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Function and malfunction of the prion protein

Periodic Reporting for period 4 - PRION2020 (Function and malfunction of the prion protein)

Reporting period: 2020-05-01 to 2020-10-31

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. Familial TSE are invariably associated with PrPC mutations, and the dearth of genetic modifiers has hampered our understanding of prion diseases.

Because these diseases affect primarily the elderly, their prevalence is steadily rising for demographic reasons, and no disease-modifying therapies exist. This bleak situation is compounded by a dearth of validated therapeutic targets, which stems from our incomplete understanding of the biochemical and cellular networks involved in pathogenesis.

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 characteristic 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?
We optimized 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. Currently, we are working on elucidating the mechanisms associated with the remainder of the hits in regulating PrPC expression. A manuscript detailing this work is in preparation.
The aim of the second part of Objective 1 is to identify genes involved in maintaining a productive prion infection and their shedding into extracellular milieu. We have identified 161 genes to specifically effect prions without altering the levels of PrPC. Validation experiments are carried out in other prion infected cell lines. This will be followed by mechanistic elucidation of pathways involved in generation and maintenance of prions from the obtained hits.

Objective 2: How do prions damage the brain?
We have characterized the mechanism associated with biogenesis of vacuoles in prion infection. A manuscript detailing this is currently being revised for submission.
The second part of Objective 2 was to set up a CRISPR platform and perform synthetic lethality screens to identify genes essential for prion induced toxicity. We have now completed amplification, quality control and lentivirus packaging of the CRISPR libraries. Cells used in Objective 1 were chosen for the purpose of the synthetic lethality screen. We estimate that the primary screen and validations will be completed by the first quarter of 2021.

Objective 3: What is the role of PrPC in cellular physiology?
We have validated the authentic phenotypes and more specifically the electrophysiological phenotypes associated with prion protein ablation using the newly generated ZH3 mice (co-isogenic prion protein ablated mice). Furthermore, we have elucidated the mechanistic details of the authentic electrophysiological phenotypes. We have also uncovered the 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. We are currently performing structural studies of the receptor and ligand interactions as a continuation of the project.

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 of the functioning of the prion protein in health and disease.
Objective 1
We have established a robust screening platform which not only enabled us to address the projects proposed in the ERC grant, but is now also being used for COVID19 research. We have obtained a list of genes affecting the biosynthesis of cellular prion protein and genes affecting the maintenance of prion infection. Using gene editing technologies such as CRISPR, biochemical assays and mouse genetics, we expect to elucidate the mechanistic details behind the modulation of the prion protein and prion infection by the individual hits obtained in the screens before the end of the grant period.

Objective 2
Part 1: We have achieved a significant understanding of the generation of vacuoles in prion infection and how vacuoles drive toxicity. As a continuation of the project, we are performing a genetic screen using CRISPR to identify genes that mediate PIKfyve induced toxicity in prion infection to identify therapeutic targets against neurodegeneration in prion disease.

Part 2: We have established a screening platform to perform genetic perturbations using CRISPR. We expect to complete the synthetic lethality screen to identify genes essential for cell viability upon prion infections before the end of the year and characterize the hits before the end of the grant period.
Objective 3
Objective 2
Objective 1