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Background: Acid-sensing ion channels (ASICs) are proton-gated cation channels that mediate acid-induced responses in neurons. ASICs are important for mechanosensation, learning and memory, fear, pain, and neuronal injury. ASIC2a is widely expressed in the nervous system and modulates ASIC channel trafficking and activity in both central and peripheral systems. Here, to better understand mechanisms regulating ASIC2a, we searched for potential protein motifs that regulate ASIC2a trafficking. Results and conclusions: We identified a LLDLL sequence in the C-terminal juxtamembrane region of ASIC2a. Deleting or mutating the LLDLL sequence increased total expression and surface levels of ASIC2a in CHO cells. Mutating either of the two LL motifs had a similar effect. We further assessed ASIC2a localization in organotypic hippocampal slice neurons. The LL motif mutants exhibited increased dendritic trafficking and elevated targeting to dendritic spines. Consistent with an efficient trafficking, the LL motif mutants increased acid-activated current density. In addition, mutating the second LL motif increased pH sensitivity of the channel. These data identify the LL motifs as a negative regulator of ASIC2a trafficking and function, and suggest novel regulatory mechanisms in acid signaling. [ABSTRACT FROM AUTHOR]

Objective : With the growing interests of bacteria as a targeting vector for cancer treatment, diverse genetically engineered Salmonella has been reported to be capable of targeting primary or metastatic tumor regions after intravenous injection into mouse tumor models. The purpose of this study was to investigate the capability of the genetically engineered Salmonella typhimurium (S. typhimurium) to access the glioma xenograft, which was monitored in mouse brain tumor models using optical bioluminescence imaging technique. Methods : U87 malignant glioma cells (U87-MG) stably transfected with firefly luciferase (Fluc) were implanted into BALB/cAnN nude mice by stereotactic injection into the striatum. After tumor formation, attenuated S. typhimurium expressing bacterial luciferase (Lux) was injected into the tail vein. Bioluminescence signals from transfected cells or bacteria were monitored using a cooled charge-coupled device camera to identify the tumor location or to trace the bacterial migration. Immunofluorescence staining was also performed in frozen sections of mouse glioma xenograft. Results : The injected S. typhimurium exclusively localized in the glioma xenograft region of U87-MG-bearing mouse. Immunofluorescence staining also demonstrated the accumulation of S. typhimurium in the brain tumors. Conclusion : The present study demonstrated that S. typhimurium can target glioma xenograft, and may provide a potentially therapeutic probe for glioma. [ABSTRACT FROM AUTHOR]

Histone deacetylases (HDACs) regulate cardiac plasticity; however, their molecular targets are unknown. As autophagy contributes to pathological cardiac remodeling, we hypothesized that HDAC inhibitors target autophagy. The prototypical HDAC inhibitor (HDACi), trichostatin A (TSA), attenuated both load- and agonist-induced hypertrophic growth and abolished the associated activation of autophagy. Phenylephrine (PE)-triggered hypertrophy and autophagy in cultured cardiomyocytes were each blocked by a panel of structurally distinct HDAC inhibitors. RNAi-mediated knockdown of either Atg5 or Beclin 1, two essential autophagy effectors, was similarly capable of suppressing ligand-induced autophagy and myocyte growth. RNAi experiments uncovered the class I isoforms HDAC1 and HDAC2 as required for the autophagic response. To test the functional requirement of autophagic activation, we studied mice that overexpress Beclin 1 in cardiomyocytes. In these animals with a fourfold amplified autophagic response to TAC, TSA abolished TAC-induced increases in autophagy and blunted load-induced hypertrophy. Finally, we subjected animals with preexisting hypertrophy to HDACi, finding that ventricular mass reverted to near-normal levels and ventricular function normalized completely. Together, these data implicate autophagy as an obligatory element in pathological cardiac remodeling and point to HDAC1/2 as required effectors. Also, these data reveal autophagy as a previously unknown target of HDAC inhibitor therapy. [ABSTRACT FROM AUTHOR]

Classic cadherins, comprising multiple subtypes, mediate selective cell-cell adhesion based on their subtype- specific binding nature. Each subtype in the brain is expressed by restricted groups of functionally connected nuclei and laminas. However, whether each subtype has any specific role in neural circuitry remains largely unknown. Here, we show that cadherin-8 (cad8), a type-II classic cadherin, is important for cold sensation, whose circuitry is established by projection of sensory neurons into the spinal cord. Cad8 was expressed by a subset of neurons in the dorsal horn (DH) of the spinal cord, as well as by a small number of neurons in the dorsal root ganglia (DRGs), and the majority of cad8-positive DRG neurons coexpressed cold temperature/menthol receptor (TRPM8). We generated cad8 knock-out mice and analyzed lacZ markers expressed by the targeted cad8 locus using heterozygous mice. LacZ/cad8-expressing sensory neurons and DH neurons were connected together, and cad8 protein was localized around the synaptic junctions formed between them. This relation was, however, not disrupted in cad8-/- mice. We performed whole-cell patch-clamp recordings from DH neurons in spinal cord slices, in combination with menthol stimulation as a tool to excite central terminals of primary afferents expressing TRPM8. LacZ-expressing DH neurons exhibited fast and slow miniature EPSCs. Menthol selectively increased the frequency of the slow mEPSCs in cad8-/-slices, but this effect was abolished in cad8-/- slices. The cad8-/- mice also showed a reduced sensitivity to cold temperature. These results demonstrate that cad8 is essential for establishing the physiological coupling between cold-sensitive sensory neurons and their target DH neurons. [ABSTRACT FROM AUTHOR]

The leading cause of death in diabetic patients is cardiovascular disease; diabetic cardiomyopathy is typified by alterations in cardiac morphology and function, independent of hypertension or coronary disease. However, the molecular mechanism that links diabetes to cardiomyopathy is incompletely understood. Insulin resistance is a hallmark feature of diabetes, and the FoxO family of transcription factors, which regulate cell size, viability, and metabolism, are established targets of insulin and growth factor signaling. Here, we set out to evaluate a possible role of FoxO proteins in diabetic cardiomyopathy. We found that FoxO proteins were persistently activated in cardiac tissue in mice with diabetes induced either genetically or by high-fat diet (HFD). FoxO activity was critically linked with development of cardiomyopathy: cardiomyocyte-specific deletion of FoxO1 rescued HFD-induced declines in cardiac function and preserved cardiomyocyte insulin responsiveness. FoxO1-depleted cells displayed a shift in their metabolic substrate usage, from free fatty acids to glucose, associated with decreased accumulation of lipids in the heart. Furthermore, we found that FoxO1-dependent downregulation of IRS1 resulted in blunted Akt signaling and insulin resistance. Together, these data suggest that activation of FoxO1 is an important mediator of diabetic cardiomyopathy and is a promising therapeutic target for the disease. [ABSTRACT FROM AUTHOR]

Background: Structural birth defects occur in approximately 3% of live births; most such defects lack defined genetic or environmental causes. Despite advances in surgical approaches, pharmacologic prevention remains largely out of reach.Methods: We queried worldwide databases of 20,248 families that included children with neurodevelopmental disorders and that were enriched for parental consanguinity. Approximately one third of affected children in these families presented with structural birth defects or microcephaly. We performed exome or genome sequencing of samples obtained from the children, their parents, or both to identify genes with biallelic pathogenic or likely pathogenic mutations present in more than one family. After identifying disease-causing variants, we generated two mouse models, each with a pathogenic variant "knocked in," to study mechanisms and test candidate treatments. We administered a small-molecule Wnt agonist to pregnant animals and assessed their offspring.Results: We identified homozygous mutations in WLS, which encodes the Wnt ligand secretion mediator (also known as Wntless or WLS) in 10 affected persons from 5 unrelated families. (The Wnt ligand secretion mediator is essential for the secretion of all Wnt proteins.) Patients had multiorgan defects, including microcephaly and facial dysmorphism as well as foot syndactyly, renal agenesis, alopecia, iris coloboma, and heart defects. The mutations affected WLS protein stability and Wnt signaling. Knock-in mice showed tissue and cell vulnerability consistent with Wnt-signaling intensity and individual and collective functions of Wnts in embryogenesis. Administration of a pharmacologic Wnt agonist partially restored embryonic development.Conclusions: Genetic variations affecting a central Wnt regulator caused syndromic structural birth defects. Results from mouse models suggest that what we have named Zaki syndrome is a potentially preventable disorder. (Funded by the National Institutes of Health and others.). [ABSTRACT FROM AUTHOR]

Thiopurines (eg, 6-mercaptopurine [MP]) are highly efficacious antileukemic agents, but they are also associated with dose-limiting toxicities. Recent studies by us and others have identified inherited NUDT15 deficiency as a novel genetic cause of thiopurine toxicity, and there is a strong rationale for NUDT15-guided dose individualization to preemptively mitigate adverse effects of these drugs. Using CRISPR-Cas9 genome editing, we established a Nudt15-/- mouse model to evaluate the effectiveness of this strategy in vivo. Across MP dosages, Nudt15-/- mice experienced severe leukopenia, rapid weight loss, earlier death resulting from toxicity, and more bone marrow hypocellularity compared with wild-type mice. Nudt15-/- mice also showed excessive accumulation of a thiopurine active metabolite (ie, DNA-incorporated thioguanine nucleotides [DNA-TG]) in an MP dose-dependent fashion, as a plausible cause of increased toxicity. MP dose reduction effectively normalized systemic exposure to DNA-TG in Nudt15-/- mice and largely eliminated Nudt15 deficiency-mediated toxicity. In 95 children with acute lymphoblastic leukemia, MP dose adjustment also directly led to alteration in DNA-TG levels, the effects of which were proportional to the degree of NUDT15 deficiency. Using leukemia-bearing mice with concordant Nudt15 genotype in leukemia and host, we also confirmed that therapeutic efficacy was preserved in Nudt15-/- mice receiving a reduced MP dose compared with Nudt15+/+ counterparts exposed to a standard dose. In conclusion, we demonstrated that NUDT15 genotype-guided MP dose individualization can preemptively mitigate toxicity without compromising therapeutic efficacy. [ABSTRACT FROM AUTHOR]