Lysosomes are crucial intersections of intracellular metabolite flux as they convert dietary and autophagy-derived macromolecules into nutrients. Combinations of genetic risk factors, environmental hazards, and metabolite deposition in the lysosomal lumen lead to the accumulation of dysfunctional lysosomes underlying rare genetic childhood dementias (e.g. Batten’s disease), neurodegeneration, aging, and metabolic syndromes. The most prevalent metabolites whose depositions lead to lysosome dysfunction and neurodegeneration are lipids and their catabolites. However, the molecular mechanisms regulating lipid flux through lysosomes are incompletely understood. In the Ebner lab we address fundamental knowledge gaps in lysosomal lipid logistics and explore their role in neurodegeneration. To do so, we use advanced imaging techniques, omics approaches, drug screening, and biochemistry to uncover lysosomal lipid regulation pathways and test their implications in neurodegeneration-relevant cellular models, specifically in microglia cells. Hyperactivation of microglia cells and the resulting chronic neuroinflammation emerged as a hallmark of many neurodegenerative disorders and lysosomal lipid pathways are crucial for activation of proinflammatory pathways.

Specifically, we explore molecular mechanisms in:

– membrane repair
– membrane biogenesis
– lipid and metabolite export
– and how all that affects pro-inflammatory pathways in microglia

The ultimate goal is to harness the findings and modulate the discovered pathways to expand and restore lysosomal health and alleviate disease phenotypes in childhood dementia and neurodegeneration.

Lysosomal lipid flux largely takes place via non-vesicular lipid transfer at membrane contact sites (MCS) with other organelles. Lysosomes entertain particularly extensive MCS with the endoplasmic reticulum (ER). These MCS are organized by phosphoinositides (PIPs) on lysosomes, lipid transfer proteins, tethering proteins, and anchoring proteins at the ER. Several proteins localizing to and organizing these sites are risk factors for neurodegenerative diseases. Examples are the ER resident MCS organizing hub VAPB, the lipid transfer proteins VPS13C and NPC1, or the tethering protein Protrudin. Interestingly, the directionality of lipid transfer between lysosomes and the ER is often not clear. However, available evidence suggests that most lipid transfer proteins at ER-lysosome MCS transfer lipids from the ER to lysosomes. Lipid transfer from the ER via VPS13D and ATG2 was recently shown to be instrumental for the biogenesis of the peroxisomal and autophagosomal membranes, respectively. However, whether lipid transfer is implicated in the biogenesis of lysosomal membranes is unknown and the lipidomic aspects of lysosomal membrane biogenesis are largely unexplored. We will fill this knowledge gap using innovative strategies for biochemical characterization of newformed and maturing lysosomes, lysosomal systems biology, and targeted screens. A cornerstone of this project will be the generation of a systems biology lysosomal coming-of-age map. The results will uncover the molecular pathways that establish the lysosomal limiting membrane and inform strategies to boost lysosome biogenesis to counteract pathologic phenotypes in lysosome-driven diseases.

Lipids and membraneous material are delivered to lysosomes via several pathways: 1. Via endocytosis and endosomal membrane trafficking to the lysosomal lumen, 2. via ESCRT-dependent intraluminal vesicle formation, 3. via autophagy-mediated delivery of mitochondria, ER, or lipid droplets including the autophagosomal membrane itself, and 4. via lipid transfer proteins that line the extensive MCS which lysosomes entertain mainly with the ER but also with other organelles. Lipid catabolites eventually need to be exported from lysosomes, i.e. across the lysosomal limiting membrane, for further processing or storage. Lysosomal membrane proteins known to export lipids and their catabolites from lysosomes include NPC1, CLN3, SPNS1, and SCARB2, all which underly neurological disorders when dysfunctional. However, which proteins function downstream of these, whether and how lipid export from lysosomes is subject to regulation, and which pathways and protein factors partake in regulation is largely unexplored. We will use innovative tools and screening approaches to uncover the mechanisms regulating lysosomal lipid export and discover tools to manipulate this disease-laden process.

A frequent consequence of lysosome dysfunction is lysosomal membrane permeabilization, the rupture of the limiting membrane and leaking of luminal content into the cytoplasm can lead to inflammation, cell death, and neurodegenerative pathology. Several cell autonomous pathways have evolved to cope with damaged lysosomal membranes, however, how they are coordinated in space and time, how they are impacted by lysosomal disease genes, and how they exactly drive inflammatory responses is not well understood. We will address these questions using microglia as model system, imaging and biochemical approaches. In addition we will use repair-associated disease phenotypes such as in juvenile Batten disease (NCL3) as a platform for high throughput drug repurposing screens.

Ebner, M., Fröhlich, F., Haucke, V. 2025. Mechanisms and functions of lysosomal lipid homeostasis. Cell Chemical Biology. https://doi.org/10.1016/j.chembiol.2025.02.003

Rawlins, L.,… Ebner, M., Baple, EL., Crosby, AH. 2024. Elucidating the clinical and genetic spectrum of inositol polyphosphate phosphatase INPP4A-related neurodevelopmental disorder. Genetics in Medicine.  https://doi.org/10.1016/j.gim.2024.101278

Lolicato, F., Nickel, W., Haucke, V., Ebner, M. 2024 Phosphoinositide switches in cell physiology – from molecular mechanisms to disease. J Biol Chem. doi: https://doi.org/10.1016/j.jbc.2024.105757

Schmied, C.E., Ebner, M. 2024. OrgaMapper: A robust and easy-to-use workflow for analyzing organelle positioning. BMC biology. https://doi.org/10.1186/s12915-024-02015-8

Ebner, M., … Haucke, V. 2023. Nutrient-regulated control of lysosome function by signaling lipid conversion. Cell. doi: https://doi.org/10.1016/j.cell.2023.09.027

Schmied, C., …, Ebner, M., …, Tischer, C., Jambor H.K., 2023. Community-developed checklists for publishing images and image analysis. 2023. Nature Methods. https://doi.org/10.1038/s41592-023-01987-9

Malek, M., Wawrzyniak, A.M., Ebner, M., Puchkov, D., Haucke, V. 2022. Inositol triphosphate-triggered calcium release from the endoplasmic reticulum induces lysosome biogenesis via TFEB/TFE3. J Biol Chem. 2022 Mar;298(3):101740. doi: 10.1016/j.jbc.2022.101740

Ebner, M., Koch, P.A., Haucke, V. 2019. Phosphoinositides in the control of lysosome function and homeostasis. Biochem Soc Trans. 47 (4) 1173-1185. doi: 10.1042/BST20190158

Ebner, M., Haucke, V. 2018. Mechanical signals regulate TORC2 activity. Nat Cell Biol. 20(9):994-995. doi: 10.1038/s41556-018-0181-5

Ebner, M., Sinkovics, B., Szczygiel, M., Ribeiro, D. W., Yudushkin, I. 2017. Localization of
mTORC2 activity inside cells. J Cell Biol. 216 (2) 343–353. DOI: 10.1083/jcb.201610060

Ebner, M., Lucic, I., Leonard, T. A., Yudushkin, I. 2017. PI(3,4,5)P3 Engagement Restricts Akt Activity to Cellular Membranes. Mol Cell 65(3): 416-431. DOI: 10.1016/j.molcel.2016.12.028

Zaffagnini, G., Savova, A., Danieli, A., Romanov, J., Tremel, S., Ebner, M., Peterbauer, T., Sztacho, M., Trapannone, R., Tarafder, AK., Sachse, C., Martens, S. 2018. p62 filaments and ubiquitin phase separate to nucleate cargos for autophagy. EMBO J. 1;37(5). pii: e98308. doi: 10.15252/embj.201798308.

Zaffagnini, G., Savova, A., Danieli, A., Romanov, J., Tremel, S., Ebner, M., Peterbauer, T., Sztacho, M., Trapannone, R., Tarafder, AK., Sachse, C., Martens, S. 2018. Phasing out the bad-How SQSTM1/p62 sequesters ubiquitinated proteins for degradation by autophagy. Autophagy. 14(7):1280-1282. doi: 10.1080/15548627.2018

Dominic Winter (Uni Essen)

Sabina Tahirovic (DZNE Munich)

Volker Haucke (FMP Berlin)

Mario Pende (Institute Necker Enfantes Malades Paris)

Michael Ebner PhD

Institut für Molekulare Biochemie
E-Mail: Michael.Ebner@i-med.ac.at