ER-transiting bacterial toxins amplify STING innate immune responses and elicit ER stress.

The cGAS/STING sensor system drives innate immune responses to intracellular microbial double-stranded DNA (dsDNA) and bacterial cyclic nucleotide second messengers (e.g., c-di-AMP). STING-dependent cell-intrinsic responses can increase resistance to microbial infection and speed pathogen clearance. Correspondingly, STING activation and signaling are known to be targeted for suppression by effectors from several bacterial pathogens. Whether STING responses are also positively regulated through sensing of specific bacterial effectors is less clear. We find that STING activation through dsDNA, by its canonical ligand 2'-3' cGAMP, or the small molecule DMXAA is potentiated following intracellular delivery of the AB5 toxin family member pertussis toxin from Bordetella pertussis or the B subunit of cholera toxin from Vibrio cholerae. Entry of pertussis toxin or cholera toxin B into mouse macrophages triggers markers of endoplasmic reticulum (ER) stress and enhances ligand-dependent STING responses at the level of STING receptor activation in a manner that is independent of toxin enzymatic activity. Our results provide an example in which STING responses integrate information about the presence of relevant ER-transiting bacterial toxins into the innate inflammatory response and may help to explain the in vivo adjuvant effects of catalytically inactive toxins.

Early Isoflurane Exposure Impairs Synaptic Development in Fmr1 KO Mice via the mTOR Pathway.

General anesthetics (GAs) may cause disruptions in brain development, and the effect of GA exposure in the setting of pre-existing neurodevelopmental disease is unknown. We tested the hypothesis that synaptic development is more vulnerable to GA-induced deficits in a mouse model of fragile X syndrome than in WT mice and asked whether they were related to the mTOR pathway, a signaling system implicated in both anesthesia toxicity and fragile X syndrome. Early postnatal WT and Fmr1-KO mice were exposed to isoflurane and brain slices were collected in adulthood. Primary neuron cultures isolated from WT and Fmr1-KO mice were exposed to isoflurane during development, in some cases treated with rapamycin, and processed for immunohistochemistry at maturity. Quantitative immunofluorescence microscopy was conducted for synaptic markers and markers of mTOR pathway activity. Isoflurane exposure caused reduction in Synpasin-1, PSD-95, and Gephyrin puncta that was significantly lower in Fmr1-KO mice than in WT mice. Similar results were found in cell culture, where synapse loss was ameliorated with rapamycin treatment. Early developmental exposure to isoflurane causes more profound synapse loss in Fmr1- KO than WT mice, and this effect is mediated by a pathologic increase in mTOR pathway activity.

Anesthetics disrupt growth cone guidance cue sensing through actions on the GABAA α2 receptor mediated by the immature chloride gradient.

BACKGROUND: General anesthetics (GAs) may exert harmful effects on the developing brain by disrupting neuronal circuit formation. Anesthetics that act on γ-aminobutyric acid (GABA) receptors can interfere with axonal growth cone guidance, a critical process in the assembly of neuronal circuitry. Here we investigate the mechanism by which isoflurane prevents sensing of the repulsive guidance cue, Semaphorin 3A (Sema3A). METHODS: Growth cone sensing was assayed by measuring growth cone collapse in dissociated neocortical cultures exposed to recombinant Sema3A in the presence or absence of isoflurane and/or a panel of reagents with specific actions on components of the GABA receptor and chloride ion systems. RESULTS: Isoflurane exposure prevents Sema3A induced growth cone collapse. A GABAA α2 specific agonist replicates this effect (36.83 ±â€¯3.417% vs 70.82 ±â€¯2.941%, in the Sema3A induced control group, p < 0.0001), but an α1-specific agonist does not. Both a Na-K-Cl cotransporter 1 antagonism (bumetanide, BUM) and a chloride ionophore (IONO) prevent isoflurane from disrupting growth cone sensing of Sema3A. (65.67 ±â€¯3.775% in Iso + BUM group vs 67.45 ±â€¯3.624% in Sema3A induced control group, 65.34 ±â€¯1.678% in Iso + IONO group vs 68.71 ±â€¯2.071% in Sema3A induced control group, no significant difference) (n = 96 growth cones per group). CONCLUSION: Our data suggest that the effects of isoflurane on growth cone sensing are mediated by the α2 subunit of the GABAA receptor and also that they are dependent on the developmental chloride gradient, in which Cl- exhibits a depolarizing effect. These findings provide a rationale for why immature neurons are particularly susceptible to anesthetic toxicity.

Early Developmental Exposure to General Anesthetic Agents in Primary Neuron Culture Disrupts Synapse Formation via Actions on the mTOR Pathway.

Human epidemiologic studies and laboratory investigations in animal models suggest that exposure to general anesthetic agents (GAs) have harmful effects on brain development. The mechanism underlying this putative iatrogenic condition is not clear and there are currently no accepted strategies for prophylaxis or treatment. Recent evidence suggests that anesthetics might cause persistent deficits in synaptogenesis by disrupting key events in neurodevelopment. Using an in vitro model consisting of dissociated primary cultured mouse neurons, we demonstrate abnormal pre- and post-synaptic marker expression after a clinically-relevant isoflurane anesthesia exposure is conducted during neuron development. We find that pharmacologic inhibition of the mechanistic target of rapamycin (mTOR) pathway can reverse the observed changes. Isoflurane exposure increases expression of phospho-S6, a marker of mTOR pathway activity, in a concentration-dependent fashion and this effect occurs throughout neuronal development. The mTOR 1 complex (mTORC1) and the mTOR 2 complex (mTORC2) branches of the pathway are both activated by isoflurane exposure and this is reversible with branch-specific inhibitors. Upregulation of mTOR is also seen with sevoflurane and propofol exposure, suggesting that this mechanism of developmental anesthetic neurotoxicity may occur with all the commonly used GAs in pediatric practice. We conclude that GAs disrupt the development of neurons during development by activating a well-defined neurodevelopmental disease pathway and that this phenotype can be reversed by pharmacologic inhibition.