Extracellular vesicle (EV)-mediated glia-to-neuron communication has been recognized in a growing number of physiological and pathological situations. They transport complex sets of molecules that can be beneficial or detrimental for the receiving cell. As in other areas of biology, their analysis is revolutionizing the field of neuroscience, since fundamental signaling processes are being re-evaluated, and applications for neurodegenerative disease therapies have emerged. Using human astrocytic and differentiated neuronal cell lines, we demonstrate that a classical neuroprotective protein, Apolipoprotein D (ApoD), expressed by glial cells and known to promote functional integrity and survival of neurons, is exclusively transported by EVs from astrocytes to neurons, where it gets internalized. Indeed, we demonstrate that conditioned media derived from ApoD-knock-out (KO) astrocytes exert only a partial autocrine protection from oxidative stress (OS) challenges, and that EVs are required for ApoD-positive astrocytic cell line derived medium to exert full neuroprotection. When subfractionation of EVs is performed, ApoD is revealed as a very specific marker of the exosome-containing fractions. These discoveries help us reframe our understanding of the neuroprotective role of this lipid binding protein and open up new research avenues to explore the use of systemically administered ApoD-loaded exosomes that can cross the blood-brain barrier to treat neurodegenerative diseases.

Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases. These results open therapeutic opportunities by providing a route of entry and a repair mechanism for lysosomes in pathological situations.

To compact the extracellular sides of myelin, an important transition must take place: from membrane sliding, while building the wraps, to membrane adhesion and water exclusion. Removal of the negatively charged glycocalyx becomes the limiting factor in such transition. What is required to ini-