Deletion of Specific Sphingolipids in Distinct Neurons Improves Spatial Memory in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by progressive neurodegeneration and a concomitant loss of synapses and cognitive abilities. Recently, we have proposed that an alteration of neuronal membrane lipid microdomains increases neuronal resistance toward amyloid-β stress in cultured neurons and protects from neurodegeneration in a mouse model of AD. Lipid microdomains are highly enriched in a specific subclass of glycosphingolipids, termed gangliosides. The enzyme glucosylceramide synthase (GCS) catalyzes the rate-limiting step in the biosynthesis of these gangliosides. The present work now demonstrates that genetic GCS deletion in subsets of adult forebrain neurons significantly improves the spatial memory and counteracts the loss of dendritic spines in the hippocampal dentate gyrus of 5x familial AD mice (5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP mice), when compared to 5xFAD//Ugcgf/f littermates (5xFAD mice). Aberrantly activated glial cells and their expression of pro-inflammatory cytokines have emerged as the major culprits for synaptic loss in AD. Typically, astrocytic activation is accompanied by a thickening of astrocytic processes, which impairs astrocytic support for neuronal synapses. In contrast to 5xFAD mice, 5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP display a less pronounced thickening of astrocytic processes and a lower expression of tumor necrosis factor-α and interleukin 1-α in the hippocampus. Thus, this work further emphasizes that GCS inhibition may constitute a potential therapeutic target against AD

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Neuronal Expression of Glucosylceramide Synthase in Central Nervous System Regulates Body Weight and Energy Homeostasis

Abstract Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis

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

Image_4_Deletion of Specific Sphingolipids in Distinct Neurons Improves Spatial Memory in a Mouse Model of Alzheimer’s Disease.TIF

<p>Alzheimer’s disease (AD) is characterized by progressive neurodegeneration and a concomitant loss of synapses and cognitive abilities. Recently, we have proposed that an alteration of neuronal membrane lipid microdomains increases neuronal resistance toward amyloid-β stress in cultured neurons and protects from neurodegeneration in a mouse model of AD. Lipid microdomains are highly enriched in a specific subclass of glycosphingolipids, termed gangliosides. The enzyme glucosylceramide synthase (GCS) catalyzes the rate-limiting step in the biosynthesis of these gangliosides. The present work now demonstrates that genetic GCS deletion in subsets of adult forebrain neurons significantly improves the spatial memory and counteracts the loss of dendritic spines in the hippocampal dentate gyrus of 5x familial AD mice (5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP mice), when compared to 5xFAD//Ugcgf/f littermates (5xFAD mice). Aberrantly activated glial cells and their expression of pro-inflammatory cytokines have emerged as the major culprits for synaptic loss in AD. Typically, astrocytic activation is accompanied by a thickening of astrocytic processes, which impairs astrocytic support for neuronal synapses. In contrast to 5xFAD mice, 5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP display a less pronounced thickening of astrocytic processes and a lower expression of tumor necrosis factor-α and interleukin 1-α in the hippocampus. Thus, this work further emphasizes that GCS inhibition may constitute a potential therapeutic target against AD.</p

Image_2_Deletion of Specific Sphingolipids in Distinct Neurons Improves Spatial Memory in a Mouse Model of Alzheimer’s Disease.TIF

<p>Alzheimer’s disease (AD) is characterized by progressive neurodegeneration and a concomitant loss of synapses and cognitive abilities. Recently, we have proposed that an alteration of neuronal membrane lipid microdomains increases neuronal resistance toward amyloid-β stress in cultured neurons and protects from neurodegeneration in a mouse model of AD. Lipid microdomains are highly enriched in a specific subclass of glycosphingolipids, termed gangliosides. The enzyme glucosylceramide synthase (GCS) catalyzes the rate-limiting step in the biosynthesis of these gangliosides. The present work now demonstrates that genetic GCS deletion in subsets of adult forebrain neurons significantly improves the spatial memory and counteracts the loss of dendritic spines in the hippocampal dentate gyrus of 5x familial AD mice (5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP mice), when compared to 5xFAD//Ugcgf/f littermates (5xFAD mice). Aberrantly activated glial cells and their expression of pro-inflammatory cytokines have emerged as the major culprits for synaptic loss in AD. Typically, astrocytic activation is accompanied by a thickening of astrocytic processes, which impairs astrocytic support for neuronal synapses. In contrast to 5xFAD mice, 5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP display a less pronounced thickening of astrocytic processes and a lower expression of tumor necrosis factor-α and interleukin 1-α in the hippocampus. Thus, this work further emphasizes that GCS inhibition may constitute a potential therapeutic target against AD.</p

Image_7_Deletion of Specific Sphingolipids in Distinct Neurons Improves Spatial Memory in a Mouse Model of Alzheimer’s Disease.TIF

<p>Alzheimer’s disease (AD) is characterized by progressive neurodegeneration and a concomitant loss of synapses and cognitive abilities. Recently, we have proposed that an alteration of neuronal membrane lipid microdomains increases neuronal resistance toward amyloid-β stress in cultured neurons and protects from neurodegeneration in a mouse model of AD. Lipid microdomains are highly enriched in a specific subclass of glycosphingolipids, termed gangliosides. The enzyme glucosylceramide synthase (GCS) catalyzes the rate-limiting step in the biosynthesis of these gangliosides. The present work now demonstrates that genetic GCS deletion in subsets of adult forebrain neurons significantly improves the spatial memory and counteracts the loss of dendritic spines in the hippocampal dentate gyrus of 5x familial AD mice (5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP mice), when compared to 5xFAD//Ugcgf/f littermates (5xFAD mice). Aberrantly activated glial cells and their expression of pro-inflammatory cytokines have emerged as the major culprits for synaptic loss in AD. Typically, astrocytic activation is accompanied by a thickening of astrocytic processes, which impairs astrocytic support for neuronal synapses. In contrast to 5xFAD mice, 5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP display a less pronounced thickening of astrocytic processes and a lower expression of tumor necrosis factor-α and interleukin 1-α in the hippocampus. Thus, this work further emphasizes that GCS inhibition may constitute a potential therapeutic target against AD.</p

Image_5_Deletion of Specific Sphingolipids in Distinct Neurons Improves Spatial Memory in a Mouse Model of Alzheimer’s Disease.TIF

<p>Alzheimer’s disease (AD) is characterized by progressive neurodegeneration and a concomitant loss of synapses and cognitive abilities. Recently, we have proposed that an alteration of neuronal membrane lipid microdomains increases neuronal resistance toward amyloid-β stress in cultured neurons and protects from neurodegeneration in a mouse model of AD. Lipid microdomains are highly enriched in a specific subclass of glycosphingolipids, termed gangliosides. The enzyme glucosylceramide synthase (GCS) catalyzes the rate-limiting step in the biosynthesis of these gangliosides. The present work now demonstrates that genetic GCS deletion in subsets of adult forebrain neurons significantly improves the spatial memory and counteracts the loss of dendritic spines in the hippocampal dentate gyrus of 5x familial AD mice (5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP mice), when compared to 5xFAD//Ugcgf/f littermates (5xFAD mice). Aberrantly activated glial cells and their expression of pro-inflammatory cytokines have emerged as the major culprits for synaptic loss in AD. Typically, astrocytic activation is accompanied by a thickening of astrocytic processes, which impairs astrocytic support for neuronal synapses. In contrast to 5xFAD mice, 5xFAD//Ugcgf/f//Thy1-CreERT2//EYFP display a less pronounced thickening of astrocytic processes and a lower expression of tumor necrosis factor-α and interleukin 1-α in the hippocampus. Thus, this work further emphasizes that GCS inhibition may constitute a potential therapeutic target against AD.</p