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Background: Aβ42 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. [ABSTRACT FROM AUTHOR]

Activation of the NLRP3 inflammasome in response to either exogenous (PAMPs) or endogenous (DAMPs) stimuli results in the production of IL-18, caspase-1 and IL-1β. These cytokines have a beneficial role in promoting inflammation, but an excessive activation of the inflammasome and the consequent constitutive inflammatory status plays a role in human pathologies, including Alzheimer's disease (AD). Autophagic removal of NLRP3 inflammasome activators can reduce inflammasome activation and inflammation. Likewise, inflammasome signaling pathways regulate autophagy, allowing the development of inflammatory responses but preventing excessive and detrimental inflammation. Nanotechnology led to the development of liposome engineered nanovectors (NVs) that can load and carry drugs. We verified in an in vitro model of AD-associated inflammation the ability of Glibenclamide-loaded NVs (GNVs) to modulate the balance between inflammasome activation and autophagy. Human THP1dM cells were LPS-primed and oligomeric Aß-stimulated in the presence/absence of GNVs. IL-1β, IL-18 and activated caspase-1 production was evaluated by the Automated Immunoassay System (ELLA); ASC speck formation (a marker of NLRP3 activation) was analyzed by FlowSight Imaging flow-cytometer (AMNIS); the expression of autophagy targets was investigated by RT-PCR and Western blot (WB); and the modulation of autophagy-related up-stream signaling pathways and Tau phosphorylation were WB-quantified. Results showed that GNVs reduce activation of the NLRP3 inflammasome and prevent the Aß-induced phosphorylation of ERK, AKT, and p70S6 kinases, potentiating autophagic flux and counteracting Tau phosphorylation. These preliminary results support the investigation of GNVs as a possible novel strategy in disease and rehabilitation to reduce inflammasome-associated inflammation. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is associated with the accumulation of amyloid-β (Aβ) within senile plaques in the brain and neuroinflammation, possibly driven by the activation of the NLRP3 inflammasome. Nucleoside reverse transcriptase inhibitors (NRTI) hamper the NLRP3 inflammasome assembly.Objective: We utilized an in vitro model reproducing the Aβ-driven inflammation seen in AD to analyze whether stavudine (D4T), a prototypical NRTI, modulates Aβ-mediated inflammasome activation and the ability of macrophages to eliminate Aβ via phagocytosis and autophagy.Methods: THP-1-derived macrophages were stimulated in vitro with Aβ42 or with Aβ42 after LPS-priming in the presence/absence of D4T. NLRP3 and TREM2 expression was analyzed by RT-PCR; phagocytosis, as well as ASC-Speck formation, was analyzed by Amnis FlowSight Imaging; NLRP3-produced cytokines were quantified by ELISA and, finally, autophagy was analyzed by measuring p-ERK1/2, p-AKT, beclin, p70-S6Kinase, and Lamp by ELISA and western blot.Results: IL-1β, IL-18, and caspase-1 were increased whereas Aβ phagocytosis and TREM2 were reduced in LPS+Aβ42-stimulated cells. D4T reduced NLRP3 assembly as well as IL-18 and caspase-1 production, but did not affect IL-1β production and TREM2 expression. Notably, whereas D4T reduced Aβ phagocytosis, Aβ autophagy by macrophages was stimulated by D4T, as witnessed by the down-modulation of ERK1/2 and AKT phosphorylation and the upregulation of beclin, LAMP, and p70-S6K, their downstream targets.Conclusion: In this in vitro model of AD, D4T reduces NLRP3 inflammasome-associated inflammation and stimulates Aβ autophagy by macrophages. It will be interesting to verify the possibly beneficial effects of D4T in the clinical scenario. [ABSTRACT FROM AUTHOR]