Is IIIG9 a New Protein with Exclusive Ciliary Function? Analysis of Its Potential Role in Cancer and Other Pathologies.

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.

Glioblastoma Invasiveness and Collagen Secretion Are Enhanced by Vitamin C.

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.

IIIG9 inhibition in adult ependymal cells changes adherens junctions structure and induces cellular detachment.

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.

SVCT2 Overexpression and Ascorbic Acid Uptake Increase Cortical Neuron Differentiation, Which Is Dependent on Vitamin C Recycling between Neurons and Astrocytes.

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.

Basal Sodium-Dependent Vitamin C Transporter 2 polarization in choroid plexus explant cells in normal or scorbutic conditions.

Vitamin C is incorporated into the cerebrospinal fluid (CSF) through choroid plexus cells. While the transfer of vitamin C from the blood to the brain has been studied functionally, the vitamin C transporter, SVCT2, has not been detected in the basolateral membrane of choroid plexus cells. Furthermore, it is unknown how its expression is induced in the developing brain and modulated in scurvy conditions. We concluded that SVCT2 is intensely expressed in the second half of embryonic brain development and postnatal stages. In postnatal and adult brain, SVCT2 is highly expressed in all choroidal plexus epithelial cells, shown by colocalization with GLUT1 in the basolateral membranes and without MCT1 colocalization, which is expressed in the apical membrane. We confirmed that choroid plexus explant cells (in vitro) form a sealed epithelial structure, which polarized basolaterally, endogenous or overexpressed SVCT2. These results are reproduced in vivo by injecting hSVCT2wt-EYFP lentivirus into the CSF. Overexpressed SVCT2 incorporates AA (intraperitoneally injected) from the blood to the CSF. Finally, we observed in Guinea pig brain under scorbutic condition, that normal distribution of SVCT2 in choroid plexus may be regulated by peripheral concentrations of vitamin C. Additionally, we observed that SVCT2 polarization also depends on the metabolic stage of the choroid plexus cells.

An in Vitro Effectiveness Evaluation of Chemical Agents for Toothbrushes Disinfection

Objective: To evaluate in the vitro effectiveness of three chemical agents for toothbrush disinfection. Material and Methods: Sixteen new toothbrushes were evaluated, previously sterilized and classified in five experimental groups (n=3) and one item as control. Three chemical agents were assessed: 0.12% Chlorhexidine gluconate (CHX), essential oil mouth rinse (Listerine) and 3.5% Sodium hypochlorite (NaOCl). The five selected strains were inoculated on toothbrushes and incubated for a 24 hours period and 37°C temperature in aerobic conditions. The incubated toothbrushes were immersed for a 15 min period into selected chemical agents and after drying in a controlled air stream, again re-cultured into enriched broth. A comparison was made between the initial and final microorganisms density recovered after chemical disinfection based on Mc Farland scale. The data obtained was compared by descriptive analysis and ANOVA methodology. Results: 3.5% NaOCl was the most effective chemical agent for toothbrush disinfection followed by CHX; Listerine was not effective to eliminate the inoculated bacteria in toothbrushes. Conclusion: 3.5% NaOCl and 0.12% CHX are the most effective chemical agents for toothbrush disinfection and Listerine was only effective against C. albicans.

SVCT2 Expression and Function in Reactive Astrocytes Is a Common Event in Different Brain Pathologies.

Ascorbic acid (AA), the reduced form of vitamin C, acts as a neuroprotector by eliminating free radicals in the brain. Sodium/vitamin C co-transporter isoform 2 (SVCT2) mediates uptake of AA by neurons. It has been reported that SVCT2 mRNA is induced in astrocytes under ischemic damage, suggesting that its expression is enhanced in pathological conditions. However, it remains to be established if SVCT expression is altered in the presence of reactive astrogliosis generated by different brain pathologies. In the present work, we demonstrate that SVCT2 expression is increased in astrocytes present at sites of neuroinflammation induced by intracerebroventricular injection of a GFP-adenovirus or the microbial enzyme, neuraminidase. A similar result was observed at 5 and 10 days after damage in a model of traumatic injury and in the hippocampus and cerebral cortex in the in vivo kindling model of epilepsy. Furthermore, we defined that cortical astrocytes maintained in culture for long periods acquire markers of reactive gliosis and express SVCT2, in a similar way as previously observed in situ. Finally, by means of second harmonic generation and 2-photon fluorescence imaging, we analyzed brain necropsied material from patients with Alzheimer's disease (AD), which presented with an accumulation of amyloid plaques. Strikingly, although AD is characterized by focalized astrogliosis surrounding amyloid plaques, SVCT2 expression at the astroglial level was not detected. We conclude that SVCT2 is heterogeneously induced in reactive astrogliosis generated in different pathologies affecting the central nervous system (CNS).