The maintenance of neuronal polarity in aging Drosophila melanogaster

Ein grundlegendes Merkmal neuronaler Zellen ist ihre stark polarisierte Morphologie, welche sich in der Ausbildung eines einzigen langen Axons und mehrerer kurzer Dendriten manifestiert. Diese polarisierten Morphologie, sowie die differentielle, polare Lokalisation von Proteinen und anderen subzellulären Bestandteilen bilden die Grundlage für die Funktion von Nervenzellen und deren Vernetzung zu funktionellen neuronalen Netzwerken. Insbesondere die selektive Lokalisation von Proteinen innerhalb spezialisierter subzellulärer Bereiche, wie dem Axon, ist essentiell für die Etablierung und Aufrechterhaltung dieser molekular und funktionell hoch spezialisierten subzellulären Kompartimente. Während neuronale Polarität, bzw. die polarisierte Verteilung von Proteinen, in jungen Säugetierneuronen relativ gut charakterisiert wurde, ist wenig über diese Aspekte in Invertebraten und alternden Neuronen bekannt. Drosophila melanogaster hat sich als hervorragender Modelorganismus zur Erforschung der Etablierung zellulärer und neuronaler Polarität erwiesen. Erst kürzlich konnte gezeigt werden, dass Drosophila Neuronen eine klare Polarität auf molekularer Ebene aufweisen, welche sich in der polarisierten Verteilung von Proteinen entweder im Axon oder den Dendriten zeigt. Dies, sowie die praktischen Vorteile von Drosophila als Modellorganismus, die Vielzahl an verfügbaren genetischen Werkzeugen und die Möglichkeit zelluläre Vorgänge in vivo zu beobachten, machen Drosophila zu einem unkomplizierten und genetisch einfach manipulierbaren Modellorganismus für die Erforschung komplexer Aspekte der Etablierung und Aufrechterhaltung neuronaler Kompartimente. In der vorliegenden Studie nutzten wir Drosophila melanogaster um die polarisierte Verteilung von Proteinen in Axonen zu bestätigen. Durch die selektive Expression transgener fluoreszierender Proteine mit vermeintlich axonaler Lokalisation im Pilzkörper von jungen Fliegen konnten wir nachweisen, dass einige Aspekte neuronaler Polarität, wie die selektive Lokalisierung von Membran- und Transportproteinen im axonalen Kompartiment, bereits in jungen Drosophila Neuronen etabliert sind. Obwohl manche Aspekte der neuronalen Polarität in jungen Neuronen weniger gut etabliert zu sein scheinen, bestätigen unsere Ergebnisse die Eignung von Drosophila als Modellsystem, um die komplexen Prozesse neuronaler Polarität zu untersuchen. Des Weiteren untersuchten wir die Aufrechterhaltung der polarisierten Verteilung von Proteinen im Alter, mittels eines vergleichenden in vivo Screens. Hinweise darauf, dass Veränderungen in der polarisierten Verteilung von Proteinen im alternden Gehirn auftreten können und zu dessen Alterungsprozess beitragen, gehen aus den Arbeiten von Niewiadomska und Kollegen hervor, die zeigen konnten das es zu altersabhängigen Veränderungen in der Lokalisation axonaler Mikrotubuli-bindender Proteine, und als Konsequenz Veränderungen im axonalen Transport [Niewiadomska et al., 2003, 2005 2006]. Durch die transgene Expression axonaler Proteine in den Pilzkörpern junger und alter Fliegen konnten wir Veränderungen in der Verteilung zweier axonaler Membranproteine zeigen, während die Lokalisation eines verwandten Proteins erhalten blieb. Dies deutet darauf hin, dass die beobachteten Veränderungen nicht das Ergebnis der langzeitigen Expression transgener Proteine sind und der Funktionalität des angelegten Screens. Die axonale Lokalisation von Transportproteinen blieb erhalten und weist darauf hin, dass es zu keinen allgemeinen Beeinträchtigung des axonalen Transports kommt. Des Weiteren fanden wir Hinweise darauf, dass präsynaptische Proteine in der neuronalen Entwicklung ihre akkurate, polarisierte Lokalisation erst etablieren, nachdem neuronale Kompartimente morphologisch klar unterscheidbar sind. Unsere Ergebnisse deuten darauf hin, dass es zur Umverteilungen spezifischer axonaler Proteine im gealterten zentralen Nervensystem kommen kann, aber um diese Fragestellung endgültig zu klären sind fortführende Forschungen hinsichtlich endogener Proteinen und der zugrunde liegenden Mechanismen erforderlich.A fundamental feature of neuronal cells is that they possess a highly polarized morphology, characterized by a single long axon and multiple short dendrites. This polarized morphology and the proper localization of proteins and other subcellular constituents form the basis for neuronal function and their assembly into functional neuronal networks. Specifically, the selective targeting of proteins to specialized subcellular domains, such as the axon, is of central importance for the establishment and maintenance of these molecularly and functionally highly specialized subcellular compartments. While neuronal polarity and respectively the polarized distribution of proteins has been thoroughly studied in the early stages of life in mammalian organisms, it is less well characterized in invertebrates and the late stages of life. Drosophila melanogaster has served as an excellent model for studying the establishment of cell and neuronal polarity. Only recently, it has become clear that Drosophila neurons exhibit a clear molecular polarization, apparent by the polarized distribution of proteins to either axons or dendrites. Taken together with the appealing properties of Drosophila as a model organism, the vast number of available genetic tools and the possibility to study cellular processes in vivo, Drosophila represents a convenient, simple and genetically tractable model organism to study the intricate aspects of the establishment and maintenance of neuronal compartments. In this study we used Drosophila melanogaster to validate the polarized distribution of proteins to the axonal compartment by the selective expression of transgenic fluorescent proteins with presumed axonal localization in the mushroom bodies of young adult flies. We could prove that several aspects of neuronal polarity, such as the selective targeting and trafficking of membrane and transport proteins to the axonal compartment are established early in the life of Drosophila neurons. Although some aspects of neuronal polarity may be less well established the early stages of life, our findings substantiate the use of Drosophila as a model to study the complex processes of neuronal polarity. Secondly, we aimed to investigate the maintenance of the polarized distribution of proteins with age by means of a comparative in vivo screen. Mechanisms that underlie the aging process and contribute to the commonly observed age-associated cognitive decline are ill defined. Indications that alterations in the polarized distribution of proteins may occur in the aged brain and contribute to the aging process come from the observations of Niewiadomska and colleagues, who could demonstrate the age-dependent redistribution of axonal microtubule binding proteins, affecting axonal transport in mammals [Niewiadomska et al., 2003, 2005 2006]. Expressing axonal proteins in the mushroom bodies of young and aged flies, we observed alterations in the distribution of two membrane proteins, whereas the localization of a related membrane protein was unaffected, suggesting that the observed rearrangements are not a consequence of the long-term expression of transgenic proteins. Transport proteins were found to maintain their highly selective localization to the axonal compartment, indicating no general impairment of axonal trafficking. Moreover we found indications that presynaptic proteins become polarized late in neuronal development, after neuronal compartments are already morphologically distinguishable. Our findings raise the possibility of re-localization of certain axonal proteins in the aged central nervous system, but further research regarding endogenous proteins and underlying mechanism is required

De novo fatty acid synthesis by Schwann cells is essential for peripheral nervous system myelination

Myelination calls for a remarkable surge in cell metabolism to facilitate lipid and membrane production. Endogenous fatty acid (FA) synthesis represents a potentially critical process in myelinating glia. Using genetically modified mice, we show that Schwann cell (SC) intrinsic activity of the enzyme essential for de novo FA synthesis, fatty acid synthase (FASN), is crucial for precise lipid composition of peripheral nerves and fundamental for the correct onset of myelination and proper myelin growth. Upon FASN depletion in SCs, epineurial adipocytes undergo lipolysis, suggestive of a compensatory role. Mechanistically, we found that a lack of FASN in SCs leads to an impairment of the peroxisome proliferator-activated receptor (PPAR) γ–regulated transcriptional program. In agreement, defects in myelination of FASN-deficient SCs could be ameliorated by treatment with the PPARγ agonist rosiglitazone ex vivo and in vivo. Our results reveal that FASN-driven de novo FA synthesis in SCs is mandatory for myelination and identify lipogenic activation of the PPARγ transcriptional network as a putative downstream functional mediator

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

The Essential Role of de Novo Fatty Acid Synthesis in CNS Myelin Regeneration

A variety of pathological conditions of the central nervous system (CNS) are associated with demyelination and loss of myelinating oligodendrocytes, e.g. spinal cord injury, stroke, and primary demyelinating diseases such as multiple sclerosis (MS). Common to all of these pathologies is the intrinsic, yet variable, capacity of the CNS to regenerate damaged myelin sheaths, resulting in the restoration of saltatory conduction and neuroprotection. Although remyelination can be initially extensive, it characteristically fails in long-standing, chronic demyelinating pathologies, such as MS. Remyelination in the CNS is primarily mediated by a pool of adult progenitor cells, the oligodendrocyte progenitor cells (OPCs). Upon demyelination, these highly proliferative and migratory progenitors are activated and recruited to the lesion site, where they differentiate into oligodendrocytes and form new myelin sheaths around denuded axons. Proliferation of adult OPCs and subsequent differentiation into myelinating cells during remyelination requires a tremendous increase in cell size and cellular membranes, hence calling for a vast surge in lipid availability. Fatty acids are the primary apolar building blocks for complex membrane lipids, and thus myelin itself. Moreover, fatty acids are critical to a variety of fundamental cellular processes, including membrane targeting of proteins, energy storage, cell signaling and transcriptional regulation. Most cells are thought to primarily rely on uptake to maintain their fatty acids pool, but highly metabolically active and proliferative, e.g. precursor/stem cells are strongly dependent on de novo synthesis mediated by fatty acid synthase (FASN). The multifunctional enzyme FASN is strictly required for de novo synthesis of fatty acids, mostly palmitate. Tissue-specific ablation of FASN in various cell types has provided valuable insights into the diverse and largely cell type- specific functions of de novo fatty acid synthesis. Given the highly proliferative nature of OPCs and the tremendous demand for lipids towards membrane synthesis during remyelination, we hypothesized endogenous fatty acid synthesis in OPCs to be critical in these processes. To this end, we induced FASN depletion in adult OPCs to assess its requirement for OPC proliferation, differentiation, and remyelination, following experimental gliotoxin-induced demyelination. We show that FASN is very low expressed in OPCs, but strongly expressed in oligodendrocytes differentiated from adult OPCs during remyelination. Consistently, FASN-activity is dispensable for adult OPC proliferation and maintenance during remyelination. Most importantly, we found FASN activity is critical for efficient CNS remyelination, an effect that is at least in part dependent on the requirement of FASN-mediated de novo fatty acid synthesis for maintaining the adult OPC-derived oligodendrocyte population during remyelination. Our results add valuable information to the understanding of the regulation of the remyelination process in demyelinating conditions, a promising currently pursued drug target

CNS myelination and remyelination depend on fatty acid synthesis by oligodendrocytes

Oligodendrocytes (OLs) support neurons and signal transmission in the central nervous system (CNS) by enwrapping axons with myelin, a lipid-rich membrane structure. We addressed the significance of fatty acid (FA) synthesis in OLs by depleting FA synthase (FASN) from OL progenitor cells (OPCs) in transgenic mice. While we detected no effects in proliferation and differentiation along the postnatal OL lineage, we found that FASN is essential for accurate myelination, including myelin growth. Increasing dietary lipid intake could partially compensate for the FASN deficiency. Furthermore, FASN contributes to correct myelin lipid composition and stability of myelinated axons. Moreover, we depleted FASN specifically in adult OPCs to examine its relevance for remyelination. Applying lysolecithin-induced focal demyelinating spinal cord lesions, we found that FA synthesis is essential to sustain adult OPC-derived OLs and efficient remyelination. We conclude that FA synthesis in OLs plays key roles in CNS myelination and remyelination.ISSN:2050-084

Circulating platelets modulate oligodendrocyte progenitor cell differentiation during remyelination.

Peer reviewed: TrueAcknowledgements: Special acknowledgments to the Comité Ético y Científico del Servicio de Salud de Valdivia (CEC-SVS) for ethical guidance and approval (ORD N° 510). The authors thank the Laboratory of Chronobiology, UACh (led by Dr. Claudia Torres-Farfán) for supporting animal experimentation. We would also like to thank Dr. Alerie Guzman de la Fuente for the Fiji/ImageJ macro for in-vitro quantification. Authors thank the funding support from Agencia Nacional de Investigación y Desarrollo (ANID, Chile)-FONDECYT Program Regular Grant Numbers 1201706 and 1161787 (both to FJR), ANID-FONDECYT Program Regular Grant Number 1201635 (to PE), ANID-PCI Program Grant N° REDES170233 (to FJR) and N° REDES180139 (to MAC), ANID-National Doctoral Fellowship N° 21170732 (to ARP), N° 21211727 (to CRR) and N° 21221559 (to CVK). In addition, the authors thank the PROFI 6 N° 336234 of the Research Council of Finland.Revealing unknown cues that regulate oligodendrocyte progenitor cell (OPC) function in remyelination is important to optimise the development of regenerative therapies for multiple sclerosis (MS). Platelets are present in chronic non-remyelinated lesions of MS and an increase in circulating platelets has been described in experimental autoimmune encephalomyelitis (EAE) mice, an animal model for MS. However, the contribution of platelets to remyelination remains unexplored. Here we show platelet aggregation in proximity to OPCs in areas of experimental demyelination. Partial depletion of circulating platelets impaired OPC differentiation and remyelination, without altering blood-brain barrier stability and neuroinflammation. Transient exposure to platelets enhanced OPC differentiation in vitro, whereas sustained exposure suppressed this effect. In a mouse model of thrombocytosis (Calr+/-), there was a sustained increase in platelet aggregation together with a reduction of newly-generated oligodendrocytes following toxin-induced demyelination. These findings reveal a complex bimodal contribution of platelet to remyelination and provide insights into remyelination failure in MS

Genotoxic and Antigenotoxic Assessment of Chios Mastic Oil by the In Vitro Micronucleus Test on Human Lymphocytes and the In Vivo Wing Somatic Test on Drosophila

International audienceChios mastic oil (CMO), the essential oil derived from Pistacia lentiscus (L.) var. chia (Duham), has generated considerable interest because of its antimicrobial, anticancer, antioxidant and other beneficial properties. In the present study, the potential genotoxic activity of CMO as well as its antigenotoxic properties against the mutagenic agent mitomycin-C (MMC) were evaluated by employing the in vitro Cytokinesis Block MicroNucleus (CBMN) assay and the in vivo Somatic Mutation And Recombination Test (SMART). In the in vitro experiments, lymphocytes were treated with 0.01, 0.05 and 0.10% (v/v) of CMO with or without 0.05 μg/ml MMC, while in the in vivo assay Drosophila larvae were fed with 0.05, 0.10, 0.50 and 1.00% (v/v) of CMO with or without 2.50 μg/ml MMC. CMO did not significantly increase the frequency of micronuclei (MN) or total wing spots, indicating lack of mutagenic or recombinogenic activity. However, the in vitro analysis suggested cytotoxic activity of CMO. The simultaneous administration of MMC with CMO did not alter considerably the frequencies of MMC-induced MN and wing spots showing that CMO doesn't exert antigenotoxic or antirecombinogenic action. Therefore, CMO could be considered as a safe product in terms of genotoxic potential. Even though it could not afford any protection against DNA damage, at least under our experimental conditions, its cytotoxic potential could be of interest

A meta-analysis of genome-wide association studies of breast cancer identifies two novel susceptibility loci at 6q14 and 20q11.

Genome-wide association studies (GWAS) of breast cancer defined by hormone receptor status have revealed loci contributing to susceptibility of estrogen receptor (ER)-negative subtypes. To identify additional genetic variants for ER-negative breast cancer, we conducted the largest meta-analysis of ER-negative disease to date, comprising 4754 ER-negative cases and 31 663 controls from three GWAS: NCI Breast and Prostate Cancer Cohort Consortium (BPC3) (2188 ER-negative cases; 25 519 controls of European ancestry), Triple Negative Breast Cancer Consortium (TNBCC) (1562 triple negative cases; 3399 controls of European ancestry) and African American Breast Cancer Consortium (AABC) (1004 ER-negative cases; 2745 controls). We performed in silico replication of 86 SNPs at P ≤ 1 × 10(-5) in an additional 11 209 breast cancer cases (946 with ER-negative disease) and 16 057 controls of Japanese, Latino and European ancestry. We identified two novel loci for breast cancer at 20q11 and 6q14. SNP rs2284378 at 20q11 was associated with ER-negative breast cancer (combined two-stage OR = 1.16; P = 1.1 × 10(-8)) but showed a weaker association with overall breast cancer (OR = 1.08, P = 1.3 × 10(-6)) based on 17 869 cases and 43 745 controls and no association with ER-positive disease (OR = 1.01, P = 0.67) based on 9965 cases and 22 902 controls. Similarly, rs17530068 at 6q14 was associated with breast cancer (OR = 1.12; P = 1.1 × 10(-9)), and with both ER-positive (OR = 1.09; P = 1.5 × 10(-5)) and ER-negative (OR = 1.16, P = 2.5 × 10(-7)) disease. We also confirmed three known loci associated with ER-negative (19p13) and both ER-negative and ER-positive breast cancer (6q25 and 12p11). Our results highlight the value of large-scale collaborative studies to identify novel breast cancer risk loci