Abstract Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate‐induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down‐regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS‐linked mutant hSOD1 G93A and wild‐type SOD1 (hSOD1 wt ) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT‐PCR it was shown that expression of either hSOD1 G93A or hSOD1 wt in astrocytes produced down‐regulation of the levels of a glutamate transporter GLT‐1, without alterations in its mRNA level. hSOD1 G93A or hSOD1 wt expression caused a decrease of the monomeric form of GLT‐1 without increasing oxidative multimers of GLT‐1. The effects were selective to GLT‐1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT‐1 protein, [ 3 H] d ‐aspartate uptake was reduced in cultures expressing hSOD1 G93A or hSOD1 wt . The hSOD1‐induced decline in GLT‐1 protein and [ 3 H] d ‐aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2′,7′‐dichlorofluorescein (DCF)‐induced fluorescence revealed that expression of hSOD1 G93A or hSOD1 wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT‐1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild‐type and G93A mutant form, but does not involve the production of intracellular oxidative stress.
SpringerPlus 2015, 4(Suppl 1):L1 MicroRNAs (miRNAs) are short, 22-25 nucleotide long transcripts that may suppress entire signaling pathways by interacting with the 3'-untranslated region (3'-UTR) of coding mRNA targets, interrupting translation and inducing degradation of these targets.The long 3'-UTRs of brain transcripts compared to other tissues predict important roles for brain miRNAs.Supporting this notion, we found that brain miRNAs co-evolved with their target transcripts, that non-coding pseudogenes with miRNA recognition elements compete with brain coding mRNAs on their miRNA interactions, and that Single Nucleotide Polymorphisms (SNPs) on such pseudogenes are enriched in mental diseases including autism and schizophrenia, but not Alzheimer's disease (AD).Focusing on evolutionarily conserved and primate-specifi c miRNA controllers of cholinergic signaling ('CholinomiRs'), we fi nd modifi ed CholinomiR levels in the brain and/or nucleated blood cells of patients with AD and Parkinson's disease, with treatment-related diff erences in their levels and prominent impact on the cognitive and anti-infl ammatory consequences of cholinergic signals.Examples include the acetylcholinesterase (AChE)-targeted evolutionarily conserved miR-132, whose levels decline drastically in the AD brain.Furthermore, we found that interruption of AChE mRNA's interaction with the primatespecifi c CholinomiR-608 in carriers of a SNP in the AChE's miR-608 binding site induces domino-like eff ects that reduce the levels of many other miR-608 targets.Young, healthy carriers of this SNP express 40% higher brain AChE activity than others, potentially aff ecting the responsiveness to AD's anti-AChE therapeutics, and show elevated trait anxiety, infl ammation and hypertension.Non-coding regions aff ecting miRNA-target interactions in neurodegenerative brains thus merit special attention.
Abstract Aquaporins (AQPs) are a family of proteins that mediate water transport across cells, but the extent to which they are involved in water transport across endothelial cells of the blood–brain barrier is not clear. Expression of AQP1 and AQP4 in rat brain microvessel endothelial cells was investigated in order to determine whether these isoforms were present and, in particular, to examine the hypothesis that brain endothelial expression of AQPs is dynamic and regulated by astrocytic influences. Reverse‐transcriptase–polymerase chain reaction (RT–PCR) and immunocytochemistry showed that AQP1 mRNA and protein are present at very low levels in primary rat brain microvessel endothelial cells, and are up‐regulated in passaged cells. Upon passage, endothelial cell expression of mdr1a mRNA is decreased, indicating loss of blood–brain barrier phenotype. In passage 4 endothelial cells, AQP1 mRNA levels are reduced by coculture above rat astrocytes, demonstrating that astrocytic influences are important in maintaining the low levels of AQP1 characteristic of the blood–brain barrier endothelium. Reverse‐transcriptase–PCR revealed very low levels of AQP1 mRNA present in the RBE4 rat brain microvessel endothelial cell line, with no expression detected in primary cultures of rat astrocytes or in the C6 rat glioma cell line. In contrast, AQP4 mRNA is strongly expressed in astrocytes, but no expression is found in primary or passaged brain microvessel endothelial cells, or in RBE4 or C6 cells. Our results support the concept that expression of AQP1, which is seen in many non‐brain endothelia, is suppressed in the specialized endothelium of the blood–brain barrier.
Evidence suggests that flavonoid-rich foods are capable of inducing improvements in memory and cognition in animals and humans. However, there is a lack of clarity concerning whether flavonoids are the causal agents in inducing such behavioral responses. Here we show that supplementation with pure anthocyanins or pure flavanols for 6 weeks, at levels similar to that found in blueberry (2% w/w), results in an enhancement of spatial memory in 18 month old rats. Pure flavanols and pure anthocyanins were observed to induce significant improvements in spatial working memory (p = 0.002 and p = 0.006 respectively), to a similar extent to that following blueberry supplementation (p = 0.002). These behavioral changes were paralleled by increases in hippocampal brain-derived neurotrophic factor (R = 0.46, p<0.01), suggesting a common mechanism for the enhancement of memory. However, unlike protein levels of BDNF, the regional enhancement of BDNF mRNA expression in the hippocampus appeared to be predominantly enhanced by anthocyanins. Our data support the claim that flavonoids are likely causal agents in mediating the cognitive effects of flavonoid-rich foods.
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