Additional file 1: Figure S1. of Lipid microdomain modification sustains neuronal viability in models of Alzheimer’s disease

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

GCS in hypothalamic neurons regulates neuronal leptin signaling at the plasma membrane.

<p>(A) Stat3 phosphorylation was markedly decreased in Arc sections of <i>Ugcg</i>^{f/f//CamKCreERT2} mice in response to peripheral leptin (5 mg/kg; 45 min) 6 wk p.i. (<i>n</i> = 16–33). Three independent animal groups were analyzed. (B) Serum leptin levels were unchanged 3 wk p.i. and increased prominently 7 wk p.i. in <i>Ugcg</i>^{f/f//CamKCreERT2} mice, reflecting increased body fat mass (<i>n</i> = 12–14). (C) mRNA expression analysis for suppressor of cytokine signaling 3 (SOCS-3) expression in Arc-enriched hypothalamic tissue was carried out 2, 6, and 9 wk p.i. <i>Socs-3</i> expression normalized to the housekeeping gene tubulin was unaltered (<i>n</i> = 3–5). (D) mRNA expression analysis for the long form of the leptin receptor, <i>Leprb</i>, in mediobasal hypothalamus was carried out 6 wk p.i. <i>Leprb</i> expression normalized to the housekeeping gene tubulin was unaltered at that time point (<i>n</i> = 4–5). (E) Immortalized mouse hypothalamic cells (N-41 cells) were analyzed for cell surface expression of ObR. Non-detergent-treated cells were fixed and simultaneously stained with two ObR antibodies. A proximity ligation assay (PLA) indicated quantifiable and unchanged ObR expression on the surface of controls and cells treated with the specific GCS inhibitor NB-DNJ (<i>n</i> = 41–47 cells). PLA principle is depicted on the right side. (F–G) N-41 cells were incubated with either saline or 100 ng/ml leptin (10 min). Close interactions between GCS-derived neuronal gangliosides GD1a/ObR (F) and GM1/ObR (G) were detected by PLA. Leptin treatment dynamically increased the GD1a/ObR and GM1/ObR PLA spots per cell (<i>n</i> = 48–67 cells). (H) Extracts from saline- and leptin-treated N-41 cells were immunoprecipitated with an ObR antibody, lipids were extracted, and GD1a and GM1 were visualized by immune overlay TLC. GD1a and GM1 co-immunoprecipitated (Co-IP) with ObR, which tended to be stronger in leptin-treated cells. Addition of a blocking peptide almost totally abolished ganglioside signals. Gangliosides GD1b and GT1b, expressed in mouse brain tissue, were not co-precipitated with ObR from hypothalamic tissue of <i>Ugcg</i>^f/f mice (5 mg/kg leptin, 45 min). (I) Co-IP showed significantly decreased leptin-induced complex formation between ObR and Jak in NB-DNJ-treated N-41 cells (<i>n</i> = 4). (J) Sustainable Jak phosphorylation could be induced in N-41 cells after 15 min of leptin treatment (0.5 µg/ml). NB-DNJ-treated cells showed a markedly delayed response to leptin. (K) Thirty minutes after leptin treatment, Jak phosphorylation was decreased in NB-DNJ-treated cells (<i>n</i> = 4). *<i>p</i>≤0.05; ***<i>p</i>≤0.001. Means ± SEM.</p

rAAV-mediated <i>Ugcg</i> gene delivery to the hypothalamic Arc ameliorates obesity and hyperleptinemia in <i>Ugcg</i>^{f/f//CamKCreERT2} mice.

<p>(A) Double immunofluorescence showed that Cre activity, indicated by beta galactosidase staining (b-gal), was targeted to Arc neurons expressing the long form of the ObR, as indicated by PStat3 staining in leptin-injected <i>R26R/Ugcg</i>^{f/+//CamKCreERT2} mice (5 mg/kg leptin, 120 min). (B) Stereotactic delivery of rAA viruses encoding <i>Ugcg</i> and <i>lacZ</i> to the Arc of <i>Ugcg</i>^{f/f//CamKCreERT2} mice resulted in a significant amelioration in body weight increase compared to rAAV-Empty/lacZ-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 6–8). (C) Serum leptin tended to be lower in rAAV-Ugcg/lacZ-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice 8 wk p.i. (<i>n</i> = 6–8). (D–F) Targeting of rAAV Ugcg/lacZ- and rAAV Empty/lacZ-injected animals that were included in the analyses. At the end of the experiments, brains were removed and stained for X-Gal to indicate vector delivery. Red marks depict exemplarily areas of strong X-Gal staining in animals considered as Arc targeted. Depicted are areas between bregma −1.9 (D), bregma −2.1 (E), and bregma −2.3 (F). (G) Restored ganglioside biosynthesis in the Arc of rAAV-Ugcg-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice, as shown by GD1a immunofluorescence 8 wk p.i. Scale bar: 18 µm. *<i>p</i>≤0.05. Means ± SEM.</p

Normal ultrastructure in ganglioside-depleted neurons.

<p>(A) Major pathway for biosynthesis of GSL including gangliosides in the brain. (B) X-Gal staining in brains of <i>R26R/Ugcg</i>^{f/+//CamKCreERT2} reporter mice revealed strong Cre activity in the hypothalamic Arc. GD1a immunofluorescence visualized ganglioside depletion in the Arc of <i>Ugcg</i>^{f/f//CamKCreERT2} mice 6 wk p.i. Scale bar: 75 µm. (C) Ceramide levels were not significantly altered in hippocampus of <i>Ugcg</i>^{f/f//CamKCreERT2} mice. Quantification from densitometry analysis of TLC results is depicted (<i>n</i> = 3). (D) Neurons in the Arc of <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed normal ultrastructural morphology of plasma membrane (pm), nucleus (N), mitochondria (M), endoplasmic reticulum (ER), golgi (G), projections (P), and myelin sheaths (my) 6 and 12 wk p.i. Scale bar: 2 µm. 3^rdv, third ventricle.</p

<i>Ugcg</i>^{f/f//CamKCreERT2} mice develop progressive obesity.

<p>Both female (A) and male (B) <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed a progressive increase in body weight after tamoxifen induction (<i>n</i> = 6–9). (C) <i>Ugcg</i>^{f/f//CamKCreERT2} mice were larger than <i>Ugcg</i>^f/f littermates (16 wk p.i.), and body fat mass was prominently elevated. (D) Enlarged adipocytes in <i>Ugcg</i>^{f/f//CamKCreERT2} mice 9 wk p.i. (E) Increased weight of epigonadal WAT 9 wk p.i. in <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 4–5). (F) NMR analysis revealed significant and progressive accumulation of body fat mass in <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 9–10). *<i>p</i>≤0.05; **<i>p</i>≤0.01;***<i>p</i>≤0.001. Means ± SEM.</p

Hypothalamic neurons of <i>Ugcg</i>^{f/f//CamKCreERT2} mice are less responsive to peripheral leptin.

<p>(A–C) Brains of leptin-stimulated mice were analyzed for neuronal activity indicated by c-Fos immunofluorescence. Detailed pictures in the upper lane indicate regions of the Arc that are outlined in overview pictures (frames). Arrowheads mark c-Fos-positive neurons located in the VMH. Axis indicators were included indicating the medial (m) and ventral (v) axes. (A) <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed leptin-induced neuronal activation comparable to <i>Ugcg</i>^f/f mice in the Arc 1–2 wk p.i. (B) Leptin response in the Arc was decreased in nonobese <i>Ugcg</i>^{f/f//CamKCreERT2} mice weight-matched to controls 3–4 weeks p.i. (C) Decreased c-Fos staining in the Arc was also observed in obese leptin-induced <i>Ugcg</i>^{f/f//CamKCreERT2} mice 6 wk p.i. The percentage of c-Fos-positive neurons per Arc section was depicted as values normalized to saline-injected <i>Ugcg</i>^f/f mice (<i>n</i> = 14–22 sections). Depicted sections are located between bregma levels −1.5 to −1.8. Quantification contains data from bregma levels −1.4 to −2.3. (D–F) <i>Ugcg</i>^{f/f//CamKCreERT2} mice retained leptin responsiveness in the VMH, as elevated c-Fos after leptin stimulation indicated (<i>n</i> = 8–20 sections). Quantification contains data from bregma levels −1.4 to −2.0. Datasets for each time point were acquired individually. Two (1–2 and 3–4 wk) or three (6 wk) independent animal groups were analyzed. Immunofluorescence and image acquisition for each dataset (treated and untreated controls and knockouts) were performed simultaneously. Scale bar: 75 µm; 3^rdv, 3^rd ventricle; *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001. Means ± SEM.</p

POMC and NPY neurons of <i>Ugcg</i>^{f/f//CamKCreERT2} mice are less responsive to leptin.

<p>(A) Leptin engages POMC neurons in the Arc of control (<i>Ugcg</i>^f/f) mice and <i>Ugcg</i>^{f/f//CamKCreERT2} mice 1–2 wk p.i., as indicated by elevated c-Fos. This response was decreased in Ugcg^{f/f//CamKCreERT2} mice 6 wk p.i. (B) Elevated leptin-induced PStat3 levels in POMC neurons of <i>Ugcg</i>^f/f mice and <i>Ugcg</i>^{f/f///CamKCreERT2} mice 1–2 wk p.i. This response was blunted in Ugcg^{f/f//CamKCreERT2} mice 6 wk p.i. (C) Leptin slightly decreased the activity of NPY neurons in <i>Ugcg</i>^f/f mice and <i>Ugcg</i>^{f/f///CamKCreERT2} mice 1–2 wk p.i. This was not detected in <i>Ugcg</i>^{f/f///CamKCreERT2} mice 6 wk p.i. (D) Unlike 1–2 wk p.i., leptin did not elevate PStat3 in NPY neurons of <i>Ugcg</i>^{f/f///CamKCreERT2} mice 6 wk p.i. Datasets for each time point were acquired individually, and quantification contains normalized data from two (1–2 wk p.i.; <i>n</i> = 4–11) or three (6 wk p.i.; <i>n</i> = 18–27) independent animal groups. Immunofluorescence and image acquisition for each dataset (treated and untreated controls and knockouts) were performed simultaneously. Scale bar: 20 µm; *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001. Means ± SEM.</p

Proposed model for GCS-derived ganglioside GD1a and GM1 regulation of hypothalamic leptin signaling and energy homeostasis.

<p>(A) GCS-derived gangliosides form complexes with ObR, thereby facilitating leptin-dependent Jak and Stat3 phosphorylation, and formation of PIP3. These pathways are crucial contributors to regulation of energy homeostasis. (B) In obese <i>Ugcg</i>^{f/f//CamKCreERT2} mice, ObR signal transduction is abolished in GCS-depleted neurons.</p