Small Molecule-Driven NLRP3 Inflammation Inhibition via Interplay between Ubiquitination and Autophagy: Implications for Parkinson Disease

Abstract

Aging-related, nonresolving inflammation in both the central nervous system (CNS) and periphery predisposes individuals to the development of neurodegenerative disorders (NDDs). Inflammasomes are thought to be especially relevant to immune homeostasis, and their dysregulation contributes to inflammation and NDDs. However, few agents have been clinically shown to reduce NDD incidence by targeting inflammasomes. Our study indicated that NLRP3 (NLR family, pyrin domain containing 3) inflammasome is involved in Parkinson disease (PD) progression in patients and various murine models. In addition, the small molecule kaempferol (Ka) protected mice against LPS- and SNCA-induced neurodegeneration by inhibiting NLRP3 inflammasome activation as evidenced by the fact that Ka reduced cleaved CASP1 expression and disrupted NLRP3-PYCARD-CASP1 complex assembly with concomitant decreased IL1B secretion. Mechanically, Ka promoted macroautophagy/autophagy in microglia, leading to reduced NLRP3 protein expression, which in turn deactivated the NLRP3 inflammasome. Intriguingly, ubiquitination was involved in Ka-induced autophagic NLRP3 degradation. These findings were further confirmed in vivo as knockdown of Atg5 expression or autophagy inhibitor treatment significantly inhibited the Ka-mediated NLRP3 inflammasome inhibition and neurodegeneration amelioration. Thus, we demonstrated that Ka promotes neuroinflammatory inhibition via the cooperation of ubiquitination and autophagy, suggesting that Ka is a promising therapeutic strategy for the treatment of NDDs.Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ACTB: actin, beta; AIF1/IBA1: allograft inflammatory factor 1; ATG5: autophagy related 5; ATG7: autophagy related 7; BafA1: bafilomycin A1; BECN1: beclin 1, autophagy related; CASP1: caspase 1; CNS: central nervous system; CQ: chloroquine; DA neurons: dopaminergic neurons; DAMPS: damage-associated molecular patterns; DAPI: 4’,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GFAP: glial fibrillary acidic protein; IP: immunoprecipitation; i.p.: intraperitoneally; Ka: kaempferol; KD: knockdown; KO: knockout; LPS: lipopolysaccharide; IL1B: interleukin 1 beta; IL6: interleukin 6; Ly: lysate; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NC: negative control; NDD: neurodegenerative diseases; NLRP3: NLR family, pyrin domain containing 3; OE: overexpression; PD: Parkinson disease; poly-Ub: poly-ubiquitin; PTM: post-translational modification; PYCARD/ASC: PYD and CARD domain containing; Rapa: rapamycin; RFP: red fluorescent protein; SN: supernatant; SNCA: synuclein alpha; SNpc: substantia nigra pars compacta; SQSTM1: sequestosome 1; TH: tyrosine hydroxylase; TNF/TNF-alpha: tumor necrosis factor; Ub: ubiquitin; WT: wild type