Neuronal cholesterol metabolism increases dendritic outgrowth and synaptic markers via a concerted action of GGTase-I and Trk

We are deeply thankful to Professor David W. Russell (University of Texas Southwestern Medical Center) for the kind gift of the anti-CYP46A1 antibody. This work was supported by FEDER (COMPETE Programme) and national funds from Fundacao para a Ciencia e a Tecnologia (FCT), Portugal, research grants iMed.ULisboa (UID/DTP/04138/2013), PTDC/SAU/NMC/110809/2009 (to E.R.), SFRH/BD/78041/2011 (to M.M.) SFRH/BPD/95855/2013 (to M.J.N), and, Swedish Research Council (J.L.R. and I.B.), Marie Curie Career Integration Grant and Novo Nordisk Fonden (J.L.R.) and Swedish Brain Power (I.B.).Cholesterol 24-hydroxylase (CYP46A1) is responsible for brain cholesterol elimination and therefore plays a crucial role in the control of brain cholesterol homeostasis. Altered CYP46A1 expression has been associated with several neurodegenerative diseases and changes in cognition. Since CYP46A1 activates small guanosine triphosphate-binding proteins (sGTPases), we hypothesized that CYP46A1 might be affecting neuronal development and function by activating tropomyosin-related kinase (Trk) receptors and promoting geranylgeranyl transferase-I (GGTase-I) prenylation activity. Our results show that CYP46A1 triggers an increase in neuronal dendritic outgrowth and dendritic protrusion density, and elicits an increase of synaptic proteins in the crude synaptosomal fraction. Strikingly, all of these effects are abolished by pharmacological inhibition of GGTase-I activity. Furthermore, CYP46A1 increases Trk phosphorylation, its interaction with GGTase-I, and the activity of GGTase-I, which is crucial for the enhanced dendritic outgrowth. Cholesterol supplementation studies indicate that cholesterol reduction by CYP46A1 is the necessary trigger for these effects. These results were confirmed in vivo, with a significant increase of p-Trk, pre- and postsynaptic proteins, Rac1, and decreased cholesterol levels, in crude synaptosomal fractions prepared from CYP46A1 transgenic mouse cortex. This work describes the molecular mechanisms by which neuronal cholesterol metabolism effectively modulates neuronal outgrowth and synaptic markers.publishersversionpublishe

Skeletal muscle PGC-1α1 reroutes kynurenine metabolism to increase energy efficiency and fatigue-resistance

The coactivator PGC-1α1 is activated by exercise training in skeletal muscle and promotes fatigue-resistance. In exercised muscle, PGC-1α1 enhances the expression of kynurenine aminotransferases (Kats), which convert kynurenine into kynurenic acid. This reduces kynurenine-associated neurotoxicity and generates glutamate as a byproduct. Here, we show that PGC-1α1 elevates aspartate and glutamate levels and increases the expression of glycolysis and malate-aspartate shuttle (MAS) genes. These interconnected processes improve energy utilization and transfer fuel-derived electrons to mitochondrial respiration. This PGC-1α1-dependent mechanism allows trained muscle to use kynurenine metabolism to increase the bioenergetic efficiency of glucose oxidation. Kat inhibition with carbidopa impairs aspartate biosynthesis, mitochondrial respiration, and reduces exercise performance and muscle force in mice. Our findings show that PGC-1α1 activates the MAS in skeletal muscle, supported by kynurenine catabolism, as part of the adaptations to endurance exercise. This crosstalk between kynurenine metabolism and the MAS may have important physiological and clinical implications

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling

Background: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. Conclusion: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases

Geoeconomic variations in epidemiology, ventilation management, and outcomes in invasively ventilated intensive care unit patients without acute respiratory distress syndrome: a pooled analysis of four observational studies

Background: Geoeconomic variations in epidemiology, the practice of ventilation, and outcome in invasively ventilated intensive care unit (ICU) patients without acute respiratory distress syndrome (ARDS) remain unexplored. In this analysis we aim to address these gaps using individual patient data of four large observational studies. Methods: In this pooled analysis we harmonised individual patient data from the ERICC, LUNG SAFE, PRoVENT, and PRoVENT-iMiC prospective observational studies, which were conducted from June, 2011, to December, 2018, in 534 ICUs in 54 countries. We used the 2016 World Bank classification to define two geoeconomic regions: middle-income countries (MICs) and high-income countries (HICs). ARDS was defined according to the Berlin criteria. Descriptive statistics were used to compare patients in MICs versus HICs. The primary outcome was the use of low tidal volume ventilation (LTVV) for the first 3 days of mechanical ventilation. Secondary outcomes were key ventilation parameters (tidal volume size, positive end-expiratory pressure, fraction of inspired oxygen, peak pressure, plateau pressure, driving pressure, and respiratory rate), patient characteristics, the risk for and actual development of acute respiratory distress syndrome after the first day of ventilation, duration of ventilation, ICU length of stay, and ICU mortality. Findings: Of the 7608 patients included in the original studies, this analysis included 3852 patients without ARDS, of whom 2345 were from MICs and 1507 were from HICs. Patients in MICs were younger, shorter and with a slightly lower body-mass index, more often had diabetes and active cancer, but less often chronic obstructive pulmonary disease and heart failure than patients from HICs. Sequential organ failure assessment scores were similar in MICs and HICs. Use of LTVV in MICs and HICs was comparable (42\ub74% vs 44\ub72%; absolute difference \u20131\ub769 [\u20139\ub758 to 6\ub711] p=0\ub767; data available in 3174 [82%] of 3852 patients). The median applied positive end expiratory pressure was lower in MICs than in HICs (5 [IQR 5\u20138] vs 6 [5\u20138] cm H2O; p=0\ub70011). ICU mortality was higher in MICs than in HICs (30\ub75% vs 19\ub79%; p=0\ub70004; adjusted effect 16\ub741% [95% CI 9\ub752\u201323\ub752]; p<0\ub70001) and was inversely associated with gross domestic product (adjusted odds ratio for a US$10 000 increase per capita 0\ub780 [95% CI 0\ub775\u20130\ub786]; p<0\ub70001). Interpretation: Despite similar disease severity and ventilation management, ICU mortality in patients without ARDS is higher in MICs than in HICs, with a strong association with country-level economic status. Funding: No funding

HIF-1α inhibitor PX-478 preserves pancreatic β cell function in diabetes

During progression of type 2 diabetes, pancreatic β cells are subjected to sustained metabolic overload. We postulated that this state mediates a hypoxic phenotype driven by hypoxia-inducible factor-1α (HIF-1α) and that treatment with the HIF-1α inhibitor PX-478 would improve β cell function. Our studies showed that the HIF-1α protein was present in pancreatic β cells of diabetic mouse models. In mouse islets with high glucose metabolism, the emergence of intracellular Ca oscillations at low glucose concentration and the abnormally high basal release of insulin were suppressed by treatment with the HIF-1α inhibitor PX-478, indicating improvement of β cell function. Treatment of db/db mice with PX-478 prevented the rise of glycemia and diabetes progression by maintenance of elevated plasma insulin concentration. In streptozotocin-induced diabetic mice, PX-478 improved the recovery of glucose homeostasis. Islets isolated from these mice showed hallmarks of improved β cell function including elevation of insulin content, increased expression of genes involved in β cell function and maturity, inhibition of dedifferentiation markers, and formation of mature insulin granules. In response to PX-478 treatment, human islet organoids chronically exposed to high glucose presented improved stimulation index of glucose-induced insulin secretion. These results suggest that the HIF-1α inhibitor PX-478 has the potential to act as an antidiabetic therapeutic agent that preserves β cell function under metabolic overload

PGC-1α4 Interacts with REST to Upregulate Neuronal Genes and Augment Energy Consumption in Developing Cardiomyocytes

Transcriptional coactivator PGC-1α is a main regulator of cardiac energy metabolism. In addition to canonical PGC-1α1, other PGC-1α isoforms have been found to exert specific biological functions in a variety of tissues. We investigated the expression patterns and the biological effects of the non-canonical isoforms in the heart. We used RNA sequencing data to identify the expression patterns of PGC-1α isoforms in the heart. To evaluate the biological effects of the alternative isoform expression, we generated a transgenic mouse with cardiac-specific overexpression of PGC-1α4 and analysed the cardiac phenotype with a wide spectrum of physiological and biophysical tools. Our results show that non-canonical isoforms are expressed in the heart, and that the main variant PGC-1α4 is induced by β-adrenergic signalling in adult cardiomyocytes. Cardiomyocyte specific PGC-1α4 overexpression in mice relieves the RE1-Silencing Transcription factor (REST)-mediated suppression of neuronal genes during foetal heart development. The resulting de-repression of REST target genes induces a cardiac phenotype with increased cellular energy consumption, resulting in postnatal dilated cardiomyopathy. These results propose a new concept for actions of the PGC-1α protein family where activation of the Pgc-1α gene, through its isoforms, induces a phenotype with concurrent supply and demand for cellular energy. These data highlight the biological roles of the different PGC-1α isoforms, which should be considered when future therapies are developed

Cell-Type-Specific Regulation of Degradation of Hypoxia-Inducible Factor 1α: Role of Subcellular Compartmentalization

The hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that mediates adaptive cellular responses to decreased oxygen availability (hypoxia). At normoxia, HIF-1α is targeted by the von Hippel-Lindau tumor suppressor protein (pVHL) for degradation by the ubiquitin-proteasome pathway. In the present study we have observed distinct cell-type-specific differences in the ability of various tested pVHL-interacting subfragments to stabilize HIF-1α and unmask its function at normoxia. These properties correlated with differences in subcellular compartmentalization and degradation of HIF-1α. We observed that the absence or presence of nuclear localization or export signals differently affected the ability of a minimal HIF-1α peptide spanning residues 559 to 573 of mouse HIF-1α to stabilize endogenous HIFα and induce HIF-driven reporter gene activity in two different cell types (primary mouse endothelial and HepG2 hepatoma cells). Degradation of HIF-1α occurred mainly in the cytoplasm of HepG2 cells, whereas it occurs with equal efficiency in nuclear and cytoplasmic compartments of primary endothelial cells. Consistent with these observations, green fluorescent protein-HIF-1α is differently distributed during hypoxia and reoxygenation in hepatoma and endothelial cells. Consequently, we propose that differential compartmentalization of degradation of HIF-1α and the subcellular distribution of HIF-1α may account for cell-type-specific differences in stabilizing HIF-1α protein levels under hypoxic conditions