Sub-toxic Ethanol Exposure Modulates Gene Expression and Enzyme Activity of Antioxidant Systems to Provide Neuroprotection in Hippocampal HT22 Cells

Ethanol is known to cause severe systemic damage often explained as secondary to oxidative stress. Brain is particularly vulnerable to ethanol-induced reactive oxygen species (ROS) because the high amounts of lipids, and because nerve cell membranes contain high amounts of peroxidable fatty acids. Usually these effects of ethanol are associated to high and/or chronic exposure to ethanol. However, as we show in this manuscript, a low and acute dose of ethanol trigger a completely different response in hippocampal cells. Thus, we have observed that 0.1% ethanol exposure to HT22 cells, a murine hippocampal-derived cell line, increases the transcriptional expression of different genes belonging to the classical, glutathione/glutaredoxin and thioredoxin/peroxiredoxin antioxidant systems, these including Sod1, Sod2, Gpx1, Gclc, and Txnrd1. Paralleling these changes, enzyme activities of total superoxide dismutase (tSOD), catalase, total glutathione peroxidase (tGPx), glutathione-S-reductase (GSR), and total thioredoxin reductase (tTXNRD), were all increased, while the generation of thiobarbituric acid reactive substances (TBARS), as indicators of lipid peroxidation, and glutathione levels remained unaltered. Ethanol exposure did not affect cell viability or cell growing as assessed by real-time cell culture monitoring, indicating that low ethanol doses are not deleterious for hippocampal cells, but rather prevented glutamate-induced excitotoxicity. In summary, we conclude that sub-toxic exposure to ethanol may well be neuroprotective against oxidative insults in hippocampal cells.Supported by grants SAF2010-22114-C02-01 and SAF2014-52582-R from Ministerio de Economía y Competitividad (Spain). VC and DQ held fellowships from Gobierno de Canarias and Fundación Cajacanarias, respectively.Peer reviewedPeer Reviewe

Lipostatic Mechanisms Preserving Cerebellar Lipids in MPTP-Treated Mice: Focus on Membrane Microdomains and Lipid-Related Gene Expression

The cerebellum is an essential component in the control of motor patterns. Despite dramatic alteration of basal ganglia morpho-functionality in Parkinson’s disease (PD), cerebellar function appears to be unaffected by the disease. Only recently this brain structure has been proposed to play compensatory roles in PD-induced motor dysfunction, particularly during the initial asymptomatic stages of PD. In PD subjects and animal models of PD, such as MPTP-treated mice, brain structures other than basal ganglia are also affected by the disease, including cortical areas not involved in motor control. Thus, it is noteworthy that the cerebellum remains unaffected. In the present study, we have analyzed the lipid composition of membrane microdomains [lipid rafts (LR) and non-raft domains] and assessed the expression levels of genes encoding enzymes synthesizing membrane-related lipids. The outcomes revealed that membrane domain lipids in cerebellum are highly preserved both in control and MPTP-treated mice as compared to control animals. Likewise, only small, mostly not significant, changes were observed in the expression of lipid-related genes in the cerebellum. Indeed, most changes were related to aging rather than to the exposure to the neurotoxin. Conversely, in the same animals, lipid composition, and gene expression were dramatically altered in the occipital cortex (OC), a brain area unrelated to the control of motor function. PCR and immunohistochemical analyses of both brain areas revealed that dopamine transporter (DAT) mRNA and protein were expressed in OC but not in the cerebellum. As MPTP neurotoxicity requires the expression of DAT to access intracellular compartments, we hypothesized that the absence of DAT in cerebellum hampers MPTP-induced toxicity. We conclude that cerebellum is endowed with efficient mechanisms to preserve nerve cell lipid homeostasis, which greatly maintain the stability of membrane microdomains involved in synaptic transmission, signal transduction, and intercellular communication, which together may participate in the compensatory role of the cerebellum in PD symptomatology

Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis

19 páginas, 6 figuras, 2 tablas.Menadione sodium bisulphite (MSB) is a water-soluble derivative of vitamin K3, or menadione, and has been previously demonstrated to function as a plant defence activator against several pathogens in several plant species. However, there are no reports of the role of this vitamin in the induction of resistance in the plant model Arabidopsis thaliana. In the current study, we demonstrate that MSB induces resistance by priming in Arabidopsis against the virulent strain Pseudomonas syringae pv. tomato DC3000 (Pto) without inducing necrosis or visible damage. Changes in gene expression in response to 0.2 mm MSB were analysed in Arabidopsis at 3, 6 and 24 h post-treatment using microarray technology. In general, the treatment with MSB does not correlate with other publicly available data, thus MSB produces a unique molecular footprint. We observed 158 differentially regulated genes among all the possible trends. More up-regulated genes are included in categories such as 'response to stress' than the background, and the behaviour of these genes in different treatments confirms their role in response to biotic and abiotic stress. In addition, there is an over-representation of the G-box in their promoters. Some interesting functions are represented among the individual up-regulated genes, such as glutathione S-transferases, transcription factors (including putative regulators of the G-box) and cytochrome P450s. This work provides a wide insight into the molecular cues underlying the effect of MSB as a plant resistance inducer.This work was partially funded by an INVESCAN, S.L. grant (No.OTT2001438) to the CSIC and by a BIO2006-02168 grant of MICINN to PT. The microarrays were funded in part by the “Genome España” Foundation. MER was supported by a research contract (ID-TF-06/002) from the Consejería de Industria, Comercio y Nuevas Tecnologías (Gobierno de Canarias). The authors thank CajaCanarias for their research support. We also thank Lorena Perales for her help in performing the bacterial growth curves, Dr. Héctor Cabrera for his useful advice on writing the manuscript, the English translation service of the Universidad Politécnica de Valencia and Mrs. Pauline Agnew whose endeavoured to edit the English translation of this paper.Peer reviewe

Differential Aggregation and Phosphorylation of Alpha Synuclein in Membrane Compartments Associated With Parkinson Disease

The aggregation of α-synuclein (α-syn) is a major factor behind the onset of Parkinson’s disease (PD). Sublocalization of this protein may be relevant for the formation of multimeric α-syn oligomeric configurations, insoluble aggregates that form Lewy bodies in PD brains. Processing of this protein aggregation is regulated by associations with distinct lipid classes. For instance, instability of lipid raft (LR) microdomains, membrane regions with a particular lipid composition, is an early event in the development of PD. However, the relevance of membrane microdomains in the regulation and trafficking of the distinct α-syn configurations associated with PD remains unexplored. In this study, using 6- and 14-month-old healthy and MPTP-treated animals as a model of PD, we have investigated the putative molecular alterations of raft membrane microstructures, and their impact on α-syn dynamics and conformation. A comparison of lipid analyses of LR microstructures and non-raft (NR) fractions showed alterations in gangliosides, cholesterol, polyunsaturated fatty acids (PUFA) and phospholipids in the midbrain and cortex of aged and MPTP-treated mice. In particular, the increase of PUFA and phosphatidylserine (PS) during aging correlated with α-syn multimeric formation in NR. In these aggregates, α-syn was phosphorylated in pSer129, the most abundant post-transductional modification of α-syn promoting toxic aggregation. Interestingly, similar variations in PUFA and PS content correlating with α-syn insoluble accumulation were also detected in membrane microstructures from the human cortex of incidental Parkinson Disease (iPD) and PD, as compared to healthy controls. Furthermore, structural changes in membrane lipid microenvironments may induce rearrangements in raft-interacting proteins involved in other neuropathologies. Therefore, we also investigated the dynamic of other protein markers involved in cognition and memory impairment such as metabotropic glutamate receptor 5 (mGluR5), ionotropic NMDA receptor (NMDAR2B), prion protein (PrPc) and amyloid precursor protein (APP), whose activity depends on membrane lipid organization. We observed a decline of these protein markers in LR fractions with the progression of aging and pathology. Overall, our findings demonstrate that lipid alterations in membranous compartments promoted by brain aging and PD-like injury may have an effect on α-syn aggregation and segregation in abnormal multimeric structures

Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis

19 páginas, 6 figuras, 2 tablas.Menadione sodium bisulphite (MSB) is a water-soluble derivative of vitamin K3, or menadione, and has been previously demonstrated to function as a plant defence activator against several pathogens in several plant species. However, there are no reports of the role of this vitamin in the induction of resistance in the plant model Arabidopsis thaliana. In the current study, we demonstrate that MSB induces resistance by priming in Arabidopsis against the virulent strain Pseudomonas syringae pv. tomato DC3000 (Pto) without inducing necrosis or visible damage. Changes in gene expression in response to 0.2 mm MSB were analysed in Arabidopsis at 3, 6 and 24 h post-treatment using microarray technology. In general, the treatment with MSB does not correlate with other publicly available data, thus MSB produces a unique molecular footprint. We observed 158 differentially regulated genes among all the possible trends. More up-regulated genes are included in categories such as 'response to stress' than the background, and the behaviour of these genes in different treatments confirms their role in response to biotic and abiotic stress. In addition, there is an over-representation of the G-box in their promoters. Some interesting functions are represented among the individual up-regulated genes, such as glutathione S-transferases, transcription factors (including putative regulators of the G-box) and cytochrome P450s. This work provides a wide insight into the molecular cues underlying the effect of MSB as a plant resistance inducer.This work was partially funded by an INVESCAN, S.L. grant (No.OTT2001438) to the CSIC and by a BIO2006-02168 grant of MICINN to PT. The microarrays were funded in part by the “Genome España” Foundation. MER was supported by a research contract (ID-TF-06/002) from the Consejería de Industria, Comercio y Nuevas Tecnologías (Gobierno de Canarias). The authors thank CajaCanarias for their research support. We also thank Lorena Perales for her help in performing the bacterial growth curves, Dr. Héctor Cabrera for his useful advice on writing the manuscript, the English translation service of the Universidad Politécnica de Valencia and Mrs. Pauline Agnew whose endeavoured to edit the English translation of this paper.Peer reviewe