WT Levels Research Articles

Taurine Partially Improves Abnormal Anxiety in Taurine-Deficient Mice.

Taurine is abundant in various tissues including the brain, muscle, heart, spleen, liver and kidney with various physiological functions. Since taurine is produced by cysteine sulfinic acid decarboxylase (CSAD) in the liver and kidney, taurine-deficient mice without CSAD have been investigated for abnormal physiological functions such as retinal development, immune, pancreatic and liver function. In this study, the behavioral effects and abnormal brain development caused by low taurine in the developing brain were examined. In neonatal brains of homozygous CSAD knockout mice (HO), taurine was reduced by 85%, compared to wild-type mice (WT). Taurine was reduced by 35% in the brains of 2month-old HO, compared to WT. Anxiety, motor coordination and autistic-like behaviors were evaluated at 2months of age using five behavioral tests: elevated plus maze, open field, social approach, marble burying and accelerating rotarod. Mice were tested from 3 groups including WT, HO and HO with oral treatment of 0.2% taurine in the drinking water (HOT). HOT were born from HO dams treated with taurine from before pregnancy and were continuously treated with taurine in the drinking water after weaning. The taurine levels in the brain and plasma of HOT were restored to WT at 2months of age. Taurine-deficiency did not lead to changes in autistic-like behaviors as the HO were not significantly different from WT in marble burying and social approach. However, taurine-deficiency increased anxiety-like behavior in HO in the elevated plus maze and open field, compared to WT. Taurine treatment significantly restored the HOT to WT levels of anxiety-like behavior in the elevated plus maze. However, changes in exploratory activity in the open field were not improved with taurine treatment. There was a slight difference in motor ability as the WT mice stayed on the accelerating rotarod longer that the HO and HOT, but the difference was significant in the HOT during the first trial only, compared to WT.These data support hypothesis that taurine is essential for the emotional development of the brain. First, taurine is remarkably low in the neonatal brain of HO, compared to the adult brain of HO. Second, taurine treatment in HO partially improves anxiety-like behavior to WT.

Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. We have identified an activity-stability trade-off and a loss in unfolding cooperativity for a variant (3M) of Escherichia coli transketolase (TK) engineered to accept aromatic substrates. Molecular dynamics simulations of 3M revealed increased flexibility in several interconnected active-site regions that also form part of the dimer interface. Mutating the newly flexible active-site residues to regain stability risked losing the new activity. We hypothesized that stabilizing mutations could be targeted to residues outside of the active site, whose dynamics were correlated with the newly flexible active-site residues. We previously stabilized WT TK by targeting mutations to highly flexible regions. These regions were much less flexible in 3M and would not have been selected a priori as targets using the same strategy based on flexibility alone. However, their dynamics were highly correlated with the newly flexible active-site regions of 3M. Introducing the previous mutations into 3M reestablished the WT level of stability and unfolding cooperativity, giving a 10.8-fold improved half-life at 55 °C, and increased midpoint and aggregation onset temperatures by 3 °C and 4.3 °C, respectively. Even the activity toward aromatic aldehydes increased up to threefold. Molecular dynamics simulations confirmed that the mutations rigidified the active-site via the correlated network. This work provides insights into the impact of rigidifying mutations within highly correlated dynamic networks that could also be useful for developing improved computational protein engineering strategies.

Additive Effects of Decreased TfR1 and Ablated Erfe Improve Both Ineffective Erythropoiesis and Iron Overload in β-Thalassemic Mice

Patients with β-thalassemias manifest anemia, ineffective erythropoiesis, extramedullary hematopoiesis, splenomegaly, and systemic iron overload. Even in non-transfusion dependent patients, iron overload in β-thalassemia develops because of increased intestinal iron absorption, leading to multiple organ dysfunction if untreated and accounts for most of the deaths in this disease. The main regulator of body iron content and distribution is hepcidin, inhibiting iron absorption in duodenal enterocytes and release of stored iron from macrophages and hepatocytes. Despite iron overload in patients and mice with β-thalassemia, hepcidin levels are insufficiently increased, as ineffective erythropoiesis dominates hepcidin regulation. Relatively low hepcidin causes iron overload in β-thalassemia. Recent evidence demonstrates that erythroferrone (ERFE), an erythroid regulator of hepcidin, is increased in bone marrow and serum from β-thalassemic patients and th3/+ mice [Kautz Nat Gen 2014] and its loss results in increased hepcidin, partially reversing iron overload in th3/+ mice [Kautz Blood 2015]. In addition, bone marrow ERFE expression normalizes in TfR1 haploinsufficient th3/+ mice [Li Blood 2017]. We hypothesize that the loss of ERFE and TfR1 influences erythropoiesis and iron metabolism in complementary ways in th3/+ mice, and therefore aim to explore iron- and erythropoiesis-related parameters in th3/+ TfR1+/- ERFE-/- (triple mutant (TM)) mice. All models are on a C57BL6 background and have been crossed to generate 4-6 mice for analysis at 6 weeks of age. We confirm our previous reports [Li Blood 2017] that th3/+TfR1+/- mice have increased RBC count and hemoglobin, decreased MCV and reticulocyte count (Table I), and reduce splenomegaly (Fig 1a and 1b) relative to th3/+ mice. We also confirm that th3/+ ERFE-/- mice do not reverse splenomegaly or improve peripheral blood circulating erythroid parameters compared to th3/+ mice [Kautz Blood 2015] (Table I) but exhibit further increase in TfR1 in late stage erythroid precursors (Fig 1c). Analysis of the bone marrow reveals that total erythroid mass is unaltered in triple mutants relative to th3/+, th3/+ ERFE-/-, and th3/+ TfR1+/- mice, but the number of late erythroblasts (poly-E and ortho-E stages) is normalized to WT levels (Fig 1d), strongly suggesting that, unlike in th3/+ erythropoiesis, where the block in differentiation occurs at the poly-E stage, th3/+ TfR1+/- and especially triple mutant mice restore differentiation at this stage to generate a higher hemoglobin. No differences in erythroblast apoptosis or ROS concentration are evident in triple mutant relative to th3/+ ERFE-/- or th3/+ TfR1+/- mice. We also analyzed markers of Epo responsiveness and demonstrate that serum Epo and EpoR expression are increased in th3/+ relative to WT mice (Fig 1e and 1f), but while serum Epo is decreased, EpoR is further increased (Fig 1f). These findings suggest that Epo responsiveness is more optimized in triple mutant erythroblasts, enabling a smaller proportion of late stage erythroblasts to produce circulating RBCs with relatively less serum Epo. Remarkably, while neither th3/+ ERFE-/- and th3/+ TfR1+/- mice reverse iron overload or impact hepcidin expression at 6 weeks of age, triple mutant mice demonstrate fully normalized ratio of hepcidin expression relative to liver iron concentration (LIC) (Fig 1g). Taken together, these experiments provide evidence of the differential and additive effects of TfR1 and ERFE loss in th3/+ mice, with a predominantly erythropoietic benefit of TfR1 loss, a predominantly iron-homeostatic benefit of ERFE loss, and synergy of both in optimizing Epo responsiveness. DisclosuresGanz:Intrinsic LifeScience: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Silarus Pharma: Consultancy, Equity Ownership; Keryx Pharma: Consultancy, Research Funding; Gilead: Consultancy; Ablynx: Consultancy; Vifor: Consultancy; Akebia: Consultancy, Research Funding; La Jolla Pharma: Consultancy, Patents & Royalties: Patent licensed to La Jolla Pharma by UCLA.

Antisense oligonucleotide-mediated ataxin-1 reduction prolongs survival in SCA1 mice and reveals disease-associated transcriptome profiles.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited ataxia caused by expansion of a translated CAG repeat encoding a glutamine tract in the ataxin-1 (ATXN1) protein. Despite advances in understanding the pathogenesis of SCA1, there are still no therapies to alter its progressive fatal course. RNA-targeting approaches have improved disease symptoms in preclinical rodent models of several neurological diseases. Here, we investigated the therapeutic capability of an antisense oligonucleotide (ASO) targeting mouse Atxn1 in Atxn1154Q/2Q-knockin mice that manifest motor deficits and premature lethality. Following a single ASO treatment at 5 weeks of age, mice demonstrated rescue of these disease-associated phenotypes. RNA-sequencing analysis of genes with expression restored to WT levels in ASO-treated Atxn1154Q/2Q mice was used to demonstrate molecular differences between SCA1 pathogenesis in the cerebellum and disease in the medulla. Finally, select neurochemical abnormalities detected by magnetic resonance spectroscopy in vehicle-treated Atxn1154Q/2Q mice were reversed in the cerebellum and brainstem (a region containing the pons and the medulla) of ASO-treated Atxn1154Q/2Q mice. Together, these findings support the efficacy and therapeutic importance of directly targeting ATXN1 RNA expression as a strategy for treating both motor deficits and lethality in SCA1.

De novo NAD synthesis is required for intracellular replication of Coxiella burnetii, the causative agent of the neglected zoonotic disease Q fever

Coxiella burnetii is an intracellular Gram-negative bacterium responsible for the important zoonotic disease Q fever. Improved genetic tools and the ability to grow this bacterium in host cell-free media has advanced the study of C. burnetii pathogenesis, but the mechanisms that allow it to survive inside the hostile phagolysosome remain incompletely understood. Previous screening of a transposon mutant library for replication within HeLa cells has suggested that nadB, encoding a putative l-aspartate oxidase required for de novo NAD synthesis, is needed for intracellular replication. Here, using genetic complementation of two independent nadB mutants and intracellular replication assays, we confirmed this finding. Untargeted metabolite analyses demonstrated key changes in metabolites in the NAD biosynthetic pathway in the nadB mutant compared with the WT, confirming the involvement of NadB in de novo NAD synthesis. Bioinformatic analysis revealed the presence of a functionally conserved arginine residue at position 275. Using site-directed mutagenesis to substitute this residue with leucine, which abolishes the activity of Escherichia coli NadB, and expression of WT and R275L GST-NadB fusion proteins in E. coli JM109, we found that purified recombinant WT GST-NadB has l-aspartate oxidase activity and that the R275L NadB variant is inactive. Complementation of the C. burnetii nadB mutant with a plasmid expressing this inactive R275L NadB failed to restore replication to WT levels, confirming the link between de novo NAD synthesis and intracellular replication of C. burnetii This suggests that targeting this prokaryotic-specific pathway could advance the development of therapeutics to combat C. burnetii infections.

Embryonic myosin is a regeneration marker to monitor utrophin-based therapies for DMD.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. Constitutive utrophin expression, a structural and functional paralogue of dystrophin, can successfully prevent the dystrophic pathology in the dystrophin-deficient mdx mouse model. In dystrophic muscles, utrophin is increased as part of the repair process and localized at the sarcolemma of regenerating myofibers. The presence of developmental myosin such as embryonic myosin (MyHC-emb) and neonatal represents a useful marker of muscle regeneration and a meaningful indicator of muscle damage, which correlates with the clinical severity of milder Becker muscular dystrophy and DMD patients. In the present study, we demonstrate that MyHC-emb is a robust marker of regeneration at different ages and in different skeletal muscles. We also evaluate the correlation between utrophin, dystrophin and MyHC-emb in wild-type (wt) and regenerating dystrophic muscles. Restoration of dystrophin significantly reduced MyHC-emb levels. Similarly, overexpression of utrophin in the transgenic mdx-Fiona mice reduced the number of MyHC-emb positive fibers to wt level, prevented the regenerative process and rescued the muscle function. In contrast, the absence of utrophin in the dystrophin-deficient double-knockout mice resulted in a higher MyHC-emb content and in a more severe dystrophic pathophysiology than in mdx mice. These data illustrate the importance of monitoring utrophin and MyHC-emb levels in the preclinical evaluation of therapies and provide translational support for the use of developmental myosin as a disease biomarker in DMD clinical trials.

Receptor recognition by the peroxisomal AAA complex depends on the presence of the ubiquitin moiety and is mediated by Pex1p

The receptor cycle of type I peroxisomal matrix protein import is completed by ubiquitination of the membrane-bound peroxisome biogenesis factor 5 (Pex5p) and its subsequent export back to the cytosol. The receptor export is the only ATP-dependent step of the whole process and is facilitated by two members of the AAA family of proteins (ATPases associated with various cellular activities), namely Pex1p and Pex6p. To gain further insight into substrate recognition by the AAA complex, we generated an N-terminally linked ubiquitin-Pex5p fusion protein. This fusion protein displayed biological activity because it is able to functionally complement a PEX5-deletion in Saccharomyces cerevisiae. In vitro assays revealed its interaction at WT level with the native cargo protein Pcs60p and Pex14p, a constituent of the receptor docking complex. We also demonstrate in vitro deubiquitination by the deubiquitinating enzyme Ubp15p. In vitro pulldown assays and cross-linking studies demonstrate that Pex5p recognition by the AAA complex depends on the presence of the ubiquitin moiety and is mediated by Pex1p.

Drug Screening Identifies Sigma-1-Receptor as a Target for the Therapy of VWM Leukodystrophy.

Vanishing white matter (VWM) disease is an autosomal genetic leukodystrophy caused by mutations in subunits of eukaryotic translation initiation factor 2B (eIF2B). The clinical symptoms exhibit progressive loss of white matter in both hemispheres of the brain, accompanied by motor functions deterioration, neurological deficits, and early death. To date there is no treatment for VWM disease. The aim of this work was to expedite rational development of a therapeutic opportunity. Our approach was to design a computer-aided strategy for an efficient and reliable screening of drug-like molecules; and to use primary cultures of fibroblasts isolated from the Eif2b5R132H/R132H VWM mouse model for screening. The abnormal mitochondria content phenotype of the mutant cells was chosen as a read-out for a simple cell-based fluorescent assay to assess the effect of the tested compounds. We obtained a hit rate of 0.04% (20 hits out of 50,000 compounds from the selected library). All primary hits decreased mitochondria content and brought it closer to WT levels. Structural similarities between our primary hits and other compounds with known targets allowed the identification of three putative cellular pathways/targets: 11β-hydroxysteroid dehydrogenase type 1, Sonic hedgehog (Shh), and Sigma-1-Receptor (S1R). In addition to initial experimental indication of Shh pathway impairment in VWM mouse brains, the current study provides evidence that S1R is a relevant target for pharmaceutical intervention for potential treatment of the disease. Specifically, we found lower expression level of S1R protein in fibroblasts, astrocytes, and whole brains isolated from Eif2b5R132H/R132H compared to WT mice, and confirmed that one of the hits is a direct binder of S1R, acting as agonist. Furthermore, we provide evidence that treatment of mutant mouse fibroblasts and astrocytes with various S1R agonists corrects the functional impairments of their mitochondria and prevents their need to increase their mitochondria content for compensation purposes. Moreover, S1R activation enhances the survival rate of mutant cells under ER stress conditions, bringing it to WT levels. This study marks S1R as a target for drug development toward treatment of VWM disease. Moreover, it further establishes the important connection between white matter well-being and S1R-mediated proper mitochondria/ER function.

Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase Pseudomonas aeruginosa

Metabolically quiescent bacteria represent a large proportion of those in natural and host environments, and they are often refractory to antibiotic treatment. Such drug tolerance is also observed in the laboratory during stationary phase, when bacteria face stress and starvation-induced growth arrest. Tolerance requires (p)ppGpp signaling, which mediates the stress and starvation stringent response (SR), but the downstream effectors that confer tolerance are unclear. We previously demonstrated that the SR is linked to increased antioxidant defenses in Pseudomonas aeruginosa We now demonstrate that superoxide dismutase (SOD) activity is a key factor in SR-mediated multidrug tolerance in stationary-phase P. aeruginosa Inactivation of the SR leads to loss of SOD activity and decreased multidrug tolerance during stationary phase. Genetic or chemical complementation of SOD activity of the ΔrelA spoT mutant (ΔSR) is sufficient to restore antibiotic tolerance to WT levels. Remarkably, we observe high membrane permeability and increased drug internalization upon ablation of SOD activity. Combined, our results highlight an unprecedented mode of SR-mediated multidrug tolerance in stationary-phase P. aeruginosa and suggest that inhibition of SOD activity may potentiate current antibiotics.

NEW THERAPEUTIC APPROACHES AND THEIR READOUT: O.16Myostatin expression is a reliable and quantifiable biomarker to monitor dose-dependent drug response in muscular dystrophy

Currently there is no cure for muscular dystrophies but recently but promising treatments have emerged, highlighting the need of robust biomarkers. Several biomarkers have been proposed but none of them can monitor drug response longitudinally in a dose-dependent fashion. We recently demonstrated that in an atrophic muscle process, including muscular dystrophies, the myostatin pathway is intrinsically down-regulated to counter balance muscle wasting. This process was reversible in MTM1-deficient myotubular myopathy upon gene transfer leading to reactivation of the myostatin pathway. Here we show that components of the myostatin pathway are robust and reliable circulating biomarkers of drug efficacy in a gene therapy approach for dystrophin deficiency. Using an AAV8-microdystrophin vector in the GRMD dog model of Duchenne muscular dystrophy we demonstrate that the intrinsic loss of myostatin production in GRMD muscle can be partially corrected by AAV8-microdystrophin transfer in a dose-dependent manner. Importantly, and in agreement with the partial rescue provided by a micro-gene transfer approach, circulating myostatin levels in treated GRMD never reached WT levels. Two-year follow-up further demonstrated a decrease of circulating myostatin levels after high-dose treatment later in the disease course. Myostatin levels are thus the first quantifyable biomarker allowing for the non-invasive monitoring of treatment efficacy by providing a measure, which can determine the overall degree of the gene therapy efficacy as well as a longitudinal monitoring tool to follow eventual decrease of the therapeutic effect. This biomarker may therefore be also useful in future to judge the effect of combined therapy approaches non-invasively. Because circulating myostatin levels represent the product of general muscle health and activity we predict that myostatin pathway monitoring can be used to judge therapy efficacy in a wide range of neuromuscular diseases, which are associated with muscle wasting.

Pathophysiology in the suprachiasmatic nucleus in mouse models of Huntington's disease.

Disturbances in sleep/wake cycle are a common complaint of individuals with Huntington's disease (HD) and are displayed by HD mouse models. The underlying mechanisms, including the possible role of the circadian timing system, are not well established. The BACHD mouse model of HD exhibits disrupted behavioral and physiological rhythms, including decreased electrical activity in the central circadian clock (suprachiasmatic nucleus, SCN). In this study, electrophysiological techniques were used to explore the ionic underpinning of the reduced spontaneous neural activity in male mice. We found that SCN neural activity rhythms were lost early in the disease progression and was accompanied by loss of the normal daily variation in resting membrane potential in the mutant SCN neurons. The low neural activity could be transiently reversed by direct current injection or application of exogenous N-methyl-d-aspartate (NMDA) thus demonstrating that the neurons have the capacity to discharge at WT levels. Exploring the potassium currents known to regulate the electrical activity of SCN neurons, our most striking finding was that these cells in the mutants exhibited an enhancement in the large-conductance calcium activated K+ (BK) currents. The expression of the pore forming subunit (Kcnma1) of the BK channel was higher in the mutant SCN. We found a similar decrease in daytime electrical activity and enhancement in the magnitude of the BK currents early in disease in another HD mouse model (Q175). These findings suggest that SCN neurons of both HD models exhibit early pathophysiology and that dysregulation of BK current may be responsible.

RNA-binding protein HuR is required for suppressive function but not development of Foxp3+ CD4+ Tregs

Abstract The RNA-binding protein (RBP) HuR (Elavl1) controls differentiation of CD4+ T cells into multiple helper subsets through regulating transcript stability of multiple T helper subset-specific targets. We generated dLck-Cre HuRfl/fl mice, in which HuR is conditionally knocked out in T cells late in thymic development. There was a 5-fold skewing toward HuR sufficient regulatory T cells (Treg) in these mice. HuR KO naïve CD4+ T cells from these mice are also less susceptible to Treg polarization than controls and fail to upregulate Foxp3 following activation. We conditionally ablated HuR in Tregs (Foxp3YFP-Cre HuRfl/fl mouse) (HuR KO). Heterozygous Foxp3YFP-Cre/WT HuRfl/fl females have normal weight gain over time. In stark contrast, both hemizygous Foxp3YFP-CreHuRfl/fl males (39% weight reduction) and homozygous Foxp3YFP-Cre/YFP-Cre HuRfl/fl females (30% reduction) have significant failure to thrive. Heterozygous HuR KO females also have a 5:1 shift in the ratio of HuR− Treg to HuR+ Treg indicating a possible survival bias toward Treg expressing WT levels of HuR. HuR KO mice have features of scurfy mice, including alopecia and stippled tails, as well as multi-organ inflammation which includes lung, skin, stomach and liver. Additionally, naïve HuR KO mice have significantly reduced peripheral Tregs and increases in activated Foxp3− effector T cells. In vitro suppression assays showed reduced suppressive capacity of HuR KO Tregs relative to WT Tregs. Together, these data suggest that HuR is critical for effective Treg function, but is dispensable for the development of Foxp3+ CD4+ Tregs.

An intersubunit electrostatic interaction in the GABAA receptor facilitates its responses to benzodiazepines

Benzodiazepines are positive allosteric modulators of the GABAA receptor (GABAAR), acting at the α-γ subunit interface to enhance GABAAR function. GABA or benzodiazepine binding induces distinct conformational changes in the GABAAR. The molecular rearrangements in the GABAAR following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABAAR, we identified electrostatic interactions between specific amino acids at the α-γ subunit interface that were broken by, or formed after, benzodiazepine binding. Using two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes, we investigated these interactions by substituting one or both amino acids of each potential pair. We found that Lys104 in the α1 subunit forms an electrostatic bond with Asp75 of the γ2 subunit after benzodiazepine binding and that this bond stabilizes the positively modified state of the receptor. Substitution of these two residues to cysteine and subsequent covalent linkage between them increased the receptor's sensitivity to low GABA concentrations and decreased its response to benzodiazepines, producing a GABAAR that resembles a benzodiazepine-bound WT GABAAR. Breaking this bond restored sensitivity to GABA to WT levels and increased the receptor's response to benzodiazepines. The α1 Lys104 and γ2 Asp75 interaction did not play a role in ethanol or neurosteroid modulation of GABAAR, suggesting that different modulators induce different conformational changes in the receptor. These findings may help explain the additive or synergistic effects of modulators acting at the GABAAR.

Chlamydia-Specific CD8+ T cells Enhance Neutrophil Infiltration into Chlamydia-Infected Female Mouse Genital Tract

Abstract We recently demonstrated that Chlamydia-specific TNF-α producing and TNF receptor 2 bearing CD8+ T cells mediate chlamydial pathogenesis in the mouse female upper genital tract (UGT). Additionally, TNF-α production from CD8+ T lymphocytes is necessary for pathogenesis; however, CD8+ T lymphocytes are not a sufficient source of TNF-α production for pathogenesis. Myeloid cells including neutrophils are important sources of TNF-α production and have been implicated in chlamydial pathogenesis. Herein, we evaluated frequency of neutrophils in Chlamydia muridarum-infected female genital tracts of C57BL/6J (WT) mice, mice deficient in Chlamydia-specific CD8+ T cells (OT-1 mice) and TNFR2 knockout (KO) mice. Frequencies of Gr-1+ cells (neutrophils) in UGT were evaluated on days 4, 9, 15, and 24 following genital Chlamydia muridarum infection. OT-1 and TNFR2 KO displayed significant reduction in neutrophil frequency compared to WT animals on day 9 following chlamydial inoculation. Furthermore, we found that OT-1 mice replete with WT CD8+ T cells at the time of chlamydial inoculation display restoration of neutrophil (Ly-6G) frequency to WT levels. These results suggest that Chlamydia-specific CD8+ T cells engage other inflammatory cells such as neutrophils in infected genital tracts in order to cause chronic pathologies in the upper reproductive tract.

Inactivation of ceramide synthase 2 catalytic activity in mice affects transcription of genes involved in lipid metabolism and cell division

The replacement of two consecutive histidine residues by alanine residues in the catalytic center of ceramide synthase 2 in a new transgenic mouse mutant (CerS2 H/A) leads to inactivation of catalytic activity and reduces protein level to 60% of the WT level. We show here by qRT-PCR and transcriptome analyses that several transcripts of genes involved in lipid metabolism and cell division are differentially regulated in livers of CerS2 H/A mice. Thus, very long chain ceramides produced by CerS2 are required for transcriptional regulation of target genes. The hepatocellular carcinomata previously described in old CerS2 KO mice were already present in 8-week-old CerS2 H/A animals and thus are caused by the loss of CerS2 catalytic activity already during early life.

Protease activated receptor 2 deficiency in alveolar macrophages impairs cAMP generation leading to NFAT‐dependent pro‐inflammatory signalling and lung injury

Endotoxin (lipopolysaccharide [LPS]) is well known to induce intravascular coagulation during sepsis which may lead to multi‐organ failure and death. Protease Activated Receptor (Par) 1 and 2 expressed on several lung cell types can mediate crosstalk between coagulation and inflammation during endotoxemia but the mechanism remains unclear. Here, we exposed Par1−/−, Par2−/− and WT mice to nebulized LPS to address the relative roles of PAR1 versus PAR2 in modulating lung vascular injury caused by endotoxin LPS. We observed that LPS failed to induce lung injury in Par1−/− mice while it resulted in unresolvable edema formation and neutrophilic injury in Par2−/− mice. Also, Par2−/− mice, showed significant increase in the expression of pro‐inflammatory cytokines in bronchoalveolar lavage. We showed that transplantation of bone marrow from WT mice into Par2−/− mice resolved inflammatory injury in Par2−/− mice indicating haematopoietic PAR2 as a critical factor mediating resolution of inflammatory lung injury. Additionally, depletion of alveolar macrophages using intratracheally (i.t) clodronate following injury augmented lung injury in WT mice indicating PAR2 functioned by dampening alveolar macrophage activity. Since PAR2 has been shown to reverse PAR1 signalling and can be activated by thrombin directly, we next assessed if LPS‐induced thrombin generation activated PAR2 that then resolved edema formation. We delivered WT‐Par2 cDNA or mutated Par2 cDNA resistant to thrombin cleavage in Par2−/− mice i.t. using liposomes and assessed lung edema following LPS challenge. We found that Par2−/− mice transducing WT‐PAR2 resolved edema formation but this response was absent in mice expressing mutant PAR2 cDNA indicating that LPS induced thrombin generation activates PAR1 to mediate lung injury which is resolved following delayed activation of PAR2 by thrombin. Because PAR2 induces the generation of cAMP and Ca2+ mobilization we assessed the generation of these second messengers in bone marrow derived macrophages (BMDM) from WT and Par2−/− mice. We found that thrombin significantly increased cAMP levels in WT‐BMDM but not in Par2−/− BMDM. This reduction was not due to insufficient activation of Gs or phosphodiesterase as foskolin and rollipram similarly altered cAMP levels in WT and Par2−/− BMDM. Additionally, we found that loss of Par2 activated Ca2+ entry into macrophages through transient receptor potential vanniloid channel (TRPV4) which reverted back to WT levels following addition of dibutryl cAMP. Since Ca2+ entry induces NFAT transcriptional activity, we assessed the role of PAR2 in dampening inflammatory signaling and noted that loss of PAR2 increased phosphorylation of p65‐NFκB via NFAT activation. Rescuing cAMP levels diminished the phosphorylation of p65‐NFκB and NFAT activity and thereby inflammatory cytokine generation. These studies demonstrate the critical role of PAR2 activation in alveolar macrophages in resolving lung injury by inducing cAMP generation which in turn suppresses Ca2+‐dependent inflammatory signalling by NFAT.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetic defects in SAPK signalling, chromatin regulation, vesicle transport and CoA-related lipid metabolism are rescued by rapamycin in fission yeast.

Rapamycin inhibits TOR (target of rapamycin) kinase, and is being used clinically to treat various diseases ranging from cancers to fibrodysplasia ossificans progressiva. To understand rapamycin mechanisms of action more comprehensively, 1014 temperature-sensitive (ts) fission yeast (Schizosaccharomyces pombe) mutants were screened in order to isolate strains in which the ts phenotype was rescued by rapamycin. Rapamycin-rescued 45 strains, among which 12 genes responsible for temperature sensitivity were identified. These genes are involved in stress-activated protein kinase (SAPK) signalling, chromatin regulation, vesicle transport, and CoA- and mevalonate-related lipid metabolism. Subsequent metabolome analyses revealed that rapamycin upregulated stress-responsive metabolites, while it downregulated purine biosynthesis intermediates and nucleotide derivatives. Rapamycin alleviated abnormalities in cell growth and cell division caused by sty1 mutants (Δsty1) of SAPK. Notably, in Δsty1, rapamycin reduced greater than 75% of overproduced metabolites (greater than 2× WT), like purine biosynthesis intermediates and nucleotide derivatives, to WT levels. This suggests that these compounds may be the points at which the SAPK/TOR balance regulates continuous cell proliferation. Rapamycin might be therapeutically useful for specific defects of these gene functions.

MTOR-dependent alterations of Kv1.1 subunit expression in the neuronal subset-specific Pten knockout mouse model of cortical dysplasia with epilepsy

Cortical dysplasia (CD) is a common cause for intractable epilepsy. Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway has been implicated in CD; however, the mechanisms by which mTOR hyperactivation contribute to the epilepsy phenotype remain elusive. Here, we investigated whether constitutive mTOR hyperactivation in the hippocampus is associated with altered voltage-gated ion channel expression in the neuronal subset-specific Pten knockout (NS-Pten KO) mouse model of CD with epilepsy. We found that the protein levels of Kv1.1, but not Kv1.2, Kv1.4, or Kvβ2, potassium channel subunits were increased, along with altered Kv1.1 distribution, within the hippocampus of NS-Pten KO mice. The aberrant Kv1.1 protein levels were present in young adult (≥postnatal week 6) but not juvenile (≤postnatal week 4) NS-Pten KO mice. No changes in hippocampal Kv1.1 mRNA levels were found between NS-Pten KO and WT mice. Interestingly, mTOR inhibition with rapamycin treatment at early and late stages of the pathology normalized Kv1.1 protein levels in NS-Pten KO mice to WT levels. Together, these studies demonstrate altered Kv1.1 protein expression in association with mTOR hyperactivation in NS-Pten KO mice and suggest a role for mTOR signaling in the modulation of voltage-gated ion channel expression in this model.

Time-Restricted Feeding Improves Circadian Dysfunction as well as Motor Symptoms in the Q175 Mouse Model of Huntington's Disease.

Huntington's disease (HD) patients suffer from a progressive neurodegeneration that results in cognitive, psychiatric, cardiovascular, and motor dysfunction. Disturbances in sleep/wake cycles are common among HD patients with reports of delayed sleep onset, frequent bedtime awakenings, and fatigue during the day. The heterozygous Q175 mouse model of HD has been shown to phenocopy many HD core symptoms including circadian dysfunctions. Because circadian dysfunction manifests early in the disease in both patients and mouse models, we sought to determine if early intervention that improve circadian rhythmicity can benefit HD and delay disease progression. We determined the effects of time-restricted feeding (TRF) on the Q175 mouse model. At six months of age, the animals were divided into two groups: ad libitum (ad lib) and TRF. The TRF-treated Q175 mice were exposed to a 6-h feeding/18-h fasting regimen that was designed to be aligned with the middle of the time when mice are normally active. After three months of treatment (when mice reached the early disease stage), the TRF-treated Q175 mice showed improvements in their locomotor activity rhythm and sleep awakening time. Furthermore, we found improved heart rate variability (HRV), suggesting that their autonomic nervous system dysfunction was improved. Importantly, treated Q175 mice exhibited improved motor performance compared to untreated Q175 controls, and the motor improvements were correlated with improved circadian output. Finally, we found that the expression of several HD-relevant markers was restored to WT levels in the striatum of the treated mice using NanoString gene expression assays.

Macrophages regulate lung ILC2 activation via Pla2g5-dependent mechanisms.

Group V phospholipase A2 (Pla2g5) is a lipid-generating enzyme necessary for macrophage effector functions in pulmonary inflammation. However, the lipid mediators involved and their cellular targets have not been identified. Mice lacking Pla2g5 showed markedly reduced lung ILC2 activation and eosinophilia following repetitive Alternaria Alternata inhalation. While Pla2g5-null mice had Wt levels of immediate IL-33 release after one Alternaria dose, they failed to upregulate IL-33 in macrophages following repeated Alternaria administration. Unexpectedly, while adoptive transfer of bone marrow-derived (BM)-macrophages restored ILC2 activation and eosinophilia in Alternaria-exposed Pla2g5-null mice, exogenous IL-33 did not. Conversely, transfers of Pla2g5-null BM-macrophages reduced inflammation in Alternaria-exposed Wt mice. Mass spectrometry analysis of free fatty acids (FFAs) demonstrated significantly reduced FFAs (including linoleic acid (LA) and oleic acid (OA)) in lung and BM-macrophages lacking Pla2g5. Exogenous administration of LA or LA+OA to Wt mice sharply potentiated IL-33-induced lung eosinophilia and ILC2 expansion in-vitro and in-vivo. In contrast, OA potentiated IL-33-induced inflammation and ILC2 expansion in Pla2g5-null mice, but LA was inactive both in-vivo and in-vitro. Notably, Pla2g5-null ILC2s showed significantly reduced expression of the FFA-receptor-1 compared to Wt ILC2s. Thus, macrophage-associated Pla2g5 contributes significantly to type-2 immunity through regulation of IL-33 induction and FFA-driven ILC2 activation.