Search Results (1,556)

Activated protein C (APC) exerts anticoagulant and cytoprotective cell signaling activities. APC’s cell signaling requires protease-activated receptor (PAR) PAR1 and PAR3, and APC’s PAR cleavages generate peptides capable of agonizing biased G-protein coupled receptor (GPCR) cytoprotective signaling, resulting in anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. The PAR-sequence-derived 34-residue “G10 peptide” comprising PAR1 residues 47–55 covalently attached by a 10-glycine linker to PAR3 residues 51–65 is an orthosteric/allosteric bivalent GPCR agonist that potently mimics APC’s anti-inflammatory activity and endothelial barrier stabilization activity. The objective of this study was to determine whether the G10 peptide mimics APC’s anti-apoptotic activity using cultured murine neurons challenged by N-methyl-d-aspartate that provokes neuronal apoptosis. In these new studies, the G10 peptide mimicked APC’s anti-apoptotic activity. Thus, the PAR-derived 34-residue G10 peptide mimics APC’s three major cytoprotective activities, namely anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. Peptides that agonize GPCRs provide promising and currently approved drugs; e.g., semaglutide and tirzepatide that contain 31 and 39 amino acid residues, respectively. Thus, this new study adds to the rationale for pursuing further studies of the G10 peptide for potential therapeutic value for multiple pathologies where APC or signaling-selective APC variants are therapeutic in preclinical animal studies. Full article

Axon polarization is a fundamental process in neuronal development, providing the structural basis for directional signaling in neural circuits. Precise control of axon specification is, thus, essential for the bottom-up construction of neuronal networks with defined architecture and connectivity. Although neurite length and elongation timing have both been implicated as determinants of axonal fate, their relative contributions have remained unresolved due to technical limitations in manipulating these factors independently in conventional culture systems. Here, we developed a constructive neuroengineering platform based on modifiable agarose gel microstructures that enables dynamic, in situ control of neurite outgrowth length and timing during neuronal cultivation. This approach allowed us to directly address whether axon polarization depends primarily on neurite length or the order of neurite extension. Using a single-neurite elongation paradigm, we quantitatively defined two length thresholds for axon specification: a critical length of 43.3 μm, corresponding to a 50% probability of axonal differentiation, and a definitive length of 95.4 μm, beyond which axonal fate was reliably established. In experiments involving simultaneous or sequential elongation of two neurites, we observed that neurite length—not elongation order—consistently predicted axonal identity, even when a second neurite was introduced after the first had already begun to grow. The presence of a competing neurite modestly elevated the effective critical length, suggesting inhibitory interactions that modulate length thresholds. These findings provide the first direct experimental confirmation that neurite length is the primary determinant of axon polarization and demonstrate the utility of constructive microfabrication approaches for dissecting fundamental principles of neuronal polarity. Our platform establishes a powerful experimental foundation for future efforts to achieve complete control over axon and dendrite orientation during the engineered construction of functional neuronal circuits. Full article

Dugongs (Dugong dugon), classified as vulnerable marine mammals, are increasingly impacted by infectious diseases, yet the role of septicemia and disseminated intravascular coagulation (DIC) in their mortality remains uncharacterized. This study aimed to investigate the pathological and microbiological features associated with an acute mortality event in a juvenile dugong during rehabilitation in southern Thailand. Comprehensive histopathological and microbiological analyses were conducted on tissue samples collected postmortem. Bacterial isolation and identification were performed using standard culture techniques and the VITEK-2 system. Histological examination revealed multisystemic lesions, including fibrin thrombi, hemorrhage, hepatocellular degeneration, pancreatic necrosis, lymphoid depletion, and neuronal damage. Oxidative stress and DNA damage were confirmed in brain tissues through immunofluorescence detection of 4-hydroxynonenal (4-HNE) and 8-hydroxy-2′-deoxyguanosine (8-oxodG). Achromobacter xylosoxidans, an opportunistic pathogen, was isolated from multiple organs, consistent with acute systemic infection. These findings represent the first evidence of septicemia-associated DIC in dugongs caused by A. xylosoxidans, highlighting a previously undocumented cause of mortality in dugongs. The results emphasize the role of opportunistic bacteria in triggering oxidative damage and coagulopathy and underscore the importance of early detection and targeted therapeutic strategies to improve survival in stranded or rehabilitated dugongs. Full article

Acute human alpha-herpesvirus 1 (HSV-1) infection culminates in a latent infection of neurons in trigeminal ganglia (TG) and the central nervous system. Following infection of mucosal epithelial cells, certain neurons survive infection and life-long latency is established. Periodically, stressful stimuli trigger reactivation from latency, which result in virus shedding, transmission to other people, and, occasionally, recurrent disease. The glucocorticoid receptor (GR) and Krüppel-like factor 15 (KLF15) comprise a feed-forward transcriptional loop that cooperatively transactivate key HSV-1 promoters that drive expression of infected cell protein 0 (ICP0), ICP4, and ICP27. Silencing KLF15 significantly reduces HSV-1 replication in cultured mouse neuroblastoma cells. Consequently, we hypothesized that KLF15 mediates certain aspects of reactivation from latency. To test this hypothesis, we compared HSV-1 replication in KLF15^−/− mice versus wild-type (wt) parental C57BL/6 mice. Virus shedding during acute infection was reduced in KLF15^−/− mice. Male KLF15^−/− mice shed higher titers of virus during late stages of reactivation from latency compared to KLF15^−/− females and wt mice regardless of sex. At 15 d after explant-induced reactivation, virus shedding was higher in male KLF15^−/− mice relative to wt mice and female KLF15^−/− mice. These studies confirm KLF15 expression enhances viral replication during acute infection and reactivation from latency. Full article

Background/Objectives: Endozepines known as the endogenous ligands of benzodiazepine-binding sites, include the diazepam binding inhibitor (DBI) and its processing products, the triakontatetraneuropeptide (TTN) and the octadecaneuropeptide (ODN). Despite indisputable evidence of the binding of ODN on GABA_AR-BZ-binding sites, their action on this receptor lacks compelling electrophysiological observations, with some studies reporting that ODN acts as a negative allosteric modulator (NAM) of GABA_AR while others suggest the opposite (positive allosteric modulation, PAM effect). All these studies were carried out in vitro with various neuronal cell types. To further elucidate the role of ODN in neuronal excitability, we tested its effect in vivo in the cerebral cortex of the anesthetized mouse. Methods: Spontaneous neuronal spikes were recorded by means of an extracellular pipette, in the vicinity of which ODN was micro-infused, either at a high dose (10⁻⁵ M) or low dose (10⁻¹¹ M). Results: ODN at a high dose induced a significant increase in neuronal spiking. This effect could be antagonized by the GABA_AR-BZ-binding site blocker flumazenil. In sharp contrast, at low concentrations, ODN reduced neuronal spiking with a magnitude similar to GABA itself. Interestingly, this decrease in neuronal activity by low dose of ODN was not flumazenil-dependent, suggesting that this effect is mediated by another receptor. Finally, we show that astrocytes in culture, known to be stimulated by picomolar doses of ODN via a GPCR, increased their export of GABA when stimulated by low dose of ODN. Conclusion: Our results confirm the versatility of ODN in the control of GABA transmission, but suggest that its PAM-like effect is, at least in part, mediated via an astrocytic non-GABA_AR ODN receptor release of GABA. Full article

Species of the Ferula genus are known for their traditional medicinal applications against diverse illnesses. Our previous study was the first to suggest the cholinesterase inhibitory activity of Ferula persica L. However, the neuroprotective efficacy of therapeutic molecules is often limited by their ability to cross the blood–brain barrier (BBB) and reach the brain. In the present study, the BBB permeability of the main molecules present in the aerial parts and roots of F. persica L. extracted under optimum conditions was assessed using two well-established methods: the parallel artificial membrane permeability assay (PAMPA) and the HBMEC cell culture in vitro model. The results demonstrated a high permeability of several neuroprotective compounds, such as apigenin, diosmetin, and α-cyperone. Additionally, the neuroprotective potential of F. persica extracts was evaluated using SH-SY5Y neuron-like cells exposed to different insults, including oxidative stress (H₂O₂), excitotoxicity (L-glutamate), and Aβ1-42 peptide toxicity. However, none of the obtained extracts provided significant protection. This study highlights the importance of in vitro cell culture models for a better understanding of BBB permeability mechanisms and reports the tentative identification of newly formed sulfated metabolites derived from the metabolism of ferulic acid, apigenin, and diosmetin by HBMEC cells. Full article

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

Osteosarcopenia is a widespread geriatric condition resulting from the coexistence of osteoporosis and sarcopenia, where the connection between bone and muscle is, in part, driven by bone–muscle crosstalk. Given the close, reciprocal influence of muscle on nerve, and vice versa, it is not surprising that there are corresponding aging changes in the biochemistry and morphology of the neuromuscular junction (NMJ). Indeed, degeneration of motor neurons and progressive disruption of the neuromuscular connectivity were observed in old age. Extracellular vesicles (EVs) derived from human amniotic fluid stem cells (hAFSC), exhibiting antioxidant properties, which can also explain their anti-aging and cytoprotective effects, can be considered as potential treatment for age-related diseases. To study cell interactions under both healthy and pathological conditions occurring in musculo–skeletal apparatus, we developed a three-culture system exploiting the use of well-known transwell supports. This system allows both myotubes and neurons, eventually treated with EVs, and osteoblasts, induced to osteoporosis, to interact physically and biochemically. Collectively, this method allowed us to understand how the modifications induced in osteoblasts during bone disorders trigger a cascade of detrimental effects in the muscle and neuron parts. Moreover, we demonstrated the efficacy of hAFSC-EVs in preventing NMJ dysfunction, muscle atrophy, and osteoblast impairment. Full article

Rafiq syndrome (RAFQS) is a rare autosomal recessive disorder that is classified as a type II congenital disorder of glycosylation (CDG-II), and caused by MAN1B1 gene mutation. To date, 24 pathogenic MAN1B1 mutations have been reported in association with MAN1B1-CDG. However, the underlying pathogenic mechanisms remain poorly understood. In this study, we recruited a consanguineous family from Pakistan with multiple affected individuals exhibiting mild facial dysmorphism, developmental delay, and intellectual disability. Utilizing exome sequencing and homozygosity mapping, we identified a novel MAN1B1 mutation (c.772_775del) that co-segregated with RAFQS in this family. Analysis of public single-cell transcriptomic data revealed that MAN1B1 is predominantly expressed in dorsal progenitors and intermediate excitatory neurons during human brain development. Knockdown of Man1b1 in primarily cultured mouse excitatory neurons disrupted axon growth, dendrite formation, and spine maturation, and could not be rescued by truncated variants identified in the family. Furthermore, in utero, electroporation experiments revealed that Man1b1 knockdown in the murine cortex impaired neural stem cells’ proliferation and differentiation, as well as cortical neuron migration. Collectively, these findings elucidate a critical role for MAN1B1 in the etiology of RAFQS and demonstrate that loss-of-function mutation in MAN1B1 disrupt neuro-developmental processes, providing mechanistic insights into the pathogenesis of this disorder. 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

Organoids allow to model healthy and diseased human tissues. and have applications in developmental biology, drug discovery, and cell therapy. Traditionally cultured in immersion/suspension, organoids face issues like lack of standardization, fusion, hypoxia-induced necrosis, continuous agitation, and high media volume requirements. To address these issues, we developed an air–liquid interface (ALi) technology for culturing organoids, termed AirLiwell. It uses non-adhesive microwells for generating and maintaining individualized organoids on an air–liquid interface. This method ensures high standardization, prevents organoid fusion, eliminates the need for agitation, simplifies media changes, reduces media volume, and is compatible with Good Manufacturing Practices. We compared the ALi method to standard immersion culture for midbrain organoids, detailing the process from human pluripotent stem cell (hPSC) culture to organoid maturation and analysis. Air–liquid interface organoids (3D-ALi) showed optimized size and shape standardization. RNA sequencing and immunostaining confirmed neural/dopaminergic specification. Single-cell RNA sequencing revealed that immersion organoids (3D-i) contained 16% fibroblast-like, 23% myeloid-like, and 61% neural cells (49% neurons), whereas 3D-ALi organoids comprised 99% neural cells (86% neurons). Functionally, 3D-ALi organoids showed a striking electrophysiological synchronization, unlike the heterogeneous activity of 3D-i organoids. This standardized organoid platform improves reproducibility and scalability, demonstrated here with midbrain organoids. The use of midbrain organoids is particularly relevant for neuroscience and neurodegenerative diseases, such as Parkinson’s disease, due to their high incidence, opening new perspectives in disease modeling and cell therapy. In addition to hPSC-derived organoids, the method’s versatility extends to cancer organoids and 3D cultures from primary human cells. Full article

The rhythm of epileptiform activity occurs in various brain injuries (ischemia, stroke, concussion, mechanical damage, AD, PD). The epileptiform rhythm is accompanied by periodic Ca²⁺ pulses, which are necessary for the neurotransmitter release, the repair of damaged connections between neurons, and the growth of new projections. The duration and amplitude of these pulses depend on intracellular calcium-binding proteins. The effect of the synthetic fast calcium buffer BAPTA on the epileptiform activity of neurons induced by the GABA(A)-receptor inhibitor, bicuculline, was investigated in a 14-DIV rat hippocampal culture. In the epileptiform activity mode, neurons periodically synchronously generate action potential (AP) bursts in the form of paroxysmal depolarization shift (PDS) clusters and their corresponding high-amplitude Ca²⁺ pulses. Changes in the paroxysmal activity and Ca²⁺ pulses were recorded continuously for 10–11 min as BAPTA accumulated. It was shown that during BAPTA accumulation, transformation of neuronal patch activity occurs. Moreover, GABAergic and glutamatergic neurons respond differently to the presence of calcium buffer. Experiments were performed on two populations of neurons: a population of GABAergic neurons that responded selectively to ATPA, a calcium-permeable GluK1 kainate receptor agonist, and a population of glutamatergic neurons with a large amplitude of cluster depolarization (greater than −20 mV). These neurons made up the majority of neurons. In the population of GABAergic neurons, during BAPTA accumulation, the amplitude of PDS clusters decreases, which leads to a switch from the PDS mode to the classical burst mode with an increase in the electrical activity of the neuron. In glutamatergic neurons, the duration of PDS clusters decreased during BAPTA accumulation. However, the amplitude changed little. The data obtained showed that endogenous calcium-binding proteins play a significant role in switching the epileptiform rhythm to the recovery rhythm and perform a neuroprotective function by reducing the duration of impulses in excitatory neurons and the amplitude of impulses in inhibitory neurons. Full article

Forty per cent of major depression patients are resistant to antidepressant medication. Thus, it is necessary to search for alternative treatments. Melatonin (N-acetyl-5-hydroxytryptamine) enhances neurogenesis and neuronal survival in the adult mouse hippocampal dentate gyrus. Additionally, melatonin stimulates the activity of Ca²⁺/Calmodulin-dependent Kinase II (CaMKII), promoting dendrite formation and neurogenic processes in human olfactory neuronal precursors and rat organotypic cultures. Similarly, ketamine, an N-methyl-D-aspartate receptor (NMDAR) antagonist, modulates CaMKII activity. Importantly, co-treatment of low doses of ketamine (10⁻⁷ M) in combination with melatonin (10⁻⁷ M) produces additive effects on neurogenic responses in olfactory neuronal precursors. Importantly, enhanced neurogenic responses are produced by conventional antidepressants like ISSRs. The goal of this study was to investigate whether hippocampal CaMKII participates in the signaling pathway elicited by combining doses of melatonin with ketamine acutely administered to mice, 30 min before being subjected to the forced swimming test. The results showed that melatonin, in conjunction with ketamine, significantly enhances CaMKII activation and changes its subcellular distribution in the dentate gyrus of the hippocampus. Remarkably, melatonin causes nuclear translocation of the active form of CaMKII. Luzindole, a non-selective MT₁ and MT₂ receptor antagonist, abolished these effects, suggesting that CaMKII is downstream of the melatonin receptor pathway that causes the antidepressant-like effects. These findings provide molecular insights into the combined effects of melatonin and ketamine on neuronal plasticity-related signaling pathways and pave the way for combating depression using combination therapy. Full article

Background/Objectives: Epilepsy is a brain condition that affects millions of people worldwide. Although there are many antiepileptic drugs with different mechanisms of action, many patients still fail to control their agonizing symptoms, a situation that highlights the need for more strategies to address this issue. In this in vitro study, we elucidated and characterized the alterations in intracellular Ca²⁺ levels in cell cultures where diazepam and repetitive transcranial magnetic stimulation were implemented, alone or in combination. Methods: Using the differentiated human-derived neuroblastoma cell line SH-SY5Y, we measured the alterations in intracellular Ca²⁺ levels under the impact of either low-frequency repetitive transcranial magnetic stimulation (1 Hz), diazepam (14 μM), or their combination. We used the Ca²⁺-sensitive fluorescent indicator Fluo-4 acetoxymethyl ester for calcium imaging, while neuronal excitation was achieved with 50 mM KCl. Results: The highest median fluorescence intensity increase (%ΔF/F = 24.80) was observed in control cell cultures, followed by rTMS cultures (%ΔF/F = 16.96) and diazepam cultures (%ΔF/F = 11.46). The lowest median fluorescence intensity value (%ΔF/F =−0.44) was observed when diazepam was used concomitantly with repetitive transcranial magnetic stimulation. Post hoc analysis assessed pairwise differences, showing statistically significant differentiation between the control group and all other groups. Additionally, statistically significant results were observed between repetitive transcranial magnetic stimulation or diazepam and their combination, but not between them. Conclusions: The combination of diazepam and repetitive transcranial magnetic stimulation resulted in the most significant reduction in intracellular Ca²⁺ levels, as indicated by the lowest fluorescence values compared with the control group. Individually, each treatment produced a notable but less pronounced effect. We conclude that both diazepam and low-frequency repetitive transcranial magnetic stimulation can control epileptiform activity in vitro, while their combination is the most effective treatment. Full article

The thalamus is an important sensory relay station. It integrates all somatic sensory pathways (excluding olfaction) and transmits information through thalamic relay neurons before projecting to the cerebral cortex via thalamocortical axons (TCAs). Emerging evidence has shown that FGF3, a member of the morphogen family, is an axon guidance molecule that repels TCAs away from the hypothalamus and into the internal capsule so that they subsequently reach different regions of the cortex. However, current studies on FGF-mediated axon guidance predominantly focus on phenomenological observations, with limited exploration of the underlying molecular mechanisms. To address this gap, we investigated both direct and indirect downstream signaling pathways mediating FGF3-dependent chemorepulsion of TCAs at later developmental stages. Firstly, we used pharmacological inhibitors to identify the signaling cascade(s) responsible for FGF3-triggered direct chemorepulsion of TCAs, in vitro and in vivo. Our results demonstrate that the PC-PLC pathway is required for FGF3 to directly stimulate the asymmetrical repellent growth of developing TCAs. Then, we found the FGF3-mediated repulsion can be indirectly induced by Slit1 because the addition of FGF3 in the culture media induced an increase in Slit1 expression in the diencephalon. Furthermore, by using downstream inhibitors, we found that the indirect repulsive effect of FGF3 is mediated through the PI3K downstream pathway of FGFR1. Full article