Protein trafficking and secretion in neurons

PhD project strategic theme: Understanding the rules of life

Protein secretion is an essential process in all living cells in order to maintain cellular homeostasis, cell-to-cell signalling, and extracellular matrix modifications (Lodish et al., 2012). It is widely accepted that there are two biochemically distinct types of protein secretion: regulated secretion and constitutive secretion. Regulated secretion occurs in a subset of specialised secretory cells, such as neurons, and refers to the highly controlled, on-demand release of specific proteins such as neurotransmitters or enzymes (Miller & Moore, 1990; Uzman, 2003). To date, regulated neuronal secretion has been extensively studied and its mechanisms are largely understood. In contrast, constitutive secretion takes place in all cell types and refers to the continuous, non-selective trafficking of soluble proteins to the plasma membrane. These proteins are exported from the cell continuously, without any mechanism of storage (Rivas & Moore, 1989; Miller & Moore, 1990). When these vesicles arrive at the cell surface, they immediately fuse with the plasma membrane and release their contents into the extracellular matrix (ECM) by exocytosis (Uzman, 2003). Comparatively, the proteins and mechanisms involved in constitutive neuronal secretion are more poorly characterised.

This project aims to further understanding of constitutive secretion and secretory routes in neurons. To do this, we will use an engineered induced pluripotent stem cell line (iPSCs) as a model system. These cells have been engineered to differentiate to i3 cortical neurons at the addition of doxycycline, in a fast and homogenous manner (Wang et al., 2017; Zhang et al., 2013). Using these cells, we aim to quantitively identify and analyse a set of proteins common to the neuronal secretome by adopting a click-chemistry approach. This strategy involves incorporating a non-canonical amino acid into nascently synthesised proteins during translation. The replacement amino acid is modified with an azido moiety that can be used to isolate all secreted proteins from the media through a "click" reaction between the azide and an alkyne or cyclooctynean. This strategy will enable us to draw comparisons between the secretomes of healthy and diseased neurons. Specific proteins-of-interest may be those that have altered secretomics in particular disease states, such as neuroserpin in Alzheimer's disease. Once we have identified proteins-of-interest by secretomics, we aim to establish a "Retention using Selective Hooks" system (RUSH) in neurons (Chen et al., 2017), in order to study the intracellular trafficking routes used to deliver these cargoes to the cell membrane for secretion. By combining molecular biology, biochemistry and super resolution imaging, we hope to decipher the molecular machinery of constitutive neuronal secretion, and how defects in this pathway can lead to inherited neurodevelopmental or neurodegenerative disorders.