Final Report Summary - MG INTERACTIONS (Towards understanding neuron-microglia communication in the brain)
Microglia are the resident macrophages of the brain and form a dynamic network with cells using their branches to actively monitor the brain parenchyma every few hours (Hanisch and Kettenmann, 2007; Nimmerjahn et al., 2005; Peri and Nusslein-Volhard, 2008). Indeed, it has been shown that microglia are able to directly sense neuronal activity and to remove apoptotic, injured and sick neurons in several disorders affecting the brain.
Interestingly, microglia are not born in the brain, and both in mouse and fish they derive from myeloid progenitors that colonize the brain during embryogenesis (Ginhaux et al., 2010).
Understanding how microglia colonize the brain during embryonic development is of great importance as this might open new avenues for the prevention and treatment of neuronal disorders.
We focused our attention on chemokines as several studies have shown that these often play important roles in leukocyte recruitment (Badolato, 2013; Bajoghli, 2013). Thus, we took a phylogenetic approach to identify zebrafish homologues of human and rodent chemokines and we analyzed their spatio-temporal expression by performing RT, Q PCRs and in situ hybridization in fish embryos at various developmental stages. In this way, we identified one interesting receptor/ligand pair, while the receptor is present in macrophages the ligand is expressed right at the time of macrophage production and during brain colonization. To understand if these factors play a role in this process, we took a knock-down approach and silenced the receptor. Strikingly, receptor knock-down results in a smaller microglial population while, tissue macrophages outside of the brain exhibit normal population size, distribution and migratory capacities. Furthermore, we showed that ectopic expression of the chemokine diverts microglia precursor cells from their migratory path towards the brain. This was achieved by placing the chemokine under a heat-shock promoter to drive strong expression everywhere in the embryo. In these embryos, the size of the microglia population was significantly reduced, suggesting that ectopic chemokine overexpression masks the native chemotactic path. To test if this factor can still attract microglia once they are in the brain, we attached a microinjection system to a spinning disc confocal microscope to inject defined protein quantities into the brain. By analyzing microglial responses in terms of migration speed and directionality we have established that this factor can indeed attract microglia. In the same way, also miss-expression of this chemokine in single neurons attracts surrounding microglia.
Together these results show that this chemokine signaling is indeed involved in microglial brain colonization. Future approaches will need to establish the route that these cells take to reach the brain and if this signaling system is also involved in the colonization of other tissues, such as Kupffer cells in the liver.
Interestingly, microglia are not born in the brain, and both in mouse and fish they derive from myeloid progenitors that colonize the brain during embryogenesis (Ginhaux et al., 2010).
Understanding how microglia colonize the brain during embryonic development is of great importance as this might open new avenues for the prevention and treatment of neuronal disorders.
We focused our attention on chemokines as several studies have shown that these often play important roles in leukocyte recruitment (Badolato, 2013; Bajoghli, 2013). Thus, we took a phylogenetic approach to identify zebrafish homologues of human and rodent chemokines and we analyzed their spatio-temporal expression by performing RT, Q PCRs and in situ hybridization in fish embryos at various developmental stages. In this way, we identified one interesting receptor/ligand pair, while the receptor is present in macrophages the ligand is expressed right at the time of macrophage production and during brain colonization. To understand if these factors play a role in this process, we took a knock-down approach and silenced the receptor. Strikingly, receptor knock-down results in a smaller microglial population while, tissue macrophages outside of the brain exhibit normal population size, distribution and migratory capacities. Furthermore, we showed that ectopic expression of the chemokine diverts microglia precursor cells from their migratory path towards the brain. This was achieved by placing the chemokine under a heat-shock promoter to drive strong expression everywhere in the embryo. In these embryos, the size of the microglia population was significantly reduced, suggesting that ectopic chemokine overexpression masks the native chemotactic path. To test if this factor can still attract microglia once they are in the brain, we attached a microinjection system to a spinning disc confocal microscope to inject defined protein quantities into the brain. By analyzing microglial responses in terms of migration speed and directionality we have established that this factor can indeed attract microglia. In the same way, also miss-expression of this chemokine in single neurons attracts surrounding microglia.
Together these results show that this chemokine signaling is indeed involved in microglial brain colonization. Future approaches will need to establish the route that these cells take to reach the brain and if this signaling system is also involved in the colonization of other tissues, such as Kupffer cells in the liver.