*Article* **Neuroprotective Effect of Apolipoprotein D in Cuprizone-Induced Cell Line Models: A Potential Therapeutic Approach for Multiple Sclerosis and Demyelinating Diseases**

**Eva Martínez-Pinilla 1,2,3,\*, Núria Rubio-Sardón 1, Rafael Peláez 4, Enrique García-Álvarez 1, Eva del Valle 1,2,3, Jorge Tolivia 1,2,3,\*, Ignacio M. Larráyoz <sup>4</sup> and Ana Navarro 1,2,3**


**Abstract:** Apolipoprotein D (Apo D) overexpression is a general finding across neurodegenerative conditions so the role of this apolipoprotein in various neuropathologies such as multiple sclerosis (MS) has aroused a great interest in last years. However, its mode of action, as a promising compound for the development of neuroprotective drugs, is unknown. The aim of this work was to address the potential of Apo D to prevent the action of cuprizone (CPZ), a toxin widely used for developing MS models, in oligodendroglial and neuroblastoma cell lines. On one hand, immunocytochemical quantifications and gene expression measures showed that CPZ compromised neural mitochondrial metabolism but did not induce the expression of Apo D, except in extremely high doses in neurons. On the other hand, assays of neuroprotection demonstrated that antipsychotic drug, clozapine, induced an increase in Apo D synthesis only in the presence of CPZ, at the same time that prevented the loss of viability caused by the toxin. The effect of the exogenous addition of human Apo D, once internalized, was also able to directly revert the loss of cell viability caused by treatment with CPZ by a reactive oxygen species (ROS)-independent mechanism of action. Taken together, our results suggest that increasing Apo D levels, in an endo- or exogenous way, moderately prevents the neurotoxic effect of CPZ in a cell model that seems to replicate some features of MS which would open new avenues in the development of interventions to afford MS-related neuroprotection.

**Keywords:** breast cystic fluid; clozapine; endocytosis; glia; neurons; ROS
