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Phenol red (PR) is a commonly used compound in culture media as a pH indicator. However, it is unknown whether this compound can interfere with the pharmacological induction of ferroptosis. Here, using high-content live-cell imaging death analysis, we determined that the presence of PR in the culture medium preconditioned normal and tumor cells to ferroptosis induced by system xc- inhibition mediated by imidazole ketone erastin (IKE) or GPX4 blockade in response to RSL-3, but had no significant effects against treatment with the endoperoxide FINO2. Mechanistically, we revealed that PR decreases the levels of the antiferroptotic genes Slc7a11, Slc3a2, and Gpx4, while promoting the overexpression de Acls4, a key inducer of ferroptosis. Additionally, through superresolution analysis, we determined that the presence of PR mislocalizes the system xc- from the plasma membrane. Thus, our results show that the presence of PR in the culture medium can be a problematic artifact for the accurate interpretation of cell sensitivity to IKE or RSL-3-mediated ferroptosis induction.

Assuntos

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Radial glia (RG) cells generate neurons and glial cells that make up the cerebral cortex. Both in rodents and humans, these stem cells remain for a specific time after birth, named late radial glia (lRG). The knowledge of lRG and molecules that may be involved in their differentiation is based on very limited data. We analyzed whether ascorbic acid (AA) and its transporter SVCT2, are involved in lRG cells differentiation. We demonstrated that lRG cells are highly present between the first and fourth postnatal days. Anatomical characterization of lRG cells, revealed that lRG cells maintained their bipolar morphology and stem-like character. When lRG cells were labeled with adenovirus-eGFP at 1 postnatal day, we detected that some cells display an obvious migratory neuronal phenotype, suggesting that lRG cells continue generating neurons postnatally. Moreover, we demonstrated that SVCT2 was apically polarized in lRG cells. In vitro studies using the transgenic mice SVCT2+/- and SVCT2tg (SVCT2-overexpressing mouse), showed that decreased SVCT2 levels led to accelerated differentiation into astrocytes, whereas both AA treatment and elevated SVCT2 expression maintain the lRG cells in an undifferentiated state. In vivo overexpression of SVCT2 in lRG cells generated cells with a rounded morphology that were migratory and positive for proliferation and neuronal markers. We also examined mediators that can be involved in AA/SVCT2-modulated signaling pathways, determining that GSK3-ß through AKT, mTORC2, and PDK1 is active in brains with high levels of SVCT2/AA. Our data provide new insights into the role of AA and SVCT2 in late RG cells.

Assuntos

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Epilepsy is a chronic condition characterized by recurrent spontaneous seizures. The interaction between astrocytes and neurons has been suggested to play a role in the abnormal neuronal activity observed in epilepsy. However, the exact way astrocytes influence neuronal activity in the epileptogenic brain remains unclear. Here, using the PTZ-induced kindling mouse model, we evaluated the interaction between astrocyte and synaptic function by measuring astrocytic Ca2+ activity, neuronal excitability, and the excitatory/inhibitory balance in the hippocampus. Compared to control mice, hippocampal slices from PTZ-kindled mice displayed an increase in glial fibrillary acidic protein (GFAP) levels and an abnormal pattern of intracellular Ca2+-oscillations, characterized by an increased frequency of prolonged spontaneous transients. PTZ-kindled hippocampal slices also showed an increase in the E/I ratio towards excitation, likely resulting from an augmented release probability of excitatory inputs without affecting inhibitory synapses. Notably, the alterations in the release probability seen in PTZ-kindled slices can be recovered by reducing astrocyte hyperactivity with the reversible toxin fluorocitrate. This suggests that astroglial hyper-reactivity enhances excitatory synaptic transmission, thereby impacting the E/I balance in the hippocampus. Altogether, our findings support the notion that abnormal astrocyte-neuron interactions are pivotal mechanisms in epileptogenesis.

Assuntos

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Since the discovery of ferroptosis, it has been postulated that this type of cell death could be utilized in treatments for cancer. Unfortunately, several highly aggressive tumor models are resistant to the pharmacological induction of ferroptosis. However, with the use of combined therapies, it is possible to recover sensitivity to ferroptosis in certain cellular models. Here, we discovered that co-treatment with the metabolically stable ferroptosis inducer imidazole ketone erastin (IKE) and the oxidized form of vitamin C, dehydroascorbic acid (DHAA), is a powerful therapy that induces ferroptosis in tumor cells previously resistant to IKE-induced ferroptosis. We determined that DHAA and IKE + DHAA delocalize and deplete GPX4 in tumor cells, specifically inducing lipid droplet peroxidation, which leads to ferroptosis. Moreover, in vivo, IKE + DHAA has high efficacy with regard to the eradication of highly aggressive tumors such as glioblastomas. Thus, the use of IKE + DHAA could be an effective and safe therapy for the eradication of difficult-to-treat cancers.

Assuntos

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Hyperglycemia increases glucose concentrations in the cerebrospinal fluid (CSF), activating glucose-sensing mechanisms and feeding behavior in the hypothalamus. Here, we discuss how hyperglycemia temporarily modifies ependymal cell ciliary beating to increase hypothalamic glucose sensing. A high level of glucose in the rat CSF stimulates glucose transporter 2 (GLUT2)-positive subcommissural organ (SCO) cells to release SCO-spondin into the dorsal third ventricle. Genetic inactivation of mice GLUT2 decreases hyperglycemia-induced SCO-spondin secretion. In addition, SCO cells secrete Wnt5a-positive vesicles; thus, Wnt5a and SCO-spondin are found at the apex of dorsal ependymal cilia to regulate ciliary beating. Frizzled-2 and ROR2 receptors, as well as specific proteoglycans, such as glypican/testican (essential for the interaction of Wnt5a with its receptors) and Cx43 coupling, were also analyzed in ependymal cells. Finally, we propose that the SCO-spondin/Wnt5a/Frizzled-2/Cx43 axis in ependymal cells regulates ciliary beating, a cyclic and adaptive signaling mechanism to control glucose sensing.

Assuntos

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Different studies have established the fundamental role of vitamin C in proliferation, differentiation, and neurogenesis in embryonic and adult brains, as well as in in vitro cell models. To fulfill these functions, the cells of the nervous system regulate the expression and sorting of sodium-dependent vitamin C transporter 2 (SVCT2), as well as the recycling of vitamin C between ascorbic acid (AA) and dehydroascorbic acid (DHA) via a bystander effect. SVCT2 is a transporter preferentially expressed in neurons and in neural precursor cells. In developmental stages, it is concentrated in the apical region of the radial glia, and in adult life, it is expressed preferentially in motor neurons of the cerebral cortex, starting on postnatal day 1. In neurogenic niches, SVCT2 is preferentially expressed in precursors with intermediate proliferation, where a scorbutic condition reduces neuronal differentiation. Vitamin C is a potent epigenetic regulator in stem cells; thus, it can induce the demethylation of DNA and histone H3K27m3 in the promoter region of genes involved in neurogenesis and differentiation, an effect mediated by Tet1 and Jmjd3 demethylases, respectively. In parallel, it has been shown that vitamin C induces the expression of stem cell-specific microRNA, including the Dlk1-Dio3 imprinting region and miR-143, which promotes stem cell self-renewal and suppresses de novo expression of the methyltransferase gene Dnmt3a. The epigenetic action of vitamin C has also been evaluated during gene reprogramming of human fibroblasts to induced pluripotent cells, where it has been shown that vitamin C substantially improves the efficiency and quality of reprogrammed cells. Thus, for a proper effect of vitamin C on neurogenesis and differentiation, its function as an enzymatic cofactor, modulator of gene expression and antioxidant is essential, as is proper recycling from DHA to AA by various supporting cells in the CNS.

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The survival of patients with solid tumors, such as prostate cancer (PCa), has been limited and fleeting with anti-angiogenic therapies. It was previously thought that the mechanism by which the vasculature regulates tumor growth was driven by a passive movement of oxygen and nutrients to the tumor tissue. However, previous evidence suggests that endothelial cells have an alternative role in changing the behavior of tumor cells and contributing to cancer progression. Determining the impact of molecular signals/growth factors released by endothelial cells (ECs) on established PCa cell lines in vitro and in vivo could help to explain the mechanism by which ECs regulate tumor growth. Using cell-conditioned media collected from HUVEC (HUVEC-CM), our data show the stimulated proliferation of all the PCa cell lines tested. However, in more aggressive PCa cell lines, HUVEC-CM selectively promoted migration and invasion in vitro and in vivo. Using a PCa-cell-line-derived xenograft model co-injected with HUVEC or preincubated with HUVEC-CM, our results are consistent with the in vitro data, showing enhanced tumor growth, increased tumor microvasculature and promoted metastasis. Gene set enrichment analyses from RNA-Seq gene expression profiles showed that HUVEC-CM induced a differential effect on gene expression when comparing low versus highly aggressive PCa cell lines, demonstrating epigenetic and migratory pathway enrichments in highly aggressive PCa cells. In summary, paracrine stimulation by HUVEC increased PCa cell proliferation and tumor growth and selectively promoted migration and metastatic potential in more aggressive PCa cell lines.

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Although scurvy, the severe form of vitamin C deficiency, has been almost eradicated, the prevalence of subclinical vitamin C deficiency is much higher than previously estimated and its impact on human health might not be fully understood. Vitamin C is an essential molecule, especially in the central nervous system where it performs numerous, varied and critical functions, including modulation of neurogenesis and neuronal differentiation. Although it was originally considered to occur only in the embryonic brain, it is now widely accepted that neurogenesis also takes place in the adult brain. The subventricular zone (SVZ) is the neurogenic niche where the largest number of new neurons are born; however, the effect of vitamin C deficiency on neurogenesis in this key region of the adult brain is unknown. Therefore, through BrdU labeling, immunohistochemistry, confocal microscopy and transmission electron microscopy, we analyzed the proliferation and cellular composition of the SVZ and the lateral ventricle (LVE) of adult guinea pigs exposed to a vitamin-C-deficient diet for 14 and 21 days. We found that neuroblasts in the SVZ and LVE were progressively and significantly decreased as the days under vitamin C deficiency elapsed. The neuroblasts in the SVZ and LVE decreased by about 50% in animals with 21 days of deficiency; this was correlated with a reduction in BrdU positive cells in the SVZ and LVE. In addition, the reduction in neuroblasts was not restricted to a particular rostro-caudal area, but was observed throughout the LVE. We also found that vitamin C deficiency altered cellular morphology at the ultrastructural level, especially the cellular and nuclear morphology of ependymal cells of the LVE. Therefore, vitamin C is essential for the maintenance of the SVZ cell populations required for normal activity of the SVZ neurogenic niche in the adult guinea pig brain. Based on our results from the guinea pig brain, we postulate that vitamin C deficiency could also affect neurogenesis in the human brain.

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The identification of new proteins that regulate the function of one of the main cellular phosphatases, protein phosphatase 1 (PP1), is essential to find possible pharmacological targets to alter phosphatase function in various cellular processes, including the initiation and development of multiple diseases. IIIG9 is a regulatory subunit of PP1 initially identified in highly polarized ciliated cells. In addition to its ciliary location in ependymal cells, we recently showed that IIIG9 has extraciliary functions that regulate the integrity of adherens junctions. In this review, we perform a detailed analysis of the expression, localization, and function of IIIG9 in adult and developing normal brains. In addition, we provide a 3D model of IIIG9 protein structure for the first time, verifying that the classic structural and conformational characteristics of the PP1 regulatory subunits are maintained. Our review is especially focused on finding evidence linking IIIG9 dysfunction with the course of some pathologies, such as ciliopathies, drug dependence, diseases based on neurological development, and the development of specific high-malignancy and -frequency brain tumors in the pediatric population. Finally, we propose that IIIG9 is a relevant regulator of PP1 function in physiological and pathological processes in the CNS.

Assuntos

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Biofilms in milking equipment on dairy farms have been associated with failures in cleaning and sanitizing protocols. These biofilms on milking equipment can be a source of contamination for bulk tank milk and a concern for animal and public health, as biofilms can become on-farm reservoirs for pathogenic bacteria that cause disease in cows and humans. This report describes a cross-sectional study on 3 dairy farms, where hoses used to divert waste milk, transition milk, and colostrum were analyzed by culture methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to assess the presence of pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella spp. In addition, the presence of biofilms was analyzed using scanning electron microscopy and confocal spectral microscopy. Biofilms composed of multispecies microbial communities were observed on the surfaces of all milk hoses. In two dairy farms, S. aureus, P. aeruginosa, Klebsiella pneumoniae, and Klebsiella oxytoca were isolated from the milk hose samples collected. Cleaning and sanitation protocols of all surfaces in contact with milk or colostrum are crucial. Hoses used to collect waste milk, colostrum, and transition milk can be a source of biofilms and hence pathogenic bacteria. Waste milk used to feed calves can constitute a biosecurity issue and a source of pathogens, therefore an increased exposure and threat for the whole herd health and, potentially, for human health.