MAss Imaging Analyzer (MAIA): A tool to visualize lipids and small molecules in tissues

Halima Hannah Schede

Institute of Bioengineering, Laboratory of Lipid Cell Biology, École Polytechnique Fédéral de Lausanne, Switzerland

MALDI mass spectrometry imaging (MALDI-MSI) is a technique that enables the spatial visualization of molecular species in an untargeted manner. MALDI-MSI is both flexible and powerful as it can be applied to a large variety of samples while quantifying the abundance of thousands of molecules in parallel, thereby also enabling the visualization of lipid species in tissues and cells. Although many advancements have been made over the years to improve the quality of images extracted by MALDI imaging techniques, methods have yet to be developed that allow for the comparison between acquisitions in a robust and reliable way. Here we present a tool which we refer to as the MAss Imaging Analyzer (MAIA) that permits this analysis by identifying molecular compounds and their mass shift distributions in individual sections of interest, and making sections comparable to one another. The latter is achieved via the matching of compounds and a rescaling of intensity values such that molecules can be properly quantified across sections.

Impact of sphingolipid profile on yeast gel domains and membrane compartments

Andreia Bento-Oliveira, Filipa C. Santos, Joaquim T. Marquês, Pedro. M.R. Paulo, Thomas Korte, Andreas Herrmann, H. Susana Marinho, Rodrigo F.M. de Almeida

Centro de Química Estrutural, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal

Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal

Department of Biology, Molecular Biophysics, IRI Life Sciences, Humboldt-Universität zu Berlin, 10115 Berlin, Germany

There are two major compartments in the plasma membrane of yeast Saccharomyces cerevisiae- membrane compartment containing the arginine permease Can1p (MCC), and membrane compartment containing the H+ ATPase Pma1p (MCP). In yeast plasma membrane, sterol-rich domains (SRDs) are distinct from sphingolipid-enriched domains (SLEDs), which are ergosterol-depleted, and biophysically unique, as they are highly rigid gel domains. The interplay between these domains and yeast membrane compartments is still unclear.

To tackle this question, we evaluated how the hydroxylation pattern or polar headgroup of complex sphingolipids impacts membrane biophysical properties, particularly SLEDs and SRDs, and Pma1p and Can1p distribution and microenvironment. To achieve this, S. cerevisiae cells, wild-type and two mutants, one lacking the main yeast complex sphingolipid M(IP)2C (ipt1D) and the other with sphingolipids lacking acyl chain 2-hydroxylation (scs7D) were compared.

Tighter packing of SLEDs, with a tendency for lower abundance, was found in both mutants when compared to the wt. However, no significant alterations could be perceived in ergosterol-enriched domains. Concomitantly, the distribution of Pma1p along the plasma membrane was considerably more heterogeneous in the mutant cells, while Can1p did not exhibit noticeable alterations. Moreover, the microenvironment surrounding Can1p was not affected by changing the sphingolipid profile, while the opposite seems to occur for Pma1p.

Since MCP and MCC have been described as spatially separated, the results obtained in this work support an independent compartmentalization of sphingolipid-enriched and sterol-enriched domains, and that these regions could be lipid domain counterparts of the MCP and the MCC, respectively.

Ceramides as drivers of kidney injury and mitochondrial dysfunction

Nicholson, R.J., Stuart, D., Li, Y., Maschek, J.A., Ramkumar, N., Holland, W.L., Summers, S.A.

Department of Nutrition and Integrative Physiology, University of Utah, USA

Kidney disease is projected to be the 5th leading cause of death in 2040. Perturbations in lipid metabolism and mitochondrial function contribute to kidney injury and progression of chronic disease endpoints, such as kidney fibrosis and functional decline. Sphingolipids such as ceramides are lipotoxic drivers of metabolic reprogramming and disease in the heart, liver, adipose, and pancreatic islet. Yet, few studies have investigated mechanisms of ceramides driving kidney pathologies. Renal ceramides are elevated in pre-clinical models of kidney fibrosis. Additionally, altered expression of ceramide metabolizing genes in human kidney tubules correlate with kidney fibrosis and eGFR decline. Genetic whole-body ceramide-lowering protects mice from acute kidney injury caused by renal ischemia and reperfusion. Furthermore, induction of ceramides in cultured proximal tubule cells impairs mitochondrial respiration. These data reveal novel mechanisms driving ceramide-dependent renal injury and dysfunction and suggest an exciting therapeutic method for combating kidney disease.

Modulation of sphingolipid signalling by herpesviruses regulates virus particle secretion

Tomasz H. Benedyk, Colin M. Crump, Stephen C. Graham

Department of Pathology, University of Cambridge, UK

Herpes simplex virus protein UL21 is conserved across α-herpesviruses and contributes to virus replication and cell-to-cell spread. Immunoprecipitation and quantitative mass spectrometry experiments performed in our laboratory have identified two cellular binding partners of pUL21: the catalytic subunit of protein phosphatase PP1 and the ceramide transfer protein CERT. PP1 is a ubiquitous highly-active cellular phosphatase, the substrate specificity of which is conferred by binding to regulatory subunits. CERT, when dephosphorylated, shuttles newly synthesized ceramide from the endoplasmic reticulum to the trans-Golgi network – the site of ceramide conversion to sphingomyelin. Using in vitro biochemical experiments, stable expression of pUL21 in cultured cells, and infection with wild-type HSV-1 or viruses carrying pUL21 mutants, we showed that pUL21 is a novel viral PP1-regulatory subunit that targets CERT for dephosphorylation, hence activation. We further established a pulse-chase assay to monitor the dynamic changes in sphingomyelin turnover using a clickable analogue of sphingosine for lipid labelling, and HPTLC for lipid quantitation by measuring the fluorescent signal. Using recombinant virus encoding a pUL21 variant unable to bind CERT, we verified pUL21-mediated upregulation of sphingomyelin synthesis in cells infected with HSV-1. Subsequent characterization of the mutant virus revealed a defect in virus secretion resulting from CERT-dependent dysregulation of protein kinase D which is known for its role in trafficking of HSV-1 virions to the cell periphery. Taken together, our work describes how

pUL21 modulates host-cell sphingolipid metabolism and signalling in order to coordinate the intracellular trafficking of the mature HSV-1 virions.

Lysosome-targeted multi-functional lipid probes reveal the lysosomal cholesterol transporters NPC1 and LIMP-2/SCARB2 as sphingosine transporters

Janathan Altuzar, Judith Notbohm, Frank Stein, Per Haberkant, Saskia Heybrock, Jutta Worsch, Paul Saftig, Doris Höglinger

Heidelberg University Biochemistry Center, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany European Molecular Biology Laboratory, Heidelberg, Germany

Institute of Biochemistry, Christian-Albrechts-Universität Kiel, Kiel, Germany

Lipid homeostasis is maintained by an intracellular network of enzymes and regulated by organelle-to-organelle communication. Within this network, the lysosome plays a central role as host for degradation, recycling and trafficking of biomolecules such as proteins, nucleic acids and lipids. Lysosomal dysfunction is associated with a variety of severe diseases, which are often associated with sphingolipid accumulation. However, lysosomal lipid egress is poorly understood due to the lack of functional tools to investigate lipids on a single-organelle and single lipid species level. To overcome this knowledge gap, we synthesised novel lysosome-targeted multifunctional sphingosine (Lyso-pacSph) and cholesterol (Lyso-pacChol) probes that allow the study of their subcellular actions. Due to their chemical modifications, we can address outstanding questions regarding post-lysosomal sphingolipid trafficking, metabolism and protein-lipid interactions. Chemoproteomic analysis revealed known cholesterol transporters NPC1 and LIMP-2/SCARB2 as sphingosine interactors. Following their post-lysosomal metabolic fate by TLC, we saw that absence of NPC1 and LIMP-2/SCARB2 impaired post-lysosomal metabolism, consistent with a potential sphingosine transport role of both proteins. Furthermore, in visualisations of timeresolved lipid localisation by confocal microscopy, NPC1-/- cells as well as pharmacological inhibition of NPC1 in WT cells displayed severe sphingosine storage inside lysosomes. Finally, we recreated an NPC1-like phenotype by releasing an excess of non-functionalized sphingosine and cholesterol and monitored kinetics of lipid export, which revealed that high sphingosine levels impaired cholesterol export, but not vice versa. This would support the idea that sphingosine storage in NPC1-/- cells is not a secondary effect of cholesterol accumulation, but that high levels of sphingosine lead to an NPC1-like defect transport.

Involvement of hypothalamic de novo ceramide synthesis in resistin induced neuronal inflammation, insulin resistance and glucose intolerance.

Jeanne Guitton, Mohammed Taouis, Yacir Benomar, Hervé Le Stunff.

Paris-Saclay Institute of Neurosciences, CNRS UMR 9197, Paris-Saclay University, France

During obesity, the adipokine resistin, like saturated fatty acids, lead to an impairment of glucose homeostasis control by the hypothalamus, a risk factor for type 2 diabetes (T2D). We investigate the involvement of hypothalamic de novo ceramide synthesis in resistin-induced neuronal inflammation and insulin resistance which lead, to glucose intolerance. Using the mHypoA mouse hypothalamic cell line, we analyzed the impact of resistin overexposure on expression levels of enzymes driving ceramide biosynthesis. Intracellular ceramide contents were quantified by lipidomic analysis. Myriocin, a pharmacological inhibitor was used to evaluate de novo ceramide synthesis involvement in resistin-induced neuronal inflammation and defect of insulin signaling. In C57BL6J mice we evaluated the impact of resistin intracerebroventricular (ICV) infusion on hypothalamic expression of enzymes involved in ceramide biosynthesis. We studied the impact of serine palmitoyl-transferase 1 (SPT1) hypothalamic invalidation by adenoviral shRNA strategy on neuronal inflammation and glucose intolerance induced by resistin ICV infusion. In mHypoA cells, we show that resistin treatment increases ceramide contents and expression levels of enzymes driving de novo ceramide synthesis. Resistin overexposure induces inflammation and inhibits insulin signaling in a de novo ceramide synthesis-dependent manner. In mice, resistin ICV infusion upregulates hypothalamic gene expression of enzymes driving de novo ceramide biosynthesis. In vivo invalidation of hypothalamic SPT1 counteracts resistin-induced inflammation and prevents glucose intolerance. These findings reveal de novo ceramide synthesis as a new regulatory pathway of neuronal inflammation and insulin resistance that drive resistin-induced glucose intolerance. This pathway may constitute a breakthrough to overcome obesity and T2D occurrence.

Sphingolipid expansion microscopy

Tobias C. Kunz, Ralph Götz, Julian Fink, Franziska Solger, Jan Schlegel, Jürgen Seibel, Vera Kozjak-Pavlovic, Thomas Rudel & Markus Sauer

Julius-Maximilians University Würzburg, Department of Microbiology, Germany

Superresolution microscopy enables to bypass the diffraction limit of fluorescence light microscopy of around 200-250nm. However, such approaches enabling a resolution <100 nm require expensive setups as well as expert knowledge and are therefore limited to highly specialized laboratories. Recently, Boyden and colleagues introduced an alternative tool, the so-called expansion microscopy (ExM). ExM enables superresolution microscopy on a conventional confocal setup by embedding the sample into a swellable hydrogel which gets isotropically expanded. Since its introduction in 2015, ExM developed rapidly with various different protocols for 4x, 10x or even 20x expansion of proteins and RNA in cells, clinical specimens and tissues. As part of my PhD, we demonstrated the applicability of ExM to study host-pathogen interactions of bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Since the importance of sphingolipids during infection of various pathogens is well established, we introduced an approach to link functionalized lipids into the hydrogel for expansion, which we termed sphingolipid expansion microscopy, and were therefore the first to enable lipid expansion. Our novel tool enabled the nanoscale visualization of ω-N3-sphingosine in the membrane of Neisseria gonorrhoeae and α-NH2-ω-N3-C6-ceramide in the membrane of Chlamydia trachomatis and Simkania negevensis. The efficient accumulation of our functionalized lipids enables in combination with tenfold expansion to study interactions between proteins and plasma membrane, organelles and bacteria. In addition, we could show the incorporation of α-NH2-ω-N3-C6-ceramide in the inner and outer membrane of individual chlamydial particles and determine their distance to 27.6 ± 7.7 nm.

References:

Kunz TC, Ralph G, Sauer M, Rudel T; Detection of Chlamydia developmental forms and secreted effectors by expansion microscopy, Front Cell Infect Microbiol, 2019, 9:276

Ralph G, Kunz TC, Fink J, Solger F, Schlegel J, Seibel J, Kozjak-Pavlovic V, Rudel T, Sauer M; Nanoscale imaging of cellular and bacterial membranes by sphingolipid expansion microscopy, Nat commun, 2020, 11:6173

Solger F, Kunz TC, Fink J, Paprotka K, Pfister P, Hagen F, Schumacher F, Kleuser B, Seibel J, Rudel T; A Role of sphingosine in the intracellular survival of Neisseria gonorrhoeae, Front Cell Infect Microbiol, 2020, 10:215

Kunz TC, Kozjak-Pavlovic V; Diverse facets of sphingolipid involvement in bacterial infections, Front Cell Dev Biol, 2019, 7:203

Kunz TC, Ruehling M, Moldovan A, Paprotka K, Kozjak-Pavlovic V, Rudel T, Fraunholz M; The expandables: Cracking the staphylococcal cell wall for expansion microscopy, Front Cell Infect Microbiol, in print

Roles of ceramide in allergic asthma exacerbation

Briana N. James, Clement Oyeniran, Jamie L. Sturgill, Jason Newton, Rebecca Martin, Erhard Bieberich, Cynthia Weigel, Melissa A. Maczis, Elisa N. D. Palladino PhD1, Joseph C. Lownik, John B. Trudeau, Joan M. Cook-Mills, Sally Wenzel, Sheldon Milstien, and Sarah Spiegel

Departments of Biochemistry and Molecular Biology and Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA, USA

Despite growing evidence supporting the involvement of sphingolipids in asthma, little is known about the mechanism by which increased ceramide contributes to allergic responses. The aim of our study was to investigate the role of ceramide in allergic asthma by utilizing mouse models that recapitulate key symptoms of human allergic asthma. Mice were sensitized and challenged intranasally with house dust mite or the fungal allergen Alternaria alternata to induce an allergic asthma response. Allergen challenge caused significant lung cell death, increased levels of reactive oxygen species, and neutrophil infiltration. Concomitantly, ceramide levels in lung and bronchoalveolar lavage were significantly increased. Moreover, specific ceramide species were increased in bronchoalveolar lavage fluid from patients with severe asthma and correlated with airway neutrophilia. Interestingly, suppression of lung ceramide levels protected against allergen-induced apoptosis, reactive oxygen species, and neutrophil infiltration. However, decreasing lung reactive oxygen species by dietary supplementation of antioxidant α-tocopherol did not affect levels of ceramide or apoptosis, suggesting that both are independent of oxidative stress. Our results suggest that ceramide elevation after allergen challenge contributes to apoptosis, reactive oxygen species generation, and neutrophilic infiltrate that characterize the severe asthmatic phenotype. Ceramide could be a biomarker to optimize diagnosis and to monitor and improve clinical outcomes in this disease.

Optical Control of Sphingosine-1-Phosphate Formation and Function

Johannes Morstein, Rose Z. Hill, Alexander J. E. Novak, Suihan Feng, Derek D. Norman, Prashant C. Donthamsetti, James A. Frank, Takeshi Harayama, Benjamin M. Williams, Abby L. Parrill, Gabor J. Tigyi, Howard Riezman, Ehud Y. Isacoff, Diana M. Bautista, Dirk Trauner

Department of Chemistry, New York University, New York, New York 10003, United States.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

Department of Biochemistry, University of Geneva, Geneva, Switzerland.

National Centre of Competence in Research (NCCR) in Chemical Biology, University of Geneva, Geneva, Switzerland.

Department of Physiology, College of Medicine, University of Tennessee Health Science Center (UTHSC), Memphis, Tennessee 39163.

Department of Chemistry and Center for Integrated Protein Science, Ludwig Maximilians University Munich, 81377 Munich, Germany.

Department of Chemistry, University of Memphis, Memphis, Tennessee 38152, United States.

Computational Research on Materials Institute, University of Memphis, Memphis, Tennessee 38152, United States.

Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, United States.

Sphingosine-1-phosphate (S1P) plays important roles as a signaling lipid in a variety of physiological and pathophysiological processes. S1P regulates angiogenesis, cell proliferation and migration, immunity, and pain. S1P signals via a family of G Protein Coupled Receptors (S1P1-5 receptors) as well as a number of intracellular proteins including HDAC, TRAF2, and PKC. Here we report on photoswitchable analogs of S1P and its precursor sphingosine, respectively termed PhotoS1P and PhotoSph. PhotoS1P enables optical control of S1P1-5 receptor activity in vitro, shown through its ability to rapidly and reversibly control S1P-sensitive currents and S1P-evoked increases in intracellular Ca2+ via engagement of S1P receptors in cultured cells. We evaluated PhotoS1P in vivo, where it reversibly controlled S1P Receptor 3-dependent pain hypersensitivity in mice via activation of nociceptive somatosensory neurons. The pain hypersensitivity induced by PhotoS1P is comparable to that induced by S1P. PhotoS1P is uniquely suited for the study of S1P biology in cultured cells and in vivo because it exhibits prolonged metabolic stability compared to rapidly metabolized S1P. Using lipidomic analysis, we constructed a comprehensive metabolic map of PhotoS1P and PhotoSph. The formation these photoswitchable lipids was found to be light-dependent, providing a novel tool to optically probe sphingolipid biology.

Sphingomyelin serves a critical role in the repair of damaged organelles

Johannes Patrick Niekamp, Joost Holthuis

Department of Chemistry, New York University, New York, New York 10003, United Molecular Cell Biology Division, University of Osnabrück, 49076 Osnabrück, Germany

Sphingomyelin (SM) displays a strict asymmetric distribution across cellular membranes, with the bulk localized to the exoplasmic leaflet of late secretory and endocytic organelles. Using an engineered SM-binding probe, we and others (Ellison et al., 2020, Curr Biol 30, 1-10) found that SM is readily exposed to the cytosolic surface of these organelles upon membrane damage inflicted by pathogenic bacteria, lysosomotropic drugs or a two-photon laser. Remarkably, cytosolic exposure of SM at sites of membrane damage precedes the recruitment of galectins, ESCRT proteins and other components of the membrane repair machinery, suggesting that a break in SM asymmetry may serve a fundamental role in the mechanism by which cells detect and repair damaged organelles. By monitoring the recovery of LysoTracker fluorescence in lysosomes transiently exposed to lysosomotropic drugs, we found that cells lacking SM displayed a significant defect in the repair of damaged lysosomal membranes in comparison to control cells. Moreover, SM-deficient cells showed an enhanced sensitivity to lysosomotropic drugs and a prolonged retention of ESCRT-III components on their damaged lysosomes. These phenotypes could be suppressed by restoring SM biosynthesis. Analogous to the role of phosphatidylserine displayed on the surface of damaged cells, we postulate that cytosolic SM serves as a key indicator of damaged organelles and actively participates in their repair. In this talk, I will present our latest findings regarding the mechanism by which SM contributes to the mending of damaged organelles.

Sphingosine-1-phoshate signaling in astrocytes is a critical modulator of stroke outcome

Matuskova H. , Matthes F., Petzold G. C. and Meissner A.

Department of Neurology, University Hospital Bonn, Germany

German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany

Department of Experimental Medical Sciences, Lund University, Sweden

Wallenberg Centre for Molecular Medicine, Lund University, Sweden

Stroke remains a leading cause of long-term disability worldwide. Due to its complexity, treatment options are sparse. The bioactive phospholipid sphingosine-1-phosphate (S1P) is involved in variety of physiological processes particularly, in vascular and immune cell responses. Moreover, altered S1P levels have been reported in several cardiovascular and inflammation-associated diseases, including stroke. As astrocytes play a critical role in the regulation of both vascular and immune responses in the injured brain, we sought to investigate astrocytic S1P signaling and its contribution to stroke. In a mouse model of transient middle cerebral artery occlusion (MCAo), we discovered significantly increased expression of one of S1P’s receptors (S1PR3) 24 hrs post-ischemia in the ipsilateral hemisphere. Vessel-parenchyma fractionation of brain tissue revealed the majority of S1PR3 protein associated to cerebral vessels. Astrocyte-specific RiboTag analysis confirmed an augmentation of ipsilateral S1PR3 expression 24 and 72 hrs post-stroke. This was further supported by colocalization of Gfap, a marker of reactive astrocytes, and S1PR3 in the ischemic hemisphere by RNA scope technique. Moreover, single administration of an S1PR3 antagonist 4 hrs after permanent MCAo revealed significant improvements of regional cerebral blood flow in the ipsilateral hemisphere 24 hrs that persisted after 72 hrs. Consequently, infarct size was markedly reduced in mice treated with S1PR3 antagonist. In conclusion, our findings point to an important involvement of the S1P/S1PR3 signaling axis during stroke, and a potential contribution of astrocytes. Modulating S1PR3-mediated vascular and inflammatory responses may emerge as viable target to improving stroke outcome.

Reciprocal regulation of cell cycle and sphingolipid metabolism

Gabriel Soares Matos, Mónica Montero Lomeli

Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro

Sphingolipids are central bioactive lipids that have important functions in cell division. However, the control sphingolipid metabolism occurs during cell cycle is not well understood. To answer this question, we used S. cerevisiae as a model. We first studied the gene expression profile of key enzymes involved in the ceramide synthesis pathway and found, by qRT-PCR experiments, that genes involved in the synthesis of long chain bases (LCBs) and ceramides are periodically expressed during the mitotic cell cycle, having a peak at G1/S. This transcription peak coincides the canonical targets of the SBF complex, which is formed by the transcription factor SWI4 and its regulator SWI6, and which play a crucial role in G1/S transition. The transcription of sphingolipid genes was decreased in SBF mutants furthermore, SBF mutants were sensitive to myriocin, which inhibits the first step of sphingolipid synthesis. In addition, HPLC- MS/MS data indicated that the swi4Δ mutant had decreased levels of dihydrosphingosine, phytosphingosine (PHS) and total ceramides. A decrease in the length of ceramide fatty acids and in the hydroxylation of its long chain bases was also observed. The reduced sphingolipid content in swi4Δ strain was mimicked in WT cells by myriocin treatment and resulted in cell cycle arrest at the G2/M phase, which was reversed by addition of PHS to the media. Our results, overall suggests that the SWI4 transcription factor is important to coordinate transcription and synthesis of sphingolipids, which may affect not just membrane architecture, but also the biological activity of ceramides which are reported to control a myriad of phenomena including cell cycle progression.

Association of Aβ with ceramide-enriched astrosomes mediates Aβ neurotoxicity

Ahmed Elsherbini, Haiyan Qin, Zhihui Zhu, Priyanka Tripathi, Simone M. Crivelli, and Erhard Bieberich

Department of Physiology, University of Kentucky, Lexington, KY

Amyloid beta (Aβ) is a pathologic hallmark of Alzheimer’s disease (AD), however, the mechanism of Aβ neurotoxicity is not fully understood. Exosomes associate with Aβ, but it is not clear how this association would affect Aβ neurotoxicity. We report that the sphingolipid ceramide mediates neurotoxicity of Aβ. We show that sera and brains from AD transgenic mouse model (5xFAD) and sera from AD patients, but not the WT or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. When taken up by Neuro2A cells and human iPS cell-derived neurons, these astrosomes are shuttled to mitochondria where they induce mitochondria clustering, evident by elevation of expression of the fission protein dynamin related protein1 (Drp1). Using proximity ligation assays (PLA), we show that Aβ associates with voltage dependent anion channel 1 (VDAC1), a key protein in mitochondria-mediated apoptosis. PLA signals colocalized with ceramide cotransported with Aβ by astrosomes. The interaction between Aβ and VDAC1 leads to caspase3 activation and subsequently apoptosis. This effect was mitigated by removal of the ceramide enriched exosomes from the exosomes pool. Interestingly, the novel ceramide analog N-oleoyl serinol (S18) prevented the aggregation of exosomes, and Aβ association with astrosomes, and reduced Aβ interaction with VDAC1. Our data suggests that association of Aβ with ceramide in astrosomes enhances Aβ interaction with VDAC1 and mediates Aβ neurotoxicity in AD, which can be prevented by novel ceramide analogs.