Modulation of MAPK- and PI3/AKT-Dependent Autophagy Signaling by Stavudine (D4T) in PBMC of Alzheimer’s Disease Patients

Background: Aβ(42) deposition plays a pivotal role in AD pathogenesis by inducing the activation of microglial cells and neuroinflammation. This process is antagonized by microglia-mediated clearance of Aβ plaques. Activation of the NLRP3 inflammasome is involved in neuroinflammation and in the impairments of Aβ-plaque clearance. On the other hand, stavudine (D4T) downregulates the NLRP3 inflammasome and stimulates autophagy-mediated Aβ-clearing in a THP-1-derived macrophages. Methods: We explored the effect of D4T on Aβ autophagy in PBMC from AD patients that were primed with LPS and stimulated with Aβ oligomers in the absence/presence of D4T. We analyzed the NLRP3 activity by measuring NLRP3-ASC complex formation by AMNIS FlowSight and pro-inflammatory cytokine (IL-1β, IL-18 and Caspase-1) production by ELISA. The phosphorylation status of p38, ERK, AKT, p70, and the protein expression of CREB, LAMP2A, beclin-1, Caspase-3 and Bcl2 were analyzed by Western blot. Results: Data showed that D4T: (1) downregulates NLRP3 inflammasome activation and the production of down-stream pro-inflammatory cytokines in PBMC; (2) stimulates the phosphorylation of AKT, ERK and p70 as well as LAMP2A, beclin-1 and Bcl2 expression and reduces Caspase-3 expression, suggesting an effect of this compound on autophagy; (3) increases phospho-CREB, which is a downstream target of p-ERK and p-AKT, inducing anti-inflammatory cytokine production and resulting in a possible decrease of Aβ-mediated cytotoxicity; and (4) reduces the phosphorylation of p38, a protein involved in the production of pro-inflammatory cytokines and tau hyperphosphorylation. Conclusions: D4T reduces the activation of the NLRP3 inflammasome, and it might stimulate autophagy as well as the molecular mechanism that modulates Aβ cytotoxicity, and D4T might reduce inflammation in the cells of AD patients. It could be very interesting to check the possible beneficial effects of D4T in the clinical scenario.