GHRH Agonists and Mesenchymal Stem Cell-derived Extracellular Vesicles as Emerging Therapies in Preclinical Models of Alzheimer’s Disease

Alzheimer’s disease (AD) is the leading cause of dementia, affecting approximately 60 million people worldwide. Its main pathological hallmarks include amyloid-β (Aβ) aggregation, tau hyperphosphorylation, and chronic neuroinflammation, leading to memory deficits, language difficulties, and impaired executive functions. Since the current therapeutic options are largely symptomatic, there is the urgent need for novel disease-modifying strategies targeting multiple aspects of AD pathology. This thesis investigates two therapeutic approaches in preclinical AD models: (1) the effects of growth hormone-releasing hormone (GHRH) and its agonist MR-409, and (2) mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) Beyond its endocrine functions, growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide which exerts significant effects within the central nervous system (CNS). Indeed, synthetic GHRH agonists have shown neuroprotective effects in several neurological conditions, thus positioning them as promising candidates for the treatment of AD. To assess cellular response to GHRH stimulation, we used neural stem cells (NSCs), and, even in the presence of Aβ fragments, we observed improved cell proliferation and viability, decreased apoptosis, and activated pro-survival and anti-inflammatory signaling pathways, including PI3K/Akt and GSK3β, which are implicated in tau phosphorylation. As for in vivo experiments, the chronic treatment of 3-month-old 5xFAD with MR-409 determined reduced Aβ plaque burden, attenuated microglial and astroglial activation, suppressed NF-κB–mediated inflammation, and preserved neuronal survival. Collectively, these results highlight GHRH agonists as pleiotropic agents capable of simultaneously targeting multiple AD hallmarks. The second projects investigates MSC-derived EVs, which mediate many of the regenerative and immunomodulatory effects of MSCs. They are small membrane-bound nanoparticles, which carry proteins, lipids, and nucleic acids and whose composition can change according to the functional state of the producing cells: this is why in this project, bone-marrow derived EVs were preconditioned via oxygen-glucose deprivation (OGD) or a cytokine cocktail (CYTO). While maintaining typical EV features and expression markers also after the preconditioning, proteomic analysis revealed distinct cargo profiles: CYTO-EVs were enriched in proteins related to anti-inflammatory pathways and extracellular matrix regulation, while OGD-EVs contained proteins involved in proteostasis and protein turnover. Following intranasal administration to 5xFAD mice, EVs reduced both neuroinflammation and amyloid deposition. Unfortunately, we did not observed cognitive rescue after the treatment. To rule out the possibility that a positive effect could actually be masked by the severity of the phenotype typical of 5xFAD, we applied the same protocol to wild-type animals: both types of EVs enhanced cognitive capabilities and lowered anxiety-related behaviours, which correlated with increased neuronal activation in different brain regions involved in memory formation and learning. These findings demonstrate that MSC-derived EVs exert immunomodulatory and neurofunctional effects in vivo and highlight the potential of EV-based therapies - particularly with non-invasive delivery routes - as promising strategies for neurodegenerative disorders. These findings demonstrate that both GHRH agonists and MSC-derived extracellular vesicles can simultaneously target multiple hallmarks of AD, including amyloid and tau pathology, neuroinflammation, and neuronal dysfunction. By modulating key molecular and cellular pathways, these approaches offer promising disease-modifying potential and emphasize the urgent need for therapies able to address the multifaceted complexity of AD.