*2.4. Neuroprotective Effect of the Exogenously Added hApo D in Oligodendroglial and Neuronal CPZ-Induced Models of MS*

The next step to assess the neuroprotective potential of Apo D was to check the impact of the exogenous addition of human Apo D (hApo D), hApo D purified from BCF or human recombinant Apo D (hrApo D), in the CPZ-based cell models of MS. On the one hand, we found that both apolipoproteins induced some improvement in mitochondrial oxidation and, consequently, an increase in OLGs (Figure 8) and neurons (Figure 9) viability under normal conditions. On the other hand, the analysis revealed that treatment with hApo D (5–100 nM) totally prevented the loss of viability caused by the addition of 500 μM CPZ for 24 h in the HOG cells (Figure 8c). Although to a lesser extent, similar results were found in cells pretreated with hrApo D (Figure 8d). Noteworthy, these findings were confirmed in the SH-SY5Y neuroblastoma cells that lack endogenous Apo D expression. Accordingly, both hApo D and hrApo D were able to prevent the toxic effect of CPZ after 24 h of treatment in neuroblastoma cells as well (Figure 9c,d).

**Figure 8.** Top panel: MTT assays in HOG cells treated with increasing concentrations (0.05–1000 nM) of hApo D (**a**) or of hrApo D during 24 h (**b**). Bottom panel: MTT assays in HOG cells treated with increasing concentrations (1–100 nM) of hApo D (**c**) or of hrApo D (**d**) for 24 h followed by 24 h with 500 μM of CPZ. Cell damage is represented as the percentage of viability versus control. Data are the mean ± SEM of five independent experiments. Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared with control; ### *p* < 0.001 compared with CPZ treatment.

Finally, and in order to test whether Apo D exerts its antioxidant activity intra- or extracellularly by sequestering/blocking CPZ or oxidative stress-induced molecules, we subjected SH-SY5Y to different pharmacological inhibitors of endocytosis. First, we examined the above-described neuroprotective effect of Apo D against CPZ upon perturbation of clathrin-mediated endocytosis (CME), or upon alteration of actin-dependent phagocytosis and micropinocytosis by pretreatment of cells with chlorpromazine (5 μg/mL) and cytochalasin D (8 μg/mL), respectively. As shown in Figure 10, these conditions did not seem to influence the effect exerted by hApo D (50–100 nM), even they significantly enhance it, as demonstrated by the MTT assay. However, when cells were pretreated with dynasore (80 μM), a compound that blocks GTPase activity of dynamin and vesicle scission, hApo D was not able to prevent the significant decrease of about 20–25% in cell viability after 24 h of treatment with 500 μM CPZ (Figure 10). Nevertheless, it should be noted that dynasore drastically magnifies, in some way, the cytotoxic effect of CPZ.

**Figure 9.** Top panel: MTT assays in SH-SY5Y cells treated with increasing concentrations (0.05–1000 nM) of hApo D (**a**) or of hrApo D (**b**) during 24 h. Bottom panel: MTT assays in SH-SY5Y cells treated with increasing concentrations (1–100 nM) of hApo D (**c**) or of hrApo D (**d**) for 24 h followed by 24 h with 500 μM of CPZ. Cell damage is represented as the percentage of viability versus control. Data are the mean ± SEM of five independent experiments. Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 compared with control; ## *p* < 0.01, ### *p* < 0.001 compared with CPZ treatment.

**Figure 10.** MTT assay in SH-SY5Y cells treated with 8 μg/mL cytochalasin D, 5 μg/mL chlorpromazine hydrochloride, or 80 μM dynasore prior to the addition of increasing concentrations (50–100 nM) of hApo D for 24 h followed by 24 h with 500 μM of CPZ. Cell damage is represented as the percentage of viability versus control. Data are the mean ± SEM of five independent experiments. Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test. \* *p* < 0.05, \*\*\* *p* < 0.001 compared with control; ### *p* < 0.001 compared with CPZ treatment.

#### *2.5. Neuroprotective Effect of hApo D is not Related to a Decrease in CPZ-Induced ROS Levels*

We previously demonstrated that CPZ affects mitochondrial function and aerobic cell respiration in neurons and glial cells which could lead to an increase in intracellular ROS production. In fact, the data summarized in Figure 11 show that treatment of HOG and SH-SY5Y cells with CPZ concentrations of 500 and 1000 μM significantly increased the levels of intracellular ROS. In the MTT assays Apo D totally prevented the loss of viability caused by CPZ so the next step was to measure ROS formation in these conditions. However, we did not find significant differences in the levels of intracellular ROS production between cells pretreated or not with hApo D (Figure 11a,b).

**Figure 11.** ROS production in HOG (**a**) and SH-SY5Y cells (**b**) treated with increasing concentrations (50–100 nM) of hApo D for 24 h followed by 24 h with 500 or 1000 μM of CPZ. Changes in ROS levels, measured with the oxidant-sensitive dye H2DCFDA, are represented as the percentage of fluorescent DCF production versus control. H2O2 500 μM was used as positive control. Data are the mean ± SEM of five independent experiments. Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test. \*\*\* *p* < 0.001 compared with control.
