**3. Discussion**

Thirty years of research have provided significant insights to unravel the function of Apo D, which has helped to elucidate its antioxidant and anti-inflammatory role and a better understanding of the mechanisms whereby this specific apolipoprotein may exert its beneficial effects. To elucidate the function of Apo D requires the development of cellular models that allow studying the actions of this protein in a physiologically relevant but simple context. The data here presented aims to make progress in the knowledge of potential neuroprotective effect of Apo D in MS and other demyelinated diseases by both indirect and direct in vitro approximations.

A common trend in multiple pathological and nonpathological conditions of the nervous system, from neural development and aging to diverse neurodegenerative processes such as those observed in MS, is the Apo D upregulation with a seemingly neuroprotective purpose [12,20,51,55,56]. Valuable information has been gained concerning the Apo D expression in MS, i.e., it is increased in the CSF of MS patients and exhibits a characteristic pattern in the brain lesions [49–51]. However, mechanisms involved in the Apo D function in this pathology have been not fully elucidated until now. By taking advantage of the CPZinduced model of MS, we aimed to analyze the expression of Apo D in HOG cells treated with CPZ. Our results showed that the changes induced by CPZ in Apo D expression are minimal despite the dose and time-dependent cytotoxic damage previously reported for CPZ in the same conditions. Unsurprisingly, a similar CPZ effect was demonstrated in the SH-SY5Y, a cell line that does not efficiently express Apo D at least in nonpathological conditions. In fact, we only found negligible levels of this apolipoprotein in the neuroblastoma cells by immunocytochemistry but not by other techniques, probably due to methodological differences. During the last decades, various authors, including us, have used in vitro assays to demonstrate that H2O2, amyloid beta-peptide, lipopolysaccharide, paraquat as well as other acute short-term oxidative stressors induce a time- and dosedependent effect on Apo D expression [55–59]. At least in astrocytes, this effect seems to be regulated by the stress responsive JNK signaling pathway [9]. Here, we found that CPZ by itself does not promote a significant increase in Apo D levels in oligodendroglioma cells. Although the exact mechanism of action of CPZ is not completely understood, we previously demonstrated that the ion chelator impacts on functional state of mitochondria and aerobic cell respiration in neurons and glial cells. Now, we have also shown that these processes are accompanied by a significant increase in intracellular levels of ROS. Interestingly, the consequent compromise of mitochondrial function, cell metabolism, and the increase in oxidative stress are not immediately apparent in in vivo CPZ models. In this regard, the toxic/demyelinating effect induced by CPZ in mice does not peak until the third week of treatment [59,60], so it is reasonable to assume that CPZ may trigger the Apo D expression in the longer term.

Neuroprotection by Apo D may be afforded by either an indirect or a direct manner. On the one hand, it has been shown that CLO, an atypical antipsychotic drug used in the treatment of schizophrenia and bipolar syndrome, is able to increase Apo D levels in the brain [53,54]. However, the exact mechanism by which CLO regulates Apo D expression is still unknown. Our findings demonstrated that CLO induces an increase in Apo D synthesis in OLGs and neurons only in the presence of CPZ, at the same time that moderately prevents the loss of viability caused by the toxin, i.e., at neuroprotective doses of CLO. An important aspect, according to the findings obtained in SH-SY5Y cells, is that low concentrations of CLO would be the ones that may exert an Apo D-related neuroprotection against CPZ. In this way, we hypothesize that the great increase in Apo D synthesis induced by the treatment with 5 μM of CLO, unable to prevent the cytotoxic effect of CPZ, may be a consequence of some cell stress/damage caused by this dose of antipsychotic drug.

Based on our data, it seems that Apo D may contribute to the protective effect of the drug. This idea is sustained by some in vivo studies postulating that the effect of CLO in pathological situations would be related to the protective function of Apo D, thanks to its ability to (i) bind hydrophobic ligands, (ii) minimize their release, (iii) prevent their peroxidative degradation, and (iv) stabilize plasma membranes [53,61]. In particular, some authors propose that mechanism of action of CLO depends on the role of Apo D in arachidonic acid metabolism [6,62]. Therefore, targeting neural cells to increase Apo D and prevent further death would be one promising choice for MS modifying approaches.

On the other hand, this study was designed to test the effect of Apo D when it is added exogenously. The results obtained, using either a purified or a recombinant version of the hApo D, were interesting as these compounds afforded some neuroprotection against the CPZ insult. In fact, our analysis revealed that the exogenous addition of hApo D, purified from BCF or produced in a mammalian expression system, induces an increase in cell viability/proliferation in normal conditions. At the same time, this apolipoprotein also completely prevents the mitochondrial damage and loss of viability caused by the treatment with moderate to high doses of CPZ in oligodendroglial cells and, more importantly, in a neuroblastoma cell line that lacks endogenous Apo D expression [52]. Moreover, and in order to check the possibility that Apo D exerts, in this case, its protective activity in an extracellular way, a chemical perturbation of endocytosis in SH-SY5Y cells was carried out. Our data showed that Apo D neuroprotection is largely independent of CME, phagocytosis and macropinocytosis, but it is significantly reduced by inhibitors of dynamin, i.e., dynamin-dependent mechanisms that are consistent with clathrin-independent endocytosis (CIE) modes (caveolae- and RhoA-dependent) [63]. The above-mentioned results may have several implications. First, these results suggest that OLGs and neurons would be able to capture and internalize hApo D from the medium, triggering an increase in the cell metabolic activity and/or proliferation rate. Apo D uptake by some cells is not an unknown phenomenon. For instance, it is clearly demonstrated that astrocytes and OLGs synthesize and secrete this protein [16,18,64] which is captured by certain neurons in some specific situations [9,64–66]. Although technically challenging, pioneering studies in last years stated that Apo D may enter cells as a clathrin-independent cargo mediated by a specific cell surface receptor, basignin [67]. The recent discovery that Apo D is located inside the endosome-lysosome-autophagosomal compartment [66] and the results here presented support this hypothesis. Second, the effect of exogenous Apo D, once internalized, turns neuroprotective in pathological situations, which is consistent across studies. For example, Najyb et al. (2017) demonstrated that hApo D internalization and accumulation in primary hippocampal neurons were accentuated by kainate treatment. In addition, these authors reported that hApo D could act by decreasing abnormally increased cholesterol levels in damaged neurons [68]. In this line, He et al. (2009) showed that hApo D purified from BCF was able to bind arachidonic acid and cholesterol, attenuating the increase in oxidants and proinflammatory derivatives as F(2)-isoprostanes and 7-ketocholesterol in similar pathological conditions [69]. These neuroprotective and antioxidant roles of Apo D may be closely associated with its capacity of reducing radical-propagating lipid hydroperoxides by three methionine (Met) residues (Met49, Met93, and Met157), highly conserved in mammals [70]. Alternatively, Apo D has an extra cysteine, Cys116, with a thiol group that can be implicated in a direct antioxidant activity [70,71]. Despite this, and according with our findings, the protective mechanism of Apo D against oxidative damage induced by CPZ may directly target mitochondrial function but would not act through the production levels of ROS.

Noteworthy, although Apo D has been generally described as a monomeric protein [4,72], it dimerizes when reducing peroxidized lipids [7,15]. Thus, small-angle X-ray scattering analysis revealed that this apolipoprotein is mainly present as a tetramer in BCF or an oligomer in CSF [15]. As a general rule, heteromers are currently considered as novel molecular entities with new ligand and signaling characteristics, and probably different antioxidant properties which could explain the greater neuroprotective effect of hApo D, compared with hrApo D, demonstrated here.

In summary, valuable information has been gained in this work concerning neuroprotective effect of Apo D against CPZ, a neurotoxin used to produce models of MS. Although

the development of simpler models, as the ones shown in this work, constitutes a way to provide reliable answers in some pathological situations, these results must be validated on more physiological models such as primary cultures in order to check whether increasing either endogenously and/or exogenously the levels of Apo D could be a feasible intervention as part of medical therapy in neurodegenerative diseases. In this regard, the origin and the native structure of this protein must be taken into account in order to design the most effective approach.
