**1. Introduction**

Apolipoprotein D (Apo D) is a well-known lipocalin family member that plays a key role in the transport, metabolism and homeostasis of some lipids due to its ability to bind cholesterol, arachidonic acid, steroids, retinoic acid or anandamide, among other small hydrophobic ligands [1–3]. In the past decades, increasing evidence at biochemical and functional level suggested that Apo D acts as an antioxidant, being part of the body's defense system against oxidative stress, and also as an endogenous neuroprotective agent. Indeed, crystallographic analysis revealed that this 29 kDa glycoprotein comprises an eight-stranded antiparallel β-barrel flanked by a singled α-helix that encloses a fat specific ligand-binding pocket. Moreover, Apo D shows various exposed hydrophobic residues located in three of its extended loops which may contribute to Apo D association with lipids and seem to explain its potential as multiligand and multifunctional protein [4,5]. Most importantly, they have been linked to the ability of Apo D to bind and reduce oxidized lipids, and thereby inhibit radical-propagation of lipid hydroperoxides [6–8].

Rubio-Sardón, N.; Peláez, R.; García-Álvarez, E.; del Valle, E.; Tolivia, J.; Larráyoz, I.M.; Navarro, A. Neuroprotective Effect of Apolipoprotein D in Cuprizone-Induced Cell Line Models: A Potential Therapeutic Approach for Multiple Sclerosis and Demyelinating Diseases. *Int. J. Mol. Sci.* **2021**, *22*, 1260. https://doi.org/10.3390/ ijms22031260

**Citation:** Martínez-Pinilla, E.;

Academic Editor: Anne Vejux Received: 20 January 2021 Accepted: 22 January 2021 Published: 27 January 2021

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Studies in cell systems reported that different stress signal pathways may modulate Apo D transcription. In fact, stressful stimuli such as H2O2, Rose Bengal, kainic acid, ultraviolet (UV) light, paraquat or lipopolysaccharide, leading to extended growth arrest and apoptosis, increase Apo D expression in a dose and time-dependent manner [9,10]. The potential neuroprotective and prosurvival roles for Apo D have also been proven in animal models where the overexpression of this protein confers greater protection against oxidative stress and contributes significantly to the regulation of longevity; the experimental lack of Apo D causes opposite results [11,12].

In humans, Apo D is expressed in neural and peripheral tissues, detected in cerebrospinal fluid (CSF), in plasma as an important component of high-density plasma lipoproteins (HDL), and in breast cyst fluid (BCF) [1,2,13–15]. In nonpathological conditions of the central and peripheral nervous system (CNS and PNS, respectively), Apo D is widely expressed in neurons, glia (astrocytes, oligodendrocytes (OLGs), and Schwann cells), perivascular cells and pericytes [13,16–18], contributing to maintain neuronal homeostasis and myelin extracellular leaflet compaction [19,20]. Remarkably, Apo D is upregulated in neural cells and CSF during aging, and in brains affected by neurodegenerative diseases characterized by cellular stress and excitotoxicity such as multiple sclerosis (MS), Spongiform encephalopathy, Parkinson's disease (PD), Niemann–Pick disease, or Alzheimer's disease (AD) as well as psychiatric disorders (schizophrenia and bipolar disorder) [8,21,22].

MS is a devastating neurodegenerative disease that affects more than 2 million young adults worldwide, mainly women, with a complex and unknown etiology [23]. This demyelinating, autoimmune and inflammatory disease is manifested clinically in the form of multiple fully or partially reversible symptomatic episodes (reviewed in [24–26]), which reflect the progressive focal degeneration of OLGs and myelin membranes around axons in both white and grey matter areas throughout the brain and spinal cord [27–30]. Classically, OLG dysfunction has been linked to an exacerbated adaptive immune response, involving the recruitment of autoreactive T cells through a defective and permeable blood– brain barrier (BBB), and the activation of B cells [31–33]. The consequent inflammatory process activates microglia, astrocytes, and infiltrated macrophages that are able, in turn, to generate oxidative stress-related molecules as reactive oxygen species (ROS) and reactive nitrogen species (RNS) [34], which promote demyelination, compromise the neuro-axonal functional unit and contribute to the progressive tissue damage in MS [26,35,36]. However and contrary to what was thought, recent evidence shows that the biochemical alteration of myelin could be the initial event that triggers a secondary autoimmune response that results in the demyelinating inflammatory reaction taking place in the diseased brains, the so-called "inside-out" model of MS pathogenesis [37–39]. In the last two decades, extensive research has been carried out to find efficacious neuroprotective therapies in an attempt to alleviate symptoms and/or slow down or delay the progression of the MS [26,40–44]. Therefore, it is essential to know the root cause of the MS pathology in order to properly select the target for developing efficacious therapeutic interventions. For this purpose, a number of neurotoxin-induced in vivo and in vitro models of demyelination and MSrelated neurodegeneration are used. Among all, neuronal and glial cell lines exposed to cuprizone (CPZ), a copper chelator that reversibly impacts on mitochondrial function, may be a convenient experimental approach instrumental in the advance of understanding of the functioning of the nervous system [45–48].

Previous studies showed that Apo D is upregulated in the CSF of MS patients [49,50], reactive astrocytes, and exhibits a characteristic expression pattern in MS lesions of the brain [51]. In this regard, levels of OLG-derived Apo D are lower in demyelinating plaques but appear to recover in areas of remyelination [51]. This study aims to assess the potential of Apo D (either by triggering its endogenous synthesis or by its exogenous addition), as well as its mechanism of action, to prevent the neurotoxic effect of CPZ in two cell models that mimic biochemical features of MS.
