Tfeb-Mediated Transcriptional Regulation of Autophagy Induces Autosis during Ischemia/Reperfusion in the Heart

Autosis is a unique form of cell death with characteristic morphological and biochemical features caused by dysregulated autophagy. Autosis is observed in the heart during the late phase of ischemia/reperfusion (I/R), when marked accumulation of autophagosomes is induced. We previously showed that the excessive accumulation of autophagosomes promotes autosis in cardiomyocytes. Although the inhibition of autophagic flux via the upregulation of Rubicon induces the accumulation of autophagosomes during I/R, it appears that additional mechanisms exacerbating autophagosome accumulation are required for the induction of autosis. Here, we show that Tfeb contributes to the induction of autosis during the late phase of I/R in the heart. During myocardial reperfusion, Tfeb is activated and translocated into the nucleus, which in turn upregulates genes involved in autophagy and lysosomal function. The overexpression of Tfeb enhanced cardiomyocyte death induced by a high dose of TAT-Beclin 1, an effect that was inhibited by the downregulation of Atg7. Conversely, the knockdown of Tfeb attenuated high-dose TAT-Beclin1-induced death in cardiomyocytes. Although the downregulation of Tfeb in the heart significantly decreased the number of autophagic vacuoles and inhibited autosis during I/R, the activation of Tfeb activity via 3,4-dimethoxychalcone, an activator of Tfeb, aggravated myocardial injury during I/R. These findings suggest that Tfeb promotes cardiomyocyte autosis during the late phase of reperfusion in the heart.

Tryptase and IL-33 in Bronchoalveolar Lavage Fluid May Predict the Types of Eosinophilic Pneumonia and Disease Recurrence

<b><i>Introduction:</i></b> Eosinophilic pneumonia (EP) is characterized by a marked accumulation of eosinophils in the lungs and blood. Eosinophils and mast cells play an important role in the pathogenesis of EP via release of biomarkers such as tryptase and interleukin (IL)-33. However, the potential role of these biomarkers is not fully understood. <b><i>Objectives:</i></b> We aimed to evaluate the differences among the levels of tryptase and IL-33 in bronchoalveolar lavage fluid (BALF) from several types of EP. We evaluated the differences between the levels of these biomarkers in the recurrent and nonrecurrent cases. <b><i>Method:</i></b> We prospectively collected the clinical data of patients with EP, diagnosed between 2006 and 2015 in our institution. Bronchoscopy was performed before steroid treatment; BALF was collected. The clinical characteristics of EP patients and the levels of tryptase and IL-33 in BALF were evaluated. <b><i>Results:</i></b> We enrolled 15 patients with chronic EP (CEP), 5 with acute EP (AEP), 10 with drug-induced EP, and 6 with angiitis-related EP. Tryptase levels in the CEP group were significantly higher than that in the drug-induced EP group (<i>p</i> = 0.048), while the AEP group had the highest IL-33 levels. Recurrence of EP was noted in 67% of patients with CEP. The levels of tryptase and IL-33 were notably higher in the recurrent cases than that in the nonrecurrent CEP group (<i>p</i> = 0.004, <i>p</i> = 0.04, respectively). Furthermore, there was a positive correlation between the levels of tryptase and IL-33 in the BALF of patients with CEP (ρ = 0.69, <i>p</i> = 0.004). <b><i>Conclusions:</i></b> Tryptase and IL-33 in BALF are useful biomarkers for the assessment of EP types. These biomarkers could be used to predict disease recurrence.

A Core Metabolome Response of Maize Leaves Subjected to Long-Duration Abiotic Stresses

Abiotic stresses reduce crop growth and yield in part by disrupting metabolic homeostasis and triggering responses that change the metabolome. Experiments designed to understand the mechanisms underlying these metabolomic responses have usually not used agriculturally relevant stress regimes. We therefore subjected maize plants to drought, salt, or heat stresses that mimic field conditions and analyzed leaf responses at metabolome and transcriptome levels. Shared features of stress metabolomes included synthesis of raffinose, a compatible solute implicated in tolerance to dehydration. In addition, a marked accumulation of amino acids including proline, arginine, and γ-aminobutyrate combined with depletion of key glycolysis and tricarboxylic acid cycle intermediates indicated a shift in balance of carbon and nitrogen metabolism in stressed leaves. Involvement of the γ-aminobutyrate shunt in this process is consistent with its previously proposed role as a workaround for stress-induced thiamin-deficiency. Although convergent metabolome shifts were correlated with gene expression changes in affected pathways, patterns of differential gene regulation induced by the three stresses indicated distinct signaling mechanisms highlighting the plasticity of plant metabolic responses to abiotic stress.

Growth and Lipidomic Analyses of Penaeus monodon Larvae Supplemented With Aurantiochytrium limacinum BCC52274

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential for growth and health of larval marine animals. Marine animals have a limited capability for LC-PUFA synthesis, and the larvae must obtain LC-PUFAs from diet. The protist Aurantiochytrium limacinum (AL) is abundant in 22:6 n-3 (docosahexaenoic acid, DHA), 22:5 n-3 (docosapentaenoic acid, DPA) and 16:0 fatty acids, which qualifies it as an LC-PUFA source for feed application. Therefore, in this study, a common feed containing lower amounts of total LC-PUFAs, Thalasiosira weissflogii, was replaced with AL at graded proportions and supplied to Penaeus monodon larvae from mysis (M) 1 to post-larval (PL) 2 stages to supplement LC-PUFAs in the diet. After that, all shrimp from PL2 to PL12 were continuously reared and subjected to the same diet regime, which was a combination of Artemia and commercial dried feed. The AL-supplemented PL2 shrimp demonstrated marked accumulation of the key fatty acids present in AL—16:0, DPA and DHA. The supplemented larvae showed no difference in growth during the supplementation period from M1 to PL2; however, average body weight and biomass were increased in PL12 shrimp that were fed earlier with AL. Lipidomic analysis revealed that profiles of fatty acids but not lipid classes/subclasses in PL shrimp reflected the supplied diet. The main saturated fatty acid (SFA, 16:0) predominantly accumulated in acylglycerols, which are energy-reserve lipids, in PL2 shrimp. Both LC-PUFAs (DHA and DPA) were preferentially deposited in phospholipids or structural lipids. Furthermore, while the amounts of both LC-PUFAs increased along with the amount of supplied AL, that of the SFA did not. This suggests that LC-PUFAs were prioritized to be stored over SFA when both types of fatty acids were present in high amounts. This analysis substantiates the importance of LC-PUFAs and provides an insight into how different types of the dietary fatty acids were differentially accumulated in lipid classes and subclasses for their biological functions.

AATF/Che-1, a new component of paraspeckles, controls R-loops formation and Interferon activation in Multiple Myeloma.

Multiple myeloma (MM) is a hematological neoplasm of plasma cells characterized by abnormal production of immunoglobulins. Che-1/AATF (Che-1) is an RNA binding protein involved in transcription regulation and is highly expressed in this malignancy. Here we experimentally show that Che-1 interacts with paraspeckle components, including the lncRNA NEAT1_2 (NEAT1), which serves as the seed for the maintenance of these structures. Che-1 and NEAT1 localize on R-loops, three-stranded RNA:DNA hybrids structures involved in DNA transcription and repair. Depletion of Che-1 produces a marked accumulation of RNA:DNA hybrids sustaining activation of a systemic inflammatory response. We provide evidence that high levels of Unfolded Protein Response (UPR) in MM cells induces RNA:DNA hybrids and an interferon (IFN) gene signature. We found that MM patients exhibit elevated R-loops levels and paraspeckle genes mRNAs increase linearly to MM progression. Strikingly, patients showing elevated IFN genes signature are associated with a marked poor prognosis. Overall, these findings delineate that elevated R-loops accumulation and inflammatory signaling may contribute to MM progression and that Che-1/NEAT1 plays an essential role in maintaining R-loops homeostasis by preventing excessive inflammatory signaling.

CIB2 regulates mTORC1 signaling and is essential for autophagy and visual function

Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aβ, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling—a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to ‘nucleotide empty’ or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders.

Loss of CIB2 causes non-canonical autophagy deficits and visual impairment

SUMMARYNon-canonical autophagy or LC3-associated phagocytosis (LAP) is essential for the maintenance and functioning of the retinal pigment epithelium (RPE) and photoreceptors. Although molecular mechanisms still remain elusive, deficits in LAP have been found to be associated with age-related retinal pathology in both mice and humans. In this study, we found that calcium and integrin-binding protein 2 (CIB2) regulates LAP in the RPE. Mice lacking CIB2, both globally and specifically within RPE, have an impaired ability to process the engulfed photoreceptor outer segments due to reduced lysosomal capacity, which leads to marked accumulation of improperly digested remnants, lipid droplets, fused phago-melanosomes in RPE, and impaired visual function. In aged mice, we also found marked accumulation of drusen markers APOE, C3, and Aβ, along with esterified cholesterol. Intriguingly, we were able to transiently rescue the photoreceptor function in Cib2 mutant mice by exogenous retinoid delivery. Our study links LAP and phagocytic clearance with CIB2, and their relevance to the sense of sight.

α-synuclein strains that cause distinct pathologies differentially inhibit proteasome

Abnormal α-synuclein aggregation has been implicated in several diseases and is known to spread in a prion-like manner. There is a relationship between protein aggregate structure (strain) and clinical phenotype in prion diseases, however, whether differences in the strains of α-synuclein aggregates account for the different pathologies remained unclear. Here, we generated two types of α-synuclein fibrils from identical monomer and investigated their seeding and propagation ability in mice and primary-cultured neurons. One α-synuclein fibril induced marked accumulation of phosphorylated α-synuclein and ubiquitinated protein aggregates, while the other did not, indicating the formation of α-synuclein two strains. Notably, the former α-synuclein strain inhibited proteasome activity and co-precipitated with 26S proteasome complex. Further examination indicated that structural differences in the C-terminal region of α-synuclein strains lead to different effects on proteasome activity. These results provide a possible molecular mechanism to account for the different pathologies induced by different α-synuclein strains.

A Novel Function of Sphingosine Kinase 2 in the Metabolism of Sphinga-4,14-Diene Lipids

The number, position, and configuration of double bonds in lipids affect membrane fluidity and the recruitment of signaling proteins. Studies on mammalian sphingolipids have focused on those with a saturated sphinganine or mono-unsaturated sphingosine long chain base. Using high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), we observed a marked accumulation of lipids containing a di-unsaturated sphingadiene base in the hippocampus of mice lacking the metabolic enzyme sphingosine kinase 2 (SphK2). The double bonds were localized to positions C4–C5 and C14–C15 of sphingadiene using ultraviolet photodissociation-tandem mass spectrometry (UVPD-MS/MS). Phosphorylation of sphingoid bases by sphingosine kinase 1 (SphK1) or SphK2 forms the penultimate step in the lysosomal catabolism of all sphingolipids. Both SphK1 and SphK2 phosphorylated sphinga-4,14-diene as efficiently as sphingosine, however deuterated tracer experiments in an oligodendrocyte cell line demonstrated that ceramides with a sphingosine base are more rapidly metabolized than those with a sphingadiene base. Since SphK2 is the dominant sphingosine kinase in brain, we propose that the accumulation of sphingadiene-based lipids in SphK2-deficient brains results from the slower catabolism of these lipids, combined with a bottleneck in the catabolic pathway created by the absence of SphK2. We have therefore uncovered a previously unappreciated role for SphK2 in lipid quality control.

Targeted Disruption of Lats1 and Lats2 in Mice Impairs Adrenal Cortex Development and Alters Adrenocortical Cell Fate

It has recently been shown that the loss of the Hippo signaling effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in adrenocortical steroidogenic cells impairs the postnatal maintenance of the adrenal gland. To further explore the role of Hippo signaling in mouse adrenocortical cells, we conditionally deleted the key Hippo kinases large tumor suppressor homolog kinases 1 and -2 (Lats1 and Lats2, two kinases that antagonize YAP and TAZ transcriptional co-regulatory activity) in steroidogenic cells using an Nr5a1-cre strain (Lats1flox/flox;Lats2flox/flox;Nr5a1-cre). We report here that developing adrenocortical cells adopt characteristics of myofibroblasts in both male and female Lats1flox/flox;Lats2flox/flox;Nr5a1-cre mice, resulting in a loss of steroidogenic gene expression, adrenal failure and death by 2 to 3 weeks of age. A marked accumulation of YAP and TAZ in the nuclei of the myofibroblast-like cell population with an accompanying increase in the expression of their transcriptional target genes in the adrenal glands of Lats1flox/flox;Lats2flox/flox;Nr5a1-cre animals suggested that the myofibroblastic differentiation could be attributed in part to YAP and TAZ. Taken together, our results suggest that Hippo signaling is required to maintain proper adrenocortical cell differentiation and suppresses their differentiation into myofibroblast-like cells.