Microscopy-based siRNA screen of microglia to identify neuroprotective drug targets

Abstract

Neuroinflammation is a fundamental process contributing to the death of neurons in neurodegenerative diseases, such as Parkinson ́s (PD) or Alzheimer ́s disease (AD). During this process, activated microglia secrete cytotoxic substances which lead to neuronal death (1). Therefore, we are looking for the molecular mechanism that reverses the inflammatory activation of microglia, since this knowledge would be essential to protect from neurodegeneration. Our previous data (2) indicate that adipose derived mesenchymal stem cells (ASCs) exert important anti-inflammatory actions on microglia. We observed that microglia exposed to ASCs or their secreted factors (conditioned medium, CM) underwent a dramatic cell shape change into a highly elongated morphology (Fig. 1A), similar to the phenotype of microglia observed in a healthy brain (3). The elongation induced by ASCs was associated with a decrease of the pro-inflammatory cytokine TNFalpha (Fig. 1B) as well as with an upregulation of neurotrophic factors (2). Thus, ASC stimulated microglia represent an ideal tool to study the intracellular events necessary for the transition from inflammatory activated to non-inflammatory neuroprotective microglia. Exploiting these anti-inflammatory properties of ASCs we set up a microscopy-based siRNA screen (Fig. 1C), identifying its hits by cell morphology (see Fig. 1A). In this light, we searched for molecules that inhibited the anti-inflammatory ASC-induced phenotype and thus are involved in the transition from neurotoxic microglia to neuroprotective ones. As changes in the cell shape are intrinsically related to changes of the cytoskeleton, we carried out the screen with the major cytoskeletal regulators. In addition, we included regulators of microglia-specific activation/inflammatory pathways as siRNA targets. Our project is the first siRNA screen performed in primary microglia and we have identified a list of molecules that are specifically implicated in the reversion from activated to neuroprotective microglia. Since our positive hits represent potential neuroprotective drug targets, the outcome of this screen opens up a variety of novel investigation lines and therapies in PD, AD or other neurodegenerative diseases.