Journal: eLife

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

doi: 10.7554/eLife.98020

Figure Lengend Snippet: ( A–F ) Adult brains stained with Coracle (CoraC) (white). In control ( Oregon R x white 1118 ), CoraC expression is pictured as the smooth line at the surface of the brain (green arrow). In sws 1 mutants ( B ) and in mutants with sws downregulation in all glia cells and specifically in subperineurial glia (SPG) cells ( repo>sws RNAi and Gli>sws RNAi , C and D , respectively), the outer glial cell layer labeled by CoraC is irregular and contains lesions and membrane clusters (magenta arrows). Animals with sws downregulation in neurons ( nSyb>sws RNAi , E ) do not have lesions and membrane clusters within the brain surface (green arrow). Expression of Drosophila NTE/SWS ( sws 1 ; moody>sws, F ) in SPG cells in mutant background results in the brain surface appearance which is similar to control (green arrow). Scale bar: 50 µm.

Article Snippet: The following primary antibodies were used: mouse anti-Repo (1:50), mouse anti-CoraC (1:50), and mouse anti-Rab7 (1:50), rat anti-DE-Cadherin (1:50) from the Developmental Studies Hybridoma Bank (DSHB); chicken anti-GFP (#ab13970, 1:1000) and rabbit anti-mCherry (#ab167453, 1:1000) from Abcam; mouse Anti-β-Galactosidase (#Z3781, 1:200) from Promega; rabbit anti-SWS (1:1000 from Doris Kretzschmar); mouse anti-CathepsinL (#1515-CY-010, 1:400) from R&D Systems; rabbit anti-NrxIV (1:1000 from Christian Klämbt); mouse anti-NimC1 (1:300 from István Andó).

Techniques: Staining, Expressing, Labeling, Membrane, Mutagenesis

Journal: eLife

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

doi: 10.7554/eLife.98020

Figure Lengend Snippet: ( 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 were used. ( B ) Bar graph shows the changed percentage of the brains of sws mutants with the glial phenotype, assayed with Coracle (CoraC), which were treated with different stress and inflammation inhibitors in comparison to untreated mutants. Two-way tables and chi-squared test were used for statistical analysis. *p<0.05, **p<0.005, ***p<0.001, number of adult brain hemispheres ≥73, at least three biological replicates (see ). ( C–Cʹ ) Adult brains of sws mutants stained with CoraC (white). In sws 1 mutants treated with control solution ( C ), the outer glial cell layer labeled by CoraC is irregular and contains membrane clusters (red arrowhead). In sws 1 mutants treated with sodium salicylate ( Cʹ ) the outer glial cell layer contains lesions (red arrowhead) and less membrane clusters. Scale bar: 50 µm. ( D–Dʹ ) Adult brains of moody mutants stained with CoraC (white). In moody mutants treated with control solution ( D ), the outer glial cell layer labeled by CoraC is irregular and contains membrane clusters (olive arrowhead). In moody mutants treated with sodium salicylate ( Dʹ ), the outer glial cell layer contains lesions (olive arrowhead) and no membrane clusters. ( E ) sws downregulation in glial cells during adulthood, after the BBB is formed, leads to the increased inflammatory response. Real-time quantitative PCR (RT-qPCR) analysis of antimicrobial peptides (AMPs) mRNA levels from control ( tub-Gal80 ts ; repo>/Oregon R , green) and tub-Gal80 ts ; repo>sws RNAi (red) fly heads shows upregulated expression of inflammatory response genes: Attacin A, Cecropin A, and Diptericin . AVE ± SEM is indicated (see ). ( F–G ) Larval and adult brains stained with mCherry (white) to reveal the macrophage entry in the brain, indicating that control ( Oregon R/srp(Hemo)3xmCherry ) brains. ( F ) show almost no macrophages marked by mCherry inside the developing and adult brains. Larval and adult brains of sws 1 ; srp(Hemo)3xmCherry mutants ( G ) show macrophages inside the larval and adult brains (yellow arrowheads). Scale bar: 50 µm.

Article Snippet: The following primary antibodies were used: mouse anti-Repo (1:50), mouse anti-CoraC (1:50), and mouse anti-Rab7 (1:50), rat anti-DE-Cadherin (1:50) from the Developmental Studies Hybridoma Bank (DSHB); chicken anti-GFP (#ab13970, 1:1000) and rabbit anti-mCherry (#ab167453, 1:1000) from Abcam; mouse Anti-β-Galactosidase (#Z3781, 1:200) from Promega; rabbit anti-SWS (1:1000 from Doris Kretzschmar); mouse anti-CathepsinL (#1515-CY-010, 1:400) from R&D Systems; rabbit anti-NrxIV (1:1000 from Christian Klämbt); mouse anti-NimC1 (1:300 from István Andó).

Techniques: Feeding Assay, Comparison, Staining, Labeling, Membrane, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

Journal: eLife

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

doi: 10.7554/eLife.98020

Figure Lengend Snippet: ( A–C ) Adult brains stained with Coracle (CoraC) (white) to reveal brain surface. CoraC expression in control brains ( Oregon R x white 1118 , A ) is depicted as the smooth line at the surface of the brain (green arrow). In moody ΔC17 mutant brains ( B ) and upon moody downregulation in subperineurial glia (SPG) cells ( moody>moody RNAi , C ), CoraC-positive outer cell layer contains lesions and membrane clusters (magenta arrows). Scale bar: 20 µm. ( D ) Bar graph shows the percentage of the brains with a defective brain surface. Two-way tables and chi-squared test were used for statistical analysis, ***p<0.001, number of adult brain hemispheres ≥20 (see ). ( E–F ) Electron microscopy images of the adult brain surface area in control ( white 1118 , E ) and moody ΔC17 mutants ( F ). As previously described , the septate junctions in moody ΔC17 mutants cannot properly stretch out during cell growth and appear abnormal when compared to controls (black arrows). Scale bar: 1 µm. ( G–H ) Adult brains stained with Rab7 (red), Neurexin IV (NrxIV) (green), and DAPI (blue) show no abnormal accumulation of Rab7 vesicles in the Oregon R control ( G ) and moody ΔC17 mutant ( H ) brains.

Article Snippet: The following primary antibodies were used: mouse anti-Repo (1:50), mouse anti-CoraC (1:50), and mouse anti-Rab7 (1:50), rat anti-DE-Cadherin (1:50) from the Developmental Studies Hybridoma Bank (DSHB); chicken anti-GFP (#ab13970, 1:1000) and rabbit anti-mCherry (#ab167453, 1:1000) from Abcam; mouse Anti-β-Galactosidase (#Z3781, 1:200) from Promega; rabbit anti-SWS (1:1000 from Doris Kretzschmar); mouse anti-CathepsinL (#1515-CY-010, 1:400) from R&D Systems; rabbit anti-NrxIV (1:1000 from Christian Klämbt); mouse anti-NimC1 (1:300 from István Andó).

Techniques: Staining, Expressing, Mutagenesis, Membrane, Electron Microscopy

Journal: eLife

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

doi: 10.7554/eLife.98020

Figure Lengend Snippet: ( A ) Bar graph shows the reduction in the percentage of the glial phenotype, assayed by Coracle (CoraC) expression pattern, in sws 1 (red) and moody ΔC17 (olive) mutants that were treated with non-steroidal anti-inflammatory drug (NSAID) and rapamycin in comparison to untreated mutants.This suggests that inflammation accelerates surface glia phenotype. Two-way tables and chi-squared test were used for statistical analysis, number of adult brain hemispheres ≥104, p<0.05, **p<0.005, ***p<0.001, at least three biological replicates (see ). ( B ) Real-time quantitative PCR (RT-qPCR) analysis of antimicrobial peptides (AMPs) mRNA levels from relevant controls (green) and sws 1 (red) and moody (olive) mutant fly heads shows significantly upregulated expression of inflammatory response genes: Attacin A, Cecropin A, and Diptericin . AVE ± SEM is indicated. Two-tailed Student’s test was used to test for statistical significance, *p<0.05, **p<0.005, ***p<0.001 (see ). ( C ) GS-MS measurements of free fatty acids (FFAs) indicate the relative increase of several FFAs in the heads of sws 1 (red) and moody ΔC17 (olive) mutants compared to relevant controls ( Oregon R and white 1118 , green). One-way ANOVA test was used for statistical analysis, *p<0.05, **p<0.005, ***p<0.001 (see ). ( D ) RT-qPCR analysis of AMP mRNA levels from the heads of 15- and 30-day-old relevant controls (green), sws 1 (red), and moody>sws RNAi (orange) mutants shows the age-dependent upregulation of the expression of inflammatory response genes ( Attacin A, Cecropin A, and Diptericin ). Moreover, expression of Drosophila NTE/SWS ( sws 1 ; moody>sws, blue) in subperineurial glia (SPG) cells in mutant background normalizes levels of AMPs. The AVE ± SEM is shown. Two-tailed Student’s test was used to test for statistical significance. p<0.05, **p<0.005, ***p<0.001 (see ). Black asterisks – sws 1 compared to Oregon R; moody>sws RNAi compared to moody>/Oregon R of the same age. Green asterisks – rescue, sws 1 ; moody>sws compared to sws 1 . Red asterisks – aging, 30-day-old compared to 15-day-old flies. ( E–F ) Adult brains stained with NimC1 (red), GFP (green), and DAPI (blue) to reveal the macrophage entry in the brain. Note that no macrophages marked by NimC1 (red) are detected in the control brain ( moody>CD8::GFP, E ), while NimC1-positive marcrophages are detected in moody>GFP, sws RNAi brain (yellow arrowheads, F ). Scale bar: 20 µm. ( G ) Mutants with defective brain barrier have upregulated innate immunity factors and exhibit elevated levels of FFAs involved in mediating the inflammatory response. Treatment with anti-inflammatory agents alleviates BBB phenotypes, suggesting that a signaling loop that links the condition of the brain barrier permeability, lipid metabolism, and inflammation. Figure 5—source data 1. GS-MS measurements of free fatty acids (FFA).

Article Snippet: The following primary antibodies were used: mouse anti-Repo (1:50), mouse anti-CoraC (1:50), and mouse anti-Rab7 (1:50), rat anti-DE-Cadherin (1:50) from the Developmental Studies Hybridoma Bank (DSHB); chicken anti-GFP (#ab13970, 1:1000) and rabbit anti-mCherry (#ab167453, 1:1000) from Abcam; mouse Anti-β-Galactosidase (#Z3781, 1:200) from Promega; rabbit anti-SWS (1:1000 from Doris Kretzschmar); mouse anti-CathepsinL (#1515-CY-010, 1:400) from R&D Systems; rabbit anti-NrxIV (1:1000 from Christian Klämbt); mouse anti-NimC1 (1:300 from István Andó).

Techniques: Expressing, Comparison, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Two Tailed Test, Staining, Permeability

Journal: eLife

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

doi: 10.7554/eLife.98020

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: mouse anti-Repo (1:50), mouse anti-CoraC (1:50), and mouse anti-Rab7 (1:50), rat anti-DE-Cadherin (1:50) from the Developmental Studies Hybridoma Bank (DSHB); chicken anti-GFP (#ab13970, 1:1000) and rabbit anti-mCherry (#ab167453, 1:1000) from Abcam; mouse Anti-β-Galactosidase (#Z3781, 1:200) from Promega; rabbit anti-SWS (1:1000 from Doris Kretzschmar); mouse anti-CathepsinL (#1515-CY-010, 1:400) from R&D Systems; rabbit anti-NrxIV (1:1000 from Christian Klämbt); mouse anti-NimC1 (1:300 from István Andó).

Techniques: Mutagenesis, Construct, Software, Labeling, Staining, Concentration Assay, Blocking Assay, Laser-Scanning Microscopy, Microscopy, Electron Microscopy