(A–B) Hematoxylin and eosin (H&E)-stained paraffin-embedded brain sections of the 30-day-old control (OregonR x white1118, A) and 30-day-old sws1/sws4 transheterozygous flies (B). Arrows indicate …
(A–D) Adult brains stained with Coracle (CoraC) (green) and DAPI (magenta). (A) In controls (Oregon R x white1118), CoraC expression is pictured as the smooth line at the surface of the brain (green …
(A–B) Adult brains stained with anti-SWS antibodies (red) and DAPI (blue). (A–Aʹ) Adult brains stained with anti-SWS antibodies (red) and DAPI (blue) show that NTE/SWS is expressed in most if not …
(A–F) Adult brains stained with Coracle (CoraC) (white). In control (Oregon R x white1118), CoraC expression is pictured as the smooth line at the surface of the brain (green arrow). In sws1 mutants …
The 3D structures of the human NTE and Drosophila NTE/SWS proteins generated using the AlphaFold2 and PyMOL tools. Both proteins contain a highly conserved patatin-like phospholipase domain known as …
(A–C) Electron microscopy images of the surface area of the adult brains. (A) In controls (white1118), glia cells that do not contain any abnormal subcellular structures. Scale bar: 1 µm. (B–Bʹ) sws1…
(A) Scheme of 10 kDa dextran dye permeability assay (see also Materials and methods for a detailed description of the procedure). (B–C) Localization of dextran dye more than 12 hr after injection in …
(A) Bar graph shows the reduction in the percentage of the glial phenotype, assayed by Coracle (CoraC) expression pattern, in sws1 (red) and moodyΔC17 (olive) mutants that were treated with …
GS-MS measurements of free fatty acids (FFA).
(A–B) Anti-inflammatory drugs partially suppress glial phenotypes in sws mutants. (A) For the drug feeding assay, vials with sugar-free food with two micropipettes filled with dyed drug solution …
(A–C) Adult brains stained with Coracle (CoraC) (white) to reveal brain surface. CoraC expression in control brains (Oregon R x white1118, A) is depicted as the smooth line at the surface of the …
(A) GC-MS measurement of arachidonic acid (C20:4) with the internal standard (IS). The peak area ratio (PAR) of m/z 303 for 20:4 to m/z 295 for the IS was linear in the range 0–4000 pmol of C20:4 at …
(A–C) Adult brains stained with a septate junction marker Neurexin IV (NrxIV) (white). Scale bar: 50 µm. (A) In control (Oregon R) brain, septate junctions formed by subperineurial glia (SPG) glia …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Anti-Repo (mouse monoclonal) | Developmental Studies Hybridoma Bank | #8D12 | IF(1:50) |
Antibody | Anti-CoraC (mouse monoclonal) | Developmental Studies Hybridoma Bank | #C566.9 | IF(1:50) |
Antibody | Anti-Rab7 (mouse monoclonal) | Developmental Studies Hybridoma Bank | #AB2722471 | IF(1:50) |
Antibody | Anti-DE-Cad (rat monoclonal) | Developmental Studies Hybridoma Bank | #DCAD2 | IF(1:50) |
Antibody | Anti-GFP (chicken polyclonal) | Abcam | #ab13970 | IF(1:1000) |
Antibody | Anti-mCherry (rabbit polyclonal) | Abcam | #ab167453 | IF(1:1000) |
Antibody | Anti-β-Galactosidase (mouse monoclonal) | Promega | #Z3781 | IF(1:200) |
Antibody | Anti-CathepsinL (mouse) | R&D Systems | #1515-CY-010 | IF(1:400) |
Antibody | Anti-NrxIV (rabbit polyclonal) | Gift from Christian Klämbt | Anti-NrxIV | IF(1:1000) |
Antibody | Anti-SWS (rabbit polyclonal) | Gift from Doris Kretzschmar | Anti-SWS | IF(1:1000) |
Antibody | Anti-NimC1 (mouse) | Gift from István Andó | Anti-NimC1 | IF(1:300) |
Antibody | Anti-chicken Alexa 488 (goat polyclonal) | Thermo Fisher Scientific | #A-11039 | Secondary antibody IF(1:500) |
Antibody | Anti-rat Alexa 488 (goat polyclonal) | Thermo Fisher Scientific | #A-11077 | Secondary antibody IF(1:500) |
Antibody | Anti-rat Alexa 647 (goat polyclonal) | Thermo Fisher Scientific | #A-21247 | Secondary antibody IF(1:500) |
Antibody | Anti-rabbit Alexa 488 (goat polyclonal) | Thermo Fisher Scientific | #A-11034 | Secondary antibody IF(1:500) |
Antibody | Anti-rabbit Alexa 568 (goat polyclonal) | Thermo Fisher Scientific | #A-11011 | Secondary antibody IF(1:500) |
Antibody | Anti-mouse IgG2a Cy3 (goat polyclonal) | Jackson ImmunoResearch | #115-165-206 | Secondary antibody IF(1:400) |
Antibody | Anti-mouse IG1 Cy3 (goat polyclonal) | Jackson ImmunoResearch | #115-165-205 | Secondary antibody IF(1:500) |
Antibody | Anti-mouse IgG1 647 (goat polyclonal) | Jackson ImmunoResearch | #115-605-205 | Secondary antibody IF(1:500) |
Genetic reagent (D. melanogaster) | w[1118] | Bloomington Drosophila Stock Center | BDSC 5905 | Wild type strain |
Genetic reagent (D. melanogaster) | Oregon-R | Bloomington Drosophila Stock Center | BDSC 5 | Wild type strain |
Genetic reagent (D. melanogaster) | sws1 | Gift from Doris Kretzschmar | sws1 | sws[1]/FM7a(null mutant) |
Genetic reagent (D. melanogaster) | sws4 | Bloomington Drosophila Stock Center | BDSC 28121 | sws[4]/C(1)DX, y[1] w[1] f[1] (Amino acid replacement: G956D) |
Genetic reagent (D. melanogaster) | UAS-sws | Gift from Doris Kretzschmar | UAS-sws | UAS-sws (sws gene under control of UAS promotor) |
Genetic reagent (D. melanogaster) | sws-Gal4 | Kyoto Stock Center | 104592 | y* w* P{GawB}swsNP4072/FM7c |
Genetic reagent (D. melanogaster) | UAS-nlsLacZ, UAS-CD8::GFP | Gift from Frank Hirth | UAS-nLacZ, UAS-GFP | UAS-nlsLacZ, UAS-CD8::GFP(nLacZ and GFP constructs under control of UAS promotor) |
Genetic reagent (D. melanogaster) | UAS-hNTE | Gift from Robert Wessells | UAS-hNTE | w[1118]; p[PUAST]-hNTE/CyO(Human NTE undercontrol of UAS promotor) |
Genetic reagent (D. melanogaster) | UAS-swsRNAi | Bloomington Drosophila Stock Center | BDSC 61338 | y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMJ23229}attP40 (sws RNAi construct under control of UAS promotor) |
Genetic reagent (D. melanogaster) | repo-Gal4, UAS-CD8::GFP/TM6B | Gift from Mikael Simons | repo-Gal4 | repo-Gal4, UAS-CD8::GFP/TM6B |
Genetic reagent (D. melanogaster) | Gliotactin-Gal4, UAS-CD8::GFP | Gift from Mikael Simons | Gli-Gal4 | Gliotactin-Gal4, UAS-CD8::GFP |
Genetic reagent (D. melanogaster) | moody-Gal4, UAS-CD8::GFP | Gift from Mikael Simons | moody-Gal4 | moody-Gal4, UAS-CD8::GFP |
Genetic reagent (D. melanogaster) | nSyb-Gal4 | Bloomington Drosophila Stock Center | BDSC 51945 | y[1] w[1118]; P{y[+t7.7] w[+mC]=nSyb-GAL4.DBD::QF.AD}attP2 |
Genetic reagent (D. melanogaster) | repo-Gal4, nSyb-Gal4, UAS-CD8::GFP/TM6B,Sb | This study | repo-Gal4, nSyb-Gal4 | repo-Gal4, nSyb-Gal4, UAS-CD8::GFP/TM6B,Sb |
Genetic reagent (D. melanogaster) | tub-Gal80ts; repo-Gal4/TM6B | This study | tub-Gal80ts; repo-Gal4/TM6B | tub-Gal80ts; repo-Gal4/TM6B(temperature sensitive) |
Genetic reagent (D. melanogaster) | moodyΔC17 | Gift from Christian Klämbt | moodyΔC17 | Null mutant |
Genetic reagent (D. melanogaster) | UAS-moodyRNAi | Bloomington Drosophila Stock Center | BDSC 66326 | y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMC06237}attP2(moody RNAi construct under control of UAS promotor) |
Genetic reagent (D. melanogaster) | UAS-DysbRNAi | Bloomington Drosophila Stock Center | BDSC 67316 | y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMC06420}attP40/CyO(Dysb RNAi construct under control of UAS promotor) |
Genetic reagent (D. melanogaster) | UAS-Npc1aRNAi | Bloomington Drosophila Stock Center | BDSC 37504 | y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01646}attP40 (Npc1a RNAi construct under control of UAS promotor) |
Genetic reagent (D. melanogaster) | UAS-PldnRNAi | Bloomington Drosophila Stock Center | BDSC 67884 | y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS05728}attP40 (Pldn RNAi construct under control of UAS promotor) |
Genetic reagent (D. melanogaster) | UAS-spinRNAi | Bloomington Drosophila Stock Center | BDSC 27702 | y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.JF02782}attP2 (spin RNAi construct under control of UAS promotor) |
Genetic reagent (D. melanogaster) | srp(Hemo) 3xmCherry | Gift from Angela Giangrande | srp(Hemo) 3xmCherry | srp(Hemo) 3xmCherry |
Software, algorithm | Microsoft Excel | Microsoft | Microsoft Excel | |
Software, algorithm | Adobe Photoshop | Adobe | Adobe CC | |
Software, algorithm | Zen 2011 (black edition) | Carl Zeiss; Emmenlauer et al., 2009 | Zen 2011 | |
Software, algorithm | AlphaFold2 v1.5.2 | Mirdita et al., 2022; https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb | AlphaFold2 | |
Software, algorithm | The PyMOL Molecular Graphics System, v2.5.5 | Schrödinger, LLC; https://pymol.org/ | PyMol | |
Software, algorithm | StepOne Software v2.3 | Applied Biosystems | StepOne | |
Chemical compound, drug | TRIzol reagent | Invitrogen | #15596018 | |
Commercial assay or kit | High Capacity cDNA Reverse Transcription kit | Applied Biosystems | #4368813 | |
Commercial assay or kit | FastSYBR Green master mix | Applied Biosystems | #435612 | |
Chemical compound, drug | TUDCA | Sigma-Aldrich | #580549 | Tauroursodeoxycholic acid |
Chemical compound, drug | 4-PBA | Sigma-Aldrich | #567616 | 4-Phenylbutyric acid |
Chemical compound, drug | Valsartan | Sigma-Aldrich | #PHR1315 | |
Chemical compound, drug | Fenofibrate | Sigma-Aldrich | #F6020 | |
Chemical compound, drug | Sodium salicylate | Sigma-Aldrich | #S3007 | |
Chemical compound, drug | Rapamycin | Sigma-Aldrich | #R0395 | |
Chemical compound, drug | Deferoxamine mesylate salt | Sigma-Aldrich | #D9533 | |
Chemical compound, drug | Liproxstatin-1 | Sigma-Aldrich | #SML1414 | |
Chemical compound, drug | Sphingosine | Sigma-Aldrich | #860025P | |
Chemical compound, drug | Brilliant Blue | Sigma-Aldrich | #80717 | |
Chemical compound, drug | Acetic acid | Sigma-Aldrich | #27225-1L-M | |
Chemical compound, drug | Chloroform | Sigma-Aldrich | #288306-2L | |
Chemical compound, drug | Glycerol | Sigma-Aldrich | #G6279-1L | |
Chemical compound, drug | Sodium azide | Sigma-Aldrich | #S2002-25G | |
Chemical compound, drug | Formaldehyde, 16% | Polysciences Inc | #18814-20 | Methanol free, ultra pure |
Other | 10 kDa Dextran | Molecular Probes | #D1864 | Dye labeled with Texas Red |
Other | DAPI stain | Sigma-Aldrich | #D9542-10MG | IF concentration used: 1 µg/mL |
Other | Normal Goat Serum | Abcam | #ab7481 | |
Other | Paraplast Plus | Sigma-Aldrich | #76258-1KG | |
Other | Casein Blocking Buffer 10× | Sigma-Aldrich | #B6429-500ML | |
Other | Hematoxylin Solution, Mayer’s | Sigma-Aldrich | #MHS16-500ML | |
Other | Eosin Y solution, aqueous | Sigma-Aldrich | #HT110232 | |
Other | DPX Mountant for histology | Sigma-Aldrich | #06522-100ML | |
Other | PBS buffer (10× Dulbecco's) | AppliChem | #A0965,9010 | |
Other | LSM700 confocal laser-scanning microscope | Carl Zeiss | LSM700 | |
Other | Hyrax M25 microtome | Carl Zeiss | Hyrax M25 | |
Other | Zeiss EM 900 microscope | Carl Zeiss | Zeiss EM 900 | |
Other | Step One Plus 96 well system | Applied Biosystems | Step One Plus | |
Chemical compound, drug | Acetonitrile | Honeywell | #34851 | |
Chemical compound, drug | Toluene | Supelco | #1.08325.1000 | |
Chemical compound, drug | Pentafluoro-benzyl bromide | Sigma-Aldrich | #101052 | |
Chemical compound, drug | Diisopropyl-ethylamine | Sigma-Aldrich | #496219 | |
Commercial assay or kit | GC-MS ISQ | Thermo Fisher | Trace 1210 series | |
Commercial assay or kit | Optima 17 | Macherey-Nagel | #MN726022.15 | |
Chemical compound, drug | C12:0; C12:1; C14:0; C14:1; C16:0; C16:1; C17:0; C17:1; C18:0; C18:1; C18:2; C18:3; C20:4; C20:5; C21:0; C22:0; C24:0 | Merck (Darmstadt, Germany) | ||
Chemical compound, drug | C19:1; C20:0; C21:1 | Larodan AB (Solna, Sweden) | ||
Chemical compound, drug | Paraformaldehyde | Merck | #1.04005.1000 | |
Chemical compound, drug | Glutaraldehyde | Merck | #1.04239.0250 | |
Chemical compound, drug | Osmiumtetroxide | Electron Microscopy Sciences | #22400-56 | |
Chemical compound, drug | HEPES | Roth | #7020.2 | |
Chemical compound, drug | Acetone puriss.p.a. ACS reagent, reag.ISO 99, 5% | Sigma-Aldrich | #:32201-2.5 | |
Chemical compound, drug | Agar 100 Premix Kit – Hard | Agar Scientific | #R1140 | |
Chemical compound, drug | Tri-Natriumcitrat-Dihydrat | Merck | #1-06448.0500 | |
Chemical compound, drug | Lead (II) nitrate for analysis | Merck | #1.07398.0100 |
Relative mRNA levels.
a – the ΔCT value is determined by subtracting the average CT value of endogenous control gene(Rpl32) from the average mRNA CT value. b –the calculation of ΔΔCT involves subtraction by the ΔCT calibrator value (ΔCT value in control). c – the range is given for relative levels determined by evaluating the expression: 2–ΔΔCT. AVE ± SEM values are reported from experiments done in at least duplicates. Two-tailed Student’s test was used to test for statistical significance. pa – compared to the relevant control. pb – compared to 15-day-old animals of the same genotype. pc – compared to sws1 mutant of the same age.
NTE/SWS expression in the surface glia is important for the integrity of Drosophila blood-brain barrier (BBB).
a – compared to control (OR x w1118). b – compared to Gal4-driver x OR. c – compared to Gal4-driver x UAS-swsRNAi. The values are reported from experiments done in triplicates. For statistical analyses of the observed phenotypes, two-way tables and chi-squared test were used.
NTE/SWS deficit in the surface glia results in the accumulation of Rab7- and CathepsinL-positive structures.
a – compared to Gal4-driver x OR animals of the same age. b – compared to 1-day-old animals of the same genotype. The values are reported from experiments done in triplicates. For statistical analyses of the observed phenotypes, two-way tables and chi-squared test were used.
NTE/SWS deficit in the surface glia results in permeable blood-brain barrier (BBB).
a – compared to control (OR x w1118). b – compared to Gal4-driver x OR. c –compared to Gal4-driver x UAS-swsRNAi. The values are reported from experiments done in triplicates. For statistical analyses of the observed phenotypes, two-way tables and chi-squared test were used.
The effect of treatment with different anti-inflammatory substances and stress suppressors on the frequency of the surface glia phenotype in sws and moody mutants.
a – compared to sws1 (no drug treatment)b – compared to moody ΔC17 (no drug treatment). The values are reported from experiments done in triplicates. For statistical analyses of the observed phenotypes, two-way tables and chi-squared test were used.
Mutants with defective BBB show upregulated levels of free fatty acids (FFA).
(a) sws and moody mutants show upregulated levels of free fatty acids (FFAs). For statistical analyses one-way ANOVA test was used. C14:1–9-cis-Tetradecenoic acid. C16:0 – Palmitic acid. C16:1 – Palmitoleic acid. C18:0 – Stearic acid. C18:1 – Oleic acid. C18:2 – Linoleic acid. C18:3 – α- and γ-Linolenic acid. C20:0 – Eicosanoic acid. C20:4 – Arachidonic acid. C20:5 – Eicosapentaenoic acid. (b) sws and moody mutants show upregulated levels of free fatty acids (FFAs). Summary of the ions monitored in the selected-ion monitoring (SIM) modeSIM#1 (12.00–14.50 min): m/z 197.4, 199.4, 225.4, 227.4, 253.4, 255.4, 267.4, 269.4. SIM#2 (14.50–15.00 min): m/z 281.4, 283.4, 279.4, 295.4, 297.4. SIM#3 (15.00–17.00 min): m/z 301.4, 303.4, 309.4, 311.4, 325.4, 337.4, 339.4, 365.4, 367.4.