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

Inhibition of virus infection by passive immunization.

<p>To assess the role of neutralizing antibodies, polyclonal serum from vaccinated animals was injected intraperitoneally one day before challenge with MnPV infectious particles. After one week, RNA was extracted from the infected area to detect the MnPV specific E1∧E4 transcript by RT-PCR. GAPDH expression was used as internal control. Three animals were analyzed per group.</p

MnPV viral load in skin biopsies.

<p><b>Panel A:</b> The red color shows viral genomes in the suprabasal layers of normal skin visualized by DNA <i>in situ</i> hybridization. The dotted line marks the basal membrane. <b>Panel B:</b> MnPV DNA <i>in situ</i> hybridization of a papilloma. Hyperproliferation of the epidermis can be observed, as well as basal and parabasal cells harboring MnPV DNA. Original magnification panel A: 80×; panel B: 40×. <b>Panel C:</b> qPCR analyses to detect viral DNA were performed by amplifying a fragment of the late L1 ORF. Data were normalized by relating the values to the copy number of β-globin and considering two copies of the gene as a cell equivalent. Tissue samples were taken from normal skin from animals from the naturally infected colony [control (n = 19), vaccinated (n = 19)] and the experimentally infected colony [control (n = 21), vaccinated (n = 22)]. Boxes span the interquartile range and contain the median as a horizontal line. Outliers (•) are depicted outside the 10th and 90th percentile (whiskers). Statistical significance was assessed by the Mann-Whitney test: ***, p<0.001; ****, p<0.0001. Median age for controls: 15.4 months (range: 8.8–20.4 months); vaccinated: 15.1 months (range: 6.7–21.3 months).</p

Overview of the vaccination study.

<p><b>Panel A:</b> Vaccination was performed on both naturally infected <i>Mastomys</i> and on virus-free animals, which were subsequently infected. A subgroup of each colony also was kept under immunosuppressive conditions. <b>Panel B:</b> Electron micrograph showing MnPV VLPs with a size of 55 nm. <b>Panel C:</b> Time course of the vaccination study. Numbers indicate the time in months. Animals were vaccinated and bled as depicted. The green asterisk marks when the virus-free animals were experimentally infected. The red line indicates the duration of the treatment with cyclosporine A for the corresponding subgroups (for details, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003924#ppat-1003924-t001" target="_blank">Table 1</a>).</p

Humoral immune response to VLP vaccination.

<p><b>Panel A:</b> Antibody titers against the major capsid protein of MnPV were measured by VLP-ELISA two weeks after the third vaccination. The end point titer was determined as the reciprocal of the highest serum dilution with an OD above the blanks. Sera were measured from animals of the naturally and experimentally infected colonies as indicated. Statistical significance was assessed by the Mann-Whitney test: ****, p<0.0001. CTR = unvaccinated controls; VAC = vaccinated animals. <b>Panel B:</b> Correlation between the titer of neutralizing antibodies and anti-L1 antibody titers measured by VLP-ELISA. Sera were obtained from animals of all groups (preimmune sera, n = 20; control animals, n = 32; vaccinated animals, n = 34; immunosuppressed control animals, n = 12; immunosuppressed vaccinated animals, n = 9). The correlation coefficient (r²) was 0.8919 and the slope of the regression line 0.9048, demonstrating a strong correlation between both methods. n: indicates the number of animals.</p

Tumor histology.

<p>A representative example of a virus-induced keratoacanthoma (A, C, E and G) and an epidermal carcinoma (B, D, F and H) is shown. Epidermal carcinomas appeared in one animal from the immunocompetent naturally infected colony and one animal from the immunocompetent experimentally infected colony. <b>Panel A:</b> Part of keratin filled craters (X) can be seen in the upper left. Irregular epidermal acanthotic proliferations (O) push into the dermis with preservation of a boundary (arrowheads). Apoptotic bodies can be seen (arrow). <b>Panel B:</b> Upper part of an epidermal carcinoma with ulceration, characterized by irregularly shaped strands of atypical keratinocytes invading the inflamed stroma. The inset shows deeper solid parts of tumor with high rates of mitosis and increased nuclear cytoplasmic ratios amongst other cellular atypical features. <b>Panel C:</b> At the lateral margin of a keratoacanthoma, a thickened highly proliferative basal layer can be seen by Ki-67 label. The upper layers also show some Ki-67 positivity. <b>Panel D:</b> In an epidermal carcinoma almost all cells are Ki-67-positive, showing the high proliferative rate of the tumor. Small tumor islets (X) invade the stroma. <b>Panel E:</b> Immunostaining against keratin 14 shows a succinct demarcation of proliferative epidermis from dermal stroma. <b>Panel F:</b> Keratin 14 immunohistology, showing the dropping off of tumor cells into the stroma (arrowheads). <b>Panel G:</b> By laminin immunohistochemistry, a separation of epidermal proliferation from the dermal stroma can be visualized. The inset shows a higher magnification. <b>Panel H:</b> Laminin staining is not continuous, constituting an additional criterion for the invasive nature of the epidermal carcinoma. Original magnification panels A, B and B-inset: 100×; panels C, D, G and H: 200×, panels E, F and G-inset: 400×.</p

Expression of GPR116 and ELTD1 in mouse embryos.

<p>(<b>A</b>) Representative fluorescent images (of 18 images from 3 examined animals) of the heart and OFT area in Gpr116-mCherry reporter mice at embryonic day 11.5–12. Endothelial cells are stained with anti-CD31 antibody. (<b>B</b>) Representative fluorescent images (of 18 images from 3 examined animals) of the heart and OFT area in <i>Eltd1</i>^{<i>lacZ/+</i>} mice at embryonic day 10–10.5. Activity of ß-galactosidase is detected by SPiDER-ßGal, endothelial cells are stained with anti-CD31 antibody. (<b>A, B</b>) Nuclei are counterstained with DAPI. As, aortic sack; Da, dorsal aorta; Ta, truncus arteriosus; Cv, cardinal vein; Aa, arch arteries; A, atrium; V, ventricle; T, trachea; O, oesophagus. Scale bars: 200 μm.</p

Defects in large arteries and the cardiac outflow tract in GPR116-ELTD1 double deficient embryos (E18.5).

<p>(<b>A</b>) H&E staining of thoracic sections from two individual <i>Gpr116</i>^<i>-/-</i><i>;Eltd1</i>^<i>-/-</i> embryonic mice with enlarged views of the areas marked by dashed lines shown below. Asterisks indicate the position of the ventricle septum defect. LV, left ventricle; RV, right ventricle. (<b>B</b>) Ventral view of large vessels in a <i>Gpr116</i>^<i>-/-</i>;<i>Eltd1</i>^-/- embryonic mouse and a control littermate (<i>Gpr116</i>^<i>-/-</i>;<i>Eltd1</i>^+/+) with large arteries marked by dashed lines. A schematic of arteries is shown below. Arrow points to the position where the right subclavian artery usually branches off. 1, Aortic arch; 2, Innominate artery; 3, Right subclavian artery; 4, Left common carotid artery; 5, Ascending thoracic aorta; 6, pulmonary artery. (<b>C</b>) Schematic of the embryonic remodeling process of the branchial arch arteries into the aortic arch and the great vessels in normal configuration (E10.5 and E18.5) as well as in disease examples showing aberrant right subclavian artery, interrupted aortic arch and double aortic arch. RSA and LSA, right and left subclavian artery; RCA and LCA, right and left common carotid artery; A, aorta; da, ductus arteriosus; PT, pulmonary trunk. (<b>D</b>) Representative ventral view of large vessels from a <i>Gpr116</i>^<i>-/-</i>;<i>Eltd1</i>^-/- embryonic mouse and a control littermate (<i>Gpr116</i>^<i>+/-</i>;<i>Eltd1</i>^-/-) with arteries indicated by dashed lines. A schematic of arteries is shown below. 1. Ascending aorta; 2. Aortic arch; 3. Pulmonary artery; 4. Right subclavian artery; 5. Right common carotid artery; 6. Left common carotid artery; 7. Left subclavian artery. 2, 3 and 7 are highlighted in yellow indicating interrupted aortic arch. (<b>E</b>) H&E staining of thoracic cross sections (from inferior (left) to superior (right)) from a <i>Gpr116</i>^<i>-/-</i>;<i>Eltd1</i>^-/- embryonic mouse with enlarged views of the dashed line areas shown below. The aorta is connected to the right ventricle, which leads to double outlet right ventricle (DORV). LV, left ventricle; RV, right ventricle. (<b>F</b>) H&E staining of thoracic cross sections (from inferior (left) to superior (right)) from a <i>Gpr116</i>^<i>-/-</i>;<i>Eltd1</i>^-/- embryonic mouse with enlarged views of the areas indicated by a dash line shown below, exhibiting a double aortic arch with a retro-esophageal segment. Os, oesophagus; T, trachea; A, aorta. Scale bars: 200 μm (<b>A, E, F</b>).</p

Frequencies of malformation observed in <i>Gpr116</i>^<i>-/-</i><i>;Eltd1</i>^<i>-/-</i>, <i>Gpr116</i>^<i>+/+</i><i>;Eltd1</i>^<i>-/-</i> and <i>Gpr116</i>^<i>-/-</i><i>;Eltd1</i>^<i>+/+</i> E18.5 embryos.

<p>Frequencies of malformation observed in <i>Gpr116</i>^<i>-/-</i><i>;Eltd1</i>^<i>-/-</i>, <i>Gpr116</i>^<i>+/+</i><i>;Eltd1</i>^<i>-/-</i> and <i>Gpr116</i>^<i>-/-</i><i>;Eltd1</i>^<i>+/+</i> E18.5 embryos.</p

Incidence of virus-induced tumors.

<p>Immunocompetent and immunosuppressed animals were monitored over a period of 20 months for the occurrence of skin tumors (for number of animals, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003924#ppat-1003924-t001" target="_blank">Table 1</a>). The Kaplan-Meier plots (<b>Panels A–D</b>) show the tumor incidence for each group in vaccinated (dashed line) and control animals (full line). Tumor-bearing animals displayed 1–4 skin tumors each (mean = 1.4). Green arrows indicate the time points of vaccination. The red arrow indicates the time of experimental infection, the blue arrow the start of the CsA feed, the purple arrow of both. The small vertical bars indicate censored animals which died for unknown reasons before tumor development. Differences between vaccinated and control animals were evaluated using the log rank test. The number of animals is given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003924#ppat-1003924-t001" target="_blank">Table 1</a>. <b>Panels E/F:</b> Papillomas and keratoacanthomas in the lower back (infection site) of control animals from the experimentally infected colony. <b>Panels G/H:</b> Papillomas and keratoacanthomas arising in control animals from the naturally infected colony. Bars: 1 cm.</p