Unraveling the link between neuropathy target esterase NTE/SWS, lysosomal storage diseases, inflammation, abnormal fatty acid metabolism, and leaky brain barrier

  1. Mariana I Tsap
  2. Andriy S Yatsenko
  3. Jan Hegermann
  4. Bibiana Beckmann
  5. Dimitrios Tsikas
  6. Halyna R Shcherbata  Is a corresponding author
  1. Institute of Cell Biochemistry, Hannover Medical School, Germany
  2. Institute of Functional and Applied Anatomy, Research Core Unit Electron Microscopy, Hannover Medical School, Germany
  3. Institute of Toxicology, Hannover Medical School, Germany
  4. Mount Desert Island Biological Laboratory, United States
6 figures, 1 table and 7 additional files

Figures

NTE/SWS is expressed in Drosophila brain and its loss leads to severe neurodegeneration.

(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 …

Figure 2 with 3 supplements
Downregulation of NTE/SWS affects surface glia architecture.

(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 …

Figure 2—figure supplement 1
NTE/SWS expression pattern, sws mRNA levels, and expression patterns of the Gal4 driver lines used in the study.

(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 …

Figure 2—figure supplement 2
sws downregulation in neurons does not result in the formation of lesions and membrane clusters within the brain surface, and expression of Drosophila NTE/SWS in glia cells rescued glial phenotype.

(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 …

Figure 2—figure supplement 3
3D structures of human NTE and Drosophila SWS.

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 …

Downregulation of NTE/SWS results in intracellular accumulations.

(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

Downregulation of NTE/SWS affects brain permeability barrier.

(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 …

Figure 5 with 3 supplements
Mutants with defective blood-brain barrier (BBB) have an increased age-dependent inflammatory response and elevated levels of free fatty acid (FFA).

(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 …

Figure 5—figure supplement 1
sws mutants show increased inflammation and macrophage entry into the brains.

(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 …

Figure 5—figure supplement 2
Moody flies with a permeable blood-brain barrier (BBB) have similar to sws mutants brain surface appearance, but distinct septate junction phenotypes, and moodyΔC17 mutant shows no accumulation of endosomal-lysosomal pathway components such as Rab7.

(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 …

Figure 5—figure supplement 3
Gas chromatography-mass spectrometry (GC-MS) analysis of free fatty acids (FFAs).

(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 …

Figure 6 with 1 supplement
Septate junction and membrane domain organization in mutants with defective brain permeability barrier.

(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 …

Figure 6—figure supplement 1
Lysosomal mutants show abnormal septate junction formation.

(A–E) Adult brains stained with Coracle (CoraC) (red), Neurexin IV (NrxIV) (green), and DAPI (blue) to reveal septate junction structures of the surface glia. In control (moody>/Oregon R, A) brain, …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-Repo (mouse monoclonal)Developmental Studies Hybridoma Bank#8D12IF(1:50)
AntibodyAnti-CoraC (mouse monoclonal)Developmental Studies Hybridoma Bank#C566.9IF(1:50)
AntibodyAnti-Rab7 (mouse monoclonal)Developmental Studies Hybridoma Bank#AB2722471IF(1:50)
AntibodyAnti-DE-Cad (rat monoclonal)Developmental Studies Hybridoma Bank#DCAD2IF(1:50)
AntibodyAnti-GFP (chicken polyclonal)Abcam#ab13970IF(1:1000)
AntibodyAnti-mCherry (rabbit polyclonal)Abcam#ab167453IF(1:1000)
AntibodyAnti-β-Galactosidase (mouse monoclonal)Promega#Z3781IF(1:200)
AntibodyAnti-CathepsinL (mouse)R&D Systems#1515-CY-010IF(1:400)
AntibodyAnti-NrxIV (rabbit polyclonal)Gift from Christian KlämbtAnti-NrxIVIF(1:1000)
AntibodyAnti-SWS (rabbit polyclonal)Gift from Doris KretzschmarAnti-SWSIF(1:1000)
AntibodyAnti-NimC1 (mouse)Gift from István AndóAnti-NimC1IF(1:300)
AntibodyAnti-chicken Alexa 488 (goat polyclonal)Thermo Fisher Scientific#A-11039Secondary antibody IF(1:500)
AntibodyAnti-rat Alexa 488 (goat polyclonal)Thermo Fisher Scientific#A-11077Secondary antibody IF(1:500)
AntibodyAnti-rat Alexa 647 (goat polyclonal)Thermo Fisher Scientific#A-21247Secondary antibody IF(1:500)
AntibodyAnti-rabbit Alexa 488 (goat polyclonal)Thermo Fisher Scientific#A-11034Secondary antibody IF(1:500)
AntibodyAnti-rabbit Alexa 568 (goat polyclonal)Thermo Fisher Scientific#A-11011Secondary antibody IF(1:500)
AntibodyAnti-mouse IgG2a Cy3 (goat polyclonal)Jackson ImmunoResearch#115-165-206Secondary antibody IF(1:400)
AntibodyAnti-mouse IG1 Cy3 (goat polyclonal)Jackson ImmunoResearch#115-165-205Secondary antibody IF(1:500)
AntibodyAnti-mouse IgG1 647 (goat polyclonal)Jackson ImmunoResearch#115-605-205Secondary antibody IF(1:500)
Genetic reagent (D. melanogaster)w[1118]Bloomington Drosophila Stock CenterBDSC 5905Wild type strain
Genetic reagent (D. melanogaster)Oregon-RBloomington Drosophila Stock CenterBDSC 5Wild type strain
Genetic reagent (D. melanogaster)sws1Gift from Doris Kretzschmarsws1sws[1]/FM7a(null mutant)
Genetic reagent (D. melanogaster)sws4Bloomington Drosophila Stock CenterBDSC 28121sws[4]/C(1)DX, y[1] w[1] f[1] (Amino acid replacement: G956D)
Genetic reagent (D. melanogaster)UAS-swsGift from Doris KretzschmarUAS-swsUAS-sws (sws gene under control of UAS promotor)
Genetic reagent (D. melanogaster)sws-Gal4Kyoto Stock Center104592y* w* P{GawB}swsNP4072/FM7c
Genetic reagent (D. melanogaster)UAS-nlsLacZ, UAS-CD8::GFPGift from Frank HirthUAS-nLacZ, UAS-GFPUAS-nlsLacZ, UAS-CD8::GFP(nLacZ and GFP constructs under control of UAS promotor)
Genetic reagent (D. melanogaster)UAS-hNTEGift from Robert WessellsUAS-hNTEw[1118]; p[PUAST]-hNTE/CyO(Human NTE undercontrol of UAS promotor)
Genetic reagent (D. melanogaster)UAS-swsRNAiBloomington Drosophila Stock CenterBDSC 61338y[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/TM6BGift from Mikael Simonsrepo-Gal4repo-Gal4, UAS-CD8::GFP/TM6B
Genetic reagent (D. melanogaster)Gliotactin-Gal4, UAS-CD8::GFPGift from Mikael SimonsGli-Gal4Gliotactin-Gal4, UAS-CD8::GFP
Genetic reagent (D. melanogaster)moody-Gal4, UAS-CD8::GFPGift from Mikael Simonsmoody-Gal4moody-Gal4, UAS-CD8::GFP
Genetic reagent (D. melanogaster)nSyb-Gal4Bloomington Drosophila Stock CenterBDSC 51945y[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,SbThis studyrepo-Gal4, nSyb-Gal4repo-Gal4, nSyb-Gal4, UAS-CD8::GFP/TM6B,Sb
Genetic reagent (D. melanogaster)tub-Gal80ts; repo-Gal4/TM6BThis studytub-Gal80ts; repo-Gal4/TM6Btub-Gal80ts; repo-Gal4/TM6B(temperature sensitive)
Genetic reagent (D. melanogaster)moodyΔC17Gift from Christian KlämbtmoodyΔC17Null mutant
Genetic reagent (D. melanogaster)UAS-moodyRNAiBloomington Drosophila Stock CenterBDSC 66326y[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-DysbRNAiBloomington Drosophila Stock CenterBDSC 67316y[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-Npc1aRNAiBloomington Drosophila Stock CenterBDSC 37504y[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-PldnRNAiBloomington Drosophila Stock CenterBDSC 67884y[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-spinRNAiBloomington Drosophila Stock CenterBDSC 27702y[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) 3xmCherryGift from Angela Giangrandesrp(Hemo)
3xmCherry
srp(Hemo) 3xmCherry
Software, algorithmMicrosoft ExcelMicrosoftMicrosoft Excel
Software, algorithmAdobe PhotoshopAdobeAdobe CC
Software, algorithmZen 2011 (black edition)Carl Zeiss; Emmenlauer et al., 2009Zen 2011
Software, algorithmAlphaFold2 v1.5.2Mirdita et al., 2022; https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynbAlphaFold2
Software, algorithmThe PyMOL Molecular Graphics System, v2.5.5Schrödinger, LLC; https://pymol.org/PyMol
Software, algorithmStepOne Software v2.3Applied BiosystemsStepOne
Chemical compound, drugTRIzol reagentInvitrogen#15596018
Commercial assay or kitHigh Capacity cDNA Reverse Transcription kitApplied Biosystems#4368813
Commercial assay or kitFastSYBR Green master mixApplied Biosystems#435612
Chemical compound, drugTUDCASigma-Aldrich#580549Tauroursodeoxycholic acid
Chemical compound, drug4-PBASigma-Aldrich#5676164-Phenylbutyric acid
Chemical compound, drugValsartanSigma-Aldrich#PHR1315
Chemical compound, drugFenofibrateSigma-Aldrich#F6020
Chemical compound, drugSodium salicylateSigma-Aldrich#S3007
Chemical compound, drugRapamycinSigma-Aldrich#R0395
Chemical compound, drugDeferoxamine mesylate saltSigma-Aldrich#D9533
Chemical compound, drugLiproxstatin-1Sigma-Aldrich#SML1414
Chemical compound, drugSphingosineSigma-Aldrich#860025P
Chemical compound, drugBrilliant BlueSigma-Aldrich#80717
Chemical compound, drugAcetic acidSigma-Aldrich#27225-1L-M
Chemical compound, drugChloroformSigma-Aldrich#288306-2L
Chemical compound, drugGlycerolSigma-Aldrich#G6279-1L
Chemical compound, drugSodium azideSigma-Aldrich#S2002-25G
Chemical compound, drugFormaldehyde, 16%Polysciences Inc#18814-20Methanol free, ultra pure
Other10 kDa DextranMolecular Probes#D1864Dye labeled with Texas Red
OtherDAPI stainSigma-Aldrich#D9542-10MGIF concentration used: 1 µg/mL
OtherNormal Goat SerumAbcam#ab7481
OtherParaplast PlusSigma-Aldrich#76258-1KG
OtherCasein Blocking Buffer 10×Sigma-Aldrich#B6429-500ML
OtherHematoxylin Solution, Mayer’sSigma-Aldrich#MHS16-500ML
OtherEosin Y solution, aqueousSigma-Aldrich#HT110232
OtherDPX Mountant for histologySigma-Aldrich#06522-100ML
OtherPBS buffer (10× Dulbecco's)AppliChem#A0965,9010
OtherLSM700 confocal laser-scanning microscopeCarl ZeissLSM700
OtherHyrax M25 microtomeCarl ZeissHyrax M25
OtherZeiss EM 900 microscopeCarl ZeissZeiss EM 900
OtherStep One Plus 96 well systemApplied BiosystemsStep One Plus
Chemical compound, drugAcetonitrileHoneywell#34851
Chemical compound, drugTolueneSupelco#1.08325.1000
Chemical compound, drugPentafluoro-benzyl bromideSigma-Aldrich#101052
Chemical compound, drugDiisopropyl-ethylamineSigma-Aldrich#496219
Commercial assay or kitGC-MS ISQThermo FisherTrace 1210 series
Commercial assay or kitOptima 17Macherey-Nagel#MN726022.15
Chemical compound, drugC12:0; 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; C24:0Merck (Darmstadt, Germany)
Chemical compound, drugC19:1; C20:0; C21:1Larodan AB (Solna, Sweden)
Chemical compound, drugParaformaldehydeMerck#1.04005.1000
Chemical compound, drugGlutaraldehydeMerck#1.04239.0250
Chemical compound, drugOsmiumtetroxideElectron Microscopy Sciences#22400-56
Chemical compound, drugHEPESRoth#7020.2
Chemical compound, drugAcetone puriss.p.a. ACS reagent, reag.ISO 99, 5%Sigma-Aldrich#:32201-2.5
Chemical compound, drugAgar 100 Premix Kit – HardAgar Scientific#R1140
Chemical compound, drugTri-Natriumcitrat-DihydratMerck#1-06448.0500
Chemical compound, drugLead (II) nitrate for analysisMerck#1.07398.0100

Additional files

Supplementary file 1

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.

https://cdn.elifesciences.org/articles/98020/elife-98020-supp1-v2.docx
Supplementary file 2

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.

https://cdn.elifesciences.org/articles/98020/elife-98020-supp2-v2.docx
Supplementary file 3

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.

https://cdn.elifesciences.org/articles/98020/elife-98020-supp3-v2.docx
Supplementary file 4

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.

https://cdn.elifesciences.org/articles/98020/elife-98020-supp4-v2.docx
Supplementary file 5

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.

https://cdn.elifesciences.org/articles/98020/elife-98020-supp5-v2.docx
Supplementary file 6

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.

https://cdn.elifesciences.org/articles/98020/elife-98020-supp6-v2.docx
MDAR checklist
https://cdn.elifesciences.org/articles/98020/elife-98020-mdarchecklist1-v2.docx

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