Skip to main content

Function and malfunction of the prion protein

Periodic Reporting for period 2 - PRION2020 (Function and malfunction of the prion protein)

Reporting period: 2017-05-01 to 2018-10-31

Transmissible spongiform encephalopathies (TSE) are caused by the ordered aggregation of PrPC into prions consisting of PrPSc. Similar pathogenetic principles operate in Alzheimer’s and Parkinson’s disease, and a growing list of further diseases whose prevalence is steadily rising. Familial TSE are invariably associated with PrPC mutations, and the dearth of genetic modifiers has hampered our understanding of prion diseases.

Therefore, the first objective utilizes a cell-based high-throughput quantitative prion replication assay (developed during my previous ERC instalment) for genome-wide unbiased screens employing new genetics tools (CRISPR, siRNA li-braries, next-gen sequencing) to identify modifiers of prion uptake, replication, and secretion.

The second objective aims at clarifying the basis of prion neurotoxicity and will be developed along two alleys: (a) we will uncover the molecular basis of spongiosis (the neuronal vacuolation charac-teristic of prion diseases), which we suspect to be a main driver of pathology, and (b) we will per-form CRISPR-based synthetic lethality screens to identify genes that become essential to prion-infected cell lines (which do not experience prion toxicity) and may not be expressed by neurons.

The third objective is to understand the function of PrPC in cellular physiology, and focuses on our evidence that (a) PrPC interacts with an orphan G-protein coupled receptor to maintain peripheral myelin integrity and (b) that PrPC may trigger cell death in response to ER stressors.

While certain pathways of degeneration will undoubtedly be specific to prion infections, I expect that some targets will prove common to a variety of protein aggregation diseases including Alzheimer’s and Parkinson’s disease, and may perhaps translate into novel diagnostics and therapeutics. Hence the proposed project may not only open new perspectives in prion biology but also yield insights applicable to much more common diseases.
Objective 1: What controls the generation of prion infectivity
In this first period, we have managed to optimize and establish the whole genome wide siRNA screening to identify the genes that modulate the expression of cellular prion protein (PrPC), which we believe will be a game changer in prion research. Using the data obtained from such screens, we hope to uncover novel physiological pathways associated with PrPC and furthermore some of these genes could become new therapeutic targets in prion infection.

Objective 2: How do prions damage the brain
Our understanding of the biogenesis of vacuoles in prion infection, which is critical for neurotoxicity is also further enriched. Using various cellular and biochemical assays we have also managed to decipher the set of events that lead to the generation of vacuoles and the downstream events once the vacuoles are generated. We believe that mechanistic details of vacuolation will provide us with novel therapeutic targets to deal with toxicity. We are also in the process of optimizing the set up for the synthetic lethality screens using lentiviral CRISPR libraries targeting the whole genome.

Objective 3: What is the role of PrPC in cellular physiology
Finally, we also managed to validate the authentic phenotypes and more specifically the electrophysiological phenotypes associated with prion protein ablation using the newly generated ZH3 (co-isogenic prion protein ablated mice). Furthermore, we have also managed to elucidate the mechanistic details of the authentic electrophysiological phenotypes. We have also uncovered the intricate details of how the N terminus of prion protein (also called flexible tail; FT) is involved in maintenance of myelin in peripheral nerves by identifying the receptor to which FT binds on Schwann cells and initiates promyelination signaling. A manuscript about the role of mGluR5 in the electrophysiological phenotype of prion protein ablated mice is currently under preparation.

The work on Objective 3 has directly resulted in a number of publications:
• Küffer, Alexander; Lakkaraju, Asvin K. K; Mogha, Amit; Petersen, Sarah C; Airich, Kristina; Doucerain, Cédric; Marpakwar, Rajlakshmi; Bakirci, Pamela; Senatore, Assunta; Monnard, Arnaud; Schiavi, Carmen; Nuvolone, Mario; Grosshans, Bianka; Hornemann, Simone; Bassilana, Frederic; Monk, Kelly R; Aguzzi, Adriano (2016). The prion protein is an agonistic ligand of the G protein-coupled receptor Adgrg6. Nature, 536(7617):464-468.
• Nuvolone, Mario; Hermann, Mario; Sorce, Silvia; Russo, Giancarlo; Tiberi, Cinzia; Schwarz, Petra; Minikel, Eric; Sanoudou, Despina; Pelczar, Pawel; Aguzzi, Adriano (2016). Strictly co-isogenic C57BL/6J-Prnp−/−mice: A rigorous resource for prion science. Journal of Experimental Medicine, 213(3):313-327.
• Nuvolone, Mario; Sorce, Silvia; Paolucci, Marta; Aguzzi, Adriano (2017). Extended characterization of the novel co-isogenic C57BL/6J Prnp−/− mouse line. Amyloid, 24(Suppl 1):36-37.
Overall, our interdisciplinary research strategy spanning from screening platforms to biochemistry to mouse genetics has helped us to gain new insights and critical understanding about the functioning of prion protein in health and disease.
Objective 1: What controls the generation of prion infectivity
The ability to perform a whole genome siRNA screen has provided us with a platform which can be now exploited to identify mechanistic aspects the prion protein functioning in a cell. A detailed bioinformatic analysis will be performed on the screen hits to identify the pathways that lead to the modulation of the prion protein. Once the analysis is completed, a more targeted approach using CRISPR, biochemical assays and mouse genetics will be performed on individual genes to elucidate the mechanistic details behind the modulation of the prion protein.

Once robust conditions for the screen are set up with appropriate cell lines we plan to proceed with the interrogation of prion infected cells using the whole genome siRNA library.

Objective 2: How do prions damage the brain
We are currently in the process of performing the rescue experiments using the analogs of the phospholipid PI(3,5)P2 by treating prion infected cells with it or directly injecting the analog into mice. In an in vivo approach we plan to use osmotic minipumps to deliver a water-soluble version of PI(3,5)P2 to the brains of prion-infected mice . We will monitor the survival and evaluate the neuropathology of brain. Vacuoles will be quantified morphometrically and subjected to statistical analyses. Simultaneously we are also using a TRPML1 analog which is also supposed to rescue the vacuolation induced by PIKfyve depletion.

We have managed to set up the pipeline for the CRSIPR screen to be performed on CAD5 cells. In the second period of the project, the entire mouse genome will be queried for the genes that modulate the expression of PrPC. The expression of PrPC will be analyzed using a FRET based assay as done for the siRNA screen. With the experience gained in setting up the screen for synthetic lethality we are now hopeful of identifying genes that become essential for cell survival upon prion infections.

Objective 3: What is the role of PrPC in cellular physiology
Currently mechanistic details of how mGluR5 activity modulated by PrPC are under investigation.