Endosome biogenesis in Niemann-Pick type C disease - ENDOGENESIS
Principal investigator
Niemann-Pick type C disease (NPC) is a fatal rare inherited lysosomal storage disorder that mainly affects children. It is caused by mutations in NPC1 or NPC2 gene leading to accumulation of free cholesterol and other lipids in late endosomes/lysosomes. The molecular details of NPC are still not fully understood and current treatments can only alleviate the symptoms.
Here, we will test the hypothesis that lipid dyshomeostasis in NPC alters biophysical properties of the membranes causing defective protein/lipid sorting and trafficking of endocytic vesicles. The goal of this project is to identify pathological changes in endosome biogenesis in NPC that contribute to the phenotype and could instruct us on key lipid/protein signals needed for this process.
- Firstly, using our recently developed fluorescent probes we will measure membrane tension in living NPC1/2-KO vs. wt HeLa cells by fluorescence-lifetime imaging microscopy (FLIM).
- Secondly, we will perform proteomics and lipidomics to identify key molecules of endosome biogenesis that are altered in NPC1/2-KO cells and are regulated by altered membrane tension.
- Thirdly, since NPC has a strong CNS component with profound neurodegeneration and neuroinflammation, we will translate the obtained results to NPC1/2-mouse neurons, microglia and astrocytes – the brain cells that are mainly affected in NPC.
- Lastly, we will validate the obtained findings in NPC1/2 patient's fibroblasts and in NPC1 human brain cells derived from CRISPR/Cas9 edited human induced pluripotent stem cells.
These studies will identify the biologically relevant lipids/proteins involved in deregulation of membrane tension and endosome biogenesis in NPC. These molecules could be further employed to design an early diagnosis and/or effective therapy against NPC. The knowledge gained through this project could have a broader impact since defects of endolysosomal pathway are shared by a number of other rare as well as more common neurodegenerative disorders.
Goal of the project
The goal of the project is to identify NPC1/2-dependent specific protein/lipid alterations that modulate membrane tension along the endocytic pathway and, thus, endosome biogenesis. In this proposal we will test the hypothesis that endosome biogenesis is altered in Niemann-Pick type C disease and that changes in membrane tension caused by specific lipid/protein changes are involved in this process.
- Specific aim 1. Characterize membrane tension in NPC: analyze membrane tension at the level of the plasma membrane, early/late endosomes and lysosomes in the cellular model of NPC disease.
- Specific aim 2. Identify key molecules of endosome biogenesis that are regulated by altered membrane tension in the cellular model of NPC.
- Specific aim 3. Analyze the results gained in NPC1/2-cell lines in different brain cells of the NPC mouse models.
- Specific aim 4. Validate the obtained findings in NPC patient's fibroblasts and in NPC human brain cells derived from CRISPR/Cas9 gene edited human iPSC cells.
Training
- Božana Blažević, Training course for people who work with experimental animals and animals for the production of biological preparations, Faculty of Veterinary Medicine, University of Zagreb, September 23 - 26, 2024.
- Lea Vidatić, Training in membrane tension analysis using flipper probes and FLIM, Department of Biochemistry, University of Geneva, 29. September – 5. October 2024.
Dissemination
- 8th Rijeka Forum on neurodegenerative diseases, Silva Katušić, invited lecture, title: Which comes first: neuroinflammation or neurodegeneration – the case of juvenile Alzheimer's diseases, 16.09.2024 - 17.09.2024, Rijeka, Croatia.
- 8th Rijeka Forum on neurodegenerative diseases, Lea Vidatić and Božana Blažević, poster presentation, title:: Neuroinflammation precedes neurodegeneration in a mouse model of Niemann-Pick type C disease, 16.09.2024 - 17.09.2024, Rijeka, Croatia.
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the Croatian Science Foundation.
Project team
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Lea Vidatić, , PhD
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Ana-Marija Bogner
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