Microglial involvement in brain physiopathology: in vitro studies using rat primary cultures

Microglial involvement in neurological disorders is well-established, being microglial activation not only associated with neurotoxic consequences, but also with neuroprotective effects. The studies presented here, based on microglia rat primary cell cultures and mainly on microglial conditioned medium (MCM), show insights into the mechanism of Superoxide dismutase 1 (SOD1) and Apolipoprotein E (ApoE) secretion by microglia as well as their neuroprotective effect towards primary cerebellar granule neurons (CGNs) exposed to the dopaminergic toxin 6-hydroxydopamine (6-OHDA). SOD1 and ApoE are released respectively through non-classical lysosomal or the classical ER/Golgi-mediated secretion pathway. Microglial conditioned medium, in which SOD1 and ApoE accumulated, protected CGNs from degeneration and these effects were replicated when exogenous SOD1 or ApoE was added to a non-conditioned medium. SOD1 neuroprotective action was mediated by increased cell calcium from an external source. ApoE release is negatively affected by microglia activation, both with lipopolysaccharide (LPS) and Benzoylbenzoyl-ATP (Bz-ATP) but is stimulated by neuronal-conditioned medium as well as in microglia-neurons co-culture conditions. This neuronal-stimulated microglial ApoE release is differently regulated by activation states (i.e. LPS vs ATP) and by 6-hydroxydopamine-induced neurodegeneration. In co-culture conditions, microglial ApoE release is essential for neuroprotection, since microglial ApoE silencing through siRNA abrogated protection of cerebellar granule neurons against 6-OHDA toxicity. Therefore, these molecules could represent a target for manipulation aimed at promoting neuroprotection in brain diseases. Considering a pathological context, and the microglial ability to adopt a neuroprotective or neurotoxic profile, we characterize the microglial M1/M2 phenotype in transgenic rats (McGill-R-Thy1-APP) which reproduce extensively the Alzheimer’s-like amyloid pathology. Here, for the first time, cortical, hippocampal and cerebellar microglia of wild type and transgenic adult rats were compared, at both early and advanced stages of the pathology. In view of possible therapeutic translations, these findings are relevant to test microglial neuroprotection, in animal models of neurodegenerative diseases.

Understanding the role of microglial extracellular vesicles (EVs) in neuroinflammation spreading: an in vitro study

Neuroinflammation is a crucial pathogenic mechanism that commonly underlies most neurodegenerative diseases. Microglia, the immune cells of the brain, play a critical role that changes depending on the stage of neuropathology: at early phases of brain diseases microglia display the neuroprotective phenotype which is switched to the classically activated pro-inflammatory subtype at later stages, contributing to neurodegeneration. The microglial phenotypic shift is characterized by a change in the release of bioactive molecules both soluble and through extracellular vesicles. Our in vitro studies aim to understand whether different types of activation could determine change in vesicles content, in particular miRNAs, and whether this could influence the activation state of control microglial cells. Microglial polarization has been induced in two different in vitro models: N9, microglial murine cell line, have been treated by using LPS towards a proinflammatory/neurotoxic phenotype or ATP towards antinflammatory/neuroprotective status; HMC3, human microglial cell line, have been activated using IFN-+ATP. We demonstrated that conditioned media/exosomes obtained from donor microglia were able to promote a pro-inflammatory phenotype in control cells, leading us to prove the existence of a neuroinflammation spreading process mediated by extracellular vesicles of microglia with a crucial role of miRNAs. Increased expression of miRNA-34a observed in N9 model underlines a possible contribution in the diffusion of proinflammatory activation of microglia. Thus, we tried to downregulate miR-34a expression using cleaving sequences of anti-mir-34a DNAzyme delivered by DNA nanostructures aimed to confirm the involvement of miR-34a in microglia polarization towards the neurotoxic phenotype. In conclusion, this thesis work reveal a new inflammation spreading mechanism that involves release of vesicles containing specific cargos by donor polarized microglia, particularly miRNAs, able to influence the phenotypic shift in unpolarized microglia: this process deserves to be deeply investigated as potential therapeutic target to counteract neurodegenerative diseases.