The Neuroprotective Lipocalin Apolipoprotein D Stably Interacts with Specific Subtypes of Detergent-Resistant Membrane Domains in a Basigin-Independent Manner

Abstract

Accumulated evidence points to the lipocalin apolipoprotein D (ApoD), one of the few genes consistently upregulated upon brain ageing and neurodegeneration, as an endogenous controller of the redox state of cellular and extracellular lipid structures. This biochemical function has downstream consequences as apparently varied as control of glycocalyx and myelin compaction, cell viability upon oxidative stress or modulation of signalling pathways. In spite of this knowledge, it is still unclear if ApoD function requires canonical receptor-mediated transductions systems. This work aims to examine ApoD-cell membrane interaction and its dependence on a proposed ApoD receptor, Basigin. Whole and fractionated membrane preparations from the brain, primary astrocytes, glial and neuronal cell lines, reveal ApoD as a very specific component of particular subtypes of detergent-resistant microdomains (DRMs). ApoD interacts in vitro with neuronal membranes and is stably associated with astrocytic membranes. ApoD associates with DRMs with specific buoyancy properties that co-fractionate with plasma or late-endosome-lysosome markers. A mass spectrometry analysis reveals that these Triton X-114 DRMs contain both plasma membrane and endosomal-lysosomal compartment lipid raft proteins. ApoD-DRM association is maintained under metabolic and acute oxidative stress conditions. However, ApoD-membrane interaction, its internalization and its lipid-antioxidant function do not require the presence of Basigin. This work supports a stable association of ApoD with membranes, independent of Basigin, and provides the basis to fully understand ApoD antioxidant neuroprotective mechanism as a mechanism taking place in specific membrane subdomains.

Dual role of Apolipoprotein D as long-term instructive factor and acute signal conditioning microglial secretory and phagocytic responses

“…As previously described for neuronal cells ( Corraliza-Gomez et al, 2022 ), membrane preparations from BV2 cells were exposed in vitro to purified human ApoD. These experiments demonstrate a dose-dependent partitioning of ApoD with microglial membranes ( Figure 6A ), a necessary interaction for the internalization demonstrated above ( Figure 2C ).…”

“…The combination of ApoD-dependent microglial genes (42 in total) was subjected to a functional enrichment analysis (fold enrichment ≥ 2; p -value EASE < 0.05) using the DAVID platform ( Sherman et al, 2022 ), which uncovered that these genes are enriched in glycoproteins, in proteins secreted or related to membranes and lysosomes, and in those involved in immunity and antigen presentation ( Figure 1B ). This is an interesting result that blindly identifies functions and subcellular locations now known to be associated with ApoD ( Pascua-Maestro et al, 2017 , 2019 ; García-Mateo et al, 2018 ; Corraliza-Gomez et al, 2022 ). In summary, these results suggest that the transcriptional response of the aging nervous system to a constitutive absence of ApoD expression is enriched in microglial genes and support the study of a functional relationship of microglia and ApoD.…”

“…It is well established that ApoD, purified from breast cystic fluid, is internalized by astrocytes and neurons ( Do Carmo et al, 2007 ; Bajo-Grañeras et al, 2011a ; Pascua-Maestro et al, 2017 , 2019 ; Corraliza-Gomez et al, 2022 ). We thus tested whether the exogenous addition of ApoD results in protein internalization in BV2 cells.…”

“…Also, the modulation of this traffic by OS is milder and slower in microglia compared to astrocytes or neurons. Therefore, ApoD roles in microglia are predicted to be mediated by triggering processes downstream of signaling cascades after interaction with particular liquid-ordered membrane domains ( Corraliza-Gomez et al, 2022 ), rather than by direct effector functions on membrane managing and preservation of lysosomal pH, as it is the case for other cell types in the nervous system. The dual signaling role uncovered by this first exploration of ApoD function on microglia might be based on the modulation of other lipid raft-dwelling proteins like APP, that can influence pathogenesis ( Cordy et al, 2003 ; Minami et al, 2011 ), or Toll-like receptors that regulate brain immune responses to disease ( Fernández-Arjona et al, 2022b ).…”

“…Fluorescence immunocytochemistry experiments with an anti-mouse ApoD-specific antibody already validated in our laboratory (Pascua-Maestro et al, 2020), demonstrate that the BV2 microglial cells do not have detectable ApoD protein levels, in comparison with a mouse astrocyte cell line (Figure 2A). The same It is well established that ApoD, purified from breast cystic fluid, is internalized by astrocytes and neurons (Do Carmo et al, 2007;Bajo-Grañeras et al, 2011a;Pascua-Maestro et al, 2017Corraliza-Gomez et al, 2022). We thus tested whether the exogenous addition of ApoD results in protein internalization in BV2 cells.…”