Additional file 1: Figure S1. of Lipid microdomain modification sustains neuronal viability in models of Alzheimer’s disease
Creators: name: Herzer, Silke
name: Meldner, Sascha
name: Rehder, Klara
name: Hermann-Josef Gröne
name: Nordström, Viola
Publisher: name: Figshare
Year: 2016
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology

description: Inhibition of ganglioside biosynthesis by GENZ123446 (GENZ) does not affect viability of mHippoE-14 neurons. (a) Immune overlay TLC with antibodies against the indicate ganglioside species confirms that mHippoE-14 cells express the a-series gangliosides GM3, GM1, and GD1a. (b) Morphology of mHippoE-14 cells after GENZ treatment (5 μM GENZ, 7 days), both depicted by phalloidin staining and bright field microscopy. (c) Western blot shows that synaptophysin expression of GENZ-treated mHippoE-14 cells is unchanged (100 nM insulin, 5 min (n = 4)). (d) Cell viability of vehicle and GENZ-treated mHippoE-14 cells shows that GENZ treatment itself does not alter cell viability. A positive control (5 % DMSO) verifies the functionality of the MTT assay (Vehicle vs. Genz: n = 6; 5 % DMSO n = 2-3). Figure S2. Generation of neurotoxic amyloid-β1-42-derived diffusible ligands (ADDLs). (a) Generation of ADDLs is monitored by electron microscopy. Aβ1-42 monomers have been incubated as described in SupplementaryMethods. The subsequent generation of ADDLs and fibrils from the Aβ1-42 monomers is shown by electron microscopy. (b) Generation of oligomeric ADDL species is verified by dot blot analysis using the oligomer-specific antibody A11. The 4G8 antibody recognizes all Aβ1-42 species. (c) Immunofluorescence depicting that ADDLs (6E10 antibody) bind to mHippoE-14 cells. Figure S3. Stimulation with 10nM insulin also increases insulin receptor (IR) tyrosine phosphorylation of GENZ-treated mHippoE-14 cells. (a) Negative control for the IR/phospho-tyrosine (pTyr) proximity ligation assay (PLA; Fig. 2) using only the IR antibody (C-19). (b) A PLA confirms that GENZ treatment enhances insulin-dependent IR tyrosine phosphorylation (IR/pTyr) upon stimulation with insulin (n = 37–45 cells). Unpaired two-tailed student’s t-test (p ≤ 0.001 is marked with (***)); 10nM insulin 3 min. Means ± SEM. Scale bars: 10 μm. Figure S4. GCS inhibition increases surface IR levels on mHippoE-14 cells upon ADDL exposure independently of the chemical nature of inhibition. (a) Western blot shows that NMDA receptor levels are not changed by GENZ treatment (n = 4). (b) Negative control for PLA stainings (Fig. 3c and e) using one IR antibody only (N-20). (c) Ganglioside expression of mHippoE-14 cells treated with either H2O (vehicle) or the GCS inhibitor NB-DNJ (100 μM, 7d, n = 4), as shown by TLC. NB-DNJ treatment results in the reduction of individual gangliosides by between approximately 30 to 50 %. (d) A PLA on non-permeabilized mHippoE-14 cells using two different IR antibodies (N-20 and D-17) enables the quantification of IR levels on the neuronal cell surface. Exposure to ADDLs (5 μM, 30 min) leads to a loss of IR in control cells (white bar), whereas surface IR levels are increased on NB-DNJ-treated cells (n = 39-101 cells). Unpaired two-tailed student’s t-test (if p ≤ 0.01 or p ≤ 0.001, results are marked with (**) or (***), respectively); 5 μM ADDLs, 30 min. Means ± SEM. Scale bars: 10 μm. Figure S5. ADDL treatment has little impact on total IR levels in mHippoE-14 cells. (a) Immunofluorescence of total IR in permeabilized mHippoE-14 cells shows that ADDL exposure has only a slight impact on total IR levels (white bar). The increased total IR levels in GENZ-treated cells reflect the observed effect of GCS inhibition (Fig. 1c) (n = 139–145 cells). (b) Negative control of surface IR PLA staining. For the negative control, only one IR antibody (N-20) was used. Unpaired two-tailed student’s t-test (if p ≤ 0.01 or p ≤ 0.001, results are marked with (**) or (***), respectively); 5 μM ADDLs, 24 h. Means ± SEM. Scale bars: 10 μm. Figure S6. Increased sphingomyelin expression in GENZ-treated mHippoE-14 neurons is not involved in caveolin-1-mediated up-regulation of surface IR. (a) Thin layer chromatography (TLC) analysis of mHippoE-14 cells shows that GENZ-treated cells display higher sphingomyelin levels (n = 4). (b) TLC analysis indicates that mHippoE-14 cells treated with caveolin-1 siRNA, which display increased surface IR levels (Fig. 4c), do not show elevated levels of sphingomyelin (n = 4). Unpaired two-tailed student’s t-test (p ≤ 0.001 was marked with (***)). Means ± SEM. Figure S7. Complex formation between IR and ADDLs at dendrites of primary hippocampal neurons involves ganglioside GD1a but not GM1. (a) Immune overlay TLC of a known brain standard confirms the specificity of the antibodies to their respective ganglioside. (b) Both primary hippocampal neurons treated with vehicle and GENZ display immunohistochemically visible synaptic contacts (co-labeling of synaptophysin and phalloidin; arrowheads). Neuronal morphology is furthermore depicted by bright field microscopy. (c) Immune fluorescence indicates that ADDLs (antibody 6E10) partially co-localize with IR on dendrites (phalloidin). (d) Immune fluorescence indicates partial co-localization of ADDLs with phalloidin (white arrowheads). (e) Immunofluorescence shows that dendritic GD1a in part co-localizes with ADDLs, and that GD1a also in part co-localizes with IR. (f) Immunofluorescence shows that dendritic GT1b in part co-localizes with ADDLs, and that GT1b also in part co-localizes with IR. (g) Immunofluorescence shows that dendritic GM1 only co-localizes very little with ADDLs and IR. (h) Negative control for PLA stainings (Fig. 6d) using only one IR antibody (N-20). (i) Combined PLA/phalloidin staining showing the PLA complexes (green labels) on a dendrite of an untreated hippocampal neuron. This staining confirms very little complex formation between IR, ADDLs and ganglioside GM1. (j) Dot blot shows that biotinylated ADDLs co-precipitate with the IR and ganglioside GD1a. (k) Negative control for dendritic caveolin-1/GD1a PLA staining on primary neurons (Fig. 7c and d) using either the caveolin-1 or the GD1a antibody only. 5 μM ADDLs, 30 min. Scale bars = 5 μm. Figure S8. Ganglioside reduction by GENZ prevents ADDL-induced IR desensitization. (a) Negative control for dendritic IR/p-Tyr PLA staining on primary neurons (Fig. 7f) using either the IR (C-19) or the p-Tyr antibody only. (b) A PLA using both an IR- and a p-Tyr-specific antibody indicates insulin-evoked dendritic IR phosphorylation (green). ADDL exposure decreases IR phosphorylation (white bar). However, GENZ treatment increases insulin sensitivity of dendritic IR upon ADDL exposure (grey bar). Quantification shows PLA spots/inch dendrite (n = 9-13 measurements). Cells were treated with either saline or 10nM insulin for 3 min. Dendrites were visualized with phalloidin. Unpaired two-tailed student’s t-test (p ≤ 0.001 was marked with (***)). Means ± SEM. Scale bars: 5 μm. Figure S9. The 5xFAD (familial Alzheimer’s disease) mouse model with inducible forebrain neuron-specific GCS deletion. (a) Breeding scheme and generation of 5xFAD//Cre mice with forebrain neuron-specific GCS deletion. (b) Negative control of total IR PLA staining on Ugcgf/f mouse brain tissue, using one IR antibody only (N-20). Depicted are cortical neurons. Scale bar: 10 μm. (PDF 13248 kb)