L-Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian CNS and five types of high-affinity Glu transporters (EAAT1-5) have been identified. The transporters EAAT1 and EAAT2 in glial cells are responsible for the majority of Glu uptake while neuronal EAATs appear to have specialized roles at particular types of synapses. Dysfunction of EAATs is specifically implicated in the pathology of neurodegenerative conditions such as amyotrophic lateral sclerosis, epilepsy, Huntington's disease, Alzheimer's disease and ischemic stroke injury, and thus treatments that can modulate EAAT function may prove beneficial in these conditions. Recent advances have been made in our understanding of the regulation of EAATs, including their trafficking, splicing and post-translational modification. This article summarises some recent developments that improve our understanding of the roles and regulation of EAATs.
Glutathione peroxidases have been thought to function in cellular antioxidant defense. However, some recent studies on Gpx1 knockout (؊/؊) mice have failed to show a role for Gpx1 under conditions of oxidative stress such as hyperbaric oxygen and the exposure of eye lenses to high levels of H 2 O 2 . These findings have, unexpectedly, raised the issue of the role of Gpx1, especially under conditions of oxidative stress. Here we demonstrate a role for Gpx1 in protection against oxidative stress by showing that Gpx1 (؊/؊) mice are highly sensitive to the oxidant paraquat. Lethality was already detected within 24 h in mice exposed to paraquat at 10 mg⅐kg ؊1 (approximately 1 ⁄7 the LD 50 of wild-type controls). The effects of paraquat were dose-related. In the 30 mg⅐kg ؊1 -treated group, 100% of mice died within 5 h, whereas the controls showed no evidence of toxicity. We further demonstrate that paraquat transcriptionally upregulates Gpx1 in normal cells, reinforcing a role for Gpx1 in protection against paraquat toxicity. Finally, we show that cortical neurons from Gpx1 (؊/؊) mice are more susceptible to H 2 O 2 ; 30% of neurons from Gpx1 (؊/؊) mice were killed when exposed to 65 M H 2 O 2 , whereas the wild-type controls were unaffected. These data establish a function for Gpx1 in protection against some oxidative stressors and in protection of neurons against H 2 O 2 . Further, they emphasize the need to elucidate the role of Gpx1 in protection against different oxidative stressors and in different disease states and suggest that Gpx1 (؊/؊) mice may be valuable for studying the role of H 2 O 2 in neurodegenerative disorders.
Lipid droplets (LDs) are increasingly recognized as critical organelles in signalling events, transient protein sequestration and inter-organelle interactions. However, the role LDs play in antiviral innate immune pathways remains unknown. Here we demonstrate that induction of LDs occurs as early as 2 h post-viral infection, is transient and returns to basal levels by 72 h. This phenomenon occurs following viral infections, both in vitro and in vivo. Virally driven in vitro LD induction is type-I interferon (IFN) independent, and dependent on Epidermal Growth Factor Receptor (EGFR) engagement, offering an alternate mechanism of LD induction in comparison to our traditional understanding of their biogenesis. Additionally, LD induction corresponds with enhanced cellular type-I and -III IFN production in infected cells, with enhanced LD accumulation decreasing viral replication of both Herpes Simplex virus 1 (HSV-1) and Zika virus (ZIKV). Here, we demonstrate, that LDs play vital roles in facilitating the magnitude of the early antiviral immune response specifically through the enhanced modulation of IFN following viral infection, and control of viral replication. By identifying LDs as a critical signalling organelle, this data represents a paradigm shift in our understanding of the molecular mechanisms which coordinate an effective antiviral response.
BACKGROUND AND PURPOSE Glutamate transporters play a major role in maintaining brain homeostasis and the astrocytic transporters, EAAT1 and EAAT2, are functionally dominant. Astrocytic excitatory amino acid transporters (EAATs) play important roles in various neuropathologies wherein astrocytes undergo cytoskeletal changes. Astrocytic plasticity is well documented, but the interface between EAAT function, actin and the astrocytic cytoskeleton is poorly understood. Because Rho kinase (ROCK) is a key determinant of actin polymerization, we investigated the effects of ROCK inhibitors on EAAT activity and astrocytic morphology.EXPERIMENTAL APPROACH The functional activity of glutamate transport was determined in murine cultured astrocytes after exposure to the ROCK inhibitors Fasudil (HA‐1077) and Y27632 using biochemical, molecular and morphological approaches. Cytochemical analyses assessed changes in astrocytic morphology, F‐/G‐actin, and localizations of EAAT1/2.RESULTS Fasudil and Y27632 increased [3H]‐d‐aspartate (d‐Asp) uptake into astrocytes, and the action of Fasudil was time‐dependent and concentration‐related. The rapid stellation of astrocytes (glial fibrillary acidic protein immunocytochemistry) induced by Fasudil was accompanied by reduced phalloidin staining of F‐actin and increased Vmax for [3H]‐d‐Asp uptake. Immunoblotting after biotinylation demonstrated that Fasudil increased the expression of EAAT1 and EAAT2 on the cell surface. Immunocytochemistry indicated that Fasudil induced prominent labelling of astrocytic processes by EAAT1/2.CONCLUSION AND IMPLICATIONS These data show for the first time that ROCK plays a major role in determining the cell surface expression of EAAT1/2, providing new evidence for an association between transporter function and astrocytic phenotype. ROCK inhibitors, via the actin cytoskeleton, effect a consequent elevation of glutamate transporter function – this activity profile may contribute to their beneficial actions in neuropathologies.
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