TNIP2 inhibits amyloidogenesis by regulating the 3'UTR of BACE1: An in vitro study.

TNFAIP3-interacting protein 2 (TNIP2) is known as a negative regulator of NF-κB signaling and inhibit inflammatory response and apoptosis, and is also involved in RNA metabolism. In this study, we investigated the potential role of TNIP2 in amyloidogenesis critically associated with Alzheimer's disease (AD). We found a significant decline of TNIP2 protein level in both mouse and cell model of AD. In SH-SY5Y and HEK cells that stably express human full-length APP695 (SY5Y-APP and HEK-APP), TNIP2 overexpression decreased the protein levels of ß-secretase (BACE1) and C99, as well as Aß peptides (including Aß40 and Aß42), while those of α-secretase (ADAM10) and the related C83 remained unchanged. We further found that TNIP2 promoted the degradation of BACE1 mRNA and was able to bound to the 3' untranslated region (3'UTR) with the reduced luciferase activity. These results indicated that TNIP2 effectively inhibited amyloidogenic processing by regulating the 3'UTR-associated mRNA decay of BACE1.

Apicidin attenuates memory deficits by reducing the Aß load in APP/PS1 mice.

AIMS: Amyloid beta (Aß) is an important pathological feature of Alzheimer's disease (AD). A disintegrin and metalloproteinase 10 (ADAM10) can reduce the production of toxic Aß by activating the nonamyloidogenic pathway of amyloid precursor protein (APP). We previously found that apicidin, which is a histone deacetylase (HDAC) inhibitor, can promote the expression of ADAM10 and reduce the production of Aß in vitro. This study was designed to determine the potential of apicidin treatment to reverse learning and memory impairments in an AD mouse model and the possible correlation of these effects with ADAM10. METHODS: Nine-month-old APP/PS1 mice and C57 mice received intraperitoneal injections of apicidin or vehicle for 2 months. At 11 months of age, we evaluated the memory performance of mice with Morris water maze (MWM) and context fear conditioning tests. The Aß levels were assessed in mouse brain using the immunohistochemical method and ELISA. The expression of corresponding protein involved in proteolytic processing of APP and the phosphorylation of tau were assessed by Western blotting. RESULTS: Apicidin reversed the deficits of spatial reference memory and contextual fear memory, attenuated the formation of Aß-enriched plaques, and decreased the levels of soluble and insoluble Aß40/42 in APP/PS1 mice. Moreover, apicidin significantly increased the expression of ADAM10, improved the level of sAPPα, and reduced the production of sAPPß, but did not affect the levels of phosphorylated tau in APP/PS1 mice. CONCLUSION: Apicidin significantly improves the AD symptoms of APP/PS1 mice by regulating the expression of ADAM10, which may contribute to decreasing the levels of Aß rather than decreasing the phosphorylation of tau.

AP2S1 regulates APP degradation through late endosome-lysosome fusion in cells and APP/PS1 mice.

AP2S1 is the sigma 2 subunit of adaptor protein 2 (AP2) that is essential for endocytosis. In this study, we investigated the potential role of AP2S1 in intracellular processing of amyloid precursor protein (APP), which contributes to the pathogenesis of Alzheimer disease (AD) by generating the toxic ß-amyloid peptide (Aß). We found that knockdown or overexpression of AP2S1 decreased or increased the protein levels of APP and Aß in cells stably expressing human full-length APP695, respectively. This effect was unrelated to endocytosis but involved lysosomal degradation. Morphological studies revealed that silencing of AP2S1 promoted the translocalization of APP from RAB9-positive late endosomes (LE) to LAMP1-positive lysosomes, which was paralleled by the enhanced LE-lysosome fusion. In support, silencing of vacuolar protein sorting-associated protein 41 (VPS41) that is implicated in LE-lyso fusion prevented AP2S1-mediated regulation of APP degradation and translocalization. In APP/PS1 mice, an animal model of AD, AAV-mediated delivery of AP2S1 shRNA in the hippocampus significantly reduced the protein levels of APP and Aß, with the concomitant APP translocalization, LE-lyso fusion and the improved cognitive functions. Taken together, these data uncover a LE-lyso fusion mechanism in APP degradation and suggest a novel role for AP2S1 in the pathophysiology of AD.

HMGCS2-Induced Autophagic Degradation of Tau Involves Ketone Body and ANKRD24.

BACKGROUND: Accumulation of hyperphosphorylated Tau (pTau) contributes to the formation of neurofibrillary tangles in Alzheimer's disease (AD), and targeting Tau/pTau metabolism has emerged as a therapeutic approach. We have previously reported that mitochondrial 3-hydroxy-3-methylglutaryl-COA synthase 2 (HMGCS2) is involved in AD by promoting autophagic clearance of amyloid-ß protein precursor via ketone body-associated mechanism, whether HMGCS2 may also regulate Tau metabolism remains elusive. OBJECTIVE: The present study was to investigate the role of HMGCS2 in Tau/p degradation. METHODS: The protein levels of Tau and pTau including pT217 and pT181, as well as autophagic markers LAMP1 and LC3-II were assessed by western blotting. The differentially regulated genes by HMGCS2 were analyzed by RNA sequencing. Autophagosomes were assessed by transmission electron microscopy. RESULTS: HMGCS2 significantly decreased Tau/pTau levels, which was paralleled by enhanced formation of autophagic vacuoles and prevented by autophagic regulators chloroquine, bafilomycin A1, 3-methyladenine, and rapamycin. Moreover, HMGCS2-induced alterations of LAMP1/LC3-II and Tau/pTau levels were mimicked by ketone body acetoacetate or ß-hydroxybutyrate. Further RNA-sequencing identified ankyrin repeat domain 24 (ANKRD24) as a target gene of HMGCS2, and silencing of ANKRD24 reduced LAMP1/LC3-II levels, which was accompanied by the altered formation of autophagic vacuoles, and diminished the effect of HMGCS2 on Tau/pTau. CONCLUSION: HMGCS2 promoted autophagic clearance of Tau/pTau, in which ketone body and ANKRD24 played an important role.

Sulfuretin exerts diversified functions in the processing of amyloid precursor protein.

Sulfuretin is a flavonoid that protects cell from damage induced by reactive oxygen species and inflammation. In this study, we investigated the role of sulfuretin in the processing of amyloid precursor protein (APP), in association with the two catalytic enzymes the α-secretase a disintegrin and metalloproteinase (ADAM10), and the beta-site APP cleaving enzyme 1 (BACE1) that play important roles in the generation of ß amyloid protein (Aß) in Alzheimer's disease (AD). We found that sulfuretin increased the levels of the immature but not the mature form of ADAM10 protein. The enhanced ADAM10 transcription by sulfuretin was mediated by the nucleotides -444 to -300 in the promoter region, and was attenuated by silencing or mutation of transcription factor retinoid X receptor (RXR) and by GW6471, a specific inhibitor of peroxisome proliferator-activated receptor α (PPAR-α). We further found that sulfuretin preferentially increased protein levels of the immature form of APP (im-APP) but significantly reduced those of BACE1, sAPPß and ß-CTF, whereas Aß1-42 levels were slightly increased. Finally, the effect of sulfuretin on BACE1 and im-APP was selectively attenuated by the translation inhibitor cycloheximide and by lysosomal inhibitor chloroquine, respectively. Taken together, (1) RXR/PPAR-α signaling was involved in sulfuretin-mediated ADAM10 transcription. (2) Alteration of Aß protein level by sulfuretin was not consistent with that of ADAM10 and BACE1 protein levels, but was consistent with the elevated level of im-APP protein, suggesting that im-APP, an isoform mainly localized to trans-Golgi network, plays an important role in Aß generation.

TUFM is involved in Alzheimer's disease-like pathologies that are associated with ROS.

Mitochondrial Tu translation elongation factor (TUFM or EF-Tu) is part of the mitochondrial translation machinery. It is reported that TUFM expression is reduced in the brain of Alzheimer's disease (AD), suggesting that TUFM might play a role in the pathophysiology. In this study, we found that TUFM protein level was decreased in the hippocampus and cortex especially in the aged APP/PS1 mice, an animal model of AD. In HEK cells that stably express full-length human amyloid-ß precursor protein (HEK-APP), TUFM knockdown or overexpression increased or reduced the protein levels of ß-amyloid protein (Aß) and ß-amyloid converting enzyme 1 (BACE1), respectively. TUFM-mediated reduction of BACE1 was attenuated by translation inhibitor cycloheximide (CHX) or α-[2-[4-(3,4-Dichlorophenyl)-2-thiazolyl]hydrazinylidene]-2-nitro-benzenepropanoic acid (4EGI1), and in cells overexpressing BACE1 constructs deleting the 5' untranslated region (5'UTR). TUFM silencing increased the half-life of BACE1 mRNA, suggesting that RNA stability was affected by TUFM. In support, transcription inhibitor Actinomycin D (ActD) and silencing of nuclear factor κB (NFκB) failed to abolish TUFM-mediated regulation of BACE1 protein and mRNA. We further found that the mitochondria-targeted antioxidant TEMPO diminished the effects of TUFM on BACE1, suggesting that reactive oxygen species (ROS) played an important role. Indeed, cellular ROS levels were affected by TUFM knockdown or overexpression, and TUFM-mediated regulation of apoptosis and Tau phosphorylation at selective sites was attenuated by TEMPO. Collectively, TUFM protein levels were decreased in APP/PS1 mice. TUFM is involved in AD pathology by regulating BACE1 translation, apoptosis, and Tau phosphorylation, in which ROS plays an important role.

Mitochondrial TXN2 attenuates amyloidogenesis via selective inhibition of BACE1 expression.

Thioredoxin-2 (TXN2) is a mitochondrial protein and represents one of the intrinsic antioxidant enzymes. It has long been recognized that mitochondrial dysfunction and oxidative stress contribute to the pathogenesis of Alzheimer's disease (AD). We hypothesized that mitochondrial TXN2 might play a role in AD-like pathology. In this study, we found that in SH-SY5Y and HEK cells stably express full-length human amyloid-ß precursor protein (HEK-APP), TXN2 silencing or over-expression selectively increased or decreased the transcription of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), respectively, without altering the protein levels of others enzymes involved in the catalytic processing of APP. As a result, ß-amyloid protein (Aß) levels were significantly decreased by TXN2. In addition, in cells treated with 3-nitropropionic acid (3-NP) that is known to increase reactive oxygen species (ROS) and promote mitochondrial dysfunction, TXN2 silencing resulted in further enhancement of BACE1 protein levels, suggesting a role of TXN2 in ROS removal. The downstream signaling might involve NFκB, as TXN2 reduced the phosphorylation of p65 and IκBα; and p65 knockdown significantly attenuated TXN2-mediated regulation of BACE1. Concomitantly, the levels of cellular ROS, apoptosis-related proteins and cell viability were altered by TXN2 silencing or over-expression. In APPswe/PS1E9 mice, an animal model of AD, the cortical and hippocampal TXN2 protein levels were decreased at 12 months but not at 6 months, suggesting an age-dependent decline. Collectively, TXN2 regulated BACE1 expression and amyloidogenesis via cellular ROS and NFκB signaling. TXN2 might serve as a potential target especially for early intervention of AD.

Mitochondrial methionine sulfoxide reductase B2 links oxidative stress to Alzheimer's disease-like pathology.

Methionine sulfoxide reductase B2 (MSRB2) is a mitochondrial protein that protects cell from oxidative stress. The antioxidant activity suggests that MSRB2 may play a role in the pathophysiology of Alzheimer's disease (AD). Here, we report that in APP/PS1 mice, an animal model of AD, MSRB2 protein levels were decreased in the hippocampus at both young (6 mon) and old (18 mon) age, and in the cortex only at an old age, respectively. In HEK293 cells that stably express human full-length ß-amyloid precursor protein (APP, HEK/APP), MSRB2 reduced the protein and mRNA levels of APP and ß-amyloid converting enzyme 1 (BACE1), and the consequent amyloid beta peptide (Aß) 1-40 and Aß1-42 levels. MSRB2 overexpression or knockdown also oppositely affected Tau phosphorylation at selective sites, with the concomitant alteration of the phosphorylated extracellular signal regulated kinase (p-ERK) and AMP-activated protein kinase (p-AMPK) levels. Moreover, in cells treated with long-term (24 h) hydrogen peroxide, the alterations of APP processing and Tau phosphorylation were reversed by MSRB2 overexpression. We further found that MSRB2-mediated regulation of APP transcription involved JNK and ERK signaling, as MSRB2 also reduced the levels of phosphorylated JNK (p-JNK), and JNK or ERK inhibitor attenuated the effect of MSRB2 on APP proteins and transcripts. Finally, MSRB2 reduced apoptosis-related proteins Bax and caspase3 and enhanced the anti-apoptotic protein Bcl2. These results indicated that the role for MSRB2 in AD-like pathology was closely associated with its antioxidant activity. By attenuating both amyloidogenesis and Tau phosphorylation, MSRB2 may serve as a potential therapeutic target for AD.