Cell Stress Pathways Research Articles

Comparative Analysis of Akebia trifoliata Fruit Softening at Different Flesh Ripening Stages Using Tandem Mass Tag Technology.

Owing to its medicinal and high nutritional values, Akebia trifoliata can be considered as a new type of medicinal and edible homologous resources, and it has begun to be widely cultivated in many areas of China. Over-softening of fruit would affect the sensorial quality, utilization rate, and consumer acceptance of the fruit postharvest. However, fruit softening has not been characterized and the molecular mechanism underlying A. trifoliata fruit softening during ripening remains unclear. A comparative proteomic analysis was performed on the fruit at three developmental stages using tandem mass tag technology. In total, 2,839 proteins and 302 differentially abundant proteins (DAPs) were identified. Bioinformatics analysis indicated that most DAPs were implicated in oxidoreductase activity, protein domain-specific binding and pyruvate metabolism. Moreover, 29 DAPs associated with cell wall metabolism, plant hormone, and stress and defense response pathways were validated using quantitative PCR. Notably, pectinesterase, pectate lyase, and β-galactosidase, which are involved in cell wall degradation, as well as gibberellin regulated protein, cysteine protease, thaumatin-like protein and heat shock proteins which is involved in plant hormone, and stress and defense response, were significantly up-regulated in softening fruit compared with the levels in non-softening fruit. This indicated that they might play key roles in A. trifoliata fruit softening. Our findings will provide new insights into potential genes influencing fruit softening traits of A. trifoliata, which will help to develop strategies to improve fruit quality and reduce softening-related losses.

Autophagy, Apoptosis, the Unfolded Protein Response, and Lung Function in Idiopathic Pulmonary Fibrosis.

Autophagy, apoptosis, and the unfolded protein response (UPR) are fundamental biological processes essential for manifold cellular functions in health and disease. Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal pulmonary disorder associated with aging that has limited therapies, reflecting our incomplete understanding. We conducted an observational study linking molecular markers of cell stress response pathways (UPR: BiP, XBP1; apoptosis: cleaved caspase-3; autophagy: LC3β) in lung tissues from IPF patients and correlated the expression of these protein markers to each subject’s lung function measures. We hypothesized that changes in lung tissue expression of apoptosis, autophagy, and UPR markers correlate with lung function deficits in IPF. The cell stress markers BiP, XBP1, LC3β puncta, and cleaved caspase-3 were found to be elevated in IPF lungs compared to non-IPF lungs, and, further, BiP and cleaved caspase-3 co-localized in IPF lungs. Considering lung function independently, we observed that increased XBP1, BiP, and cleaved caspase-3 were each associated with reduced lung function (FEV1, FVC, TLC, RV). However, increased lung tissue expression of LC3β puncta was significantly associated with increased diffusion capacity (DLCO), an indicator of alveolar–capillary membrane function. Similarly, the co-localization of UPR (XBP1, BiP) and autophagy (LC3β puncta) markers was positively correlated with increased lung function (FEV1, FVC, TLC, DLCO). However, the presence of LC3β puncta can indicate either autophagy flux inhibition or activation. While the nature of our observational cross-sectional study design does not allow conclusions regarding causal links between increased expression of these cell stress markers, lung fibrosis, and lung function decline, it does provide some insights that are hypothesis-generating and suggests that within the milieu of active UPR, changes in autophagy flux may play an important role in determining lung function. Further research is necessary to investigate the mechanisms linking UPR and autophagy in IPF and how an imbalance in these cell stress pathways can lead to progressive fibrosis and loss of lung function. We conclude by presenting five testable hypotheses that build on the research presented here. Such an understanding could eventually lead to the development of much-needed therapies for IPF.

The Unfolded Protein Response As a Predictor of Chemosensitivity in Pediatric Acute Myeloid Leukemia

Background: Survival in newly diagnosed pediatric patients with acute myeloid leukemia (AML) is approximately 65% with a much lower survival in patients with relapsed disease. Leukemia cells are exposed to proteotoxic stress due to their rapid, inefficient metabolism and accumulation of reactive oxygen species. This proteotoxic stress results in the production of misfolded proteins, which leads to activation of cell stress pathways, such as the unfolded protein response (UPR). We hypothesized that UPR induction correlates with a better response to chemotherapy.Methods: Peripheral blood samples from pediatric patients with AML were collected prior to initiation of induction chemotherapy, then at ~6 hours (h) and 24 h post initiation of chemotherapy. Peripheral blood mononuclear cells were frozen as pellets after depletion of non-tumor cells using magnetic bead selection (Miltenyi Biotech). The expression of UPR proteins was determined by chemiluminescence using an automated capillary electrophoresis system, ProteinSimple WesTM. The expressions of 5 UPR proteins were examined including Grp78, IRE1, total and phosphorylated (active) eIF2-α, and ATF6-α. Protein expression was normalized to actin expression and fold differences were compared between samples and within each sample at different time points.Results: Within each group of UPR proteins, there was a wide variability in the level of baseline expression, with 6 of 38 samples expressing >4 fold increase in Grp78, compared to the median level in healthy pediatric and adult controls (Figure 1). Basal IRE1 levels were also significantly higher in pediatric controls vs. adult controls. A trend towards lower overall survival (OS) and event-free survival (EFS) was noted in patients who had a low baseline Grp78 and a low baseline IRE1 (low baseline IRE1 was defined as <2 x below the median level of healthy pediatric controls). Post chemotherapy, UPR induction (defined as >2 x above baseline level) was seen in a subset of patients, most notably for ATF6α. However, post chemotherapy UPR induction was not associated with differences in either event-free survival or overall survival.Conclusion: UPR has a variable expression at baseline in pediatric AML patients, with a trend for lower overall survival in patients with a low basal Grp78 and IRE1 expression, suggesting chemoresistance in this subset of patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Long-Term Exposure to Nanosized TiO2 Triggers Stress Responses and Cell Death Pathways in Pulmonary Epithelial Cells.

There is little in vitro data available on long-term effects of TiO2 exposure. Such data are important for improving the understanding of underlying mechanisms of adverse health effects of TiO2. Here, we exposed pulmonary epithelial cells to two doses (0.96 and 1.92 µg/cm2) of TiO2 for 13 weeks and effects on cell cycle and cell death mechanisms, i.e., apoptosis and autophagy were determined after 4, 8 and 13 weeks of exposure. Changes in telomere length, cellular protein levels and lipid classes were also analyzed at 13 weeks of exposure. We observed that the TiO2 exposure increased the fraction of cells in G1-phase and reduced the fraction of cells in G2-phase, which was accompanied by an increase in the fraction of late apoptotic/necrotic cells. This corresponded with an induced expression of key apoptotic proteins i.e., BAD and BAX, and an accumulation of several lipid classes involved in cellular stress and apoptosis. These findings were further supported by quantitative proteome profiling data showing an increase in proteins involved in cell stress and genomic maintenance pathways following TiO2 exposure. Altogether, we suggest that cell stress response and cell death pathways may be important molecular events in long-term health effects of TiO2.

The endoplasmic reticulum unfolded protein response - homeostasis, cell death and evolution in virus infections.

ABSTRACTViruses elicit cell and organismic stress, and offset homeostasis. They trigger intrinsic, innate and adaptive immune responses, which limit infection. Viruses restore homeostasis by harnessing evolutionary conserved stress responses, such as the endoplasmic reticulum (ER) unfolded protein response (UPRER). The canonical UPRER restores homeostasis based on a cell-autonomous signalling network modulating transcriptional and translational output. The UPRER remedies cell damage, but upon severe and chronic stress leads to cell death. Signals from the UPRER flow along three branches with distinct stress sensors, the inositol requiring enzyme (Ire) 1, protein kinase R (PKR)-like ER kinase (PERK), and the activating transcription factor 6 (ATF6). This review shows how both enveloped and non-enveloped viruses use the UPRER to control cell stress and metabolic pathways, and thereby enhance infection and progeny formation, or undergo cell death. We highlight how the Ire1 axis bypasses apoptosis, boosts viral transcription and maintains dormant viral genomes during latency and persistence periods concurrent with long term survival of infected cells. These considerations open new options for oncolytic virus therapies against cancer cells where the UPRER is frequently upregulated. We conclude with a discussion of the evolutionary impact that viruses, in particular retroviruses, and anti-viral defense has on the UPRER.

Complementary and divergent functions of zebrafish Tango1 and Ctage5 in tissue development and homeostasis

Coat protein complex II (COPII) factors mediate cargo export from the endoplasmic reticulum (ER), but bulky collagens and lipoproteins are too large for traditional COPII vesicles. Mammalian CTAGE5 and TANGO1 have been well characterized individually as specialized cargo receptors at the ER that function with COPII coats to facilitate trafficking of bulky cargoes. Here, we present a genetic interaction study in zebrafish of deletions in ctage5, tango1, or both to investigate their distinct and complementary potential functions. We found that Ctage5 and Tango1 have different roles related to organogenesis, collagen versus lipoprotein trafficking, stress-pathway activation, and survival. While disruption of both ctage5 and tango1 compounded phenotype severity, mutation of either factor alone revealed novel tissue-specific defects in the building of heart, muscle, lens, and intestine, in addition to previously described roles in the development of neural and cartilage tissues. Together, our results demonstrate that Ctage5 and Tango1 have overlapping functions, but also suggest divergent roles in tissue development and homeostasis.

Cell-cycle phospho-regulation of the kinetochore.

The kinetochore is a mega-dalton protein assembly that forms within centromeric regions of chromosomes and directs their segregation during cell division. Here we review cell cycle-mediated phosphorylation events at the kinetochore, with a focus on the budding yeast Saccharomyces cerevisiae and the insight gained from forced associations of kinases and phosphatases. The point centromeres found in the budding yeast S. cerevisiae are one of the simplest such structures found in eukaryotes. The S. cerevisiae kinetochore comprises a single nucleosome, containing a centromere-specific H3 variant Cse4CENP-A, bound to a set of kinetochore proteins that connect to a single microtubule. Despite the simplicity of the budding yeast kinetochore, the proteins are mostly homologous with their mammalian counterparts. In some cases, human proteins can complement their yeast orthologs. Like its mammalian equivalent, the regulation of the budding yeast kinetochore is complex: integrating signals from the cell cycle, checkpoints, error correction, and stress pathways. The regulatory signals from these diverse pathways are integrated at the kinetochore by post-translational modifications, notably phosphorylation and dephosphorylation, to control chromosome segregation. Here we highlight the complex interplay between the activity of the different cell-cycle kinases and phosphatases at the kinetochore, emphasizing how much more we have to understand this essential structure.

F13A1 transglutaminase expression in human adipose tissue increases in acquired excess weight and associates with inflammatory status of adipocytes.

F13A1/FXIII-A transglutaminase has been linked to adipogenesis in cells and to obesity in humans and mice, however, its role and associated molecular pathways in human acquired excess weight have not been explored. We examined F13A1 expression and association to human weight gain in weight-discordant monozygotic twins (Heavy-Lean difference (ΔWeight, 16.8 kg ± 7.16 for n = 12). The twin pairs were examined for body composition (by dual-energy X-ray absorptiometry), abdominal body fat distribution (by magnetic resonance imaging), liver fat content (by magnetic resonance spectroscopy), circulating adipocytokines, leptin and adiponectin, as well as serum lipids. Affymetrix full transcriptome mRNA analysis was performed from adipose tissue and adipocyte-enriched fractions from subcutaneous abdominal adipose tissue biopsies. F13A1 differential expression between the heavy and lean co-twins was examined and its correlation transcriptome changes between co-twins were performed. F13A1 mRNA showed significant increase in adipose tissue (p < 0.0001) and an adipocyte-enriched fraction (p = 0.0012) of the heavier co-twin. F13A1 differential expression in adipose tissue (Heavy-Lean ΔF13A1) showed significant negative correlation with circulating adiponectin (p = 0.0195) and a positive correlation with ΔWeight (p = 0.034), ΔBodyFat (0.044) and ΔAdipocyte size (volume, p = 0.012;) in adipocyte-enriched fraction. A whole transcriptome-wide association study (TWAS) on ΔF13A1 vs weight-correlated ΔTranscriptome identified 182 F13A1-associated genes (r > 0.7, p = 0.05) with functions in several biological pathways including cell stress, inflammatory response, activation of cells/leukocytes, angiogenesis and extracellular matrix remodeling. F13A1 did not associate with liver fat accumulation. F13A1 levels in adipose tissue increase with acquired excess weight and associate with pro-inflammatory, cell stress and tissue remodeling pathways. This supports its role in expansion and inflammation of adipose tissue in obesity.

Abstract IA13: An mTOR/eIF4E-independent translation mechanism promotes breast cancer metastasis

Abstract While translation initiation of most mammalian mRNAs is mediated by a 5' cap structure that binds eIF4E, inactivation/silencing of eIF4E, or quantitative sequestration of eIF4E by 4E-BPs through mTORC1 inhibition, does not impair translation of many capped mRNAs. This suggests an alternate mechanism may exist for cap-dependent but eIF4E/mTORC1-independent translation. DAP5 (NAT1, eIF4G2, p97) is an eIF4G family member that lacks the N-terminal binding domains for eIF4E and PABP. DAP5 was shown to mediate translation initiation through internal ribosome entry sites (IRES elements). We recently showed that DAP5 utilizes the cap binding activity of eIF3d to facilitate cap-dependent translation of a large number of capped mRNAs, making it a previous unknown major mechanism of cap-dependent but eIF4E/mTORC1-independent mRNA translation. Genome-wide transcriptomic and translatomic analyses indicate that DAP5 is required for translation of many transcription factor and cell receptor capped mRNAs and their mRNA targets involved in cell survival, motility, DNA repair, oncogenesis, translation initiation, and cancer cell metastasis. Up to 20% of cellular mRNAs utilize DAP5-eIF3d for cap-dependent mRNA translation. Mass spectrometry and in vivo cross-linking studies found that eIF3d is a direct binding partner of DAP5 but does not bind strongly to eIF4GI or II family members that bind eIF4E. In vitro translation and ribosome complex studies demonstrate that DAP5 and eIF3d are both essential for eIF4E and mTORC1-independent capped-mRNA translation. Given the prevalence of oncogenic and survival mRNAs directed in translation by DAP5-eIF3d, we analyzed the NCI human tissue genome cancer atlas (TCGA) and found that high expression of DAP5 mRNA is strongly associated with estrogen receptor-negative (ER-) breast cancer metastasis and poor survival. We therefore investigated the role of DAP5 in preclinical mouse models of highly metastatic ER- breast cancer. Reduction of DAP5 expression by ~70% almost eliminated metastasis to the lung and significantly increased survival with no impact on primary tumor growth. These and other studies indicate that the DAP5-eIF3d complex is a significant alternate mechanism for cap-dependent, eIF4E/mTORC1-independent mRNA translation despite lacking eIF4E and PABP binding, that DAP5 directs translation of many capped mRNAs that are involved in cell stress and survival pathways, is essential for promoting breast cancer metastasis, and that most mRNAs translated by DAP5 do not utilize an IRES. Citation Format: Columba de la Parra, Amanda Ernlund, Amandine Alard, Kelly Ruggles, Beatrix Ueberheide, Olga Katsara, Robert J. Schneider. An mTOR/eIF4E-independent translation mechanism promotes breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA13.

Macrophage dysfunction in cystic fibrosis: Nature or nurture?

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) affect the homeostasis of chloride flux by epithelial cells. This has deleterious consequences, especially in respiratory epithelia, where the defect results in mucus accumulation distinctive of cystic fibrosis. CFTR is, however, also expressed in phagocytic cells, like macrophages. Immune cells are highly sensitive to conditioning by their environment; thus, CFTR dysfunction in epithelia influences macrophages by affecting the lung milieu, but the mutations also appear to be directly consequential for intrinsic macrophage functions. Particular mutations can alter CFTR's folding, traffic of the protein to the membrane and function. As such, understanding the intrinsic effects of CFTR mutation requires distinguishing the secondary effects of misfolded CFTR on cell stress pathways from the primary defect of CFTR dysfunction/absence. Investigations into CFTR's role in macrophages have exploited various models, each with their own advantages and limitations. This review summarizes these methodologic approaches, discussing their physiological correspondence and highlighting key findings. The controversy surrounding CFTR-dependent acidification is used as a case study to highlight difficulties in commensurability across model systems. Recent work in macrophage biology, including polarization and host-pathogen interaction studies, brought into the context of CFTR research, offers potential explanations for observed discrepancies between studies. Moreover, the rapid advancement of novel gene editing technologies and new macrophage model systems makes this assessment of the field's models and methodologies timely.

Signatures of cell stress and altered bioenergetics in skin fibroblasts from patients with multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system inflammatory demyelinating disease and the most common cause of non-traumatic disability in young adults. Despite progress in the treatment of the active relapsing disease, therapeutic options targeting irreversible progressive decline remain limited. Studies using skin fibroblasts derived from patients with neurodegenerative disorders demonstrate that cell stress pathways and bioenergetics are altered when compared to healthy individuals. However, findings in MS skin fibroblasts are limited. Here, we collected skin fibroblasts from 24 healthy control individuals, 30 patients with MS, and ten with amyotrophic lateral sclerosis (ALS) to investigate altered cell stress profiles. We observed endoplasmic reticulum swelling in MS skin fibroblasts, and increased gene expression of cell stress markers including BIP, ATF4, CHOP, GRP94, P53, and P21. When challenged against hydrogen peroxide, MS skin fibroblasts had reduced resiliency compared to ALS and controls. Mitochondrial and glycolytic functions were perturbed in MS skin fibroblasts while exhibiting a significant increase in lactate production over ALS and controls. Our results suggest that MS skin fibroblasts have an underlying stress phenotype, which may be disease specific. Interrogating MS skin fibroblasts may provide patient specific molecular insights and aid in prognosis, diagnosis, and therapeutic testing enhancing individualized medicine.

Microwaves can reverse the tumour phenotype of human papillomavirus type 16 (HPV16)-positive keratinocytes in 3D cell culture models: a novel therapy for HPV-associated disease?

High-risk human papillomavirus (HPV) is the causative agent of benign, precancerous and cancerous lesions, in both anogenital and oropharyngeal sites. Increased expression of the viral oncoproteins E6 and E7 are responsible for tumour progression. The treatment of these precancerous and cancerous lesions is invasive, painful and with long-term side effects. Localised microwaves have been used successfully in the clinic for the treatment of verrucas, which are caused by low-risk HPV genotypes (&gt;75% success rate versus &gt;33% for cryotherapy). Moreover, local hyperthermia is known to have anti-tumour effects. Ten-second microwave treatment of 3D in vitro-grown cervical tumour tissues (HPV16-positive SiHa cell) resulted in cell death in the treated zone while the tissue integrity was disrupted in the adjacent area. Microwaves induced apoptosis (induction of cleaved caspase 3) and autophagy (induction of LC3) and inhibited cell proliferation (loss of Ki67 and MCM2) in the entire tissue. Furthermore, HPV16 E6 and E7 expression was reduced in cells in the treated and transition zones, with subsequent induction of expression of the apoptosis-regulator, p53 over a 24 hour period following microwave treatment. Thermal stress, identified with the Heat Shock Protein 70 (HSP70) and translational stress identified by G3BP expression, was observed in the transition zone. In conclusion, we demonstrate that the microwave treatment induces cell stress pathways and inhibits HPV oncoprotein expression that causes tumour progression. Induction of apoptosis and reduced cell proliferation suggest a reversal of the cervical tumour phenotype in the 3D tissues.

N-Methyl-D-Aspartate Receptors in Hematopoietic Cells: What Have We Learned?

The N-methyl-D-aspartate receptor (NMDAR) provides a pathway for glutamate-mediated inter-cellular communication, best known for its role in the brain but with multiple examples of functionality in non-neuronal cells. Data previously published by others and us provided ex vivo evidence that NMDARs regulate platelet and red blood cell (RBC) production. Here, we summarize what is known about these hematopoietic roles of the NMDAR. Types of NMDAR subunits expressed in megakaryocytes (platelet precursors) and erythroid cells are more commonly found in the developing rather than adult brain, suggesting trophic functions. Nevertheless, similar to their neuronal counterparts, hematopoietic NMDARs function as ion channels, and are permeable to calcium ions (Ca2+). Inhibitors that block open NMDAR (memantine and MK-801) interfere with megakaryocytic maturation and proplatelet formation in primary culture. The effect on proplatelet formation appears to involve Ca2+ influx-dependent regulation of the cytoskeletal remodeling. In contrast to normal megakaryocytes, NMDAR effects in leukemic Meg-01 cells are diverted away from differentiation to increase proliferation. NMDAR hypofunction triggers differentiation of Meg-01 cells with the bias toward erythropoiesis. The underlying mechanism involves changes in the intracellular Ca2+ homeostasis, cell stress pathways, and hematopoietic transcription factors that upon NMDAR inhibition shift from the predominance of megakaryocytic toward erythroid regulators. This ability of NMDAR to balance both megakaryocytic and erythroid cell fates suggests receptor involvement at the level of a bipotential megakaryocyte-erythroid progenitor. In human erythroid precursors and circulating RBCs, NMDAR regulates intracellular Ca2+ homeostasis. NMDAR activity supports survival of early proerythroblasts, and in mature RBCs NMDARs impact cellular hydration state, hemoglobin oxygen affinity, and nitric oxide synthase activity. Overexcitation of NMDAR in mature RBCs leads to Ca2+ overload, K+ loss, RBC dehydration, and oxidative stress, which may contribute to the pathogenesis of sickle cell disease. In summary, there is growing evidence that glutamate-NMDAR signaling regulates megakaryocytic and erythroid cells at different stages of maturation, with some intriguing differences emerging in NMDAR expression and function between normal and diseased cells. NMDAR signaling may provide new therapeutic opportunities in hematological disease, but in vivo applicability needs to be confirmed.

Examination of Protein Folding and Metabolic Activity in a Saccharomyces cerevisiae Model for TPI Deficiency

Triose Phosphate Isomerase (TPI) is a glycolytic enzyme that catalyzes the inter‐conversion of DHAP to G3P. Mutations in TPI have been identified that are associated with a neurodegenerative disease in humans known as TPI deficiency. To study the cause of TPI deficiency pathogenesis, we constructed Saccharomyces cerevisiae yeast strains expressing six different TPI alleles that are associated with the development of TPI deficiency in humans and Drosophila melanogaster. Interestingly, not all of the TPI alleles exhibit similar characteristics in terms of stability, temperature sensitivity and activity. Yeast with the M80T allele exhibits significant changes in growth and metabolic activity at all temperatures. Yeast with the E104D allele exhibit an acute temperature sensitive phenotype for growth and metabolic activity at 37C. All of the alleles except C41Y demonstrate protein instability at 37C. This study further examines the role of protein folding in TPI deficiency disease pathogenesis. Yeast expressing the mutant TPI alleles were examined for activation of HSF, a cytoplasmic protein folding stress response transcription factor. Understanding the activation of cell stress pathways in TPI deficient yeast may lead to understanding of disease pathogenesis in TPI deficiency patients and provide potential pharmacological targets.Support or Funding InformationFaculty Research Development Grant from Slippery Rock University

The Multi-Modal Effect of the Anti-fibrotic Drug Pirfenidone on NSCLC.

Although immune checkpoint and targeted therapies offer remarkable benefits for lung cancer treatment, some patients do not qualify for these regimens or do not exhibit consistent benefit. Provided that lung cancer appears to be driven by transforming growth factor beta signaling, we investigated the single drug potency of Pirfenidone, an approved drug for the treatment of lung fibrosis. Five human lung cancer cell lines and one murine line were investigated for transforming growth factor beta inhibition via Pirfenidone by using flow cytometry, In-Cell western analysis, proliferation assays as well as comprehensive analyses of the transcriptome with subsequent bioinformatics analysis. Overall, Pirfenidone induced cell cycle arrest, down-regulated SMAD expression and reduced proliferation in lung cancer. Furthermore, cell stress pathways and pro-apoptotic signaling may be mediated by reduced expression of Survivin. A murine subcutaneous model was used to assess the in vivo drug efficacy of Pirfenidone and showed reduced tumor growth and increased infiltration of T cells and NK cells. This data warrant further clinical evaluation of Pirfenidone with advanced non-small cell lung cancer. The observed in vitro and in vivo effects point to a substantial benefit for using Pirfenidone to reactivate the local immune response and possible application in conjunction with current immunotherapies.

Neuronal ablation of mt-AspRS in mice induces immune pathway activation prior to severe and progressive cortical and behavioral disruption

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare, slowly progressive white matter disease caused by mutations in the mitochondrial aspartyl-tRNA synthetase (mt-AspRS, or DARS2). While patients show characteristic MRI T2 signal abnormalities throughout the cerebral white matter, brainstem, and spinal cord, the phenotypic spectrum is broad and a multitude of gene variants have been associated with the disease. Here, Dars2 disruption in CamKIIα-expressing cortical and hippocampal neurons results in slowly progressive increases in behavioral activity at five months, and culminating by nine months as severe brain atrophy, behavioral dysfunction, reduced corpus callosum thickness, and microglial morphology indicative of neuroinflammation. Interestingly, RNAseq based gene expression studies performed prior to the presentation of this severe phenotype reveal the upregulation of several pathways involved in immune activation, cytokine production and signaling, and defense response regulation. RNA transcript analysis demonstrates that activation of immune and cell stress pathways are initiated in advance of a behavioral phenotype and cerebral deficits. An understanding of these pathways and their contribution to significant neuronal loss in CamKII-Dars2 deficient mice may aid in deciphering mechanisms of LBSL pathology.

Inflammation associated with noise-induced hearing loss.

Inflammation is a complex biological response to harmful stimuli including infection, tissue damage, and toxins. Thus, it is not surprising that cochlear damage by noise includes an inflammatory component. One mechanism by which inflammation is generated by tissue damage is the activation of damage-associated molecular patterns (DAMPs). Many of the cellular receptors for DAMPS, including Toll-like receptors, NOD-like receptors, and DNA receptors, are also receptors for pathogens, and function in the innate immune system. DAMP receptors are known to be expressed by cochlear cells, and binding of molecules released by damaged cells to these receptors result in the activation of cell stress pathways. This leads to the generation of pro-inflammatory cytokines and chemokines that recruit pro-inflammatory leukocytes. Extensive evidence indicates pro-inflammatory cytokines including TNF alpha and interleukin 1 beta, and chemokines including CCL2, are induced in the cochlea after noise exposure. The recruitment of macrophages into the cochlea has also been demonstrated. These provide substrates for noise damage to be enhanced by inflammation. Evidence is provided by the effectiveness of anti-inflammatory drugs in ameliorating noise-induced hearing loss. Involvement of inflammation provides a wide variety of additional anti-inflammatory and pro-resolution agents as potential pharmacological interventions in noise-induced hearing loss.

Marine macroalga Caulerpa: role of its metabolites in modulating cancer signaling.

Cancer, the leading causes of death worldwide, causes multiple metabolic and physiological alterations, leading to an unregulated proliferation of cells. The existing anticancer therapies are usually nonspecific with side effects and or are extremely expensive, thus hunt for better therapeutics is still on, specially efforts are made to look for naturally occurring molecules. Sea harbors several organisms which are unexplored for their biological potentials. Green macroalga genus, Caulerpa, is one such invaluable repository of bioactive metabolites like alkaloids, terpenoids, flavonoids, steroids and tannins with reported bioactivities against many diseases including cancer. Anti-cancerous metabolites of Caulerpa like caulerpenyne (Cyn), caulerpin, caulersin, and racemosin C, possess unique structural moieties and are known to exhibit distinct effects on cancer cells. Theses metabolites are reported to affect microtubule dynamics, unfolded protein response, mitochondrial health, cell cycle progression, metabolic and stress pathways by their cross-talk with signalling proteins like AMPK, GRP78, GADD153, Bid, Bax, AIF, Bcl2, P21, cyclin D, cyclin E, caspase 9, and PTP1B. Targeting of multiple cancer hallmarks by Caulerpa metabolites, with concomitant modulations of multiple signalling cascades, displays its multifactorial approach against cancer. Evaluation of anti-cancer properties of this genus is particularly important as Caulerpa species are widely edible and utilized in several delicacies in the coastal countries. This is the first review article providing a consolidated information about the role of Caulerpa in cancer with major contributing metabolites and plausible modulations in cancer signaling and prospects.

Examination of HSF Activation in a Saccharomyces cerevisiae Model for TPI Deficiency

Triose Phosphate Isomerase (TPI) is a glycolytic enzyme that catalyzes the inter‐conversion of DHAP to G3P. Mutations in TPI have been identified that are associated with a neurodegenerative disease in humans known as TPI deficiency. To study the cause of TPI deficiency pathogenesis, we constructed Saccharomyces cerevisiae yeast strains expressing six different TPI alleles that are associated with the development of TPI deficiency in humans and Drosophila melanogaster. Interestingly, not all of the TPI alleles exhibit similar characteristics in terms of stability, temperature sensitivity and activity. Yeast with the M80T allele exhibits significant changes in growth and metabolic activity at all temperatures. Yeast with the E104D allele exhibit an acute temperature sensitive phenotype for growth and metabolic activity at 37C. All of the alleles except C41Y demonstrate protein instability at 37C. This study further examines the role of protein folding in TPI deficiency disease pathogenesis. Yeast expressing the mutant TPI alleles were examined for activation of HSF, a cytoplasmic protein folding stress response transcription factor. Understanding the activation of cell stress pathways in TPI deficient yeast may lead to understanding of disease pathogenesis in TPI deficiency patients and provide potential pharmacological targets.Support or Funding InformationResearch is supported by an SRU Student Faculty Research GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Limited Fatty Acid Oxidation (FAO) in CPT2 Knockout Myocytes Associates with Insulin Resistance and Cell Stress: possible role of acylcarnitine lipotoxicity

Lipotoxicity (the accumulation of fat‐derived metabolites that activate cell stress pathways) has been implicated in muscle insulin resistance. We have proposed that lipotoxicity, and in particular accumulation of long‐chain acylcarnitines (LCACs), also contributes to the episodic myopathy and rhabdomyolysis that is evident in persons with inherited disorders of long‐chain FAO. Many studies in the arena of LCAC bioactivity have employed exogenous application of LCACs to cells; few studies have modified endogenously‐produced LCACs to determine myocyte outcomes. Thus, conclusions about the (patho)physiological relevance of LCAC‐associated lipotoxicity have been difficult to determine. Herein, we used CRISPR‐Cas9 technology to knock out carnitine palmitoyltransferase‐2 (CPT2) in murine C2C12 myotubes: as expected, this maneuver led to almost complete inhibition of FAO, plus significant cellular and media accumulation of palmitoylcarnitine in palmitate‐treated cells. In CPT2 KO myotubes, treatment with 500 μM palmitate led to ~2‐fold higher production of IL‐6 (a myocyte cell stress marker) and a ~1.5 to 2‐fold increased release of adenylate kinase (cell death and permeability marker) at 3 and 6 hr post‐treatment (when compared to palmitate‐treated wildtype control cells; n=4/treatment). Furthermore, CPT2 KO myotubes displayed higher sensitivity to palmitate‐induced insulin resistance: the % inhibition in terms of insulin‐induced p‐Akt abundance was 60%, 80%, and 95% in CPT2 KO cells (at 200, 500, and 750 μM palmitate, respectively), vs. 38%, 53%, and 67% in controls (p&lt;0.01, n=8/treatment). The results indicate that limitation of long‐chain FAO by loss of CPT2 in muscle cells elicits cell stress and insulin resistance, consistent with a working model in which lipotoxicity and accumulation of LCAC trigger these outcomes. Further studies are needed to determine if endogenously‐produced LCACs contribute to the phenotypes observed in CPT2 KO myotubes, or if LCAC‐independent mechanisms are involved. It remains to be established whether these phenomena underlie the cardiac and skeletal muscle myopathies typical of inherited disorders of FAO, or contribute to the insulin resistance accompanying type 2 diabetes.Support or Funding InformationThis work was supported by USDA‐ARS Project 6026‐51000‐010‐05S.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.