Search Results (136)

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

Opioid use induces neurobiological adaptations throughout mesolimbic brain regions, such as the orbitofrontal cortex (OFC), which mediates decision-making and emotional–cognitive regulation. Previously, we showed that a circular RNA (circRNA) species, rno_circGrin2b_011731 (circGrin2b), is upregulated in the OFC of rats following chronic self-administration (SA) of the opioid heroin. circGrin2b is derived from Grin2b, which encodes the regulatory subunit of the glutamate ionotropic NMDA receptor, GluN2B. However, the upstream regulatory mechanisms of circGrin2b biogenesis and the downstream consequences of circGrin2b dysregulation remain unknown. We hypothesized that opioid-induced elevation of circGrin2b is accompanied by regulation of circRNA biogenesis enzymes, and that circGrin2b may sponge microRNAs (miRNAs), as miRNA sponging is a well-described characteristic of circRNAs. To test these hypotheses, we established an in vitro primary cortical cell culture model to examine alterations in circGrin2b expression following exposure to the opioid morphine. We measured mRNA expression of known circRNA splicing factors and observed significant downregulation of Fused in Sarcoma (Fus), a negative regulator of circRNA biogenesis, following 90 min or 24 h of morphine exposure. Downregulation of Fus at 24 h post-morphine was accompanied by upregulation of circGrin2b and downregulation of miR-26b-3p, a predicted miRNA target of circGrin2b. Luciferase reporter assays confirmed interaction of miR-26b-3p with circGrin2b. Finally, we report a significant negative relationship between circGrin2b and miR-26b-3p expression in the OFC of rats following heroin SA. We conclude that regulation of circGrin2b is an opioid-induced neuroadaptation that may impact downstream signaling of miRNA pathways in the frontal cortex. Full article

Excitotoxicity is a pathological process that occurs in many neurological diseases, such as stroke or epilepsy, and is characterized by the extracellular accumulation of high concentrations of glutamate or other excitatory amino acids (EAAs). Nicotinamide adenine dinucleotide (NAD) depletion is an early event following excitotoxicity in many in vitro and in vivo excitotoxic-related models and contributes to the deregulation of energy homeostasis. However, the interplay between glutamate excitotoxicity and the NAD biosynthetic pathway is not fully understood. To address this question, we used a primary culture of rat cortical neurons and found that an excitotoxic glutamate insult alters the expression of the NAD biosynthetic enzymes. Additionally, using a fluorescent NAD mitochondrial sensor, we observed that glutamate induces a significant decrease in the mitochondrial NAD pool, which was reversed when exogenous NAD was added. We also show that exogenous NAD protects against the glutamate-induced decrease in mitochondrial membrane potential (MMP). Glutamate excitotoxicity changed mitochondrial retrograde transport in neurites, which seems to be reversed by NAD addition. Finally, we show that NAD and NAD precursors protect against glutamate-induced cell death. Together, our results demonstrate that glutamate-induced excitotoxicity acts by compromising the NAD biosynthetic pathway, particularly in the mitochondria. These results also uncover a potential role for mitochondrial NAD as a tool for central nervous system (CNS) regenerative therapies. Full article

Parkinson’s disease (PD) is a neurodegenerative disorder affecting nearly 10 million people worldwide, and for which no cure is currently known. Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene, age, as well as environmental factors such as neurotoxin exposure and stress, are known to increase the risk of developing the disease in humans. To investigate the role of a specific Asian variant of the LRRK2 gene to induce susceptibility to stress and trigger PD phenotypes with time, knock-in (KI) mice bearing the human LRRK2 R1628P risk variant have been generated and studied from 2 to 16 months of age in the presence (or absence) of stress insults, including neurotoxin injections and chronic mild stress applied at 3 months of age. Pathophysiological and behavioural phenotypes have been measured at different ages and primary neurons and fibroblast cells were cultured from the KI mouse line and treated with H₂O₂ to study susceptibility towards oxidative stress in vitro. KI mice displayed specific PD features and these phenotypes were aggravated by environmental stresses. In particular, KI mice developed locomotion impairment and increased constipation. In addition, dopamine-related proteins were dysregulated in KI mice brains: Dopamine transporter (DAT) was decreased in the midbrain and striatum and dopamine levels were increased. Primary fibroblast cells and cortical neurons from KI mice also displayed increased susceptibility to oxidative stress. Therefore, the LRRK2 R1628P KI mice are an excellent model to study the progressive development of PD. Full article

Neuromimetic in vitro models, simulating in vivo architecture/organization, are urgently needed to reduce experimental reliance on live animals. Our group recently reported a novel brain tissue derivation protocol, simultaneously deriving all major cortical cell types (including immune cells) in a facile protocol, generating a network of neurons in a single growth medium, which was interfaced with nanomaterials. This represents a significant advance, as tissue engineers overwhelmingly use diverse methods to derive and combine individual brain cells for materials-interfacing. However, this multicellular model lacked cellular directionality/structural organization (unlike the highly organized cortical circuits in vivo). Synthetic nanofiber constructs are of high value in tissue engineering, providing directional cues for cells. Most neuro-nanofiber studies employ simple monocultures of astrocytes/neurons and commonly use peripheral neurons rather than central nervous system populations. Here, we have interfaced our complex brain model (neurons/astrocytes derived simultaneously) with randomly oriented or aligned polycaprolactone (PCL) fiber meshes. Both cell types showed targeted extension along aligned fibers versus coverslips or random fibers. A new analysis method developed in-house demonstrated that peak orientations for astrocytes and neurons correlated with aligned nanofibers. Our data support the concept that nanofiber scaffolds can achieve organized growth of mixed cortical neural cell populations, mimicking neural architecture. Full article

A key principle of guided bone regeneration (GBR) is the use of a barrier membrane to prevent cells from non-osteogenic tissues from interfering with bone regeneration in patients with hard tissue deficiencies. The aim of the study was to investigate whether the osteoinductive properties of bone-conditioned medium (BCM) obtained from cortical bone chips harvested at the surgical site can be transferred to a native bilayer collagen membrane (nbCM). BCM extracted within 20 or 40 min, which corresponds to a typical implant surgical procedure, and BCM extracted within 24 h, which corresponds to BCM released from the autologous bone chips in situ, contained significant and comparable amounts of TGF-β1, IGF-1, FGF-2, VEGF-A, and IL-11. Significant (p < 0.001) quantities of BMP-2 were only detected in the 24-h BCM preparation. The bioactive substances contained in the BCM were adsorbed to the nbCMs with almost 100% efficiency. A fast but sequential release of all investigated proteins occurred within 6–72 h, reflecting their stepwise involvement in the natural regeneration process. BCM-coated nbCM significantly (p < 0.05) increased the migratory, adhesive, and proliferative capacity of primary human bone-derived cells (hBC), primary human periodontal ligament cells (hPDLC), and an osteosarcoma-derived osteoblastic cell line (MG-63) compared to cells cultured on BCM-free nbCM. The high proliferative rates of MG-63 cells cultured on BCM-free nbCM were not further potentiated by BCM, indicating that BCM-coated nbCM has no detrimental effects on cancer cell growth. BCM-coated nbCM caused significant (p < 0.05) induction of early osteogenic marker gene expression and alkaline phosphatase activity, suggesting an important role of BCM-functionalized nbCM in the initiation of osteogenesis. The 24-h BCM loaded on the nbCM was the only BCM preparation that caused significant induction of late osteogenic marker gene expression. Altogether, our data define the pre-activation of collagen membranes with short-term-extracted BCM as a potential superior modality for treating hard tissue deficiencies via GBR. Full article

Astrocytes produce and export glutathione (GSH), an important thiol antioxidant essential for protecting neural cells from oxidative stress and maintaining optimal brain health. While it has been established that oxidative stress increases GSH production in astrocytes, with Nrf2 acting as a critical transcription factor regulating key components of the GSH synthetic pathway, the role of Nrf2 in controlling constitutive GSH synthetic and release mechanisms remains incompletely investigated. Our data show that naïve primary mouse astrocytes cultured from the cerebral cortices of Nrf2 knockout (Nrf2^−/−) pups have significantly less intracellular and extracellular GSH levels when compared to astrocytes cultured from Nrf2 wild-type (Nrf2^+/+) pups. Key components of the GSH synthetic pathway, including xCT (the substrate-specific light chain of the substrate-importing transporter, system x_c⁻), glutamate-cysteine ligase [catalytic (GCLc) and modifying (GCLm) subunits], were affected. To wit: qRT-PCR analysis demonstrates that naïve Nrf2^−/− astrocytes have significantly lower basal mRNA levels of xCT and both GCL subunits compared to naïve Nrf2^+/+ astrocytes. No change in mRNA levels of glutathione synthetase (GS) or the GSH exporting transporter, Mrp1, was found. Western blot analysis reveals reduced protein levels of both subunits of GCL, while (seleno)cystine uptake into Nrf2^−/− astrocytes was reduced compared to Nrf2^+/+ astrocytes, confirming decreased system x_c⁻ activity. These findings suggest that Nrf2 regulates the basal production of GSH in astrocytes through constitutive transcriptional regulation of GCL and xCT. Full article

We previously reported that membrane-type 5-matrix metalloproteinase (MT5-MMP) deficiency not only reduces pathological hallmarks of Alzheimer’s disease (AD) in 5xFAD (Tg) mice in vivo but also impairs interleukin-1 beta (IL-1β)-mediated neuroinflammation and Aβ production in primary Tg immature neural cell cultures after 11 days in vitro. We now investigate the effect of MT5-MMP on incipient pathogenic pathways that are activated in cortical primary cultures at 21–24 days in vitro (DIV), during which time neurons are organized into a functional mature network. Using wild-type (WT), MT5-MMP^−/− (MT5^−/−), 5xFAD (Tg), and 5xFADxMT5-MMP^−/− (TgMT5^−/−) mice, we generated primary neuronal cultures that were exposed to IL-1β and/or different proteolytic system inhibitors. We assessed neuroinflammation, APP metabolism, synaptic integrity, and electrophysiological properties using biochemical, imaging and whole-cell patch-clamp approaches. The absence of MT5-MMP impaired the IL-1β-mediated induction of inflammatory genes in TgMT5^−/− cells compared to Tg cells. Furthermore, the reduced density of dendritic spines in Tg neurons was also prevented in TgMT5^−/− neurons. IL-1β caused a strong decrease in the dendritic spine density of WT neurons, which was prevented in MT5^−/− neurons. However, the latter exhibited fewer spines than the WT under untreated conditions. The spontaneous rhythmic firing frequency of the network was increased in MT5^−/− neurons, but not in TgMT5^−/− neurons, and IL-1β increased this parameter only in Tg neurons. In terms of induced somatic excitability, Tg and TgMT5^−/− neurons exhibited lower excitability than WT and MT5^−/−, while IL-1β impaired excitability only in non-AD backgrounds. The synaptic strength of miniature global synaptic currents was equivalent in all genotypes but increased dramatically in WT and MT5^−/− neurons after IL-1β. MT5-MMP deficiency decreased endogenous and overexpressed C83 and C99 levels but did not affect Aβ levels. C99 appears to be cleared by several pathways, including γ-secretase, the autophagolysosomal system, and also α-secretase, via its conversion to C83. In summary, this study confirms that MT5-MMP is a pivotal factor affecting not only neuroinflammation and APP metabolism but also synaptogenesis and synaptic activity at early stages of the pathology, and reinforces the relevance of targeting MT5-MMP to fight AD. 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

This study explores how select microRNAs (miRNAs) influence bone structure in humans and in transgenic mice. In trabecular bone biopsies from 84 postmenopausal women (healthy, osteopenic, and osteoporotic), we demonstrate that DLEU2 (deleted in lymphocytic leukemia 2)-encoded miR-15a-5p is strongly positively associated with bone mineral density (BMD) at different skeletal sites. In bone transcriptome analyses, miR-15a-5p levels correlated positively with the osteocyte characteristic transcripts SOST (encoding sclerostin) and MEPE (Matrix Extracellular Phosphoglycoprotein), while the related miR-15b-5p showed a negative association with BMD and osteoblast markers. The data imply that these miRNAs have opposite roles in bone remodeling with distinct actions on bone cells. Expression quantitative trait loci (eQTL) variants confirmed earlier DLEU2 associations. Furthermore, a novel variant (rs12585295) showed high localization with transcriptionally active chromatin states in osteoblast primary cell cultures. The supposition that DLEU2-encoded miRNAs have an important regulatory role in bone remodeling was further confirmed in a transgenic mice model showing that miR-15a/16-1-deleted mice had significantly higher percentage bone volume and trabecular number than the wild type, possibly due to prenatal actions. However, the three-point mechanical break force test of mice femurs showed a positive correlation between strength and miR-15a-5p/miR-16-5p levels, indicating differential effects on cortical and trabecular bone. Moreover, these miRNAs appear to have distinct and complex actions in mice prenatally and in adult humans, impacting BMD and microstructure by regulating bone cell transcription. However, detailed interactions between these miRNAs and their downstream mechanisms in health and disease need further clarification. 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

Failure of myelin regeneration by oligodendrocytes contributes to progressive decline in many neurological diseases. Here, using in vitro and in vivo rodent models, functional blockade, and mouse brain demyelination, we demonstrate that Sonic hedgehog (Shh) expression in a subset of oligodendrocyte progenitor cells precedes the expression of myelin basic protein (MBP), a major myelin sheath protein. Primary cultures of rodent cortical oligodendrocytes show that Shh mRNA and protein are upregulated during oligodendrocyte maturation before the upregulation of MBP expression. Importantly, almost all MBP-positive cells are Shh positive during differentiation. During remyelination, we identify a rapid induction of Shh mRNA and peptide in oligodendroglial cells present in the demyelinated corpus callosum of mice, including a population of PDGFRα-expressing cells. Shh invalidation by an adeno-associated virus strategy demonstrates that the downregulation of Shh impairs the differentiation of oligodendrocytes in vitro and decreases MBP and myelin proteolipid protein expression in the demyelinated mouse brain at late stages of remyelination. We also report a parallel expression of Shh and MBP in oligodendroglial cells during early post-natal myelination of the mouse brain. Thus, we identify a crucial Shh signal involved in oligodendroglial cell differentiation and remyelination, with potential interest in the design of better-targeted remyelinating therapeutic strategies. Full article

This study explores the neuroprotective effects of neuropeptide FF (NPFF, FLFQPQRFamide) in the context of ischemic injury. Based on transcriptomic analysis in stroke models treated with 5-Aza-dC and task-specific training, we identified significant gene expression changes, particularly involving NPFF. To further explore NPFF’s role in promoting neuronal recovery, recombinant NPFF protein (rNPFF) was used in primary mixed cortical cultures subjected to oxygen-glucose deprivation and reoxygenation. Our results demonstrated that rNPFF significantly reduced lactate dehydrogenase release, indicating decreased cellular damage. It also significantly increased the expression of TUJ1 and MAP2, markers of neuronal survival and dendritic integrity. Additionally, rNPFF significantly upregulated key synaptic proteins, including GAP43, PSD95, and synaptophysin, which are essential for synaptic repair and plasticity. Post-injury rNPFF treatment led to a significant upregulation of pro-brain-derived neurotrophic factor (BDNF) and mature BDNF, which play critical roles in neuronal survival, growth, and synaptic plasticity. Moreover, rNPFF activated the protein kinase Cε isoform, Sirtuin 1, and peroxisome proliferator-activated receptor gamma pathways, which are crucial for regulating cellular stress responses, synaptic plasticity, and energy homeostasis, further promoting neuronal survival and recovery. These findings suggest that rNPFF may play a pivotal role in enhancing neuronal survival and synaptic plasticity after ischemic injury, highlighting its potential as a therapeutic target for stroke recovery. Full article

ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER. Full article

Neurodegenerative disorders, including traumatic injuries to the central nervous system (CNS) and neurodegenerative diseases, are characterized by early axonal damage, which does not regenerate in the adult mammalian CNS, leading to permanent neurological deficits. One of the primary causes of the loss of regenerative ability is thought to be a developmental decline in neurons’ intrinsic capability for axon growth. Different molecules are involved in the developmental loss of the ability for axon regeneration, including many transcription factors. However, the function of microRNAs (miRNAs), which are also modulators of gene expression, in axon re-growth is still unclear. Among the various miRNAs recently identified with roles in the CNS, miR-17, which is highly expressed during early development, emerges as a promising target to promote axon regeneration. Here, we used adeno-associated viral (AAV) vectors to overexpress miR-17 (AAV.miR-17) in primary cortical neurons and evaluate its effects on neurite and axon regeneration in vitro. Although AAV.miR-17 had no significant effect on neurite outgrowth and arborization, it significantly enhances neurite regeneration after scratch lesion and axon regeneration after axotomy of neurons cultured in microfluidic chambers. Target prediction and functional annotation analyses suggest that miR-17 regulates gene expression associated with autophagy and cell metabolism. Our findings suggest that miR-17 promotes regenerative response and thus could mitigate neurodegenerative effects. Full article