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MRC Prion Unit
From fundamental research to prevention and cure
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Cell biology: Cellular functions of the prion protein

Prion Research

Prion diseases such as Creutzfeldt-Jakob diseases (CJD) and bovine spongiform encephalopathy are severe, devastating intractable diseases.  They are caused by infectious agents found in many animal species including sheep, cows, and deer, as well as humans.  The infectious agent is composed of aggregates of an abnormally form of the cellular prion protein (PrPC).  This is unique as normally a pathogen carries genetic material in the form of DNA or RNA for replicating infectivity, whereas in prion disease, the disease associated prion protein acts as a template to convert PrPC to the disease state.  

PrPC is a highly conserved cell-surface glycoprotein, expressed in most cell types but without a precise unitary cellular function, although many functions have been proposed.  The wide variety of activities and functions ascribed to PrPC suggest that it does not act exclusively in a single pathway, but instead function as a dynamic scaffold for the assembly of a variety of multicomponent signalling complexes while it moves between the cell surface and the endocytic compartment.  One very important function of PrPC is that it is absolutely required for prion propagation: mice devoid of PrPC are resistant to prion infection.

Cell lines are an invaluable tool to study complex cell processes and diseases including aspects of prion biology.

However, the use of cell culture systems to study prion propagation is limited to a few cell lines susceptible to either mouse-adapted or sheep prion strains, with none yet described able to stably propagate human prions, despite worldwide efforts.  Currently human prions are propagated in mice that lack mouse PrPC but have been engineered to express human PrPC, however propagating human prions in vitro remains a challenge.  The development of PK1 cells by colleagues within the MRC Prion Unit, by extensive serial sub-cloning of mouse neuroblastoma N2a cells, that can efficiently propagate Rocky Mountain Laboratory (RML) mouse prions has permitted the development of a cell culture based bioassay for mouse prions and revolutionised their study. 

We aim to develop cells that can propagate human prions, both variant (v) and sporadic (s) CJD prions.  We use a “silencing-reconstitution strategy” that recapitulates the approach used to make mouse models of human and other prion diseases to derive a susceptible cell line capable of supporting a robust infection.  This will enable us to develop robust, highly sensitive, automated cell culture assays for measuring human prion infectivity in vitro.

PJfig 1

Figure 1 Silencing reconstitution strategy.  PK1 cells, a derivative of mouse neuroblastoma cells that are highly susceptible to infection by RML mouse prions, become resistant to infection when the endogenous prion protein is silenced as in PK1-KD cells. However they regain full susceptibility to infection upon reconstitution with the wild type mouse prion protein (moPrPWT). 

Recent advances within the Unit and elsewhere suggest that prions themselves are not directly toxic, but rather their propagation involves production of a toxic species uncoupled from infectious prions.  In fact, clinical disease is only observed after prion infectivity peaks followed by a time lag which is inversely proportional to the level of PrPC expression.  To identify the toxic species in prion disease, we have developed a multi-parametric high content imaging assay of prion-induced neurotoxic phenotypes such as neuronal death, neurite fragmentation and dendritic atrophy.  Our aim is to examine toxicity through the course of a prion infection and ultimately to isolate the toxic species and investigate the mechanism of action.

PJ fig2
Figure 2 A multi-parametric high content imaging assay of dendritic spine loss induced by RML-infected brain homogenate.  Mouse embryonic neuronal cultures are exposed to RML-infected brain homogenates and resulting changes in neuronal phenotype determined by high-through put microscopy using the Opera Phenix microscope.

Cancer Research: Dissecting the molecular mechanisms for the infinite proliferative potential of cancer cells

The aim of this research is to identify the underlying molecular basis for the finite proliferative life span of normal somatic cells.  Normal cells undergo a finite number of divisions and then cease dividing and undergo cellular senescence whereas cancer cells are able to proliferate indefinitely.

Cellular senescence is a stable cell cycle arrest that can be triggered in response to a variety of intrinsic and extrinsic stimuli.  It can compromise tissue repair and regeneration and contribute to tissue and organismal ageing due to depletion of stem/progenitor cell compartments.  It can also lead to removal of defective and potentially cancerous cells from the proliferating pool thereby preventing tumour development.  The acquisition of an infinite proliferative potential is one of the key events that are required for cancer and one of the least understood since the underlying pathways that controls cellular senescence are poorly understood. 

To facilitate the study of senescence, we have developed conditionally immortalised human mammary fibroblasts that are stringently temperature sensitive.  These cells are immortal if grown at 34°C but upon shift up 38°C; they undergo senescence upon activation of the p53-p21 and p16-pRB tumour suppressor pathways.

Although these two tumour suppressor pathways are clearly implicated in senescence, the critical downstream targets are unknown.  As transcription factors (TFs) are key to the establishment and maintenance of specific cell fates, our aim is to identify TFs that act downstream of the p53-p21 and p16-pRB pathways and determine if they have a causative role in senescence and cancer.

Peer reviewed articles:
2016

Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous WT1 Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15.
Brandt A, Löhers K, Beier M, Leube B, de Torres C, Mora J, Arora P, Jat PS, Royer-Pokora B.PLoS One. 2016 May 23;11(5):e0155561. doi: 10.1371/journal.pone.0155561. eCollection 2016. PMID: 27213811

Novel highly specific anti-periostin antibodies uncover the functional importance of the fascilin 1-1 domain and highlight preferential expression of periostin in aggressive breast cancer.
Field S, Uyttenhove C, Stroobant V, Cheou P, Donckers D, Coutelier JP, Simpson PT, Cummings MC, Saunus JM, Reid LE, Kutasovic JR, McNicol AM, Kim BR, Kim JH, Lakhani SR, Neville AM, Van Snick J, Jat PS. Int J Cancer. 2016 Apr 15;138(8):1959-70. doi: 10.1002/ijc.29946. Epub 2015 Dec 21. PMID:  26619948

2015

A systematic investigation of production of synthetic prions from recombinant prion protein.
Schmidt C, Fizet J, Properzi F, Batchelor M, Sandberg MK, Edgeworth JA, Afran L, Ho S, Badhan A, Klier S, Linehan JM, Brandner S, Hosszu LL, Tattum MH, Jat P, Clarke AR, Klöhn PC, Wadsworth JD, Jackson GS, Collinge J. Open Biol. 2015 Dec;5(12):150165. doi: 10.1098/rsob.150165. PMID:  26631378

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance.
Saunus JM, Quinn MC, Patch AM, Pearson JV, Bailey PJ, Nones K, McCart Reed AE, Miller D, Wilson PJ, Al-Ejeh F, Mariasegaram M, Lau Q, Withers T, Jeffree RL, Reid LE, Da Silva L, Matsika A, Niland CM, Cummings MC, Bruxner TJ, Christ AN, Harliwong I, Idrisoglu S, Manning S, Nourse C, Nourbakhsh E, Wani S, Anderson MJ, Fink JL, Holmes O, Kazakoff S, Leonard C, Newell F, Taylor D, Waddell N, Wood S, Xu Q, Kassahn KS, Narayanan V, Taib NA, Teo SH, Chow YP, kConFab, Jat PS, Brandner S, Flanagan AM, Khanna KK, Chenevix-Trench G, Grimmond SM, Simpson PT, Waddell N, Lakhani SR. J Pathol. 2015 Nov;237(3):363-78. doi: 10.1002/path.4583. Epub 2015 Aug 19. PMID: 26172396 

Tumour suppressors and cellular senescence.
Chan AS, Mowla SN, Arora P, Jat PS. IUBMB Life. 2014 Dec;66(12):812-22. doi: 10.1002/iub.1335. Epub 2014 Dec 29. Review. PMID: 25557529

Identification of clinical target areas in the brainstem of prion-infected mice.
Mirabile I, Jat PS, Brandner S, Collinge J. Neuropathol Appl Neurobiol. 2015 Aug;41(5):613-30. doi: 10.1111/nan.12189. Epub 2015 Apr 23. PMID: 25311251

Identification of small proline-rich repeat protein 3 as a novel atheroprotective factor that promotes adaptive Akt signaling in vascular smooth muscle cells.
Segedy AK, Pyle AL, Li B, Zhang Y, Babaev VR, Jat P, Fazio S, Atkinson JB, Linton MF, Young PP. Arterioscler Thromb Vasc Biol. 2014 Dec;34(12):2527-36. doi: 10.1161/ATVBAHA.114.303644. Epub 2014 Oct 2.PMID: 25278290

2014

Regulation of p53 and Rb links the alternative NF-κB pathway to EZH2 expression and cell senescence.
Iannetti A, Ledoux AC, Tudhope SJ, Sellier H, Zhao B, Mowla S, Moore A, Hummerich H, Gewurz BE, Cockell SJ, Jat PS, Willmore E, Perkins ND. PLoS Genet. 2014 Sep 25;10(9):e1004642. doi: 10.1371/journal.pgen.1004642. eCollection 2014 Sep. PMID: 25255445

Cellular senescence and aging: the role of B-MYB.
Mowla SN, Lam EW, Jat PS. Aging Cell. 2014 Oct;13(5):773-9. doi: 10.1111/acel.12242. Epub 2014 Jul 1. Review. PMID: 24981831

Identification of a gene regulatory network associated with prion replication.
Marbiah MM, Harvey A, West BT, Louzolo A, Banerjee P, Alden J, Grigoriadis A, Hummerich H, Kan HM, Cai Y, Bloom GS, Jat P, Collinge J, Klöhn PC. EMBO J. 2014 Jul 17;33(14):1527-47. doi: 10.15252/embj.201387150. Epub 2014 May 19. PMID: 24843046

In vitro screen of prion disease susceptibility genes using the scrapie cell assay.
Brown CA, Schmidt C, Poulter M, Hummerich H, Klöhn PC, Jat P, Mead S, Collinge J, Lloyd SE. Hum Mol Genet. 2014 Oct 1;23(19):5102-8. doi: 10.1093/hmg/ddu233. Epub 2014 May 15. PMID: 24833721

The miR-106b~25 cluster promotes bypass of doxorubicin-induced senescence and increase in motility and invasion by targeting the E-cadherin transcriptional activator EP300.
Zhou Y, Hu Y, Yang M, Jat P, Li K, Lombardo Y, Xiong D, Coombes RC, Raguz S, Yagüe E.Cell Death Differ. 2014 Mar;21(3):462-74. doi: 10.1038/cdd.2013.167. Epub 2013 Nov 22. PMID: 24270410

2013

Friend or foe: emerging role of nuclear factor kappa-light-chain-enhancer of activated B cells in cell senescence.
Mowla SN, Perkins ND, Jat PS. Onco Targets Ther. 2013 Sep 4;6:1221-9. doi: 10.2147/OTT.S36160. Review. PMID: 24043947

Effects of CT-Xp gene knock down in melanoma cell lines.
Caballero OL, Cohen T, Gurung S, Chua R, Lee P, Chen YT, Jat P, Simpson AJ. Oncotarget. 2013 Apr;4(4):531-41. PMID: 23625514

The expression of podocyte-specific proteins in parietal epithelial cells is regulated by protein degradation.
Guhr SS, Sachs M, Wegner A, Becker JU, Meyer TN, Kietzmann L, Schlossarek S, Carrier L, Braig M, Jat PS, Stahl RA, Meyer-Schwesinger C. Kidney Int. 2013 Sep;84(3):532-44. doi: 10.1038/ki.2013.115. Epub 2013 Apr 24. PMID: 23615505

Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients.
Gorvin CM, Wilmer MJ, Piret SE, Harding B, van den Heuvel LP, Wrong O, Jat PS, Lippiat JD, Levtchenko EN, Thakker RV Proc Natl Acad Sci U S A. 2013 Apr 23;110(17):7014-9. doi: 10.1073/pnas.1302063110. Epub 2013 Apr 9. PMID:  23572577

Deciphering the role of nuclear factor-κB in cellular senescence.
Vaughan S, Jat PS. Aging (Albany NY). 2011 Oct;3(10):913-9. PMID:  21990145

An RNA interference screen for identifying downstream effectors of the p53 and pRB tumour suppressor pathways involved in senescence.
Rovillain E, Mansfield L, Lord CJ, Ashworth A, Jat PS. BMC Genomics. 2011 Jul 8;12:355. doi: 10.1186/1471-2164-12-355. PMID:  21740549

2011

Rapid cell-surface prion protein conversion revealed using a novel cell system.
Goold R, Rabbanian S, Sutton L, Andre R, Arora P, Moonga J, Clarke AR, Schiavo G, Jat P, Collinge J, Tabrizi SJ. Nat Commun. 2011;2:281. doi: 10.1038/ncomms1282. PMID:  21505437

Activation of nuclear factor-kappa B signalling promotes cellular senescence.
Rovillain E, Mansfield L, Caetano C, Alvarez-Fernandez M, Caballero OL, Medema RH, Hummerich H, Jat PS. Oncogene. 2011 May 19;30(20):2356-66. doi: 10.1038/onc.2010.611. Epub 2011 Jan 17. PMID:  21242976

2010

Proteome profiling of immortalization-to-senescence transition of human breast epithelial cells identified MAP2K3 as a senescence-promoting protein which is downregulated in human breast cancer.
Jia M, Souchelnytskyi N, Hellman U, O'Hare M, Jat PS, Souchelnytskyi S. Proteomics Clin Appl. 2010 Nov;4(10-11):816-28. doi: 10.1002/prca.201000006. PMID:  21137025

Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes.
Sakairi T, Abe Y, Jat PS, Kopp JB. Am J Physiol Renal Physiol. 2010 Oct;299(4):F802-9. doi: 10.1152/ajprenal.00205.2010. Epub 2010 Jul 28. PMID:  20668098

Dissecting the transcriptional networks underlying breast cancer: NR4A1 reduces the migration of normal and breast cancer cell lines.
Alexopoulou AN, Leao M, Caballero OL, Da Silva L, Reid L, Lakhani SR, Simpson AJ, Marshall JF, Neville AM, Jat PS. Breast Cancer Res. 2010;12(4):R51. doi: 10.1186/bcr2610. Epub 2010 Jul 19. PMID:  20642837

Conditionally immortalized human podocyte cell lines established from urine.
Sakairi T, Abe Y, Kajiyama H, Bartlett LD, Howard LV, Jat PS, Kopp JB. Am J Physiol Renal Physiol. 2010 Mar;298(3):F557-67. doi: 10.1152/ajprenal.00509.2009. Epub 2009 Dec 2. PMID:  19955187