Project Outline

mTOR signalling is an important metabolic signalling pathway that is implicated in aging and many diseases like cancer and neurodevelopmental diseases. The mTOR pathway regulates many important physiological pathways; including inflammation and immune response. Recently, Dr. Chan’s lab has found that lysine, an essential amino acid, is an important regulator of the mTOR pathway. Much is not known about the physiological role of lysine; but several studies varying lysine in animal’s diet changes the inflammatory and immune response, possibly linked to the mTOR pathway (Han et al, 2018; Huang et al, 2021).

Dr. Chan’s lab studies a rare neurodevelopmental disease with epilepsy called tuberous sclerosis complex (TSC) where the genetic mutation causes hyperactivity in mTOR. We have pilot data showing that lysine metabolism is increased in TSC and also, signatures of inflammation in the TSC brain. Specifically, we observed an increase in CCL5 expression, a receptor that has recently been linked in microglia-neuronal communication (Festa et al, 2023). This suggests that microglial immune response could drive the neuronal activity in the brain and potentially be linked to the epilepsy in TSC. Dr. Chan’s pilot data has also demonstrated that accumulation of lysine metabolite in the TSC brain lead to neuronal hyperactivity; however, it is unclear if this increase in lysine metabolism is driven by or drives the microglial or immune response in the brain.

Thus, this project aims to understand the link between immune response, lysine metabolism, and mTOR signalling. While our ultimate goal is to understand the physiology of this immune-metabolic signalling, results from this project may have relevance towards understanding the disease TSC. The cellular models of TSC in Dr. Chan’s lab; including the induced pluripotent stem cell models; will be invaluable tools to understand the influence of mTOR activity on these fundamental processes.

This project has two main objectives:

1. To understand bidirectional relationship between lysine metabolism on mTOR signaling in immune cells of macrophage and microglial lineage

2. To characterize the effect of immune signalling via CCL5 on neuronal activity and lysine metabolism in the brain

This project uses in vitro cell culture models developed in Dr. Chan and Dr. Ross’s lab. The student will work on primary cell culture models (mouse neurons and glia) as well as immortalized cell culture models (macrophage lines). Pharmacological tools will be used to manipulate lysine metabolism, mTOR signalling, and/or immune response (CCL5 signalling) in the cells. To validate some of these findings, we will also use genetic modification tools like CRISPR-Cas9 or siRNA to understand the effect of genetic knockdown/knockout on the immunometabolic signalling. Finally, the last stage of this project will validate the fundamental immunometabolic signalling we characterise on a human induced pluripotent stem cell (iPSC) model to understand human neuron-microglia immunometabolic signalling.

Techniques

• Cell culture (primary and immortalized cell culture, iPSC cell culture)
• Molecular biology (qPCR, Western Blot, etc)
• Genetic modification (CRISPR-Cas9 or siRNA)
• Flow cytometry