Functional Lipidomics analysis of retrovirus envelopes

Ph.DDOCTOR OF PHILOSOPH

Phospholipase D functional ablation has a protective effect in an Alzheimer's disease Caenorhabditis elegans model

Phospholipase D (PLD) is a key player in the modulation of multiple aspects of cell physiology and has been proposed as a therapeutic target for Alzheimer's disease (AD). Here, we characterize a PLD mutant, pld-1, using the Caenorhabditis elegans animal model. We show that pld-1 animals present decreased phosphatidic acid levels, that PLD is the only source of total PLD activity and that pld-1 animals are more sensitive to the acute effects of ethanol. We further show that PLD is not essential for survival or for the normal performance in a battery of behavioral tests. Interestingly, pld-1 animals present both increased size and lipid stores levels. While ablation of PLD has no important effect in worm behavior, its ablation in an AD-like model that overexpresses amyloid-beta (Aβ), markedly improves various phenotypes such as motor tasks, prevents susceptibility to a proconvulsivant drug, has a protective effect upon serotonin treatment and reverts the biometric changes in the Aβ animals, leading to the normalization of the worm body size. Overall, this work proposes the C. elegans model as a relevant tool to study the functions of PLD and further supports the notion that PLD has a significant role in neurodegeneration.We would like to thank members of the Oliveira and Maciel labs for discussions, for critical analysis of data and discussions on the manuscript. Ricardo Rosa for his technical assistance in lifespan assays and Carlos Bessa for his technical suggestions. Thanks to the Caenorhabditis Genetics Center (CGC), which is funded by the National Institutes of Health – National Center for Research Resources, for some of the nematode strains. Costs with acquisition and transfer of genetic C. elegans models were covered by Tiago Gil Oliveira. This work was supported by grants from the Portuguese North Regional Operational Program (ON.2 – O Novo Norte) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER), the Portuguese Foundation for Science and Technology PD/BD/52286/2013 (Francisca Vaz Bravo) as well as NIH ADRC grant P50 AG008702 to Scott A. Small (project G.D.P.) and NIH grant R21 AG045020 to G.D.P.info:eu-repo/semantics/publishedVersio

Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism

Small GTPases play a critical role in membrane traffic. Among them, Arf6 mediates transport to and from the plasma membrane, as well as phosphoinositide signalling and cholesterol homeostasis. Here we delineate the molecular basis for the link between Arf6 and cholesterol homeostasis using an inducible knockout (KO) model of mouse embryonic fibroblasts (MEFs). We find that accumulation of free cholesterol in the late endosomes/lysosomes of Arf6 KO MEFs results from mistrafficking of Niemann–Pick type C protein NPC2, a cargo of the cation-independent mannose-6-phosphate receptor (CI-M6PR). This is caused by a selective increase in an endosomal pool of phosphatidylinositol-4-phosphate (PI4P) and a perturbation of retromer, which controls the retrograde transport of CI-M6PR via sorting nexins, including the PI4P effector SNX6. Finally, reducing PI4P levels in KO MEFs through independent mechanisms rescues aberrant retromer tubulation and cholesterol mistrafficking. Our study highlights a phosphoinositide-based mechanism for control of cholesterol distribution via retromer

Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity

Elevated GM3 plasma concentration in idiopathic Parkinson’s disease: A lipidomic analysis

Parkinson’s disease (PD) is a common neurodegenerative disease whose pathological hallmark is the accumulation of intracellular α-synuclein aggregates in Lewy bodies. Lipid metabolism dysregulation may play a significant role in PD pathogenesis; however, large plasma lipidomic studies in PD are lacking. In the current study, we analyzed the lipidomic profile of plasma obtained from 150 idiopathic PD patients and 100 controls, taken from the ‘Spot’ study at Columbia University Medical Center in New York. Our mass spectrometry based analytical panel consisted of 520 lipid species from 39 lipid subclasses including all major classes of glycerophospholipids, sphingolipids, glycerolipids and sterols. Each lipid species was analyzed using a logistic regression model. The plasma concentrations of two lipid subclasses, triglycerides and monosialodihexosylganglioside (GM3), were different between PD and control participants. GM3 ganglioside concentration had the most significant difference between PD and controls (1.531±0.037 pmol/μl versus 1.337±0.040 pmol/μl respectively; p-value = 5.96E-04; q-value = 0.048; when normalized to total lipid: p-value = 2.890E-05; q-value = 2.933E-03). Next, we used a collection of 20 GM3 and glucosylceramide (GlcCer) species concentrations normalized to total lipid to perform a ROC curve analysis, and found that these lipids compare favorably with biomarkers reported in previous studies (AUC = 0.742 for males, AUC = 0.644 for females). Our results suggest that higher plasma GM3 levels are associated with PD. GM3 lies in the same glycosphingolipid metabolic pathway as GlcCer, a substrate of the enzyme glucocerebrosidase, which has been associated with PD. These findings are consistent with previous reports implicating lower glucocerebrosidase activity with PD risk

Gene-Wise Association of Variants in Four Lysosomal Storage Disorder Genes in Neuropathologically Confirmed Lewy Body Disease

Objective Variants in GBA are associated with Lewy Body (LB) pathology. We investigated whether variants in other lysosomal storage disorder (LSD) genes also contribute to disease pathogenesis. Methods We performed a genetic analysis of four LSD genes including GBA, HEXA, SMPD1, and MCOLN1 in 231 brain autopsies. Brain autopsies included neuropathologically defined LBD without Alzheimer Disease (AD) changes (n = 59), AD without significant LB pathology (n = 71), Alzheimer disease and lewy body variant (ADLBV) (n = 68), and control brains without LB or AD neuropathology (n = 33). Sequencing of HEXA, SMPD1, MCOLN1 and GBA followed by ‘gene wise’ genetic association analysis was performed. To determine the functional effect, a biochemical analysis of GBA in a subset of brains was also performed. GCase activity was measured in a subset of brain samples (n = 64) that included LBD brains, with or without GBA mutations, and control brains. A lipidomic analysis was also performed in brain autopsies (n = 67) which included LBD (n = 34), ADLBV (n = 3), AD (n = 4), PD (n = 9) and control brains (n = 17), comparing GBA mutation carriers to non-carriers. Results In a ‘gene-wise’ analysis, variants in GBA, SMPD1 and MCOLN1 were significantly associated with LB pathology (p range: 0.03–4.14 x10-5). Overall, the mean levels of GCase activity were significantly lower in GBA mutation carriers compared to non-carriers (p<0.001). A significant increase and accumulation of several species for the lipid classes, ceramides and sphingolipids, was observed in LBD brains carrying GBA mutations compared to controls (p range: p<0.05-p<0.01). Interpretation Our study indicates that variants in GBA, SMPD1 and MCOLN1 are associated with LB pathology. Biochemical data comparing GBA mutation carrier to non-carriers support these findings, which have important implications for biomarker development and therapeutic strategies

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin