A novel role for SHARPIN in amyloid-β phagocytosis and inflammation by peripheral blood-derived macrophages in Alzheimer’s disease

Abstract

23 INTRODUCTION: Defective immune cell-mediated clearance of amyloid-beta (Aβ) and 24 Aβ-associated inflammatory activation of immune cells are key contributors of Aβ 25 accumulation and neurodegeneration in Alzheimer's disease (AD), however, the underlying 26 mechanisms remain elusive. 27 METHODS: Differentiated THP-1 cells treated with Aβ and AD patient-derived 28 macrophages were used as in-vitro model. The role of SHARPIN was analysed in 29 differentiated THP-1 cells using siRNA-mediated knockdown followed by immunoblotting, 30 ELISA, real-time PCR, immunoprecipitation and flow cytometry. Differentiated SHSY5Y 31cells were used to study inflammation-mediated apoptosis. 32RESULTS: SHARPIN was found to regulate Aβphagocytosis and NLRP3 expression in 33 THP-1 derived macrophages. Further, it was found to promote macrophage polarization to an 34 M1 (pro-inflammatory) phenotype resulting in enhanced inflammation and associated 35 neuronal death, demonstrated using in-vitro culture systems. SHARPIN expression by blood-36 derived macrophages was further found to be higher in the early stages of AD, which 37 correlates with Aβ 40/42 concentration in the plasma and age of the study subjects. 38DISCUSSION: The novel protein, SHARPIN has been shown to play critical roles in 39 regulation of Aβ-phagocytosis and inflammation in AD and the mechanism by which 40 SHARPIN is activated by Aβ in macrophages has been elucidated. 41 42 Keywords: SHARPIN, amyloid-beta, Alzheimer's disease, mild cognitive impairment, 43 macrophage, NLRP3, inflammation, phagocytosis, oxidative stress. 44 3 Abbreviations: AD, Alzheimer's disease; Aβ, amyloid-beta; MCI, Mild Cognitive 45 Impairment; SHARPIN, Shank-associated RH domain-interacting protein; NLRP3, 46 nucleotide-binding domain (NOD)-like receptor protein 3; ASC, Apoptosis-associated Speck-47 like protein containing a caspase-recruitment domain (CARD); LUBAC, linear 48 ubiquitination assembly complex; iNOS, induced Nitric Oxide Synthase; IL-1β, Interleukin-49 1beta; TGF-β, Transforming Growth Factor-1beta; TNF-α, Tumor Necrosis Factor-alpha; 50 PBMC, Peripheral Blood Mononuclear cells; NF-κB, Nuclear Factor kappa-light chain-51 enhancer of activated B cells; ROS, reactive oxygen species; CRP, C-Reactive Protein; PBS, 52 Phosphate-buffered saline; RPMI, Roswell Park Memorial Institute; FBS, fetal bovine 53 serum; THP-1, Tohoku Hospital Pediatrics-1; NH 4 OH, Ammonium hydroxide; FITC, 54 Fluorescein isothiocyanate. 55 56 57 58 59 60 61 62 63 64 4 polarization to M1 (pro-inflammatory) phenotype in differentiated THP-1 cell line as an in-89 vitro model and SHARPIN silencing protects neurons from Aβ-induced inflammatory 90 damage using differentiated SHSY5Y cell line. Further, utilizing control, mild cognitively 91 impaired (MCI) and AD patient-derived macrophages, we show that SHARPIN plays a 92 significant role the progression of AD. 93 2. Materials and methods 94 2.1. Inclusion of study subjects 95 AD and MCI patients were recruited from the Memory & Neurobehavioral Clinic (MNC) at 96 the S...

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“…Phagocytosis by macrophages is a critical process for the uptake and degradation of infectious agents and senescent cells, and it participates in tissue remodeling, immune response, and inflammation [14]. A defective phagocytic ability of macrophages is considered as a critical determinant in the progression of different diseases because this fact is related to a suppressive adaptive immune response [15,16].…”

Erythropoietin signaling in peripheral macrophages is required for systemic β‐amyloid clearance

“…To exclude this potential disturbance, an EPO‐derived peptide, ARA290, that is nonerythrogenic but retains other functions of EPO was further applied in our experiments. In AD patients, while peripheral blood‐derived macrophages have been reported to demonstrate ineffective phagocytosis of Aβ compared with the age‐matched control subjects (Jairani et al , 2019; Krishnan et al , 2020), contributions of peripheral tissue macrophages to the development of human AD and therapeutic effects of targeting peripheral tissue macrophages in treating human AD warrants further investigation.…”

“…Aβ also dose‐dependently activates serum complement, and complement‐opsonized Aβ are eventually captured by erythrocytes (Brubaker et al , 2017). In terms of monocytes/macrophages, Aβ can promote itself clearance through activate monocytes/macrophages (Condic et al , 2014; Krishnan et al , 2020). However, molecules that coordinate expression of Aβ phagocytic genes remain unknown.…”

Genome-Wide Meta-Analysis of Late-Onset Alzheimer’s Disease Using Rare Variant Imputation in 65,602 Subjects Identifies Novel Rare Variant Locus NCK2: The International Genomics of Alzheimer’s Project (IGAP)

“…Among the novel loci, TSPAN14 , encoding tetraspanin 14, is one of a family of proteins involved in proteolytic processing of the amyloid precursor protein (APP) 53 through physical interactions with APP secretases, and appears to regulate ADAM10 cleavage of substrates 54 , which may alter levels of Aβ production 55 . SHARPIN , which encodes Shank-associated RH domain-interacting protein, is a key regulator of neuroinflammatory response, stimulating macrophage activity and promoting Aβ phagocytosis in the presence of circulating Aβ 56 . A rare variant in SHARPIN have been shown to limit immune response 57 , which may promote LOAD development.…”