solely with lactate and become more susceptible to glutamate toxicity. Full article