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Roflupram, a phosphodiesterase 4 inhibitior, suppresses inflammasome activation through autophagy in microglial cells Tingting You, Yu-Fang Cheng, Jiahong Zhong, Bingtian Bi, Bingqing Zeng, Wenhua Zheng, Hai-Tao Wang, and Jiang-Ping Xu ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017
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ACS Chemical Neuroscience
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Roflupram, a phosphodiesterase 4 inhibitior, suppresses inflammasome
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activation through autophagy in microglial cells
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Tingting Youa,b, Yufang Chenga, Jiahong Zhonga, Bingtian Bia,c, Bingqing Zenga,
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Wenhua Zhengd, Haitao Wanga,*, Jiangping Xua, *
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a
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Sciences, Southern Medical University, Guangzhou 510515, China
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b
Department of Neuropharmacology and Drug Discovery, School of Pharmaceutical
Department of Pharmacology, Guangdong Key Laboratory for R&D of Natural Drug,
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Guangdong Medical University, Zhanjiang 524023, China
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c
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Guangzhou 510060, China
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d
Department of Clinical Trial Center, Cancer Center, Sun Yat-sen University,
Faculty of Health Sciences, University of Macau , Taipa, Macau, China
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*Correspondence:
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Prof. Jiangping Xu, M.D., Ph.D.
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Department of Neuropharmacology and Drug Discovery,
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School of Pharmaceutical Sciences,
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Southern Medical University, Guangzhou 510515, China.
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Tel.: +86-20-61648236, Fax: +86-20-61648236
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E-mail:
[email protected] 23 24
Dr. Haitao Wang, Ph.D.
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Department of Neuropharmacology and Drug Discovery,
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School of Pharmaceutical Sciences,
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Southern Medical University, Guangzhou 510515, China.
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Tel.: +86-20-61648594, Fax: +86-20-61648236
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E-mail:
[email protected] 30 1
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Abstract
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Inhibition of phosphodiesterase 4 (PDE4) suppressed the inflammatory responses in
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the brain. However, the underlying mechanisms are poorly understood. Roflupram
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(ROF) is a novel PDE4 inhibitor. In the present study, we found that ROF enhanced
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the level of microtubule-associated protein 1 light chain 3 II (LC3-II) and decreased
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p62 in microglial BV-2 cells. Enhanced fluorescent signals were observed in BV-2
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cells treated with ROF by Lysotracker red and acridine orange staining. In addition,
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immunofluorescence indicated a significant increase in punctate LC3. Moreover, β
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amyloid25-35 (Aβ25-35) or lipopolysaccharide (LPS) with ATP were used to activate
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inflammasome. We found that both LPS plus ATP and Aβ25-35 enhanced the
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conversion of pro-caspase-1 to cleaved-caspase-1 and increased the production of
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mature IL-1β in BV-2 cells. Interestingly, these effects were blocked by the treatment
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of ROF. Consistently, Knocking down the expression of PDE4B in primary microglial
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cells led to enhanced level of LC-3Ⅱ and decreased activation of inflammasome.
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What's more, Hoechst staining showed that ROF decreased the apoptosis of neuronal
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N2a cells in conditioned media from microglia. Our data also showed that ROF
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dose-dependently enhanced autophagy, reduced the activation of inflammasome and
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suppressed the production of IL-1β in mice injected with LPS. These effects were
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reversed by inhibition of microglial autophagy. These results put together demonstrate
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that ROF inhibits inflammasome activities and reduces the release of IL-1β by
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inducing autophagy. Therefore, ROF could be used as a potential therapeutic
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compound for the intervention of inflammation-associated diseases in the brain.
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Keywords: Phosphodiesterase 4; Roflupram; Autophagy; Inflammasome; Microglia;
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Neuroinflammation.
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Abbreviations
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3-MA, 3-methyladenine; Aβ, β amyloid protein; AD, Alzheimer’s disease; AO,
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acridine orange; ATG, autophagy-related gene; ATG16L1, autophagy-related gene 16
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like 1; ATG7, autophagy related protein 7; ATP, adenosine triphosphate; cAMP,
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3',5'-cyclic monophosphate; CASP1, caspase-1; CASP3, caspase-3; cGMP, cyclic
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guanosine monophosphate; CM, conditioned media; CNS, central nervous system;
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CQ, lysosome inhibitor chloroquine; ELISA, enzyme linked immunosorbent assay;
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fAβ, amyloid β-protein fibrils; FORS, forskolin; IL-1β, interleukin beta; LC3,
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microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; LYT,
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lysotraker red; NLR, NOD-like receptor; NLRP3, pyrin domain containing 3;
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SQSTM1,
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phosphodiesterase-4; PD, Parkinson’s disease; pro-IL-18, precursor interleukin-18;
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pro-IL-1β, precursors interleukin-1β; PSD95, postsynaptic density-95; RAPA,
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rapamycin; ROF, Roflupram; ROLI, Rolipram; TNF-α, tumor necrosis factor alpha.
sequestosome
1
protein;
PDEs,
phosphodiesterases;
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p62/ PDE4,
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Introduction
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Sustained neuroinflammation contributes to the pathogenesis of various neurological
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and psychiatric disorders, including Alzheimer’s disease (AD), Parkinson’s disease
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(PD), and depression1. A variety of factors, such as toxic metabolites, infection, and
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traumatic brain injury, potentially activate the glial cells that secrete proinflammatory
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cytokines and eventually contribute to the initiation and progression of
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neuroinflammation 2. However, activated glia can initiate both protective and toxic
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inflammatory responses according to the severity and duration of the activation 3.
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When inflammation is rampant, persistent activation of glial cells leads to neuronal
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injury 4. Hence, limiting the inflammatory responses is useful in ameliorating chronic
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inflammation-related diseases.
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Autophagy is a critical quality control system for cellular homeostasis regulation 5.
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Recent studies have established that autophagy plays a pivotal role in regulating
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immune responses and controlling excessive inflammation
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effects of autophagy in the pathology of inflammation is the modulation of the
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inflammasome activation and the production of interleukin-1β (IL-1β)
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inflammasome is a multimeric protein complex composed of a sensor protein (such as
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an NOD-like receptor, NLR), an adaptor protein ASC, and the inflammatory protease
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caspase-1 that enzymatically processes pro-IL-1β and pro-IL-18 into their mature
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forms9. Autophagy deficiency might contribute to the exaggeration of inflammation 10.
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For example, fetal macrophages obtained from autophagocytosis-deficient ATG16L1-/-
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(autophagy-related gene 16 like 1) mice, produced excessive amounts of IL-1β and
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IL-18 in response to endotoxin stimulation, and interestingly, the enhanced levels of
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pro-inflammatory factors were found to be associated with higher level of caspase-1 11.
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These
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pharmacological inhibition or genetic deletion
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mainly focused on the autophagy-mediated anti-inflammatory effect in macrophages.
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In the central nervous system (CNS), microglia is the resident macrophage along with
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primary immune cells exhibiting a key role in neuroinflammation
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regulatory role of microglial autophagy in inflammasome has been the focus of
findings
have
been
confirmed
by
6, 7
. One of the major
manipulating
autophagy
8
. The
with
12, 13
. However, most of these studies
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. Recently, the
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intense research. Increased inflammation was observed following the disruption of
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microglial autophagy with Atg7 small interfering RNA (siRNA), while promoting
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autophagy reduced the IL-1β release in Aβ-treated microglial cells
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molecular machinery remains to be determined, these data postulate that promoting
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microglial
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neuroinflammation-related diseases.
7
autophagy
might
be
a
potential
15
. Although the
therapeutic
strategy
for
On the other hand, intracellular adenosine 3',5'-cyclic monophosphate (cAMP) 16-18
8
increase the autophagic activities reported in various cells in the past few years
.
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cAMP is a well-established ubiquitous secondary messenger that plays a major role in
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the regulation of various cellular processes. It is synthesized from ATP catalyzed by
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adenylate cyclase and decomposed into AMP catalyzed by phosphodiesterases (PDEs).
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Ugland et al. reported that cAMP-mediated induction of autophagy in mesenchymal
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stem cells might provide new targets for the intervention of neurological disorders 17.
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PDE type 4 (PDE4) inhibitor resveratrol could induce autophagy in endothelial cells
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by upregulating the cAMP signaling pathway
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Resveratrol induced autophagy through cAMP in HepG2 cells
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suggested that cAMP might mediate the induction of autophagy in certain types of
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cell. However, whether cAMP or PDE4 inhibitor exert similar roles on microglia
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autophagy was unknown.
16
. A recent study also reported that 18
. These studies
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PDEs are a ubiquitous superfamily of enzymes involved in the regulation of cAMP
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and/or cyclic guanosine monophosphate (cGMP) by degrading these secondary
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messengers to inactive nucleotides 19. Their vital role in cell signaling renders them as
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therapeutic targets for many diseases, and the development of PDE inhibitors is under
24
study
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enzyme, whose expression was detected in various cells including immune cells,
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proinflammatory cells, and airway smooth muscle cells
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inhibitors are considered as promising novel drugs for the treatment of chronic
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inflammatory diseases based on the anti-inflammatory effect 22. Our previous studies
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showed that inhibition of PDE4 can adequately attenuate neuroinflammation in
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neurodegenerative and mental disorders 23-25. Roflupram (ROF, also known as Zl-n-91
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. Among the 11 identified isoenzymes, PDE4 is a major cAMP-specific
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. Remarkably, PDE4
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or FFPM), a selective PDE4 inhibitor, was designed based on the structures of
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PDE4D2 and rolipram
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anti-inflammatory effect and ameliorated the behavior of AD model in APP/PS1
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transgenic
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anti-neuroinflammatory effect are yet to be elucidated. Coupled with autophagy in the
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regulation of inflammatory response, the role of ROF in microglial autophagy and the
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putative correlation between the anti-inflammatory effect and autophagy is yet an
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enigma. Therefore, the present study aimed to investigate the regulatory effect of ROF
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on autophagic activities, as well as, the relevant contribution of autophagy to the
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anti-inflammatory effect by emphasizing on the NLRP3 inflammasome pathway in
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microglial cells.
mice27.
26
. Our recent studies also showed that ROF exerted an
Nevertheless,
the
mechanisms
underlying
the
12 13
RESULTS AND DISCUSSION
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Effect of ROF on cell viability in microglial BV-2 cells. The chemical structure of
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ROF was shown in Figure 1A. This compound was kindly provided by Dr. Hengming
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Ke (University of North Carolina, Chapel Hill, USA). Methyl thiazolyl tetrazolium
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(MTT) assay was performed to determine the cellular compatibility of ROF with
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BV-2 cells. As shown in Figure 1B, ROF (0.625–20 µM) did not exhibit toxicity on
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BV-2 cells after 24 h incubation. ROF also showed adequate compatibility with
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activated microglial BV-2 cells triggered by lipopolysaccharide (LPS) plus ATP, as
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well as, Aβ25-35 exposure for 6 h (Figure 5A).
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ROF induced autophagy in microglial BV-2 cells. Autophagy is a highly 28
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conserved process for degradation in all types of eukaryotic cells
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altered LC3 expression in microglial BV-2 cells in two isoforms, LC3-I and LC3-II.
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During
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phospholipid-conjugated LC3-II, which is a valuable indicator of altered autophagic
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activity29. The present study focused on whether PDE4 inhibitor ROF could induce
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microglial autophagy. Thus, we firstly examined the expression of LC3 in BV-2 cells
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treated with ROF. As shown in Figure 1C–G, ROF increased the protein level of
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LC3-II in BV-2 cells treated with ROF while the that of p62/sequestosome 1 (p62), an
the
formation
of
autophagosome,
cytosolic
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LC3-I
. and found an
converts
into
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autophagy substrate, was significantly decreased in a concentration- and
2
time-dependent manner. These results indicated that ROF had an active role in the
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autophagic activity.
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However, it is notable that both induction and inhibition of autophagy will lead 30
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to an increase in LC3-II
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reflects the entire process of autophagy, was used to determine the source of enhanced
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LC3-II level. As autophagic flux can be evaluated based on the LC3-II turnover
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through the use of lysosomal inhibitors
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presence or absence of lysosomal inhibitor CQ. CQ suppresses the fusion of
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autophagosome and lysosome at the late stage of autophagy, thereby resulting in the
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impairment of LC3-II autophagic degradation and subsequent accumulation(29). As
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shown in Figure 1H-I, compared to the cells treated with ROF alone, co-treatment
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with CQ and ROF resulted in increased LC3-II accumulation, suggesting that
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autophagic flux was enhanced after ROF treatment. Collectively, these data indicate
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that ROF enhances the autophagic activities and stimulates the autophagic flux in
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BV-2 cells.
17
. Therefore, further assessment of autophagic flux, which
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, BV-2 cells were treated with ROF in the
In addition to LC3-II and p62, the quantity of autophagy-lysosome, formed at the 32
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late stage of autophagy, is also an indicator of the autophagic activity
. Hence, we
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labeled the lysosomes in BV-2 cells with acridine orange (AO) and lysotraker red
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(LYT) stain to monitor the effect of ROF on the late stages of autophagic activity. As
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shown in Figure 2A and 2B, compared to the cells in the vehicle control group, the
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enhanced fluorescence signal was observed in cells treated with ROF. The typical
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autophagy inducer rapamycin (RAPA, as a positive control) also induced fluorescence
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signal of lysosome (Figure 2A-B), which further confirmed the induction effect of
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ROF on autophagy. Notably, the classic PDE4 inhibitor rolipram (ROLI) and an
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adenosine cyclase activator forskolin (FORS) could also induce high fluorescence
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signal of lysosome (Figure 2A-B). These parallel experiments suggested that cAMP
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contributes to microglial autophagy.
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Furthermore, immunofluorescence staining showed that the increased LC3
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punctuate staining in response to ROF was inhibited by class III PI3K inhibitor, 7
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3-methyladenine (3-MA), which is known to block autophagy at the early stage
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(Figure 3A). The immunofluorescence images were consistent with results of Western
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blot analysis for LC3-II (Figure 3B). Together, these results suggested that ROF
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induces microglial autophagy involving the class III PI3K-dependent autophagy
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signaling pathway. Although the underlying mechanisms require further study, it was
6
interesting to find that PDE4 inhibitor ROF could induce microglial autophagy.
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Blocking autophagy enhanced the activation of NLRP3 inflammasome in BV-2
8
cells. Next, we investigated the effect of ROF-induced microglial autophagy on
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neuroinflammation focusing on NLRP3 inflammasome/caspase-1/IL-1β axis, which is 33, 34
10
critical in inflammatory response
, and recently, this axis was found to be
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regulated by autophagic activities
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NLRP3 inflammasome activity and microglia-mediated inflammation, NLRP3
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inflammasome activators including LPS (1 µg/ml) plus ATP (1 mM) as well as Aβ25-35
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(5 µM) were used to trigger BV-2 cells. As shown in Figure 4A, BV-2 cells were in
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the ‘resting’ state in the normal culture medium. After stimulation for 4–6 h, BV-2
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cells were in the ‘activated’ state with morphological changes, including bigger cell
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body and more branches. To initiate and amplify the neuroinflammatory response,
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activated microglia not only displayed a characteristic change in cell morphology but
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also showed functional alterations including secretion of proinflammatory cytokines 36.
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IL-1β is one of the main factors mediating inflammatory responses 37. In the present
21
study, mature-IL-1β (IL-1β p17) was detectable by in the supernatant of BV-2
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microglial cell 4 h after LPS plus ATP as well as Aβ25-35 simulation (Figure 4B and
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4C). Intriguingly, IL-1β production requires caspase-1 (CASP1) activation by
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inflammasome, which is a molecular platform responsible for the processing of
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caspase-1 and subsequent maturation and secretion of IL-1β and IL-18
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blot analysis of caspase-1 showed a gradual increase in cleaved-caspase-1 (CASP1
27
p10) after stimulation for 4–6 h and the changes in CASP1 p10 were similar to that of
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IL-1β p17 (Figure 4B and 4C). Together, these data show that inflammasome was
29
distinctly activated 6 h after LPS plus ATP as well as Aβ25-35 stimulation in BV-2 cells.
30
Based on the above cell model, we further investigated the interaction between
35
. In order to study the effect of autophagy on
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. Western
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autophagy and microglial inflammation focusing on the modified NLRP3
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inflammasome activities and LC3-II expression in BV-2 cells. As shown in Figure 4D,
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4G, 4J, and 4K, the level of LC3-II was significantly increased in response to LPS
4
plus ATP as well as Aβ25-35 stimulation as compared to the control. These observations
5
were consistent with the previous reports that inflammatory stimuli could also
6
enhance the autophagic activities
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in the negative feedback regulation thereby, restricting the inflammatory response 6.
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Strikingly, as a response to 3-MA, a significant increase in CASP1 p10 and IL-1β p17
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was observed in activated BV-2 cells as compared to cells treated without 3-MA
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(Figure 4D–K). These results demonstrated that the blockage of microglial autophagy
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amplified NLRP3 inflammasome activation and neuroinflammation. Moreover, the
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protein levels of NLRP3 and precursor of interleukin-1β (pro-IL-1β) were increased
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as a result of 3-MA treatment (Figure 4D–K). These data suggested that blockage of
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autophagy promotes IL-1β processing that is associated with reduced autophagic
15
degradation of NLRP3 inflammasome component and IL-1β precursor.
15, 39
. The enhanced autophagy, in turn, participates
16
ROF-induced autophagy suppressed the activation of NLRP3 inflammasome
17
in BV-2 cells. In our previous in vivo studies, ROF showed an anti-inflammatory
18
effect in APP/PS1 transgenic AD mice27; however, the underlying mechanism was not
19
clarified. Although ROF-induced microglial autophagy was found in the present study,
20
the link between its anti-inflammatory effect and autophagy was yet to be determined.
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Here, we found that ROF significantly increased the level of LC3-II in activated BV-2
22
cells (p
