Location: Produce Safety and Microbiology ResearchTitle: Cellular prion protein mediates a-synuclein uptake, localization and toxicity in vitro and in vivo
|THOM, TOBIAS - Gottingen University|
|SCHMITZ, MATTHIAS - Gottingen University|
|FISCHER, ANNA-LISA - Gottingen University|
|CORREIA, ANGELA - Gottingen University|
|CORREIA, SUSANA - Gottingen University|
|LLORENS, FRANC - Gottingen University|
|PIQUE, ANNA-VILLAR - Gottingen University|
|MOBIUS, WIEBKE - Gottingen University|
|DOMINGUES, RENATO - Gottingen University|
|ZAFAR, SAIMA - Gottingen University|
|STOOP, ERIK - Adx Neurosciences|
|Silva, Christopher - Chris|
|FISCHER, ANDRE - Gottingen University|
|OUTEIRO, TIAGO - Gottingen University|
|ZERR, INGA - Gottingen University|
Submitted to: Movement Disorders
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/4/2021
Publication Date: 8/27/2021
Citation: Thom, T., Schmitz, M., Fischer, A., Correia, A., Correia, S., Llorens, F., Pique, A., Mobius, W., Domingues, R., Zafar, S., Stoop, E., Silva, C.J., Fischer, A., Outeiro, T.F., Zerr, I. 2021. Cellular prion protein mediates a-synuclein uptake, localization and toxicity in vitro and in vivo. Movement Disorders. Available:https://movementdisorders.onlinelibrary.wiley.com/doi/10.1002/mds.28774. https://doi.org/10.1002/mds.28774.
Interpretive Summary: Mammals make a highly conserved normal cellular prion protein (PrPC) whose biological role in brain tissue is only partially understood. PrPC is made within a cell, is transported to the cell’s surface, performs its role on the cell’s surface and is then internalized into the cell. Recent studies indicate that misfolded proteins can bind to PrPC, which allows those proteins to enter the cell, accumulate, and eventually exert a toxic effect. Examples of misfolded proteins include prions (the infectious shape of PrPC = PrPSc), and amyloid beta. We wanted to determine if misfolded alpha-synuclein could bind PrPC to facilitate its internalization, accumulation, and consequent pathology. We bred two transgenic mouse models that display different alpha-synuclein-related diseases, but are unable to make PrPC, developed cell models that produce excessive amounts of PrPC, and used microscopic imagery to visualize the proximity of alpha-synuclein and PrPC. Surface plasmon resonance (SPR) was used to measure the binding of PrPC to alpha-synuclein. Mass spectrometry was used to identify other proteins that were associated with PrPC and alpha-synuclein. The behavioral deficits (memory, anxiety, locomotion, etc.) and reduced lifespan traits were reversed in the transgenic mice unable to produce PrPC. When the cell models were examined by microscopy, we found that PrPC colocalized with and facilitated alpha-synuclein internalization. Our SPR-based analysis showed significant binding between PrPC and alpha-synuclein. Mass spectrometry-based analysis revealed that clathrin was a common PrPC/alpha-synuclein binding partner. Clathrin is a protein that creates structures, clathrin-coated pits, that facilitate internalization of other proteins from a cell’s surface. When we disrupted the clathrin-coated vesicles, alpha-synuclein internalization was significantly decreased. Thus, misfolded alpha-synuclein can bind to PrPC and, by using clathrin-based internalization, accumulate within a cell to cause disease. Disrupting the binding of PrPC and alpha-synuclein may help prevent disease.
Technical Abstract: Background: The cellular prion protein (PrPC ) is a membrane-bound, multifunctional protein mainly expressed in neuronal tissues. Recent studies indicate that the native trafficking of PrPC can be misused to internalize misfolded amyloid beta and a-synuclein (aSyn) oligomers. Objectives: We define PrPC 's role in internalizing misfolded aSyn in a-synucleinopathies and identify further involved proteins. Methods: We performed comprehensive behavioral studies on four transgenic mouse models (ThySyn and ThySynPrP00, TgM83 and TgMPrP00) at different ages. We developed PrPC -(over)-expressing cell models (cell line and primary cortical neurons), used confocal laser microscopy to perform colocalization studies, applied mass spectrometry to identify interactomes, and determined disassociation constants using surface plasmon resonance (SPR) spectroscopy. Results: Behavioral deficits (memory, anxiety, locomotion, etc.), reduced lifespans, and higher oligomeric aSyn levels were observed in PrPC -expressing mice (ThySyn and TgM83), but not in homologous Prnp ablated mice (ThySynPrP00 and TgMPrP00). PrPC colocalized with and facilitated aSyn (oligomeric and monomeric) internalization in our cell-based models. Glimepiride treatment of PrPC -overexpressing cells reduced aSyn internalization in a dose-dependent manner. SPR analysis showed that the binding affinity of PrPC to monomeric aSyn was lower than to oligomeric aSyn. Mass spectrometry-based proteomic studies identified clathrin in the immunoprecipitates of PrPC and aSyn. SPR was used to show that clathrin binds to recombinant PrP, but not aSyn. Experimental disruption of clathrin-coated vesicles significantly decreased aSyn internalization. Conclusion: PrPC 's native trafficking can be misused to internalize misfolded aSyn through a clathrin-based mechanism, which may facilitate the spreading of pathological aSyn. Disruption of aSyn-PrPC binding is, therefore, an appealing therapeutic target in a-synucleinopathies.