Cell-Based Assays to Assess Neuroprotective Activity

The most common neurodegenerative diseases (NDDs), i.e. Alzheimer’s disease (AD), Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS), share common features both at molecular and cellular level: at molecular level, all these disorders are characterized by conformational changes of specific proteins leading to aggregates formation and mitochondrial dysfunctions together with impairment in ubiquitin-proteasome system and autophagy to this pathogenic mechanism; at cellular level, NDDs affect all brain cell types, leading to their dysfunction, which contributes to neurodegeneration. In fact, at early stages, all NDDs are characterized by degeneration and death of neurons in specific brain areas, often related to neural precursors cells (NPCs) proliferation/differentiation dysfunctions, making difficult the replacement of degenerated neurons. In the meanwhile, activation of microglia occurs, which initially acts to protect neurons and to support neuroregeneration, but, when activation is wide and/or prolonged, it contributes to neurodegeneration, to neurogenesis impairment and, together with astrocytes, to neuroinflammation. Finally, at later stages, most of NDDs are also characterized by white matter dysfunction, as a consequence of defects in oligodendrocytes functions or precursors cells (OPCs) proliferation/differentiation processes. Therefore, considering the cellular mechanisms involved in the complex process of neurodegeneration, to exactly characterize the neuroprotective potential of candidate molecules for central nervous system (CNS) drug discovery there is a need to use different brain cells-based assays. To this aim, here we describe the drug screening pipeline that we have developed in more than 10 years of research and that involves the use of different in vitro brain cell models. First, rat primary cultures of cerebellar granule neurons (CGNs) are used to evaluate the potential dose-dependent toxicity of all molecules to be tested; then, the potential neuroprotective effect of selected, non-toxic molecules is evaluated on specific in vitro models of neurodegeneration (age-related, AD, PD or ALS). Then, the use of neurospheres isolated from the sub-ventricular zone (SVZ) of adult mice allows to investigate the potential neuroregenerative effects of selected molecules of interest. Cultures of rat primary microglial cells or microglial cell lines (murine N9 or human HMC3), previously activated by commonly-used inflammatory stimuli (LPS or INFγ), are then set up, together with murine astrocytes cell line (IMA 2.1) or primary rat astrocytes, to assess immunomodulation, i.e. the shift from neurotoxic to neuroprotective phenotypes, of further selected compounds. In parallel, proliferation and differentiation mechanisms need to be analyzed in immortalized murine oligodendrocytes precursor cells (Oli-Neu cells) to evaluate effects on remyelination processes. Finally, to confirm the specific neuroprotective, neuroregenerative and immunomodulatory effects of selected molecules, different brain cells derived by human induced pluripotent stem cells (hiPSCs) from healthy donors or of patients with specific NDDs can be used to confirm the translational potential of the interesting candidates, as well as a strategy for the identification of more personalized and effective treatments for neuroprotection.