Stavudine Reduces NLRP3 Inflammasome Activation and Modulates Amyloid-β Autophagy

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.