Programme 4: Investigating the prion secretory pathway in neuronal cells
Aims We recently provided first experimental evidence on how prions are replicated in neuronal cells. Our results show that aggregates of disease-associated PrP (PrPd) are transported from perinuclear regions of prion-infected neuronal cells to the plasma membrane, where they trigger conversion of the cellular prion protein to nascent full-length PrPd (FL-PrPd). While this data provides important insight into prion replication, the secretory route of PrPd to the plasma membrane remains elusive. This important aspect of PrPd trafficking will be addressed in this PhD studentship proposal using a transcriptomics approach. We recently isolated rare clones of chronically infected cells that showed abundant PrPd deposits at perinuclear sites, but failed to produce FL-PrPd at the plasma membrane, suggesting that the transport of PrPd to the plasma membrane is compromised. Such cell clones thus provide a compelling model to identify critical genetic factors that facilitate the transport of PrPd to the plasma membrane. In this study we will use transcriptome analysis, gene perturbation by CRISPR-Cas9 and gene silencing as well as pathway analysis to identify the critical secretion machinery of prions. The pursuit of this project benefits from the broad expertise in genetic analysis, molecular cloning and cellular imaging of neuronal cells within Programme 4.
Methods In collaboration with Cambridge Genomic Services and Dr Holger Hummerich, we will identify differentially expressed genes between wild-type and secretion-compromised neuroblastoma cells and thoroughly characterise the underlying defects in protein secretory pathways. Using gene deletion or overexpression, we will modify the expression levels of gene candidates and monitor effects on seeded aggregation and secretion, respectively.
Rotation project In a three-month rotation project, the student will identify the most suitable candidate cell clones for transcriptome analysis. This includes, but is not restricted to determination of secretion rates of prions from a variety of wild-type and secretion-compromised neuronal cells and use of small inhibitory molecules to further confirm defects in protein secretion. Dissipation of the vesicular pH gradient with Bafilomycin A1, a specific inhibitor of the vesicular ATPase (V-ATPase), will provide a clue whether the pH regulation of exocytosis is compromised. In wild-type cells, Bafilomycin A1 led to a strong stimulation of prion secretion and seeded aggregation at the plasma membrane. The student will also learn techniques in molecular imaging.
PhD project The PhD project is structured into three phases. In the first phase, the panel of isogenic secretion-competent and -compromised cells will be expanded and fully characterised to document the observed cellular phenotypes. A minimum of six isogenic cell clones per phenotype will be isolated to provide sufficient power for the subsequent transcriptome analysis. RNA will be isolated and sent to Cambridge Genomic Services for cDNA hybridisation, data acquisition and normalisation. The second phase of the project will focus on array and pathway analysis in collaboration with Holger Hummerich. The goal here is to scrutinise the obtained gene expression signatures on the context of known exocytosis pathways and protein interaction links to infer potential hypotheses. In the third phase, we will validate critical gene candidates of the gene signature by gene deletion and overexpression, respectively. This study will be supported by our collaborator, Prof Sharon Tooze from the Francis Crick Institute.
References
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 (2014) Identification of a gene regulatory network associated with prion replication. EMBO J. 14,1527-47.