Summary Nuclear factor κB (NF-κB) is an anti-apoptotic transcription factor. We show that the anti-apoptotic actions of NF-κB are mediated by hydrogen sulfide (H2S) synthesized by cystathionine gamma-lyase (CSE). TNFα treatment triples H2S generation by stimulating binding of SP1 to the CSE promoter. H2S generated by CSE stimulates DNA binding and gene activation of NF-κB, processes that are abolished in CSE deleted mice. As CSE deletion leads to decreased glutathione levels, resultant oxidative stress may contribute to alterations in CSE mutant mice. H2S acts by sulfhydrating the p65 subunit of NF-κB at cysteine-38, which promotes its binding to the co-activator ribosomal protein S3 (RPS3). Sulfhydration of p65 predominates early following TNFα treatment, then declines and is succeeded by a reciprocal enhancement of p65 nitrosylation. Anti-apoptotic influences of NF-κB, which are markedly diminished in CSE mutant mice. Thus, sulfhydration of NF-κB appears to be a physiologic determinant of its anti-apoptotic transcriptional activity.

The parasites of the order kinetoplastidae including Leishmania spp. emerge from most ancient phylogenic branches of unicellular eukaryotic lineages. In their life cycle, topoisomerase I plays a significant role in carrying out vital cellular processes. Camptothecin (CPT), an inhibitor of DNA topoisomerase I, induces programmed cell death (PCD) both in the amastigotes and promastigotes form of L. donovani parasites. CPT-induced cellular dysfunction in L. donovani promastigotes is characterized by several cytoplasmic and nuclear features of apoptosis. CPT inhibits cellular respiration that results in mitochondrial hyperpolarization taking place by oligomycin-sensitive F0-F1 ATPase-like protein in leishmanial cells. During the early phase of activation, there is an increase in reactive oxygen species (ROS) inside cells, which causes subsequent elevation in the level of lipid peroxidation and decrease in reducing equivalents like GSH. Endogenous ROS formation and lipid peroxidation cause eventual loss of mitochondrial membrane potential. Furthermore, cytochrome c is released into the cytosol in a manner independent of involvement of CED3/CPP32 group of proteases and unlike mammalian cells it is insensitive to cyclosporin A. These events are followed by activation of both CED3/CPP32 and ICE group of proteases in PCD of Leishmania. Taken together, our study indicates that different biochemical events leading to apoptosis in leishmanial cells provide information that could be exploited to develop newer potential therapeutic targets.

Obesity is one of the major health issues in the United States. Consumption of diets rich in energy, notably from fats and sugars (high-fat/high-sugar diet: HF/HSD) is linked to the development of obesity and a popular dietary approach for weight loss is to reduce fat intake. Obesity research traditionally uses low and high fat diets and there has been limited investigation of the potential detrimental effects of a low-fat/high-sugar diet (LF/HSD) on body fat accumulation and health. Therefore, in the present study, we investigated the effects of HF/HSD and LF/HSD on microbiota composition, gut inflammation, gut-brain vagal communication and body fat accumulation. Specifically, we tested the hypothesis that LF/HSD changes the gut microbiota, induces gut inflammation and alters vagal gut-brain communication, associated with increased body fat accumulation. Sprague-Dawley rats were fed an HF/HSD, LF/HSD or control low-fat/low-sugar diet (LF/LSD) for 4 weeks. Body weight, caloric intake, and body composition were monitored daily and fecal samples were collected at baseline, 1, 6 and 27 days after the dietary switch. After four weeks, blood and tissues (gut, brain, liver and nodose ganglia) were sampled. Both HF/HSD and LF/HSD-fed rats displayed significant increases in body weight and body fat compared to LF/LSD-fed rats. 16S rRNA sequencing showed that both HF/HSD and LF/HSD-fed animals exhibited gut microbiota dysbiosis characterized by an overall decrease in bacterial diversity and an increase in Firmicutes/Bacteriodetes ratio. Dysbiosis was typified by a bloom in Clostridia and Bacilli and a marked decrease in Lactobacillus spp. LF/HSD-fed animals showed a specific increase in Sutterella and Bilophila, both Proteobacteria, abundances of which have been associated with liver damage. Expression of pro-inflammatory cytokines, such as IL-6, IL-1β and TNFα was upregulated in the cecum while levels of tight junction protein occludin were downregulated in both HF/HSD and LF/HSD fed rats. HF/HSD and LF/HSD-fed rats also exhibited an increase in cecum and serum levels of lipopolysaccharide (LPS), a pro-inflammatory bacterial product. Immunofluorescence revealed the withdrawal of vagal afferents from the gut and at their site of termination the nucleus of the solitary tract (NTS) in both the HF/HSD and LF/HSD rats. Moreover, there was significant microglia activation in the nodose ganglia, which contain the vagal afferent neuron cell bodies, of HF/HSD and LF/HSD rats. Taken together, these data indicate that, similar to HF/HSD, consumption of an LF/HSD induces dysbiosis of gut microbiota, increases gut inflammation and alters vagal gut-brain communication. These changes are associated with an increase in body fat accumulation.

Head and neck squamous cell carcinoma (HNSCC) cells exposed to cisplatin (CIS) displayed a dramatic ATM-dependent phosphorylation of DNp63a that leads to the transcriptional regulation of downstream mRNAs. Here, we report that phospho (p)-DNp63a transcriptionally deregulates miRNA expression after CIS treatment. Several p-DNp63a-dependent microRNA species (miRNAs) were deregulated in HNSCC cells upon CIS exposure, including miR-181a, miR-519a, and miR-374a (downregulated) and miR-630 (upregulated). Deregulation of miRNA expression led to subsequent modulation of mRNA expression of several targets (TP53-S46, HIPK2, ATM, CDKN1A and 1B, CASP3, PARP1 and 2, DDIT1 and 4, BCL2 and BCL2L2, TP73, YES1, and YAP1) that are involved in the apoptotic process. Our data support the notion that miRNAs are critical downstream targets of p-DNp63a and mediate key pathways implicated in the response of cancer cells to chemotherapeutic drugs.

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of DNp63a associated with its increased phosphorylated status. Since DNp63a usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of DNp63a in HNSCC cells upon cisplatin exposure. We found that DNp63a is phosphorylated in the time-dependent fashion at the following positions: S385, T397 and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of DNp63a in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of DNp63a residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of DNp63a degradation. Finally, we demonstrated that the DNp63a protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of DNp63a following DNA damage.

Obesity is associated with consumption of energy-dense diets and development of systemic inflammation. Gut microbiota play a role in energy harvest and inflammation and can influence the change from lean to obese phenotypes. The nucleus of the solitary tract (NTS) is a brain target for gastrointestinal signals modulating satiety and alterations in gut-brain vagal pathway may promote overeating and obesity. Therefore, we tested the hypothesis that high-fat diet-induced changes in gut microbiota alter vagal gut-brain communication associated with increased body fat accumulation. Sprague-Dawley rats consumed a low energy-dense rodent diet (LFD; 3.1 kcal/g) or high energy-dense diet (HFD, 5.24 kcal/g). Minocycline was used to manipulate gut microbiota composition. 16S Sequencing was used to determine microbiota composition. Immunofluorescence against IB4 and Iba1 was used to determine NTS reorganization and microglia activation. Nodose ganglia from LFD rats were isolated and co-cultured with different bacteria strains to determine neurotoxicity. HFD altered gut microbiota with increases in Firmicutes/Bacteriodetes ratio and in pro-inflammatory Proteobacteria proliferation. HFD triggered reorganization of vagal afferents and microglia activation in the NTS, associated with weight gain. Minocycline-treated HFD rats exhibited microbiota profile comparable to LFD animals. Minocycline suppressed HFD-induced reorganization of vagal afferents and microglia activation in the NTS, and reduced body fat accumulation. Proteobacteria isolated from cecum of HFD rats were toxic to vagal afferent neurons in culture. Our findings show that diet-induced shift in gut microbiome may disrupt vagal gut-brain communication resulting in microglia activation and increased body fat accumulation.

The PKR-like ER kinase (PERK), a transmembrane protein, resides in the endoplasmic reticulum (ER). Its activation serves as a key sensor of ER stress, which has been implicated in traumatic brain injury (TBI). The loss of memory is one of the most common symptoms after TBI, but the precise role of PERK activation in memory impairment after TBI has not been well elucidated. Here, we have shown that blocking the activation of PERK using GSK2656157 prevents the loss of dendritic spines and rescues memory deficits after TBI. To elucidate the molecular mechanism, we found that activated PERK phosphorylates CAMP response element binding protein (CREB) and PSD95 directly at the S129 and T19 residues, respectively. Phosphorylation of CREB protein prevents its interaction with a coactivator, CREB-binding protein, and subsequently reduces the BDNF level after TBI. Conversely, phosphorylation of PSD95 leads to its downregulation in pericontusional cortex after TBI in male mice. Treatment with either GSK2656157 or overexpression of a kinase-dead mutant of PERK (PERK-K618A) rescues BDNF and PSD95 levels in the pericontusional cortex by reducing phosphorylation of CREB and PSD95 proteins after TBI. Similarly, administration of either GSK2656157 or overexpression of PERK-K618A in primary neurons rescues the loss of dendritic outgrowth and number of synapses after treatment with a PERK activator, tunicamycin. Therefore, our study suggests that inhibition of PERK phosphorylation could be a potential therapeutic target to restore memory deficits after TBI.

Persistent endoplasmic reticulum (ER) stress in neurons is associated with activation of inflammatory cells and subsequent neuroinflammation following traumatic brain injury (TBI); however, the underlying mechanism remains elusive. We found that induction of neuronal-ER stress, which was mostly characterized by an increase in phosphorylation of a protein kinase R-like ER kinase (PERK) leads to release of excess interferon (IFN)␤ due to atypical activation of the neuronal-STING signaling pathway. IFN␤ enforced activation and polarization of the primary microglial cells to inflammatory M1 phenotype with the secretion of a proinflammatory chemokine CXCL10 due to activation of STAT1 signaling. The secreted CXCL10, in turn, stimulated the T-cell infiltration by serving as the ligand and chemoattractant for CXCR3 ϩ T-helper 1 (Th1) cells. The activation of microglial cells and infiltration of Th1 cells resulted in white matter injury, characterized by impaired myelin basic protein and neurofilament NF200, the reduced thickness of corpus callosum and external capsule, and decline of mature oligodendrocytes and oligodendrocyte precursor cells. Intranasal delivery of CXCL10 siRNA blocked Th1 infiltration but did not fully rescue microglial activation and white matter injury after TBI. However, impeding PERK-phosphorylation through the administration of GSK2656157 abrogated neuronal induction of IFN␤, switched microglial polarization to M2 phenotype, prevented Th1 infiltration, and increased Th2 and Treg levels. These events ultimately attenuated the white matter injury and improved anxiety and depressive-like behavior following TBI.