Cellular Functions of the PrP

Lead Research Organisation: University College London

Abstract

Prion diseases are fatal progressive neurodegenerative disorders that, like Alzheimer’s and Huntington’s disease, involve accumulation of a misfolded protein. This protein, the prion protein, is normally expressed within most cells of the body but the highest expression is within the brain and in the immune system. It is predominantly found on the surface of cells attached to the plasma membrane via a carbohydrate containing fatty acid linker. The prion protein has been proposed to have many cellular functions but is has been found that mice that are null for this protein are viable, do not have any overt symptoms but are completely resistant to prion disease. Even though the cellular prion protein is a prerequisite for prion disease, it is not known which parts of this protein are required for prion propagation. We aim to investigate which regions of the prion protein are required for propagating prions. We also aim to develop cells which can be used to propagate human prions since there are currently no cell lines which can propagate human prions. We also aim set up a primary cell assay to demonstrate that prions can kill neurones and use it to isolate and characterise the toxic species in prion disease.

Technical Summary

According to the widely accepted protein-only hypothesis, prions comprise aggregated forms of misfolded host-encoded cellular prion protein (PrPC) generally referred to as PrPSc. PrPC is a glycosyl phosphatidylinositol (GPI)-anchored cell-surface glycoprotein, expressed in most cell types and tissues, but at the highest levels in the CNS and immune system. PrPC is highly conserved in mammals but a precise cellular function remains unclear although multiple functions have been proposed. However mice, in which the PrP gene has been knocked out, have no overt phenotype and a normal lifespan, but do not propagate prions and are completely resistant to prion disease, indicating that PrPC expression is absolutely required for prion propagation and prion-induced neurotoxicity. Like most GPI-anchored proteins, PrPC is predominantly found at the cell surface in lipid rafts which has led to the suggestion that it may be involved in signal transduction by acting as a cell-surface receptor. Since PrPC lacks a transmembrane domain for transmitting signals from the cell surface to the inside of the cell, much work has been done to identify binding partners that could transmit the signal from the cell surface to the cell interior. A number of putative binding partners have been identified but the biological significance of many of them is unclear since some are only present within the cytoplasm, whereas others are nuclear. Recent evidence has suggested that PrPC acts as a cell surface receptor for A?-oligomers, the molecular species that mediate toxicity in Alzheimer’s disease. PrPC is also widely expressed in the immune system where it has been suggested to play a role in homeostasis as well as in the immune synapse and signalling. We have undertaken a reverse genetic approach, utilising alanine mutagenesis, to investigate the regions of PrPC that are required for efficient prion propagation in PK1 cells, a derivative of N2a neuroblastoma cells. This has identified three regions within the unstructured N-terminus as critical for prion propagation: residues 23-25 (KKR, Charge Cluster 1), glutamine 41 and residues 90-111 (Charge Cluster 2). Our aim is now to identify the proteins that interact with these regions. We also aim to develop cells that can efficiently propagate human sCJD and vCJD prions to set up an automated bioassay for human prions that can be used as a therapeutic biomarker. We will also develop a primary neuronal cell assay for prion neurotoxicity that will be applied to isolate and characterise the putative toxic PrP species.

Publications

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Benilova I (2020) Highly infectious prions are not directly neurotoxic. in Proceedings of the National Academy of Sciences of the United States of America

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Jones E (2020) Genetic risk factors for Creutzfeldt-Jakob disease. in Neurobiology of disease

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Rayner MLD (2020) Quantifying regeneration in patients following peripheral nerve injury. in Journal of plastic, reconstructive & aesthetic surgery : JPRAS

 
Description Policy Research Programme
Amount £493,145 (GBP)
Funding ID PR-R17-0916-23004 
Organisation Department of Health (DH) 
Sector Public
Country United Kingdom
Start 05/2018 
End 03/2022
 
Description Immortalisation of IPSCs 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution The team leader is the secondary supervisor for the PhD student conducting this research and has a great contribution with his expertise in engineering cells and intellectual input into the forward direction of the project. Furthermore, the team leader provided the training for the team members and the collaborating PhD student and has provided the facilities and equipment for the student to be able to conduct this work effectively. The other team members have then trained and supervised the student within the labs and had intellectual input in the planning of the project.
Collaborator Contribution The partner has provided the cells and other key consumables for the expansion of these cells.
Impact There are currently no outputs or ouotcomes.
Start Year 2019
 
Description Therapies for nerve regeneration 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution As a part of this collaboration members of the team (Prof Jat and Dr Rayner) are involved in projects that are developing cell and drug therapies for peripheral nerve regeneration. The team members have a role in supervising and training students (PhD, masters and undergraduates) which involves providing expertise; particular specialised laboratory techniques and having intellectual input into their research projects. This role requires us to have regular meetings with the students and provide support and guidance for the progression of the research. Furthermore, Dr Rayner was a co-applicant for EPSRC IAA Discovery-to-use funding and has been a co-supervisor to the post-doc working on the project looking to develop biomaterials for a drug delivery platform.
Collaborator Contribution The partners in this collaboration have provided the funding for these projects to be taken forward through PhD doctoral schemes and the facilities for the students to conduct this research. The partner also acts as the primary supervisor for these students.
Impact The outputs from this collaboration to date is a pending patent application through UCLB and journal publications. The collaboration includes many disciplines such as tissue engineering, pharmaceutics, cell engineering and mathematical modelling.
Start Year 2019
 
Description Keystone Symposia, Keystone, Colorado, USA, 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Madeleine Reilly attended the Keystone Symposia, Keystone, Colorado, USA on Advances in neurodegenerative disease research and therapeutics and presented a poster on her PhD research. She has developed a multiparametric cell based imaging assay for prion induced neurotoxicity.
Year(s) Of Engagement Activity 2018
 
Description Big Questions in Neuroscience, Cell Symposia/ Society for Neuroscience 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented a poster at the satellite symposium on Big Questions in Neuroscience, Cell Symposia / SFN 2017 in November 2017, Washington, USA.
SFN is the annual meeting of the Society for Neuroscience- a very large meeting. This was a small satellite symposium. The aim was to promote discussion.
Year(s) Of Engagement Activity 2017
 
Description EMBL Course: Assay Development for Drug Discovery and Characterisation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This wa san EMBL Course in Heidelberg. It wa sattended by Iryna Benilova. The aim

The European Molecular Biology Laboratory and Sartorius training course was held in Heidelberg, Germany October 23-25th 2018. The programme involved lectures from both faculty and industry experts to share technologies and expertise on drug development. The course also explored the utilisation of cell-based technologies in the drug discovery process for small molecules, biologics and cell therapy. The program also provided hands-on experience of both the IncuCyte® S3 Live-Cell Analysis System and the iQue Screener PLUS platform to enable insight into cell-based experimental design and analysis.

The course was targeted at researchers working in the fields of pharmacology, chemical biology, biologicals, cell therapy, etc. who wish to learn more about the basics and challenges of high-throughput screening, assay development and lead profiling and characterisation.
Year(s) Of Engagement Activity 2018
 
Description National Prion Clinic Open Day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact The National Prion Clinic (NPC) is the national referral centre for prion disease and is part of the University College London Hospitals NHS Foundation Trust. It is funded by the NHS to provide diagnosis and care for patients with, or suspected to have, any form of human prion disease (inherited, iatrogenic, sporadic and variant CJD). The NPC is integrally linked with the Prion Unit.

The Open Day is an annual public engagement event in which its stakeholders - patients, patients' relatives, health and allied health professionals are invited to attend. The Open Day seeks to inform and update stakeholders on the Unit's prion research; focus group discussions are also held to interact with the stakeholders and gauge their views about our research. Laboratory tours are also offered.
Year(s) Of Engagement Activity 2017
 
Description Neurodegeneration activity for MRC Festival of Medical research 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We designed and organised a one day workshop/stall for the MRC Festival of Medical research. The activity educated the public about the brain and neurodegenerative diseases. Visitors experienced what it was like living with neurodegenerative diseases and the difficulty doing simple tasks through hands-on activities.
Year(s) Of Engagement Activity 2018
 
Description Poetry collaboration with Experimental words 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Poetry collaboration with Experimental Words. Performance art- poem about Prion disease

Scientists performed on stage with poets and actors delivering a poem about prion diseases.
Year(s) Of Engagement Activity 2017
 
Description Prion 2018 Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact "Prion 2018: Back to basics, understanding the biology of prions", was held in Santiago de Compostela, Spain. Focus of the meeting was on the biology of prions, without neglecting other very important aspects of human and animal prion disease. There was also a workshop on structural biology of prions and a round table on the structure of PrPSc. Aim was a deeper understanding of how prions in general, and PrPSc in particular, propagate, and what are the possible structural and molecular targets that can be exploited to find a therapy for prion diseases.
Year(s) Of Engagement Activity 2018
 
Description Queen Sqaure Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact The Queen Square symposium is an annual event organised by the Postgraduate students studying at the Queen Square UCL Institute of Neurology. All graduate students (PhD, MSc and MRes) students attend and present posters. The posters are judged by a panel of Senior Academic staff selected by the student organising committee. There are some short talks by the students. There is also an External speaker selected by the students.
Year(s) Of Engagement Activity 2017