And then there was light: Combining photocaging of a TLR2 ligand with optogenetic tools to study brain immune responses

Abstract

The nervous system is a complex neural network consisting of the central and peripheral nervous system. The central nervous system (CNS) plays an important role since it receives and interprets information enabling neural communication. In recent years, there has been growing interest in providing insights into interactions between the CNS and the immune system. The cross-talk between the CNS and the immune system has been shown to play a crucial role in neural activity and development of neurodegenerative diseases. Microglial cells, also known as brain macrophages, are the first and main form of immune response in the CNS. They express toll-like receptors (TLRs) that are specialized in recognising pathogens. Once activated, TLRs initiate signalling cascades resulting in the activation of proinflammatory pathways. TLR2 is highly expressed on the surface of microglial cells and is thought to be one of the most studied TLRs regarding neurodegenerative diseases. There is a growing body of evidence suggesting the role of TLR2 activation in the pathophysiology of multiple sclerosis (MS), however the exact mechanism still remains elusive. Multiple sclerosis is a neuro-autoimmune disease caused by immune cells infiltrating the CNS and damaging the neurons. We believe that a better understanding of the pathophysiology of MS would contribute to the development of more effective treatment options. In this study, we propose to combine photocaging of a known TLR2 ligand, P2K4, with optogenetic tools to gain more understanding of the mechanisms underlying the pathophysiology of MS. Photocaging involves introducing a photoactivatable protecting group that can be cleaved off upon illumination with a certain wavelength. In this study, we chose a boron-dipyrromethene (BODIPY) group due to its exceptional photochemical properties. BODIPY-based photoprotecting groups can be cleaved off using red light which enables deep tissue penetration without inducing cellular toxicity. Optogenetic approaches utilize light to control activity of genetically modified populations of cells. In our study, we will make use of a CX3CR1creER/+: R26LSL-ReaChR/+ transgenic mouse model. This mouse strain has been genetically modified to express red light-sensitive protein, channelrhodopsin, which will enable us to study microglial activation in complex neuronal circuits using red light. First, the feasibility of photorelease of P2K4 and its ability to induce TLR2-mediated inflammatory responses will be tested in several in vitro assays in a BV-2 microglial cell line. Next, photoactivation of TLR2 will be investigated in a C57BL/6J mouse model. Lastly, optogenetic tools will be used to study the effect of microglial activation on the development of MS phenotype in mice. To our knowledge, this is the first study employing a PPG cleavable with visible light in an animal model and the first time when optogenetic approaches are used to study the role of activated microglia in the development of MS. Combining the results from both approaches will enable to elucidate the exact role of TLR2 activation in the pathogenesis of MS.