RESUMO

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

RESUMO

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

RESUMO

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.

RESUMO

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.

RESUMO

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

RESUMO

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.

RESUMO

Neuroblastomas are the main extracranial tumors that affect children, while glioblastomas are the most lethal brain tumors, with a median survival time of less than 12 months, and the prognosis of these tumors is poor due to multidrug resistance. Thus, the development of new therapies for the treatment of these types of tumors is urgently needed. In this context, a new type of cell death with strong antitumor potential, called ferroptosis, has recently been described. Ferroptosis is molecularly, morphologically and biochemically different from the other types of cell death described to date because it continues in the absence of classical effectors of apoptosis and does not require the necroptotic machinery. In contrast, ferroptosis has been defined as an iron-dependent form of cell death that is inhibited by glutathione peroxidase 4 (GPX4) activity. Interestingly, ferroptosis can be induced pharmacologically, with potential antitumor activity in vivo and eventual application prospects in translational medicine. Here, we summarize the main pathways of pharmacological ferroptosis induction in tumor cells known to date, along with the limitations of, perspectives on and possible applications of this in the treatment of these tumors.

RESUMO

Aims: Glioblastoma (GB) is one of the most aggressive brain tumors. These tumors modify their metabolism, increasing the expression of glucose transporters, GLUTs, which incorporate glucose and the oxidized form of vitamin C, dehydroascorbic acid (DHA). We hypothesized that GB cells preferentially take up DHA, which is intracellularly reduced and compartmentalized into the endoplasmic reticulum (ER), promoting collagen biosynthesis and an aggressive phenotype. Results: Our results showed that GB cells take up DHA using GLUT1, while GLUT3 and sodium-dependent vitamin C transporter 2 (SVCT2) are preferably intracellular. Using a baculoviral system and reticulum-enriched extracts, we determined that SVCT2 is mainly located in the ER and corresponds to a short isoform. Ascorbic acid (AA) was compartmentalized, stimulating collagen IV secretion and increasing in vitro and in situ cell migration. Finally, orthotopic xenografts induced in immunocompetent guinea pigs showed that vitamin C deficiency retained collagen, reduced blood vessel invasion, and affected glomeruloid vasculature formation, all pathological conditions associated with malignancy. Innovation and Conclusion: We propose a functional role for vitamin C in GB development and progression. Vitamin C is incorporated into the ER of GB cells, where it favors the synthesis of collagen, thus impacting tumor development. Collagen secreted by tumor cells favors the formation of the glomeruloid vasculature and enhances perivascular invasion. Antioxid. Redox Signal. 37, 538-559.

Assuntos

RESUMO

During brain development, sodium-vitamin C transporter (SVCT2) has been detected primarily in radial glial cells in situ, with low-to-absent expression in cerebral cortex neuroblasts. However, strong SVCT2 expression is observed during the first postnatal days, resulting in increased intracellular concentration of vitamin C. Hippocampal neurons isolated from SVCT2 knockout mice showed shorter neurites and low clustering of glutamate receptors. Other studies have shown that vitamin C-deprived guinea pigs have reduced spatial memory, suggesting that ascorbic acid (AA) and SVCT2 have important roles in postnatal neuronal differentiation and neurite formation. In this study, SVCT2 lentiviral overexpression induced branching and increased synaptic proteins expression in primary cultures of cortical neurons. Analysis in neuroblastoma 2a (Neuro2a) and human subventricular tumor C3 (HSVT-C3) cells showed similar branching results. SVCT2 was mainly observed in the cell membrane and endoplasmic reticulum; however, it was not detected in the mitochondria. Cellular branching in neuronal cells and in a previously standardized neurosphere assay is dependent on the recycling of vitamin C or reduction in dehydroascorbic acid (DHA, produced by neurons) by glial cells. The effect of WZB117, a selective glucose/DHA transporter 1 (GLUT1) inhibitor expressed in glial cells, was also studied. By inhibiting GLUT1 glial cells, a loss of branching is observed in vitro, which is reproduced in the cerebral cortex in situ. We concluded that vitamin C recycling between neurons and astrocyte-like cells is fundamental to maintain neuronal differentiation in vitro and in vivo. The recycling activity begins at the cerebral postnatal cortex when neurons increase SVCT2 expression and concomitantly, GLUT1 is expressed in glial cells.

RESUMO

Ependymal cells have multiple apical cilia that line the ventricular surfaces and the central canal of spinal cord. In cancer, the loss of ependymal cell polarity promotes the formation of different types of tumors, such as supratentorial anaplastic ependymomas, which are highly aggressive in children. IIIG9 (PPP1R32) is a protein restricted to adult ependymal cells located in cilia and in the apical cytoplasm and has unknown function. In this work, we studied the expression and localization of IIIG9 in the adherens junctions (cadherin/ß-catenin-positive junctions) of adult brain ependymal cells using confocal and transmission electron microscopy. Through in vivo loss-of-function studies, ependymal denudation (single-dose injection experiments of inhibitory adenovirus) was observed, inducing the formation of ependymal cells with a "balloon-like" morphology. These cells had reduced cadherin expression (and/or delocalization) and cleavage of the cell death marker caspase-3, with "cilia rigidity" morphology (probably vibrational beating activity) and ventriculomegaly occurring prior to these events. Finally, after performing continuous infusions of adenovirus for 14 days, we observed total cell denudation and reactive parenchymal astrogliosis. Our data confirmed that IIIG9 is essential for the maintenance of adherens junctions of polarized ependymal cells. Eventually, altered levels of this protein in ependymal cell differentiation may increase ventricular pathologies, such as hydrocephalus or neoplastic transformation.

Assuntos