*2.2. Clozapine Prevents Loss of Mitochondrial Functionality and Cell Viability in Oligodendroglial and Neuronal CPZ-Induced Models of MS*

The atypical antipsychotic drug, clozapine (CLO), widely used in the treatment of schizophrenia, among other psychiatric disorders, is considered as a therapeutic agent that seems to exert its beneficial effects by its ability to increase Apo D levels in the brain [53,54]. Therefore, we first evaluated the potential neuroprotective effect of CLO in the CPZ-induced cell models. For this purpose, a wide range of CLO concentrations, from 0.1 to 100 μM, was used to treat HOG or SH-SY5Y cells during 24 and 48 h in absence of CPZ. Once it was established that CLO did not cause loss of cell viability, except in extremely high doses and/or prolonged exposures (Figures A1 and A2), we assessed whether the addition of CLO could avoid the CPZ cytotoxicity. Of note, the two cell lines were differentially affected by CLO, being neurons more sensitive than glial cells to the same concentrations. Our findings demonstrated that CLO was able to prevent the mitochondrial dysfunction caused by the toxic in both HOG and SH-SY5Y cells. As shown in Figure 3, cell viability assessed by the MTT assay revealed that CLO (0.1–1 μM) prevented about 15–30% loss of cell viability when added 24 h before 500 μM of CPZ (Figure 3a,b). Similar results were obtained when cells were treated with CLO and CPZ at the same time. In contrast, this neuroprotective effect was not noticeable when cells were incubated with 500 μM of CPZ for 24 h and subsequently with increasing concentrations of CLO for, at least, another 24 h (data not shown).

**Figure 3.** MTT assay in HOG (**a**) and SH-SY5Y cells (**b**) treated with increasing concentrations of CLO (0.1–5 μM) 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* < 0.01, \*\*\* *p* < 0.001 compared with control; # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 compared with CPZ treatment.
