Tuberous Sclerosis Complex Activity Is Required to Control Neuronal Stress Responses in an mTOR-Dependent Manner

Tuberous Sclerosis Complex (TSC) is a neurogenetic disorder caused by loss-of-function mutations in either the TSC1 or TSC2 genes and frequently results in prominent CNS manifestations including epilepsy, mental retardation, and autism spectrum disorder. The TSC1/TSC2 protein complex plays a major role in controlling the Ser/Thr kinase mTOR, which is a master regulator of protein synthesis and cell growth. In this study, we show that endoplasmic reticulum (ER) stress regulates TSC1/TSC2 complex to limit mTOR activity. In addition, Tsc2-deficient rat hippocampal neurons and brain lysates from a Tsc1-deficient mouse model both demonstrate elevated ER and oxidative stress. In Tsc2-deficient neurons, the expression of stress markers such as CHOP and HO-1 is increased, and this increase is completely reversed by the mTOR inhibitor rapamycin both in vitro and in vivo. Neurons lacking a functional TSC1/TSC2 complex have increased vulnerability to ER stress-induced cell death via the activation of the mitochondrial death pathway. Importantly, knockdown of CHOP reduces oxidative stress and apoptosis in Tsc2-deficient neurons. These observations indicate that ER stress modulates mTOR activity through the TSC protein complex and that ER stress is elevated in cells lacking this complex. They also suggest that some of the neuronal dysfunction and neurocognitive deficits seen in TSC patients may be due to ER and oxidative stress, and therefore potentially responsive to agents moderating these pathways

Response of a neuronal model of tuberous sclerosis to mammalian target of rapamycin (mTOR) inhibitors: Effects on mTORC1 and Akt signaling lead to improved survival and function

Tuberous sclerosis (TSC) is a hamartoma syndrome due to mutations in either TSC1 or TSC2 in which brain involvement causes epilepsy, mental retardation, and autism. We have recently reported (Meikle et al., J Neurosci 2007) a mouse neuronal model of TSC in which Tsc1 is ablated in most neurons during cortical development. We have tested rapamycin and RAD001, both mTORC1 inhibitors, as potential therapeutic agents in this model. Median survival is improved from 33 days to over 100 days; behavior, phenotype, and weight gain are all also markedly improved. There is brain penetration of both drugs, with accumulation over time with repetitive treatment, and effective reduction of levels of phospho-S6, a downstream target of mTORC1. In addition, there is restoration of phospho-Akt and phospho-GSK3 levels in the treated mice, consistent with restoration of Akt function. Neurofilament abnormalities, myelination, and cell enlargement are all improved by the treatment. However, dysplastic neuronal features persist, and there are only modest changes in dendritic spine density and length. Strikingly, mice treated with rapamycin or RAD001 for 23 days only (P7 — P30) displayed a persistent improvement in phenotype with median survival of 78 days. In summary, rapamycin/RAD001 are highly effective therapies for this neuronal model of TSC, with benefit apparently due to effects on mTORC1 and Akt signaling, and consequently cell size and myelination. Although caution is appropriate, the results suggest the possibility that rapamycin/RAD001 may have benefit in the treatment of TSC brain disease, including infantile spasms

PF-431396 hydrate inhibition of kinase phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn’s disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease and a regulator of intestinal inflammation. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while the inhibitor PF-431396 hydrate, which blocks Pyk2 activation, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD

SipA activation of caspase-3 is a decisive mediator of host cell survival at early stages of Salmonella enterica serovar Typhimurium infection

Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors

Genomic diversity of novel strains of mammalian gut microbiome derived Clostridium XIVa strains is driven by mobile genetic element acquisition

Despite advances in sequencing technologies that enable a greater understanding of mammalian gut microbiome composition, our ability to determine a role for individual strains is hampered by our inability to isolate, culture and study such microbes. Here we describe highly unusual Clostridium XIVa group strains isolated from the murine gut. Genome sequencing indicates that these strains, Clostridium symbiosum LM19B and LM19R and Clostridium clostridioforme LM41 and LM42, have significantly larger genomes than most closely related strains. Genomic evidence indicates that the isolated LM41 and LM42 strains diverge from most other Clostridium XIVa strains and supports reassignment of these groups at genus-level. We attribute increased C. clostridioforme LM41 and LM42 genome size to acquisition of mobile genetic elements including dozens of prophages, integrative elements, putative group II introns and numerous transposons including 29 identical copies of the IS66 transposase, and a very large 192 Kb plasmid. antiSmash analysis determines a greater number of biosynthetic gene clusters within LM41 and LM42 than in related strains, encoding a diverse array of potential novel antimicrobial compounds. Together these strains highlight the potential untapped microbial diversity that remains to be discovered within the gut microbiome and indicate that, despite our ability to get a top down view of microbial diversity, we remain significantly blinded to microbe capabilities at the strain level

Propionic acid promotes the virulent phenotype of Crohn's disease-associated adherent-invasive Escherichia coli

Propionic acid (PA) is a bacterium-derived intestinal antimicrobial and immune modulator used widely in food production and agriculture. Passage of Crohn’s disease-associated adherent-invasive Escherichia coli (AIEC) through a murine model, in which intestinal PA levels are increased to mimic the human intestine, leads to the recovery of AIEC with significantly increased virulence. Similar phenotypic changes are observed outside the murine model when AIEC is grown in culture with PA as the sole carbon source; such PA exposure also results in AIEC that persists at 20-fold higher levels in vivo. RNA sequencing identifies an upregulation of genes involved in biofilm formation, stress response, metabolism, membrane integrity, and alternative carbon source utilization. PA exposure also increases virulence in a number of E. coli isolates from Crohn’s disease patients. Removal of PA is sufficient to reverse these phenotypic changes. Our data indicate that exposure to PA results in AIEC resistance and increased virulence in its presence

Microbiome-derived carnitine mimics as previously unknown mediators of gut-brain axis communication

Alterations to the gut microbiome are associated with various neurological diseases, yet evidence of causality and identity of microbiome-derived compounds that mediate gut-brain axis interaction remain elusive. Here, we identify two previously unknown bacterial metabolites 3-methyl-4-(trimethylammonio)butanoate and 4-(trimethylammonio)pentanoate, structural analogs of carnitine that are present in both gut and brain of specific pathogen–free mice but absent in germ-free mice. We demonstrate that these compounds are produced by anaerobic commensal bacteria from the family Lachnospiraceae (Clostridiales) family, colocalize with carnitine in brain white matter, and inhibit carnitine-mediated fatty acid oxidation in a murine cell culture model of central nervous system white matter. This is the first description of direct molecular inter-kingdom exchange between gut prokaryotes and mammalian brain cells, leading to inhibition of brain cell function

Microbiome-derived carnitine mimics as previously unknown mediators of gut-brain axis communication

Alterations to the gut microbiome are associated with various neurological diseases, yet evidence of causality and identity of microbiome-derived compounds that mediate gut-brain axis interaction remain elusive. Here, we identify two previously unknown bacterial metabolites 3-methyl-4-(trimethylammonio)butanoate and 4-(trimethylammonio)pentanoate, structural analogs of carnitine that are present in both gut and brain of specific pathogen-free mice but absent in germ-free mice. We demonstrate that these compounds are produced by anaerobic commensal bacteria from the family Lachnospiraceae (Clostridiales) family, colocalize with carnitine in brain white matter, and inhibit carnitine-mediated fatty acid oxidation in a murine cell culture model of central nervous system white matter. This is the first description of direct molecular inter-kingdom exchange between gut prokaryotes and mammalian brain cells, leading to inhibition of brain cell function.Additional co-authors: Emily K. Osterweil, Andrew S. MacDonald, Chris J. Schofield, Saverio Tardito, Josephine Bunch, Gillian Douce, Julia M. Edgar, RuAngelie Edrada-Ebel, Richard J. A. Goodwin, Richard Burchmore, Daniel M. Wal

Propionic Acid Promotes the Virulent Phenotype of Crohn’s Disease-Associated Adherent-Invasive Escherichia coli

The short chain fatty acid propionic acid is a bacterium-derived human intestinal antimicrobial and immune modulator used widely in Western food production and agriculture. Here, Ormsby et al. demonstrate that exposure to propionic acid induces virulence-associated phenotypic changes in Crohn's disease-associated adherent-invasive Escherichia coli (AIEC)