The Role of 18kDa Translocator Protein (TSPO) in cellular bioenergetics and microglial activation

The brain contains cells of the immune system called "microglia". When nerve cells are damaged, microglia spring into action and become "activated". Microglia can become activated in different ways. For example, they can activate in a way designed to protect the nerve cells from damage, or they can activate in a way designed to kill an invading organism, like bacteria or viruses. Many researchers now believe that when microglia become activated in neurodegenerative disease (eg Alzheimer's disease, Parkinson's disease and even multiple sclerosis) they "choose the wrong" activation state. In other words, they become activated in a way that does not benefit the patient. In this state, not only do they fail to protect nerve cells, but they actually make the nerve cell damage worse. Therefore, many people are trying to develop drugs which change the way the microglia become activated, making them more likely to be activated in a way that supports and protects the nerve cells, rather than making the damage worse. This is important, because even though we don't fully understand the causes of these diseases, we might still be able to protecting nerve cells from damage by changing the way microglia activate. It so, this would be a great stride forward, because it might slow the underlying disease progression in diseases such as Alzheimer's disease or Parkinson's disease, and currently we do not have any medications which do this.

Laboratory and animal experiments have shown that drugs which bind a protein called Translocator Protein (or "TSPO") can cause a favourable change in activation state in microglia and protect nerve cells. However, we do not understand the way the drugs do this. The reason we do not understand how the drugs work is because we do not even know what the role of the protein is. It is important to understand this. If we can understand these details it will help us to turn these drugs into effective medicines. It will also help us identify other potential drug targets.

The aim of this project, therefore, is to understand the role of TSPO in microglia and to understand how drugs which bind TSPO change microglial activation.

This project will be entirely laboratory based. I will be carrying out this work in the Alavian laboratory at Imperial College London. I will be using microglial cells derived from stem cells from the laboratory of Professor Allen at Cardiff University. These cells offer great advantages compared to other microglial cells which are available. For example they are derived from humans, and therefore are more likely to reflect how human microglia function compared to animal cells.

Aims: Understand the mechanism by which TSPO ligands modulate microglial phenotype, and investigate effect of rs6971 polymorphism

Objectives
1. Establish IPSC derived microglia in my lab and characterise the cellular bioenergetic profile of these and primary human microglia
2. Characterise protein-protein interactions of microglial TSPO
3. Test the hypothesis that modulation of microglial immunophenotype by TSPO ligands is mediated by an effect on cellular bioenergetics.

Methods
Aims 1 and 3:

The respiratory chain complexes will be analyzed using Western blot and in vitro activity assays of the native and denatured whole mitochondrial protein samples. The complex enzymatic activity will be assessed in vitro. The activity of Complex-V will be assessed using the NADH or the luciferase assays. To determine the coupling of mitochondrial oxidation to phosphorylation, the leak currents will be measured by patch clamp electrophysiology on the mitochondrial inner membrane vesicles. On-organelle patch clamp recordings at holding voltages between -100 mV and +100 mV will be obtained. ROS will be measured with CellROX Green and Mitosox

Aim 2:

Myc and FLAG-tagged constructs for the wildtype and rs6971 mutant TSPO will be overexpressed in control and activated IPSC-derived microglia. Mitochondria will be isolated. TSPO will be immunoprecipitated and candidate proteins (already identified by the phylogenetic profiling) will be assessed by using LC-MS/MS


Scientific/Medical value of results
A treatment capable of protecting against neurodegeneration would have clear medical benefit

This work will foster new research, not only in neurodegenerative diseases, but also in aging, stroke, encephalitides and certain psychiatric diseases (eg schizophrenia), all characterised by increased TSPO expression

The primary purpose of the project is to better understand a potential novel therapeutic pathway for modulation of microglial phenotype and therefore a potential slowing of neurodegeneration. I will work closely with staff of Imperial Innovations to secure patent protection for therapeutic concepts where possible. With success of this project, I would look forward to subsequent new funding for experimental medicine proof of concept studies and ultimate development of a medicine. As highlighted in the application, there are no marketed modern drugs that primarily modulate the microglia for direct impact on this compartmentalized brain inflammation. Successful validation of TSPO as a target will immediately identify a broad range of chemical structures from which an optimised therapeutic candidate could be developed. While the chemical space is broadly covered by patents, chemical novelty is achievable, especially with exploitation of the human genetic variation in TSPO for screening and design, uses of which are covered broadly by a patent application that I have previously filed with Imperial (WO2012168697 A1).