Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress

Abstract

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.

Myelin extracellular leaflet compaction requires apolipoprotein D membrane management to optimize lysosomal‐dependent recycling and glycocalyx removal

“…ApoD‐KO and WT neonatal (1–2 days old) mice were used for primary astrocytes cultures as described (Bajo‐Grañeras, Ganfornina, Martin‐Tejedor, & Sanchez, ; Pascua‐Maestro et al, ). Cell extracts from cerebral cortices were obtained by a combination of enzymatic and mechanical dissociation, resuspended in Dulbecco's modified Eagle medium with 10% fetal bovine serum, 1% l ‐glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 0.25 μg/ml amphoterycin B plated on culture flasks and incubated at 37°C in 5% CO 2 with 90%–95% humidity.…”

“…A lysosomal population with these properties would be the perfect candidate to manage glycocalyx remodeling and myelin membrane recycling. This unexpected relationship of ApoD with such a subset of lysosomes, and the evidences that myelin built in an ApoD‐KO background must have different properties altering myelin recognition and digestion by phagocytic cells (Garcia‐Mateo et al, ; Pascua‐Maestro et al, ), led us to study the role of ApoD in the development and maturation of myelin.…”

“…We have recently discovered that the Lipocalin Apolipoprotein D (ApoD), already known to be required for adequate myelin management after PNS injury (Ganfornina et al, ; Garcia‐Mateo et al, ), is targeted to lysosomes in astrocytes and neurons, and is required for lysosomal membrane stability and pH homeostasis (Pascua‐Maestro, Diez‐Hermano, Lillo, Ganfornina, & Sanchez, ). The pH distribution of the subset of ApoD‐positive lysosomes is in the range of optimal pH for neuraminidases, also coincident with that of secretory lysosomes.…”

Oxidised protein metabolism: recent insights

“…Oxidised proteins can also cause lysosomal membrane permeabilisation, increase lysosomal pH, and trigger a cascade of pro-apoptotic events (Dunlop et al, 2011). A very important study recently identified Apolipoprotein D (ApoD), an extracellular lipid-binding lipoprotein, as having a lysosomal membrane stabilising function (Pascua-Maestro et al, 2017). ApoD can reduce radical-propagating lipid hydroperoxides (Bhatia et al, 2012) and was shown to be endocytosed and to inhibit lysosomal lipid peroxidation in vitro (Pascua-Maestro et al, 2017).…”

“…A very important study recently identified Apolipoprotein D (ApoD), an extracellular lipid-binding lipoprotein, as having a lysosomal membrane stabilising function (Pascua-Maestro et al, 2017). ApoD can reduce radical-propagating lipid hydroperoxides (Bhatia et al, 2012) and was shown to be endocytosed and to inhibit lysosomal lipid peroxidation in vitro (Pascua-Maestro et al, 2017). The authors suggested that ApoD could play an important role in the brain by protecting neurones against oxidative damage and could have important implications in neurodegenerative disorders (Pascua-Maestro et al, 2017).…”