Unravelling novel players in astrocyte-mediated brain clearance

Under certain conditions, astrocytes demonstrate phagocytic capability and cooperate with microglia as an ancillary clearance system to clear up the brain. Astrocyte processes are in close association with synapses and contribute to synaptic health at several levels. Noteworthy, astrocytes are efficient sensors of synaptic dysfunction or degeneration. Indeed, they intervene by eliminating neuronal terminals, engulfing debris and internalizing neuronal-released aggregated proteins. However, the molecular machinery recruited for the recognition of specific targets is only in part clarified. The main aim of this PhD Project is to molecularly characterize astrocyte-mediated brain clearance both in health and disease, from different perspectives and through several approaches. In the first part of this PhD project, we identified a novel molecular mechanism behind astrocyte-mediated synapse elimination. We used a high throughput system to study the role of 3000 genes in the internalization kinetic of astrocytes We validated the most promising modulator: the Atypical Chemokine Receptor 3 (Ackr3). For the first time we demonstrated that the absence of Ackr3 abolishes synaptosome internalization in primary astrocytes. By employing biochemical and imaging approaches we also confirmed a direct involvement of Ackr3 in neuronal terminal recognition and internalization and identified CXCL12 as a possible novel “eat/find me” signal. Specifically, we detected that CXCL12 is attached to the synaptosomal membrane and increased CXCL12 enhance astrocyte clearance capacity. Of note, we revealed that CXCL12 recognizes phosphatidylethanolamine at the plasma membrane, a lipid that increases its externalization in the early phase of neuronal death. Finally, we demonstrate the involvement of Ackr3 in synaptosomes internalization in human iPSC-derived astrocytes, suggesting conserved mechanisms across species. Although additional data are required, these results generate new fundamental knowledge on the molecular machinery involved in astrocyte-mediated synaptic elimination. Studies on Ackr3 dysfunction in astrocytes as possible contributor in human diseases, such as glioma, are now on-going. In the second part of this PhD project, we described a novel mechanism by which astrocytes internalize the toxic form of α-syn, a protein that accumulates in many neurodegenerative diseases, including Parkinson’s disease. In collaboration with the University of Uppsala, we showed an impaired α-syn clearance in primary astrocytes harboring the G2019S pathogenic mutation in the LRRK2, a kinase involved in the pathogenesis of PD. This clearance deficit correlates with deleterious changes in the architecture of the endosomal/lysosomal organelles of astrocytes. We identified ANXA2 as a LRRK2 interactor and showed that decreased levels of Anxa2 in primary astrocytes are associated to an impairment of α-syn clearance. We also demonstrated that in the presence of the G2019S mutation, there is a decrease expression of AnxA2 both in cell culture and in human post-mortem brains of PD patients. Moreover, in the pathological background, Anxa2 fails to re-localize into puncta in close proximity to internalized α-syn particles. Notably, we proved that this phenotype could be completely reverted a selective LRRK2-inhibitor. Altogether, our results offer a better understanding of the molecular mechanisms behind impaired α-syn clearance in G2019S astrocytes, pointing to Anxa2 as a novel target for astrocytes’ loss of function in neurodegenerative diseases. In conclusion, we identified i) Ackr3 as a novel receptor involved in astrocyte-mediated synapses engulfment and ii) Anxa2 as a novel critical player in astrocyte-mediated α-syn clearance. Given the importance of astrocyte-mediated clearance to maintain brain homeostasis, these data offer the opportunity to investigate cell-targeted therapies as a novel approach in neurological disorders.