P38 - Enfermedades humanas

Neuron-derived vesicles as therapeutic vectors: modulation of M6a content in EVs and their administration in a chronic stress model

Chmiel, Agustina* ^1,2 - Bühler, María Victoria* ^1,2 - Cosenza, Maximiliano ^1,2- Brocco, Marcela ^1,2 - Monteleone, Melisa ^1,2

1 Instituto de Investigaciones Biotecnológicas (IIB), Universidad Nacional de San Martín (UNSAM) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), San Martín, Argentina.
2 Escuela de Bio y Nanotecnología (EByN), Universidad Nacional de San Martín, San Martín, Argentina.
Contacto: chmielagustina01@gmail.com

Chronic stress is a key factor in the development of neuropsychiatric disorders such as depression. Current treatments are often limited in efficacy and show adverse effects. We propose an alternative therapeutic strategy based on extracellular vesicles (EVs), which due to their small size and high biocompatibility can facilitate biomolecule delivery to the brain. 

Our focus is on the neuronal glycoprotein M6a, which is involved in neuronal connectivity and whose levels are altered by chronic stress. EVs were obtained from the neural cell line HT22 through differential centrifugation and filtering, and characterized by TEM and nanoparticle analysis (NTA). An exogenous loading protocol was used to load M6a-GFP or GFP (control) plasmids into the EVs, using PEI as a transfection agent, followed by DNAse and PEG precipitation. The loading was confirmed by plasmid DNA extraction and by NTA analysis, which revealed changes in the concentration and size of the loaded EVs compared to the unloaded ones.

In a murine model of restriction induced stress, treatment with loaded EVs partially reversed weight loss and restored hippocampal M6a levels, although no significant behavioral changes were observed. These effects were independent of the loaded plasmid, suggesting a possible neuroprotective role of endogenous factors present in HT22-derived EVs.

We are working to optimize the loading efficacy of M6a-GFP through overexpression in HT22 cells. To assess this, loading efficiency will be quantified using fluorescence based NTA, and the zeta potential will be evaluated as a measure of: (1) stability for subsequent applications such as animal administration, (2) the M6a loading process (zeta potential sensitivity to modifications in the vesicular membrane). Changes in M6a expression will also be analyzed in 3D spheroids, as well as in differentiated HT22 cells.

We hope to obtain HT22-derived EVs with an increased proportion of M6a to
test their neuroprotective effect in vivo.

Palabras clave: EXTRACELLULAR VESICLES, M6A, NEURAL CELL LINE HT22