Position-Specific Secondary Acylation Determines Detection of Lipid A by Murine TLR4 and Caspase-11.

Immune sensing of the Gram-negative bacterial membrane glycolipid lipopolysaccharide (LPS) is both a critical component of host defense against bacterial infection and a contributor to the hyperinflammatory response, potentially leading to sepsis and death. Innate immune activation by LPS is due to the lipid A moiety, an acylated di-glucosamine molecule that can activate inflammatory responses via the extracellular sensor Toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) or the cytosolic sensor caspase-11 (Casp11). The number and length of acyl chains present on bacterial lipid A structures vary across bacterial species and strains, which affects the magnitude of TLR4 and Casp11 activation. TLR4 and Casp11 are thought to respond similarly to various lipid A structures, as tetra-acylated lipid A structures do not activate either sensor, whereas hexa-acylated structures activate both sensors. However, the precise features of lipid A that determine the differential activation of each receptor remain poorly defined, as direct analysis of extracellular and cytosolic responses to the same sources and preparations of LPS/lipid A structures have been limited. To address this question, we used rationally engineered lipid A isolated from a series of bacterial acyl-transferase mutants that produce novel, structurally defined molecules. Intriguingly, we found that the location of specific secondary acyl chains on lipid A resulted in differential recognition by TLR4 or Casp11, providing new insight into the structural features of lipid A required to activate either TLR4 or Casp11. Our findings indicate that TLR4 and Casp11 sense nonoverlapping areas of lipid A chemical space, thereby constraining the ability of Gram-negative pathogens to evade innate immunity.

KCNH2-3.1 expression impairs cognition and alters neuronal function in a model of molecular pathology associated with schizophrenia.

Overexpression in humans of KCNH2-3.1, which encodes a primate-specific and brain-selective isoform of the human ether-a-go-go-related potassium channel, is associated with impaired cognition, inefficient neural processing and schizophrenia. Here, we describe a new mouse model that incorporates the KCNH2-3.1 molecular phenotype. KCNH2-3.1 transgenic mice are viable and display normal sensorimotor behaviors. However, they show alterations in neuronal structure and microcircuit function in the hippocampus and prefrontal cortex, areas affected in schizophrenia. Specifically, in slice preparations from the CA1 region of the hippocampus, KCNH2-3.1 transgenic mice have fewer mature dendrites and impaired theta burst stimulation long-term potentiation. Abnormal neuronal firing patterns characteristic of the fast deactivation kinetics of the KCNH2-3.1 isoform were also observed in prefrontal cortex. Transgenic mice showed significant deficits in a hippocampal-dependent object location task and a prefrontal cortex-dependent T-maze working memory task. Interestingly, the hippocampal-dependent alterations were not present in juvenile transgenic mice, suggesting a developmental trajectory to the phenotype. Suppressing KCNH2-3.1 expression in adult mice rescues both the behavioral and physiological phenotypes. These data provide insight into the mechanism of association of KCNH2-3.1 with variation in human cognition and neuronal physiology and may explain its role in schizophrenia.

Differential expression of putative sodium-dependent cation-chloride cotransporters in Aedes aegypti.

The yellow fever mosquito, Aedes aegypti, has three genes that code for proteins with sequence similarity to vertebrate Na+-K+-Cl- cotransporters (NKCCs) of the solute-linked carrier 12 superfamily of cation-chloride cotransporters (CCCs). We hypothesized that these mosquito NKCC orthologues have diverged to perform distinct roles in salt secretion and absorption. In phylogenetic analyses, one protein (aeNKCC1) groups with a Drosophila melanogaster NKCC that mediates salt secretion whereas two others (aeCCC2 and aeCCC3) group with a Drosophila transporter that is not functionally characterized. The aeCCC2 and aeCCC3 genes probably result from a tandem gene duplication in the mosquito lineage; they have similar exon structures and are consecutive in genomic DNA. Predicted aeCCC2 and aeCCC3 proteins differ from aeNKCC1 and vertebrate NKCCs in residues from the third transmembrane domain known to influence ion and inhibitor binding. Quantitative PCR revealed that aeNKCC1 and aeCCC2 were approximately equally expressed in larvae and adults, whereas aeCCC3 was approximately 100-fold more abundant in larvae than in adults. In larval tissues, aeCCC2 was approximately 2-fold more abundant in Malpighian tubules compared to anal papillae. In contrast, aeCCC3 was nearly 100-fold more abundant in larval anal papillae compared to Malpighian tubules, suggesting a role in absorption. Western blots with polyclonal antibodies against isoform-specific peptides revealed stronger aeCCC2 immunoreactivity in adults versus larvae, whereas aeCCC3 immunoreactivity was stronger in larvae versus adults. The differential expression pattern of aeCCC2 and aeCCC3, and their sequence divergence in transmembrane domains, suggests that they may have different roles in transepithelial salt transport.

Catalytic activity and autoprocessing of murine caspase-11 mediate noncanonical inflammasome assembly in response to cytosolic LPS.

Inflammatory caspases are cysteine protease zymogens whose activation following infection or cellular damage occurs within supramolecular organizing centers (SMOCs) known as inflammasomes. Inflammasomes recruit caspases to undergo proximity-induced autoprocessing into an enzymatically active form that cleaves downstream targets. Binding of bacterial LPS to its cytosolic sensor, caspase-11 (Casp11), promotes Casp11 aggregation within a high-molecular-weight complex known as the noncanonical inflammasome, where it is activated to cleave gasdermin D and induce pyroptosis. However, the cellular correlates of Casp11 oligomerization and whether Casp11 forms an LPS-induced SMOC within cells remain unknown. Expression of fluorescently labeled Casp11 in macrophages revealed that cytosolic LPS induced Casp11 speck formation. Unexpectedly, catalytic activity and autoprocessing were required for Casp11 to form LPS-induced specks in macrophages. Furthermore, both catalytic activity and autoprocessing were required for Casp11 speck formation in an ectopic expression system, and processing of Casp11 via ectopically expressed TEV protease was sufficient to induce Casp11 speck formation. These data reveal a previously undescribed role for Casp11 catalytic activity and autoprocessing in noncanonical inflammasome assembly, and shed new light on the molecular requirements for noncanonical inflammasome assembly in response to cytosolic LPS.

Novel, non-nitrocatechol catechol-O-methyltransferase inhibitors modulate dopamine neurotransmission in the frontal cortex and improve cognitive flexibility.

RATIONALE: Cognitive impairment is a primary feature of many neuropsychiatric disorders and there is a need for new therapeutic options. Catechol-O-methyltransferase (COMT) inhibitors modulate cortical dopaminergic function and have been proposed as potential cognitive enhancers. Unfortunately, currently available COMT inhibitors are not good candidates due to either poor blood-brain barrier penetration or severe toxicity. OBJECTIVES: To address the need for safe, brain-penetrant COMT inhibitors, we tested multiple novel compounds in a set of preclinical in vivo efficacy assays in rats to determine their ability to inhibit COMT function and viability as potential clinical candidates. METHODS: We measured the change in concentration of dopamine (DA) metabolites in cerebrospinal fluid (CSF) from the cisterna magna and extracellular fluid (ECF) from the frontal cortex produced by our novel compounds. Additionally, we tested the effects of our brain-penetrant COMT inhibitors in an attentional set-shifting assay (ASST). We benchmarked the performance of the novel COMT inhibitors to the effects produced by the known COMT inhibitor tolcapone. RESULTS: We found that multiple COMT inhibitors, exemplified by LIBD-1 and LIBD-3, significantly modulated dopaminergic function measured as decreases in homovanillic acid (HVA) and increases in 3,4-Dihydroxyphenylacetic acid (DOPAC), two DA metabolites, in CSF and the frontal cortex. Additionally, we found that LIBD-1 significantly improved cognitive flexibility in the ASST, an effect previously reported following tolcapone administration. CONCLUSIONS: These results demonstrate that LIBD-1 is a novel COMT inhibitor with promising in vivo activity and the potential to serve as a new therapy for cognitive impairment.

Synthesis and Evaluation of Bicyclic Hydroxypyridones as Inhibitors of Catechol O-Methyltransferase.

A series of bicyclic pyridones were identified as potent inhibitors of catechol O-methyltransferase (COMT). Substituted benzyl groups attached to the basic nitrogen of the core scaffold gave the most potent inhibitors within this series. Rat pharmacokinetic studies showed medium to high levels of clearance for this series, but with high free fraction due to remarkably low levels of protein and tissue binding. In rat biomarker studies, levels of unbound drug exposure are seen in the brain, which exceed their respective IC50s, leading to changes in the levels of dopamine metabolites in a manner consistent with COMT inhibition.

Optimization of 8-Hydroxyquinolines as Inhibitors of Catechol O-Methyltransferase.

A series of 8-hydroxy quinolines were identified as potent inhibitors of catechol O-methyltransferase (COMT) with selectivity for the membrane-bound form of the enzyme. Small substituents at the 7-position of the quinoline were found to increase metabolic stability without sacrificing potency. Compounds with good pharmacokinetics and brain penetration were identified and demonstrated in vivo modulation of dopamine metabolites in the brain. An X-ray cocrystal structure of compound 21 in the S-COMT active site shows chelation of the active site magnesium similar to catechol-based inhibitors. These compounds should prove useful for treatment of many neurological and psychiatric conditions associated with compromised cortical dopamine signaling.