Lipid Reshaping and Lipophagy Are Induced in a Modeled Ischemia-Reperfusion Injury of Blood Brain Barrier

Ischemic-reperfusion (I/R) injury induced a remodeling of protein and lipid homeostasis, under oxidative stress and inflammatory status. Starvation occurring during I/R is a condition leading to autophagy activation, which allows abnormal material clearance or amino acid, or both, and fatty acid (FA) recycling essential for survival. This study investigated the lipid reshaping, peroxidation, and related-signaling pathways, in rat brain endothelial cells (RBE4) subjected to 3 h of oxygen and glucose deprivation (OGD) and restoration of standard condition (I/R in vitro model). Lipids and proteins were analyzed after 1 or 24 h of oxygen and nutrient restoration. Together with the oxidative stress and inflammatory status, I/R injury induced a reshaping of neutral lipids and biogenesis of lipid droplets (LD) with excessive lipid storage. The increase of LC3-II/LC3-I ratio, an autophagy marker, and LC3 co-localization with LD suggest the activation of lipophagy machinery to counteract the cell engulfment. Lipophagy leads to cholesterol ester (CE) hydrolysis, increasing free cholesterol (FC) secretion, which occurred by specific transporters or unconventional exocytosis pathways, or both. Here, we propose that an unconventional spreading of FC and other lipid metabolites may influence the neurovascular unit (NVU) cells, contributing to Blood brain barrier (BBB) alteration or adaptation, or both, to the cumulative effects of several transient ischemia

In Vivo Comparative Study on Acute and Sub-acute Biological Effects Induced by Ultrafine Particles of Different Anthropogenic Sources in BALB/c Mice

Exposure to ultrafine particles (UFPs) leads to adverse effects on health caused by an unbalanced ratio between UFPs deposition and clearance efficacy. Since air pollution toxicity is first direct to cardiorespiratory system, we compared the acute and sub-acute effects of diesel exhaust particles (DEP) and biomass burning-derived particles (BB) on bronchoalveolar Lavage Fluid (BALf), lung and heart parenchyma. Markers of cytotoxicity, oxidative stress and inflammation were analysed in male BALB/c mice submitted to single and repeated intra-tracheal instillations of 50 g UFPs. This in-vivo study showed the activation of inflammatory response (COX-2 and MPO) after exposure to UFPs, both in respiratory and cardiovascular systems. Exposure to DEP results also in pro- and anti-oxidant (HO-1, iNOS, Cyp1b1, Hsp70) protein levels increase, although, stress persist only in cardiac tissue under repeated instillations. Statistical correlations suggest that stress marker variation was probably due to soluble components and/or mediators translocation of from first deposition site. This mechanism, appears more important after repeated instillations, since inflammation and oxidative stress endure only in heart. In summary, chemical composition of UFPs influenced the activation of different responses mediated by their components or pro-inflammatory and pro-oxidative molecules, indicating DEP as the most damaging pollutant in the comparison

Impact of COVID-19 lockdowns on adolescent pregnancy and school dropout among secondary schoolgirls in Kenya

Introduction Secondary school closures aimed at limiting the number of infections and deaths due to COVID-19 may have amplified the negative sexual and reproductive health (SRH) and schooling outcomes of vulnerable adolescent girls. This study aimed to measure pandemic-related effects on adolescent pregnancy and school dropout among school-going girls in Kenya. Methods We report longitudinal findings of 910 girls in their last 2 years of secondary school. The study took place in 12 secondary day schools in rural western Kenya between 2018 and 2021. Using a causal-comparative design, we compared SRH and schooling outcomes among 403 girls who graduated after completion of their final school examinations in November 2019 pre-pandemic with 507 girls who experienced disrupted schooling due to COVID-19 and sat examinations in March 2021. Unadjusted and adjusted generalised linear mixed models were used to investigate the effect of COVID-19-related school closures and restrictions on all outcomes of interest and on incident pregnancy. Results At study initiation, the mean age of participants was 17.2 (IQR: 16.4–17.9) for girls in the pre-COVID-19 cohort and 17.5 (IQR: 16.5–18.4) for girls in the COVID-19 cohort. Girls experiencing COVID-19 containment measures had twice the risk of falling pregnant prior to completing secondary school after adjustment for age, household wealth and orphanhood status (adjusted risk ratio (aRR)=2.11; 95% CI:1.13 to 3.95, p=0.019); three times the risk of school dropout (aRR=3.03; 95% CI: 1.55 to 5.95, p=0.001) and 3.4 times the risk of school transfer prior to examinations (aRR=3.39; 95% CI: 1.70 to 6.77, p=0.001) relative to pre-COVID-19 learners. Girls in the COVID-19 cohort were more likely to be sexually active (aRR=1.28; 95% CI: 1.09 to 1.51, p=0.002) and less likely to report their first sex as desired (aRR=0.49; 95% CI: 0.37 to 0.65, p<0.001). These girls reported increased hours of non-school-related work (3.32 hours per day vs 2.63 hours per day in the pre-COVID-19 cohort, aRR=1.92; 95% CI: 1.92 to 2.99, p=0.004). In the COVID-19 cohort, 80.5% reported worsening household economic status and COVID-19-related stress was common. Conclusion The COVID-19 pandemic deleteriously affected the SRH of girls and amplified school transfer and dropout. Appropriate programmes and interventions that help buffer the effects of population-level emergencies on school-going adolescents are warranted

The Golgi Localization of GOLPH2 (GP73/GOLM1) Is Determined by the Transmembrane and Cytoplamic Sequences

Golgi phosphoprotein 2 (GOLPH2) is a resident Golgi type-II membrane protein upregulated in liver disease. Given that GOLPH2 traffics through endosomes and can be secreted into the circulation, it is a promising serum marker for liver diseases. The structure of GOLPH2 and the functions of its different protein domains are not known. In the current study, we investigated the structural determinants for Golgi localization using a panel of GOLPH2 truncation mutants. The Golgi localization of GOLPH2 was not affected by the deletion of the C-terminal part of the protein. A truncated mutant containing the N-terminal portion (the cytoplasmic tail and transmembrane domain (TMD)) localized to the Golgi. Sequential deletion analysis of the N-terminal indicated that the TMD with a positively charged residue in the cytoplasmic N-terminal tail were sufficient to support Golgi localization. We also showed that both endogenous and secreted GOLPH2 exist as a disulfide-bonded dimer, and the coiled-coil domain was sufficient for dimerization. This structural knowledge is important for the understanding the pathogenic role of GOLPH2 in liver diseases, and the development of GOLPH2-based hepatocellular cancer diagnostic methods

Alternative Splicing of Spg7, a Gene Involved in Hereditary Spastic Paraplegia, Encodes a Variant of Paraplegin Targeted to the Endoplasmic Reticulum

BACKGROUND: Hereditary spastic paraplegia defines a group of genetically heterogeneous diseases characterized by weakness and spasticity of the lower limbs owing to retrograde degeneration of corticospinal axons. One autosomal recessive form of the disease is caused by mutation in the SPG7 gene. Paraplegin, the product of SPG7, is a component of the m-AAA protease, a high molecular weight complex that resides in the mitochondrial inner membrane, and performs crucial quality control and biogenesis functions in mitochondria. PRINCIPAL FINDINGS: Here we show the existence in the mouse of a novel isoform of paraplegin, which we name paraplegin-2, encoded by alternative splicing of Spg7 through usage of an alternative first exon. Paraplegin-2 lacks the mitochondrial targeting sequence, and is identical to the mature mitochondrial protein. Remarkably, paraplegin-2 is targeted to the endoplasmic reticulum. We find that paraplegin-2 exposes the catalytic domains to the lumen of the endoplasmic reticulum. Moreover, endogenous paraplegin-2 accumulates in microsomal fractions prepared from mouse brain and retina. Finally, we show that the previously generated mouse model of Spg7-linked hereditary spastic paraplegia is an isoform-specific knock-out, in which mitochondrial paraplegin is specifically ablated, while expression of paraplegin-2 is retained. CONCLUSIONS/SIGNIFICANCE: These data suggest a possible additional role of AAA proteases outside mitochondria and open the question of their implication in neurodegeneration

Assembly, organization, and function of the COPII coat

A full mechanistic understanding of how secretory cargo proteins are exported from the endoplasmic reticulum for passage through the early secretory pathway is essential for us to comprehend how cells are organized, maintain compartment identity, as well as how they selectively secrete proteins and other macromolecules to the extracellular space. This process depends on the function of a multi-subunit complex, the COPII coat. Here we describe progress towards a full mechanistic understanding of COPII coat function, including the latest findings in this area. Much of our understanding of how COPII functions and is regulated comes from studies of yeast genetics, biochemical reconstitution and single cell microscopy. New developments arising from clinical cases and model organism biology and genetics enable us to gain far greater insight in to the role of membrane traffic in the context of a whole organism as well as during embryogenesis and development. A significant outcome of such a full understanding is to reveal how the machinery and processes of membrane trafficking through the early secretory pathway fail in disease states

Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized epithelial cells

The g-aminobutyric acid transporter (GAT-1) isoform of the g-aminobutyric acid and the betaine (BGT) transporters exhibit distinct apical and basolateral distributions when introduced into Madin-Darby canine kidney cells (Pietrini, G., Suh, Y. J., Edelman, L., Rudnick, G.,and Caplan, M. J. (1994) J. Biol. Chem. 269, 4668\u20134674). We have investigated the presence of sorting signals in their COOH-terminal cytosolic domains by expression in Madin-Darby canine kidney cells of mutated and chimeric transporters. Whereas truncated GAT-1 (DC-GAT) maintained the original functional activity and apicallocalization, either the removal (DC-myc BGT) or the substitution (BGS chimera) of the cytosolic tail of BGT generated proteins that accumulated in the endoplasmic reticulum. Moreover, we have found that the cytosolic tail of BGT redirected apical proteins, the polytopic GAT-1 (GBS chimera) and the monotopic human nerve growth factor receptor, to the basolateral surface.These results suggest the presence of basolateral sorting information in the cytosolic tail of BGT. We have further shown that information necessary for the exit of BGT from the endoplasmic reticulum and for the basolateral localization of the GBS chimera is contained in a short segment, rich in basic residues, within the cytosolic tail of BGT

Ischemic conditions and beta-secretase activation: The impact of membrane cholesterol enrichment as triggering factor in rat brain endothelial cells

Among harmful conditions damaging the blood-brain barrier, cerebral stroke and reperfusion injuries were proposed as contributing factors to Alzheimer's disease etiology. Indeed it was reported that ischemic conditions promote beta-amyloid peptide production in brain endothelial cells, although implicated mechanisms are yet not fully understood. Oxidative injury related to ischemia affects membrane-lipids profile by altering their biochemical properties and structural dynamics, which are also believed to play significant role in the amyloid precursor protein processing, suggesting a link between alterations in lipid membrane composition and beta-amyloid peptide production enhancement. Using brain microvascular endothelial cells, here we demonstrate how oxygen and glucose deprivation followed by normal conditions restoration, mimicking ischemic environment, increases cell cholesterol amount (+20%), reduces membrane fluidity and results in strong activation (+40%) of beta-secretase 1 enzymatic activity. Moreover, we observed an increase of amyloid precursor protein and beta-secretase 1 protein levels with altered localization in non-discrete (Triton X-100 soluble) membrane domains, leading to an enhanced production of amyloid precursor protein beta-carboxyl-terminal fragment. Therefore, lipid alterations induced by oxygen and glucose deprivation enhance beta-secretase 1 activity, favor its proximity to amyloid precursor protein and may concur to increased amyloidogenic cleavage. The latter, represents a detrimental event that may contribute to beta-amyloid homeostasis impairment in the brain and to Alzheimer's disease-related BBB dysfunctions