*2.3. Neuroprotective Doses of Clozapine Increase Apo D Expression in the CPZ-Induced Cell Models of MS*

Then, and in order to check the possible link between the neuroprotective effect observed for CLO and the endogenous Apo D levels, the expression of this apolipoprotein was analyzed in HOG cells upon CLO treatment. qPCR and immunocytochemical analyses demonstrated that this antipsychotic drug did not produce changes in Apo D expression by itself, at least in the tested concentrations and times of treatment (Figure 4). However, CLO (0.1–3 μM) induced an increase in Apo D synthesis when it was coadministrated with CPZ in OLGs at the same concentrations that prevented the loss of viability caused by the toxin. As shown in Figure 5, the increase in Apo D signal was higher than the control values when added 24 h before 500 μM of CPZ.

**Figure 4.** Relative Apo D gene expression in HOG cells treated or not with 5 μM of CLO during 24 h. Data represent the quotient between the gene and the expression of the housekeeping gene 18S rRNA. Bars represent the mean ± SEM of all measurements (*n* = 6–8) (**a**). Representative fluorescence microscopy images of Apo D expression in HOG cells treated or not with 3 μM of CLO during 24 and 48 h. 40× magnification (**b**). Densitometric quantification of Apo D immunocytochemical signal after 24 (**c**) and 48 h (**d**) of treatment with increasing concentrations of CLO (0.1–5 μM) in HOG cells (*n* = 6). Bars represent mean density per cell ± SEM (over control) in a 40× field.

Similar results, but with some nuances, were obtained in the neuroblastoma cell line. In fact, immunocytochemical assays revealed that CLO induced changes in Apo D expression in SH-SY5Y cells but only at the highest concentration (5 μM), at 24 and 48 h of treatment, as observed in the images (Figure 6a) and the immunocytochemical quantification (Figure 6b,c). When CLO was added 24 h before 500 μM of CPZ the Apo D immunosignal increased from 1.5 to 2-fold (compared to control) in the concentrations of the antipsychotic drug associated with the neuroprotective effects (Figure 7a,b). Interestingly, the treatment with 5 μM of CLO, which almost doubled Apo D levels in SH-SY5Y cells (Figure 7), was unable to prevent the cytotoxic effect of CPZ (Figure 3b).

**Figure 5.** Relative Apo D gene expression in HOG cells treated with increasing concentrations of CLO (0.1–3 μM) during 24 h followed by 24 h with 500 μM of CPZ. Data represent the quotient between the gene and the expression of the housekeeping gene 18S rRNA. Bars represent the mean ± SEM of all measurements (*n* = 6–8) (**a**). Densitometric quantification of Apo D immunocytochemical signal after 24 h of treatment with increasing concentrations of CLO (0.1–3 μM) followed by 24 h with 500 μM of CPZ (*n* = 6). Bars represent mean density per cell in a 40× field ± SEM (over control) (**b**). Representative fluorescence microscopy images of Apo D expression in HOG cells treated with increasing concentrations of CLO (0.1–3 μM) followed by 24 h with 500 μM of CPZ. 40× magnification (**c**). Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test. \*\*\* *p* < 0.001 compared with control.

**Figure 6.** Representative fluorescence microscopy images of Apo D expression in SH-SY5Y cells treated or not with 5 μM of CLO during 24 and 48 h. 40× magnification (**a**). Densitometric quantification of Apo D immunocytochemical signal after 24 (**b**) and 48 h (**c**) of treatment with increasing concentrations of CLO (0.1–5 μM) in SH-SY5Y cells (*n* = 6). Bars represent mean density per cell ± SEM (% versus control) in a 40× field. Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey's test. \*\*\* *p* < 0.001 compared with control.
