Secretome from SH-SY5Y APPSwe cells trigger time-dependent CHME3 microglia activation phenotypes, ultimately leading to miR-21 exosome shuttling

Accepted Manuscript Secretome from SH-SY5Y APPSwe cells trigger time-dependent CHME3 microglia activation phenotypes, ultimately leading to miR-21 exosome shuttling Adelaide Fernandes, Ana Rita Ribeiro, Mafalda Monteiro, Gonçalo Garcia, Ana Rita Vaz, Dora Brites PII:

S0300-9084(18)30149-4

DOI:

10.1016/j.biochi.2018.05.015

Reference:

BIOCHI 5427

To appear in:

Biochimie

Received Date: 14 February 2018 Accepted Date: 27 May 2018

Please cite this article as: A. Fernandes, A.R. Ribeiro, M. Monteiro, G. Garcia, A.R. Vaz, D. Brites, Secretome from SH-SY5Y APPSwe cells trigger time-dependent CHME3 microglia activation phenotypes, ultimately leading to miR-21 exosome shuttling, Biochimie (2018), doi: 10.1016/ j.biochi.2018.05.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ABSTRACT Exosome-mediated intercellular communication has been increasingly recognized as having a broad impact on Alzheimer’s disease (AD) pathogenesis. Still, limited information exists regarding their “modus operandi”, as it critically depends on exosomal cargo,

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environmental context and target cells. Therefore, a more thorough understanding of the role of exosomes from different cell types as mediators of neuroinflammation in AD context is a decisive step to open avenues for innovative and efficient therapies. In this study, we demonstrate that SH-SY5Y cells transfected with the Swedish mutant of APP695 (SHSwe)

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remarkably express increased inflammatory markers, combined with higher APP and Aβ1production, when compared to naïve SH-SY5Y (SH) cells. Although exerting an early

clearance effect on extracellular APP and Aβ accumulation when in co-culture with SHSwe

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cells, human CHME3 microglia gradually lose such property, and express both proinflammatory (iNOS, IL-1, TNF-α, MHC class II, IL-6) and pro-resolving genes (IL-10 and Arginase 1), while also evidence increased senescence-associated β-galactosidase activity. Interestingly, upregulation of inflammatory-associated miRNA(miR)-155, miR-146a and miR-124 by SHSwe secretome was shown to be time-dependent and to inversely correlate with their respective targets (SOCS1, IRAK1 and C/EBPα). We report that

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microglia also internalize exosomes released from SHSwe cells, which are enriched in miR155, miR-1464a, miR-124, miR-21 and miR-125b and recapitulate the cells of origin. Furthermore, we show that SHSwe-derived exosomes are capable of inducing acute and delayed microglial upregulation of TNF-α, HMGB1 and S100B pro-inflammatory markers, from which only S100B is found on their derived exosomes. Most importantly, our data

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reveal that miR-21 is a consistent biomarker that is found not only in SHSwe cells and in released exosomes, but also in the recipient CHME3 microglia and their exosomes. This

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work contributes to the increased understanding of neuron-microglia communication and exosome-mediated neuroinflammation in AD, and highlights miR-21 as a promising biomarker/target for therapeutic intervention.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Secretome from SH-SY5Y APPSwe cells trigger time-dependent CHME3 microglia activation phenotypes, ultimately leading to miR-21 exosome shuttling

Adelaide Fernandes1,2, Ana Rita Ribeiro1, Mafalda Monteiro1, Gonçalo Garcia1, Ana

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Rita Vaz1,2, Dora Brites1,2*

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Neuron Glia Biology in Health and Disease, Research Institute for Medicines

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(iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal; 2

Department of Biochemistry and Human Biology, Faculty of Pharmacy, Universidade

*Corresponding author: Dora Brites ([email protected])

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de Lisboa, Lisboa, Portugal.

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Faculty of Pharmacy, Universidade de Lisboa Avenida Professor Gama Pinto

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Portugal

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1649-003 Lisboa

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ACCEPTED MANUSCRIPT Highligths:

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SHSwe cells show increased inflammatory-associated markers

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CHME3 microglia activation occurs by early exposure to SHSwe cells

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• SHSwe cells induce delayed upregulation of pro-resolving markers in CHME3 microglia Exosomes from SHSwe cells recapitulate their inflamma-miRNA profile

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• Exosomes from SHSwe cells rise S100B/HMGB1/TNF-α mRNA and miR-21 in CHME3 microglia

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ACCEPTED MANUSCRIPT Abstract (300 words)

Exosome-mediated intercellular communication has been increasingly recognized as having a broad impact on Alzheimer’s disease (AD) pathogenesis. Still, limited information exists regarding their “modus operandi”, as it critically depends on exosomal cargo, environmental context and target cells. Therefore, a more thorough

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understanding of the role of exosomes from different cell types as mediators of neuroinflammation in AD context is a decisive step to open avenues for innovative and efficient therapies. In this study, we demonstrate that SH-SY5Y cells transfected with the Swedish mutant of APP695 (SHSwe) remarkably express increased inflammatory

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markers, combined with higher APP and Aβ1-40 production, when compared to naïve SH-SY5Y (SH) cells. Although exerting an early clearance effect on extracellular APP and Aβ accumulation when in co-culture with SHSwe cells, human CHME3 microglia

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gradually lose such property, and express both pro-inflammatory (iNOS, IL-1, TNF-α, MHC class II, IL-6) and pro-resolving genes (IL-10 and Arginase 1), while also evidence increased senescence-associated β-galactosidase activity. Interestingly, upregulation of inflammatory-associated miRNA(miR)-155, miR-146a and miR-124 by SHSwe secretome was shown to be time-dependent and to inversely correlate with their respective targets (SOCS1, IRAK1 and C/EBPα). We report that microglia also

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internalize exosomes released from SHSwe cells, which are enriched in miR-155, miR1464a, miR-124, miR-21 and miR-125b and recapitulate the cells of origin. Furthermore, we show that SHSwe-derived exosomes are capable of inducing acute and delayed microglial upregulation of TNF-α, HMGB1 and S100B pro-inflammatory

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markers, from which only S100B is found on their derived exosomes. Most importantly, our data reveal that miR-21 is a consistent biomarker that is found not only in SHSwe

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cells and in released exosomes, but also in the recipient CHME3 microglia and their exosomes. This work contributes to the increased understanding of neuron-microglia communication and exosome-mediated neuroinflammation in AD, and highlights miR21 as a promising biomarker/target for therapeutic intervention.

Keywords (max 6): Alzheimer’s

disease;

Exosomes;

Inflammatory-associated

miRNAs;

Microglia

activation; Neuroinflammation; Neuron-microglia communication

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ACCEPTED MANUSCRIPT Abbreviations: AB/AM, Antibiotic/antimycotic; AD, Alzheimer’s disease; ALS, Amyotrophic lateral sclerosis; Apo E, Apolipoprotein E; APP, Amyloid precursor protein; Arg1, Arginase 1; Aβ, Amyloid-beta; BBB, Blood-brain barrier; BSA, Bovine serum albumin; CEBP-α, CCAAT/Enhancer-Binding Protein alpha; CNS, Central nervous system; DAMPs,

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Danger-associated molecular patterns; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, Enzyme-Linked Immunosorbent Assay; FBS, Foetal bovine serum; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; HMGB1, High-mobility group box 1; IL, Interleukin; iNOS, inducible Nitric Oxide Synthase; IRAK1, Interleukin-1 ReceptorAssociated Kinase 1; LC3, Microtubule-associated protein 1A/1B-light chain 3; L-glu, L-

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glutamine; MHC class II, Major Histocompatibility Complex – class II; miR, MiRNA; miRNAs, MicroRNAs; mRNA, Messenger RNA; MVB, Multivesicular bodies; NTA, Nanoparticle Tracking Analysis; PBS, Phosphate buffer saline; qRealTime-PCR,

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quantitative real time-polymerase chain reaction; RA, Retinoic acid; RAGE, Receptor for advanced glycation end-products; RT, Room temperature; S100B, S100 calciumbinding protein B; SA-β-gal, Senescence-associated β-galactosidase; SH, SH-SY5Y; SHSwe, SH-SY5Y expressing APP695 Swedish mutation; SOCS-1, Suppressor Of Cytokine Signalling 1; TGF-β, Transforming growth factor-β; TLR, Toll-like receptor; TNF-α, Tumour Necrosis Factor-alpha; RAGE, Receptor for Advanced glycation End-

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products; S100B, S100 calcium-binding protein; SEM, Standard Error of Measurement;

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Swe, Swedish; wt, Wild type.

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ACCEPTED MANUSCRIPT 1. Introduction Alzheimer’s disease (AD) is the most common, progressive and irreversible neurodegenerative disorder, characterized by memory loss, cognitive impairment and behavioral abnormalities. Following advanced age, family history is the second major risk factor for AD whereas the presence of specific genetic mutations correlate with enhanced susceptibility to develop rare early onset familial AD (~45-65 years) or most

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commonly late onset AD (> 65 years) [1, 2]. Among those, the Swedish (Swe) mutation is a specific modification in the amyloid precursor protein (APP) gene, at the APP βsecretase cleavage site (codons 595 and 596 in APP695), making APP a preferable substrate for β-secretase. Consequently, it occurs the enhancement of the

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amyloidogenic processing of APP leading to the secretion of exacerbated amounts of Amyloid-β (Aβ) forms and abnormal intracellular Aβ accumulation [3, 4], one of the hallmarks of AD.

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Several authors have used primary neuronal cultures or neuroblastoma cell lines bearing this Swe mutation as AD cell models, namely to study APP processing [5] or the impact of different agents in this phenomenon [6]. However, a more complete characterization of the role of the Swe mutation in neuronal response has not been reported. Most attractively, it has been described that microglial activation enhance Aβ burden either by supporting APP amyloidogenic processing or due to inefficient Aβ

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degradation [7]. On the other hand, we showed that the presence of Aβ in different assembly states interacts with microglia leading to an inflammatory response in young cells that is lost in aged ones, suggesting a differential response along the progression of AD disease [8]. So it becomes crucial to understand the interplay between neurons

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and microglial cells in AD pathogenesis.

Neuron-glia communication can be mediated through direct cell-to-cell contact, by the action of secreted molecules or by extracellular vesicles [9]. Exosomes, small

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vesicles (40–100 nm) derived from multivesicular bodies (MVB) [10], have gain an increasing relevance in AD pathogenesis [11]. Exosomes are secreted from almost all types of cells, including central nervous system (CNS) ones [12] and may be retrieved in vitro in culture media or in vivo in several body fluids [13]. Exosomes have a particular composition due to their endosomal origin containing membrane transporter and fusion proteins, cytoskeleton, lipids and genetic material including significant amounts of microRNAs (miRNAs) as well as messenger RNA [14]. In AD context, it has been described that Aβ accumulates in MVB and is released to the extracellular space via exosomes [15], while specific exosome proteins have been found surrounding the senile plaques [16]. Moreover, besides Aβ, also APP and β-secretase have been detected in exosomes [17], suggesting that exosomes may be involved in AD 5

ACCEPTED MANUSCRIPT spreading not only by shuttling Aβ but also by being able to process APP exacerbating long distance Aβ load. However, no further characterization of this exosome content has been described. On the other hand, it has been shown that exosomes, although promoting Aβ amyloidogenesis in extracellular space, facilitate microglia incorporation of Aβ fibrils in a phosphatidylserine-dependent manner leading to Aβ degradation [18]. Yet, the

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response of microglia to these exosomes or Aβ was not further explored. To note, we have previously shown that, in an amyotrophic lateral sclerosis neuron-microglia coculture system, microglia are the main recipient of exosomes, and that exosomes derived from mutated motor neurons induce a pro-inflammatory microglia phenotype

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[19]. So, here we sought to first characterize the response of human neuroblastoma SH-SY5Y cells expressing the APP695 with Swedish mutation (SHSwe) in terms of inflammatory markers and second to assess how human microglia CHME3 would

2. Material and methods

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respond to SHSwe secretome and in particular to SHSwe derived exosomes.

2.1. Culture of neuroblastoma cell line and differentiation

Human neuroblastoma cells SH-SY5Y (SH), SH-SY5Y stably expressing wild-type

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APP695 (SHwt) and SH-SY5Y stably expressing APP695 Swedish mutation (SHSwe) were a gift from Professor Anthony Turner [5]. Cells were cultured in T75, in Dulbecco Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Biochrom) and 2% Antibiotic/Antimicotic (AB/AM,

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Sigma-Aldrich). Cells were maintained in a humidified atmosphere containing 5% CO2 at 37ºC and medium changed every 2 to 3 days. For experiments, cells were seeded at

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a final concentration of 5x104 cells/well onto 12-well plates coated with poly-D-lysine (100 µg/mL, Sigma-Aldrich) and laminin (4 µg/mL, Gibco). After 24h proliferation, differentiation into a neuronal phenotype was induced by adding retinoic acid (RA, Sigma-Aldrich) at a final concentration of 10 µM in culture medium and maintaining cells for 7 days, as previously described [20]. RA-containing culture medium was changed every 2 days. Then, prior to co-culture medium was changed to DMEM basal medium (FBS free).

2.2. Culture of human CHME3 microglia cell line Human CHME3 microglial cells were kindly provided by Professor Marc Tardieu [21]. Cells were cultured in T75 culture flasks in DMEM supplemented with 10% FBS, 6

ACCEPTED MANUSCRIPT 2% AB/AM (Sigma-Aldrich) and 1% L-glu (Sigma-Aldrich) in a humidified atmosphere containing 5% CO2 at 37ºC. Medium was changed every 2 to 3 days. For co-cultures, cells were seeded onto 12-well non-coated plates containing hydrochloric acid-washed coverslips with 3-4 paraffin dots at a final concentration of 5x104 cells. Prior to coculture medium was changed to FBS-free medium, similarly to neuroblastoma cells.

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2.3. Neuroblastoma-microglia co-culture

At day 8 of neuroblastoma differentiation, CHME3 cells seeded onto paraffin dotscontaining coverslips were placed on top of RA-differentiated SH and SHSwe containing wells, in order to establish co-cultures for 24 h, 48 h and 72 h in DMEM with 1% L-glu

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and 2% AB/AM. Because no differences were obtained between SH and SHwt relatively to the upregulation of inflammatory miRNAs, iNOS or M2 markers in microglia, further

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experiments with SHwt cells were abandoned, despite their ability to generate mature and immature APP695 and to release sAPPα and Aβ1-40 into the extracellular media, although in lower levels than SHSwe cells do.

2.4. Isolation, characterization and labeling of exosomes

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Exosomes were isolated from cell media of SH, SHSwe and CHME3 cells as previously described [2]. Briefly, cell supernatant was centrifuged at 1 000 g for 10 min to pellet cell debris, then supernatant was transferred into another tube and centrifuged at 16 000 g for 1 hour, to pellet microvesicles. Next, remaining supernatant was filtered

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in a 0.22 µm pore filter to remove particles above 220 nm. Thereafter, the suspension was ultra-centrifuged at 100 000 g for 2 hours, using a Beckman OptimaTM L-100 XP ultracentrifuge, with a type 90 Ti rotor (fixed angle), from Beckman Coulter, and

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washed once in phosphate buffer saline (PBS). Transmission electron microscopy (TEM) technique used the Jeol JEM 1400 Transmission Electron Microscope (Peabody, MA, USA). To analyze size distribution and concentration of exosomes, collected particles were subjected to the Nanoparticle Tracking Analysis (NTA) using the NanoSight equipment (Malvern). To monitor the incorporation of neuronal-derived exosomes by CHME3 cells, exosomes were labeled using the PKH67 Fluorescent Linker Kit (Sigma Aldrich), according with manufacturer specifications, resuspended in DMEM with 1% AB/AM and incubated on CHME3 microglia cultures. Fluorescence was visualized using an AxioCam HRm camera adapted to an AxioSkope® microscope (Zeiss). Green-fluorescence images of ten random microscopic fields (original

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ACCEPTED MANUSCRIPT magnification: 400X) were acquired per sample and exosome fluorescence intensity measured using ImageJ® software. Results are presented as arbitrary units (± SEM).

2.5. Evaluation of cell viability To determine viability from adherent and floating cells, these were collected from

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culture medium or detached with trypsin, mixed and spun down at 500 g for 5 min. Pellet was resuspended in 1% bovine serum albumin (BSA) in PBS and stained with phycoerythrin-conjugated annexin V (V-PE) and 7-amino-actinomycin D (7-AAD), using the Guava Nexin Reagent (Milipore). Stained cells were analyzed on a flow cytometer (Guava easyCyte 5HT, Merck-Millipore) using the Guava Nexin Software. Four cellular

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populations were distinguished: viable cells (V-PE and 7-AAD double-negative), earlyapoptotic cells (V-PE positive and 7-AAD negative), late stages of apoptosis cells (V-

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PE and 7-AAD double-positive) and necrotic/debris (annexin V-PE negative and 7-AAD positive).

2.6. Detection of Aβ by enzyme-linked immunosorbent assay

Determination of Aβ1-40 and Aβ1-42 release by neuroblastoma cells was performed

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by enzyme-linked immunosorbent assay (ELISA) using Human Amyloidβ (1-40) Assay Kit (IBL, 27713) and Human Amyloidβ (1-42) Assay Kit (IBL, 27711), respectively. All the procedures were performed in accordance to the manufacturer’s guidelines and absorbance was measured at 450 nm in a Bio-Rad microplate spectrophotometer (Bio-

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Rad) and results are presented as pg/ml.

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2.7. Microglial phagocytic capacity To evaluate CHME3 microglial phagocytic capacity, cells were incubated for 75 min at 37ºC with 0.0025% (v/v) 1 µm fluorescent latex beads (SigmaChemical) at 24 h, 48 h and 72 h post co-culturing with neuroblastoma cells and then fixed with 4% (w/v) paraformaldehyde in PBS, according with previous studies from our lab [2,3]. Nuclei were stained with Hoechst dye, and fluorescence was visualized under 630x magnification in an AxioSkope.A1 fluorescence microscope coupled with AxioCam HR camera (Zeiss). At least ten random images from different microscopic fields were acquired per sample. Bright field images were also captured to co-localize beads within the microglial cytoplasm. The number of ingested beads per cell was counted and data

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ACCEPTED MANUSCRIPT ranked into three classes, presented as number of cells that phagocytosed [≤ 5], [6-10] or [> 10] beads, respectively.

2.8. Cellular senescence determination CHME3 microglial senescence was analyzed by measuring the activity of

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senescence-associated β-galactosidase (SA-β-gal) using the Cellular senescence assay kit (#KAA002RF, Millipore), according to the manufacturer instructions. Hematoxylin was used to counterstain the microglial nuclei. At least ten bright field images per sample were acquired using a Leica DC 100 camera (Leica, Wetzlar, Germany) adapted to an Axioskop microscope (Zeiss, Germany). The number of

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turquoise stained microglia (SA-β-gal-positive cells) was counted using ImageJ® software, and results are presented as percentage of senescent cells as previously

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described [22].

2.9. Lysosome labeling

To track microglial lysosomal activity, cells were labelled with LysoTracker™ Red DND-99 fluorescent probe for 30 min after incubation and fixed with 4% (w/v) paraformaldehyde in PBS, and nuclei were stained with Hoechst 33258 dye (1:1000 in

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PBS). Fluorescence was visualized as described above. Pairs of U.V. and redfluorescence images of ten random microscopic fields (original magnification: 400X) were acquired per sample. Lysotracker fluorescence intensity was measured using

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ImageJ® software, and results are presented as arbitrary units.

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2.10. Protein extraction and western blot analysis Protein content secreted into culture media by neuroblastoma cells was collected before co-culturing (0 h) and after 24 h, 48 h and 72 h of co-culture with CHME3 microglia. Total protein precipitation was performed by adding 10% trichloroacetic acid (TCA) in acetone, followed by 2-4 washing cycles with acetone containing 20 mM Dithiothreitol (DTT) and centrifuged at 15000 g for 10 min. Protein pellet was dissolved in a buffer containing 8M urea, 1% SDS (1:1) and proteases inhibitor (1:25) and resuspended by sonication, followed by centrifugation at 3200 g for 10 min to remove insoluble particles. For intracellular protein content, cells were collected in Cell Lysis Buffer® (Cell Signaling) and protein concentration measured as usual in our lab [8]. Equal amounts of protein were separated in Tris-Tricine gel, transferred into 9

ACCEPTED MANUSCRIPT nitrocellulose membranes (Amersham) and incubated in blocking buffer [5% (w/v) nonfat dried milk in Tween 20 (0.1%) tween-tris buffer saline (T-TBS)] at room temperature for 1 h. Membranes were incubated at 4ºC overnight with primary antibody 6E10 antibody (mouse, 1:200, BioLegend) diluted in T-TBS and 5% BSA. Then, membranes were incubated with secondary antibody HRP-conjugated anti-mouse (1:2000, Santa Cruz Biotechnology) diluted in blocking buffer at room temperature for 1 h.

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WesternBright™ Sirius (Advansta) was used as chemiluminescent substrate and signal acquired in ChemiDoc Imaging System (Bio-Rad). Relative intensity of protein bands was analyzed using the Image Lab analysis software (Bio-Rad Laboratories).

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2.11. Total RNA extraction, reverse transcription and RealTime-PCR

Inflammatory gene and miRNA expression levels were determined by quantitative

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real time-PCR (qRealTime-PCR) according with previous studies [8]. Total RNA was extracted from CHME3, SH or SHSwe using TRIzol® reagent according to the manufacturer instructions. Extracted RNA was quantified using Nanodrop® ND-100 Spectrophotometer (NanoDrop Technologies). For mRNA expression, total RNA was reverse transcribed using the SensiFAST cDNA Synthesis Kit (Bioline), under manufacturer’s instructions and qRT-PCR conditions were: 50ºC for 2 min, 95ºC for 2 min followed by 40 cycles at 95ºC for 5 s and 62ºC for 30 s. The specificity of

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amplification was ensured by melt-curve analysis after the amplification protocol (95ºC for 15 s, followed by 60ºC for 30 s and 95ºC for 15 s). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as reference. Primer sequences are listed in Supplementary Table 1. Regarding miRNA expression, cDNA was synthetized using

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Universal cDNA Synthesis Kit (Exiqon, Woburn, MA, USA. For qRT-PCR, miRCURY LNA™ Universal RT microRNA PCR Kit (Exiqon) was used with predesigned (Exiqon)

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primers (Supplementary Table 2) to target specific miRNA sequences. U6 and a kit spike in were used as references. qRealTime-PCR was performed on a 7300 RealTime PCR System. Each sample was performed in duplicate, and a non-template control was included for miRNA analysis. The expression fold change vs. respective controls was determined by the 2-∆∆CT method.

2.12. Statistical analysis Results were expressed as mean ± SEM. Comparisons between two different groups were performed using one-tailed Student's t-test for equal or unequal variance,

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ACCEPTED MANUSCRIPT as appropriate. Analysis was performed in PRISM 7.0 software (GraphPad Software)

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and p < 0.05 was considered statistically significant.

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ACCEPTED MANUSCRIPT 3. Results 3.1. Differentiated SHSwe show increased inflammatory mediators and elevated production of APP and Aβ1-40 that do not compromise cell viability Human neuroblastoma SH-SY5Y cell line (SH) is an immortalized and proliferative cell line obtained from a bone marrow biopsy of a neuroblastoma patient [23]. These

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cells acquire a neuronal phenotype after differentiation with retinoic acid, which induces an extensive outgrowth of neurites, the expression of synaptic proteins, and in general, higher tolerance by up-regulating survival signaling [24]. SH cells transfected with the single-mutant amyloid precursor protein (APP) Swedish mutation (SHSwe) have been

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frequently used to investigate the pathological events leading to AD, the aberrant Aβ production, and the therapeutic potential of promising compounds [5, 25, 26]. As depicted in Figure 1A, SHSwe cells differently express common inflammatory mediators,

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such as S100B and HMGB1 when compared to SH cells (6.3-fold and 1.9-fold, respectively, p < 0.05). In addition, besides the upregulation of RAGE mRNA expression (2.2-fold, p < 0.05), which is a S100B and HMGB1 receptor, as well as an Aβ peptide transporter [27], we also observed an elevated gene expression of the first line pro-inflammatory cytokine TNF-α (4.6-fold, p < 0.05).

Previous works have documented that the transcriptionally active APP intracellular

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domain is preferentially produced from the 695 isoform of APP [5]. These Authors confirmed that SHSwe cells show increased Aβ levels, as compared to SHwt cells. Other studies additionally demonstrated that the increase in APP was smaller in SHwt cells than in SHSwe cells and that Aβ secretion was much higher for Aβ1-40, than for Aβ1-42 [6].

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Therefore, we then evaluated the expression of APP and the secretion of Aβ by SH, SHwt and SHSwe cells after 24 h incubation. In SHSwe cell lysates we observed the presence of both mature and immature APP695 forms, the last almost absent in SH

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cells, based on the western blot analysis using the 6E10 antibody (Fig. 1B). Similar results were obtained for SHwt cells (data not shown). In the cell culture media, we detected the presence of sAPPα with the same band molecular weight distribution as in cells, confirming the predominant mature form in SH supernatants and the immature one in those of SHSwe cells. Again, similar results were obtained for SHwt cells (data not shown). When we evaluated secreted Aβ by ELISA, we observed increased levels for Aβ1-40, but not for Aβ1-42, in the supernatants of SHSwe cells, as compared with those of SH cells (at least 10-fold, p < 0.05) (Fig. 1B,C). Similar data, although at lower levels, were obtained for SHwt cells (data not shown). Thus, increased levels of immature APP and Aβ1-40 are produced and released by the mutated cells, as compared to naïve ones. However, by using the Guava Nexin® Reagent, as described in Material and 12

ACCEPTED MANUSCRIPT methods section, we observed that the APPSwe mutation did not significantly influence cell viability relatively to the naïve cells (Fig. 1D). Therefore, we hypothesized that, although not affected in viability, SHSwe cells behave as stressed neurons, unable to manage excessive APP695 accumulation and Aβ1-40 release, and showing upregulated inflammatory markers.

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3.2. Preventive effects of human CHME3 microglia on the extracellular accumulation of sAPPα and Aβ1-40, when co-incubated with differentiated SHSwe cells, are followed by increased levels that course with elevated microglial SA-β-gal activity

Microglia were shown to extensively participate in the clearance of Aβ [28].

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Therefore, before evaluating the effects produced by the SHSwe secretome on CHME3 microglia, we decided to assess whether APP and Aβ1-40 levels in the cell culture media

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were modified by microglia presence after 24 h, 48 h and 72 h of co-culturing. When comparing to the results obtained for pure neuroblastoma cultures (Fig. 1B), we found that both the single-band of APP expression in SH cells and the two-band pattern of APP expression in SHSwe cells were retained, although with slight differences along time (Fig. 2A). However, we noticed a marked reduction in sAPPα signal from incubation medium after 24 h post co-culturing neuroblastoma cells with CHME3 microglia, as compared with the one previously indicated for the neuroblastoma cell

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cultures in the absence of microglia (Fig. 1B), though an increased accumulation in a time-dependent manner was later observed. Similarly, when we evaluated secreted Aβ1-40 we observed that the levels of this peptide in the incubation media of CHME3/SHSwe co-cultures were also efficiently reduced at 24 h, if compared with the

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330 pg/mL detected in SHSwe cultures (p < 0.01) (Fig. 1B). Nevertheless, accumulation of Aβ1-40 occurred in CHME3/ SHSwe incubation media, as compared with matched

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controls, after 48 h and 72 h co-incubation periods (p < 0.01) (Fig. 2B), indicating a saturated efficacy of microglia in Aβ clearance. Defective microglial clearance mechanisms have been indicated by several studies

and are believed to result from imbalance between Aβ production and removal [29, 30]. To note, however, that CHME3 microglia, when co-incubated with SHSwe cells, only showed a small and not significant reduction in the phagocytic ability to ingest ≤ 5 beads per cell, (2.0% at 24 h, 4.5% at 48 h and 2.1% at 72 h, data not shown), in comparison with microglia co-incubated with SH cells. Interestingly, brains of AD patients were shown to harbor microglia that appear to be senescent and dysfunctional [31, 32], and we have recently demonstrated that Aβ upregulates senescenceassociated biomarkers in primary cortical microglia isolated from mouse pups [8]. 13

ACCEPTED MANUSCRIPT Accordingly, exposure to SHSwe cells that showed to release elevated amounts of Aβ triplicated the number of CHME3 senescent-like cells, as compared to microglial cells exposed to the naïve SH, at 48 h (p < 0.05) and 72 h (p < 0.01) of co-incubation periods (Fig. 2C). Such findings corroborate our previous data supporting that 1 µM of mixed Aβ1-42 oligomers and fibrils enhance the number of SA-β-gal positively stained microglia [8].

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These results confirm the participation of microglia in the early elimination of APP and Aβ from the cell culture media, being such protective property somehow lost in the chronic stressed condition, where a significant extracellular accumulation of Aβ1-40 is

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observed.

3.3. Secretome from SHSwe cells activate human CHME3 microglia leading to

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upregulation of concurrent pro-inflammatory and pro-resolving genes

Most of published studies evaluated the influence of microglial activation on neuronal function in AD [33-35] and only a few centered the attention on the involvement of dysfunctional neurons and their secretome on microglia induced phenotypic alterations [19, 36]. Given the marked expression of inflammatory markers by SHSwe cells, when compared to naïve SH, we next decided to evaluate the response of human CHME3 microglia response to their secretome using a co-culture system.

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As depicted in Figure 3, CHME3 microglia co-cultured with SHSwe markedly overexpress the innate immune marker iNOS at 24h, when compared to CHME3 cocultured with SH, that was maintained for prolonged time-points although with a lower magnitude (p < 0.01, for all time points, Fig. 3A). A marked impact of SHSwe secretome

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was also observed in the gene expression of the pro-inflammatory cytokines IL-1β and TNF-α by the CHME3 microglia, with the highest levels being achieved at 72 h of co-

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incubation (at least p < 0.05, Fig. 3A). To note that IL-6, considered to be a late cytokine with both pro- and anti-inflammatory properties [37] was found elevated only at 72 h of co-incubation with SHSwe cells when compared with co-incubation with SH cells. Then, we focused on the gene expression of the adaptive immune marker MHC class II, which revealed to be upregulated in CHME3 microglia co-cultured with SHSwe cells at all time points (at least p < 0.05, Fig. 3A) and exhibit a profile close to that of TNF-α. Considering the involvement of Arginase 1 positive microglia in Aβ plaque reduction upon IL-1β stimulation [38], we next evaluated this marker of alternatively activated microglia. Secretome from SHSwe cells led to a sustained increase of Arginase 1 mRNA expression by microglial CHME3 cells for all time points (at least p < 0.05, Fig. 3B), probably to reverse the M1 phenotype into a M2 one. Therefore, we then 14

ACCEPTED MANUSCRIPT assessed IL-10 expression, a cytokine known to counteract inflammation [39]. Interestingly, we also observed that IL-10 mRNA expression increased along the incubation time in the microglia co-cultured with the SHSwe cells when compared to cells co-cultured with SH (p < 0.01, Fig. 3B), possibly counteracting M1 cytokine expression as an attempt to re-establish homeostasis. These results highlight the presence of

in different stages of AD progression [34, 40].

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heterogeneous microglia population upon exposure to SHSwe secretome, as described

3.4. Secretome from SHSwe cells impact on the sequential expression of inflammatory-

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related miRNAs and their targets in human CHME3 microglia

To further characterize the microglia population upon the interaction with the SHSwe

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cells, we evaluated the expression of inflammatory-associated miRNAs (in short inflamma-miRs) known to influence microglia activation states [41], as well as some of their directed targets. One miRNA mostly associated with microglia activation is miR155, recognized as essential for IL-6 induction [42] and SOCS-1 downregulation [43]. We observed that miR-155 was preferentially upregulated in CHME3 microglia cocultured with SHSwe at 24 h (near 2-fold, p < 0.01) as depicted in Figure 4A. Curiously, instead of being repressed, SOCS-1 was more than 5-fold upregulated in CHME3

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exposed to SHSwe secretome at 24 h (p < 0.01, Fig. 1B), with a continuous increase for later time points (p < 0.01), where CHME3 expression of miR-155 was lower. So it is possible that other miRNAs may be regulating SOCS-1 expression rather than miR155, which may account for the elevation of the pro-resolution mediators previously

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observed (Fig. 3). Since miR-155 may also target other molecules such as the C/EBPβ [44], we also assessed its mRNA expression, but no significant changes were

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obtained (data not shown).

MiR-146a is known to mediate CNS inflammation [41] and to be increased in N9 microglia upon incubation with the Aβ peptide [45]. Alike miR-155, we observed its upregulation, but only at 24 h incubation (p < 0.01, Fig. 4A). Since its expression was described to be inversely correlated with IRAK1 we further assessed the expression of this adaptor protein in the microglia incubated with the SHSwe cells. We noticed that IRAK1 expression was significantly increased in CHME3 microglia from 48 h onward upon SHSwe secretome exposure (p < 0.01, Fig. 4B), when levels of miR-146a have normalized (48 h), or even decreased (72 h, p < 0.05) when compared to microglia cocultured with SH naïve cells. However, no effects were produced in TRAF6, also indicated as its direct target [46] (data not shown). 15

ACCEPTED MANUSCRIPT Considering the early upregulation of miR-155 and miR-146a, we next evaluated the expression profile of miR-124 known to modulate microglia polarization toward the M2 phenotype via the C/EBP-α pathway [47]. We observed that expression of miR-124 was decreased in CHME3 microglia exposed to SHSwe secretome at 24 h (p < 0.05, Fig. 4A), but significantly increased at 48 h (p < 0.05), and even more at 72 (p < 0.01, Fig. 4A). In agreement with this profile, gene expression of the target C/EBP-α was

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only found elevated at 24 h (p < 0.01, Fig. 4B), when miR-124 was elevated. Altogether, our results highlighted that miRNAs and their respective targets correlate inversely to some extent, namely for miR-146a/IRAK1 and miR-124/ C/EBP-α, as expected, suggesting a shift of microglia phenotype from an initial inflammatory state to

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a more anti-inflammatory phenotype.

into their derived exosomes

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3.5. SHSwe cells contain upregulated expression of inflamma-miRs that are transferred

In a previous work we demonstrated that exosomes from NSC-34 motor neuron-like cells transfected with hSOD1-G93A recapitulate the elevated expression of miR-124 in cells and determine alterations in microglia activation phenotype [19]. Exosomes are extracellular vesicles released from cells, which contain a diversity of proteins and

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different nucleic acids, such as miRNAs, that are delivered to neighboring cells [48]. Since dysfunctional exosomal miRNAs were suggested to influence AD progression and to initiate inflammation, their presence in exosomes have been indicated as potential diagnostic markers in AD [49]. Based on aforementioned findings indicating

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that secretome from SHSwe cells determine upregulation of inflammatory mediators and miRNAs in the human CHME3 microglia, in a co-culturing system, we decided to

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evaluate the specific role of neuronal-derived exosomes in such activation profile. For that we isolated exosomes from the extracellular media of SH and SHSwe cells, as previously reported by us [19], and evaluated their morphology by TEM. As shown in Fig. 5A SHSwe–derived exosomes show a more electron-dense material in their content than SH-derived ones. We also analyzed their number and diameter size using the NTA technique to ascertain possible differences in the concentration and/or diameter of these vesicles. No differences were found between exosomes derived from SH and from SHSwe cells relatively to the number of particles/mL or diameter size average (Fig. 5B-C), showing in both samples the presence of two main populations (~100 and 150 nm sorts).

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ACCEPTED MANUSCRIPT Inflamma-miRs have gained special attention due to their ability of modulating cell activation and consequently neuroinflammation [41, 50], as shown above. However, information on the expression of such miRNA in dysfunctional neurons, as such associated to AD, is scarce. Therefore, we assessed their expression in both SH and SHSwe cells, and their presence in respective cell exosomes. As shown in Figure 6, SHSwe cells express significantly higher levels of miR-124 (3.6-fold, p < 0.05), miR-155

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(9.0-fold, p < 0.05), miR-146a (6.5-fold, p < 0.01), miR-21 (7.9-fold, p < 0.05), and miR125b (6.9-fold, p < 0.01), than their counterpart SH naïve cells. Accordingly, exosomal cargo recapitulated such inflamma-miRNA upregulation (miR-124, 11.6-fold, p < 0.01; miR-155, 5.0-fold, p < 0.05; miR-146a, 19.0-fold, p < 0.05; miR-21, 3.4-fold, p < 0.05;

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miR-125b, 3.6-fold, p < 0.05). To note, however, that in contrast with SHSwe cell expression (Fig. 1) their derived exosomes did not show elevated RAGE, S100B or

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TNF-α gene expression, and that HMGB1 was under detection limits (data not shown).

3.6. Exosomes from SHSwe cells that are uptaken by CHME3 microglia, co-localize with lysosomes

Considering that exosomes can be uptaken by neighboring or distant cells, as microglia, and subsequently modulate gene expression on such cells, thus playing an

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important role in disease progression [51, 52], we next evaluated if exosomes were transferred into microglia and, if so, how they impact on cell viability and function/dysfunction, with focus on autophagy-lysosomal effects [53]. So, CHME3 microglia were exposed to exosomes from SH and SHSwe cells for 24 h, and incubated

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for an additional period of 24 h after the renewal of the culture medium to assess possible recovery.

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As shown in Figure 7, exosomes from SH and SHSwe cells were similarly internalized by CHME3 microglia upon 24 h incubation, as indicated by the density of PKH67 (green) labeled exosomes inside the cells (Fig. 7A), namely at the perinuclear area, where staining for lysosomes (LysoTrackerTM, red) are particular evident. However, we observed that CHME3 cells treated with SHSwe-exosomes have a reduced exosomal staining when cultured for an additional period of 24 h after cell medium replacement (p < 0.05) (Fig. 7B). Interestingly, also a significant reduction on the exosome/lysosome co-localization was only observed in CHME3 microglial cells exposed to SHSwe–derived exosomes after post renewed media (p < 0.01) (Fig. 7C). Further, lysosome intensity in CHME3 cells, either exposed to SH- or SHSwe-derived exosomes, markedly decreased following the 24 h of renewed media (p < 0.05), with a 17

ACCEPTED MANUSCRIPT higher effect on SHSwe-exosomes treated microglia (p < 0.01) (Fig. 7D), suggesting a reduced lysosomal activity. Next we evaluated if exosomes, mainly those from SHSwe cells, were able to compromise CHME3 cell viability, by flow cytometry using the Guava Nexin® Reagent. In Figure 8 it is shown that CHME3 microglia exposed during 24 h to exosomes from SHSwe cells have a slight but significant increase in necrotic cells/bodies population (p <

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0.05) when compared to microglia treated with SH-derived exosomes. This increased cell death was maintained after the removal of exosomes and following a 24 h recovery period (p < 0.05), where also late apoptosis was detected (p < 0.05). These results suggest that exosomes derived from SHSwe cells have a slighter toxicity for CHME3

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microglia than those released by SH naïve cells

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3.7. Exosomes from SHSwe cells enriched in inflamma-miRs trigger the expression of inflammatory mediators in CHME3 microglia and lead to exosome-mediated miR-21 shuttling.

In order to understand whether exosomes from SHSwe cells activate microglia in a similar way to their secretome, and if they cause lasting alterations, we evaluated the expression of inflammatory mediators and miRNAs in the recipient CHME3 microglia

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and their derived exosomes.

Our results indicate that microglia is activated by SHSwe-exosomes when evaluated at both time periods, showing elevated gene expression of S100B and HMGB1 (S100B, 2.0-fold and 2.4-fold respectively, p < 0.05; HMGB1, 16.6–fold and 13.9-fold, p

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< 0.01 and p < 0.05, respectively), when compared with microglia response to SHexosomes (Fig. 9A and B). This initial response to exosomes and later reactivity

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following media renewal, although mediated by SHSwe-exosomes, do not directly derive from a transfer of exosomal cargo in such genes to the recipient cells. Indeed, as previously indicated, SHSwe-exosomes did not have increased levels of either S100B or HMGB1 mRNA in contrast with their donor cells (data not shown). We next assessed the RNA cargo of exosomes released by microglia exposed to exosomes and it was only possible to observe an upregulated S100B mRNA (p < 0.05) in vesicles collected from the supernatants of cells previously exposed to SHSwe-exosomes or from the one of cells that were allowed to recover for additional 24 h with fresh media. As before, HMGB1 mRNA was under detection levels. Similarly, TNF-α gene expression was significantly upregulated in CHME3 cells treated with SHSwe-derived exosomes at 24 h and after the additional 24 h recovery period (14.1-fold and 5.9-fold, respectively, p < 18

ACCEPTED MANUSCRIPT 0.01), when compared to CHME3 cells incubated with SH-exosomes. Intriguingly, TNFα mRNA was reduced (p < 0.05) in exosomes released from microglia exposed to SHSwe-exosomes, relatively to their matched controls, at both time periods. Additionally, none of the inflamma-miRs were found overexpressed in the exosometreated microglia, or in their release exosomes (data not shown), except for miR-21. Besides miR-21 upregulation in SHSwe cells and in released exosomes (Fig. 6), SHSwe-

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exosomes exposure also triggered miR-21 expression in CHME3 microglia in a sustained way (Fig. 9A and B). Increased miR-21 expression was found both after the first 24 h incubation period and following the additional period of 24 h recovery (from 0.5 to 1.3-fold and from 0.5 to 1.5-fold, respectively; p < 0.05) when compared to

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microglial cells incubated with SH naïve exosomes. Notoriously, it was recapitulated in the population of exosomes collected from these CHME3 microglia stimulated with SHSwe-exosomes, after exosomal incubation and following a 24 h of fresh media

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incubation (from 0.4 to 1.3-fold and from 0.5 to 1.5-fold, respectively; p<0.01). Overall our study clearly shows that not only SHSwe secretome but also their derived exosomes are able to induce an inflammatory microglia response, further elucidating the role of neuronal derived molecules and vesicles in glia-related neuroinflammation

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during AD course.

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ACCEPTED MANUSCRIPT 4. Discussion

In this study, we demonstrated that SHSwe, besides producing higher levels of APP and Aβ1-40 than SH cells, also expressed increased values of inflammatory markers, and led to a higher reactivity of human CHME3 microglia when in co-culture and a later senescence-associated β-galactosidase activity. Moreover, we showed that CHME3

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microglia was able to internalize exosomes released from neuroblastoma cells that recapitulated miRNAs cargo of the cells of origin, inducing a higher microglia inflammatory response when derived from SHSwe cells. Interestingly, miR-21 was identified as a consistent biomarker expressed not only by SHSwe cells and respective

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exosomes, but also in the recipient CHME3 microglia and derived exosomes.

SHSwe cells have been widely used as AD cellular models [5, 6] and here we further

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demonstrated that these cells are also able to mount an inflammatory response with augmented gene expression of S100B, HMGB1, their receptor RAGE and TNF-α. High levels of S100B was first described to promote neuroblastoma apoptosis [54], while more recently it was described that at nanomolar levels S100B counteracts Aβ-induced neuroblastoma toxicity but when present in micromolar doses S100B exerts an additive toxic effect over that of Aβ [55]. Although we see no marked cell death in our model this

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SHSwe expression of S100B may contribute to the higher microglia reactivity in the coculture system. Furthermore, RAGE expression was previously observed in neurons and related with the generation of ROS and induction of either cell survival and differentiation, or death, depending on the activation intensity [56], whereas its

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activation was already associated with AD pathogenesis (AD) [57]. Expression of TNFα was previously identified in the murine brain cells that expressed neuronal-specific enolase, indicating that in addition to glial cells, neuronal cells also express constitutive

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TNF-α [58]. Moreover, these Authors showed that such neuronal TNF-α expression was increased upon LPS injection, while others obtained similar results during hyperalgesia [59] and ischemia [60], indicative of neuronal response upon injury. Our present data are therefore indicative that SHSwe cells exhibit a response of stressed cells that derive from APPSwe transfection and consequent production of higher levels of sAPP and Aβ1-40. Being phagocytic cells, microglia have been implicated in Aβ clearance [29, 30]. Nevertheless, a study in a transgenic animal model of AD showed that while in young mice microglial efficiently promotes Aβ clearance, in old mice microglia release proinflammatory cytokines in response to Aβ deposition downregulating genes involved in Aβ clearance, leading to Aβ accumulation [61]. In accordance, we observed that with 20

ACCEPTED MANUSCRIPT time CHME3 microglia is no longer able to reduce the levels of both sAPP and Aβ1-40 released by SHSwe to the culture medium, which parallels with an increased production of inflammatory cytokines by these cells. However, no changes were observed in microglia phagocytic abilities as measured at the end of the co-culture period. Also other microglial cell lines were not very effective in showing decreased microglia phagocytic ability upon Aβ interaction [62, 63]. Interestingly, co-culture of CHME3

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microglia with SHSwe induced microglia senescence, which may also justify, at least in part, the reduced clearance of sAPP and Aβ1-40. Indeed, senescent and dysfunctional microglia have been described in brain samples of AD patients [31, 32], and we recently showed that Aβ increases senescence-associated markers in primary cortical

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microglia cultures [8].

Distinct microglia phenotypes have been described in different animal models of AD [40] or even in AD patient samples [64], which may vary with disease progression and

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ageing [40]. In accordance, we observed that SHSwe secretome promotes an upregulation of pro- and anti-inflammatory genes, with a variable expression intensity from 24 to 72 h of co-culture. The simultaneous upregulation of pro- and antiinflammatory markers is characteristic of a microglial intermediate status during a polarization shift (presumably from M1 into M2). Neuronal expression of damageassociated signals, as S100B, HMGB1 and TNF-α that were upregulated in SHSwe

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cells, have been reported to contribute to microglia functional changes leading to a parainflammatory state, which may be triggered to re-establish homeostasis [65]. Additionally, we may hypothesize that multiple microglial subsets may be induced by the co-culture, as this model is closer than what happens in vivo. Indeed, a mixed and

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heterogeneous microglia population is believed to maintain the capacity to change among activation states during AD progression [30, 66, 67].

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Inflamma-miRs, namely miR-155 and miR-124 have been implicated in microglia polarization into M1 and M2 phenotype, respectively [50]. More recently also miR-146a was described in CNS inflammation [41] and to be increased in microglia following Aβ challenge [45]. Our results indicate that CHME3 microglia respond to stress signals from SHSwe cells by acquiring an initial pro-inflammatory behavior, with upregulation of miR-155 and miR-146a, that later shifts to a transition/deactivated state for repairing with miR-124 overexpression, highlighting the prominent role of miRNAs in regulating gene expression during inflammation [41]. Interestingly, we observed an increasing expression of inflammatory cytokines for later time-points where miR-146a is reduced but miR-155 is still elevated, which is in accordance with a previous study showing a

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ACCEPTED MANUSCRIPT dominant function of miR-155 in promoting inflammation overriding miR-146a effect [68]. Besides the inflamma-miRs described above, also miR-21 and miR-125b have gained special attention in neurodegenerative disorders modulating cell activation in a neuroinflammatory context [41, 50]. Yet, only a few reports address the expression of these miRNAs by dysfunctional neurons in relation to neurodegenerative disorders

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[69]. Here we showed that not only SHSwe cells but also their derived exosomes have an increased content of miR-124, miR-146a miR-155, miR-21 and miR-125b in comparison to SH naïve cells and respective exosomes. These results highlight once again a response of stressed cells upon APPSwe transfection that can mediate a

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paracrine effect by a vesicle mediated mechanism.

We observed that microglia is able to uptake both SH- and SHSwe-derived exosomes, in accordance with previous reports showing that microglia internalizes

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exosomes derived from oligodendrocytes [70] and motor neurons [19]. Interestingly these exosomes co-localize with lysosomes as previously reported by others [71, 72], questioning whether exosome internalization will favor intercellular communication or will serve as a means of elimination. Curiously we observed that CHME3 microglia show a reduced lysosome staining, namely if exposed to exosomes from SHSwe cells, upregulated with inflamma-miRs. This may suggest an inhibitory role in lysosomal

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activity, thus compromising lysosome status for protein degradation that may contribute to Aβ accumulation, as proposed for α-synuclein aggregation [73]. In accordance, we also observed a reduction of LC3-II staining in microglial cells exposed to SHSweexosomes (data not shown), as described in APP transgenic mice and associated with

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impaired autophagy and Aβ accumulation [74]. However, further studies should be performed to better understand the effect of SHSwe-exosomes on microglial

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autophagic/lysosomal degradation pathway. Accumulating evidence suggests that secreted exosomes influence bystander cells by the transfer of dysregulated miRNAs and toxic forms of aggregated proteins, such as Aβ, initiating an inflammatory cascade [75]. Previous data from the group using an in vitro ALS model showed that mouse microglial cell line N9 was able to uptake exosomes delivered by SOD1 mutated motor neurons inducing an earlier M1 microglia phenotypes that switched to mixed M1 and M2 subtypes [19]. Here, we also observed that SHSwe–exosomes elicit a CHME3 microglia inflammatory response with the upregulation of S100B, HMGB1 and TNF-α gene expression, indicative of M1 phenotype. To note that this mRNAs were not found in SHSwe–exosomes suggesting

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ACCEPTED MANUSCRIPT that other types of exosomal cargo may be triggering this exosome-mediated microglia activation. Earlier studies also demonstrated that upon ATP stimulation, microglia release extracellular vesicles containing IL-1β and caspase-1 [76]. In addition, in an experimental model of multiple sclerosis it was shown that the number of exosomes increase relative to microglia activation, and that some of them may have a beneficial

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action protecting against disease [77]. Here we observed that exosomes released by CHME3 microglia exposed to SHSwe–exosomes contain increased levels of S100B mRNA but decreased values of TNF-α mRNA, and no upregulated miRNA except for miR-21. MiR-21 is one of the most common miRNAs indicated to be a key mediator of

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the anti-inflammatory response [78] and a negative regulator of toll-like receptor (TLR)4 signalling [79]. Recent studies have highlighted the beneficial role of microglia miR-21 in protecting neurons from cell death in hypoxia/ischemia conditions [80]. However, it

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was reported harmful effects of exosomal miR-21 in causing neurotoxicity via TLR signalling, during simian immunodeficiency virus induced CNS disease [81]. In a model of traumatic brain injury, increased levels of miR-21 were found in neurons of the injury boundary zone together with reactive microglia, as well as in extracellular vesicles isolated from the brain after controlled cortical impact [82]. Authors suggested that vesicles containing miR-21 may transmigrate into adjacent microglia and cause their

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activation. Here we observed that miR-21 upregulation is constantly upregulated in SHSwe cells and derived exosomes, and in CHME3 microglia in response to both SHSwe secretome and exosomes, being further present in microglia released exosomes, which

pathogenesis. 5. Conclusion

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may indicate an important role in neuron-microglia communication during AD

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Overall, our study demonstrates that SHSwe cells, showing increased immature APP695 and release of sAPPα and Aβ1-40, naturally express high inflammatory markers, if compared with SH cells. Curiously, we show that co-culture of SHSwe cells with CHME3 microglia determines upregulation of pro- (iNOS, IL-1β, TNF-α, MHC class II), anti- (IL-10 and Arginase 1) and pro-/anti-inflammatory (IL-6) gene associated markers. Simultaneous microglial elevation of miR-155, miR-146a and miR-124 further corroborates the existence of mixed activated cell subsets after interaction with SHSwe cells. Further, we were able to identify the coincident increase of miR-124, miR-146a miR-155, miR-21 and miR-125b in both SHSwe cells and derived exosomes, in opposing to SH naïve cells. Interestingly, once internalized, SHSwe-derived exosomes co-localize with microglial lysosomes, while causing a sustained microglia sensitization mediated 23

ACCEPTED MANUSCRIPT by overexpression of pro-inflammatory gene markers. Intriguingly, excluding miR-21, no other inflammatory-associated miRNAs were induced in microglia or in their derived vesicles by SHSwe-exosomes, indicating a potential role in dysfunctional neuronmicroglia signalling associated to AD pathological processes. Such promising data should be confirmed in neurons and microglia derived from induced pluripotent stem cells generated from patients with AD, which were shown to recapitulate several

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pathological features of AD in vitro [83], to establish if miR-21 is a common biomarker or a first-line standard of a specific group of AD patients. With this work, we contribute for a more integrative overview of the AD pathogenesis by unveiling relevant aspects of exosome trafficking from neurons to microglia, as well as by disclosing miR-21 as a

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potential biomarker or therapeutic target, which may lead to advances in the development of AD personalized medicine.

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Conflict of interest

Authors have no conflicts of interest to declare.

Acknowledgements

This work was supported by the EU Joint Programme-Neurodegenerative Disease

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Research (JPND) project with funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 643417 and Fundação para a Ciência e Tecnologia (FCT), Lisboa, Portugal: JPco-fuND/0003/2015 to DB, FCT-EXPL/NEU-NMC/1003/2013 to AF and, in part, UID/DTP/04138/2013 to ARV

is

recipient

EP

iMed.ULisboa.

of

a

postdoctoral

research

fellowship

(SFRH/BPD/76590/2011) and GG of a doctoral grant (FRH/BD/128738/2017) from

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FCT. The funding organization had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Supplementary Table 1 - List of primer sequences used to assess gene expression in qRealTime-PCR Forward primer (5’-3’)

Reverse primer (5’-3’)

Arg1

TGGAAACTTGCATGGACA

AAGTCCGAAACAAGCCAA

CEBP-α

CAAAGCCAAGAAGTCGGTGGACAA

TCATTGTGACTGGTCAACTCCAGC

GAPDH

CGCTCTCTGCTCCTCCTGTT

CCATGGTGTCTGAGCGATGT

IL-10

CCTGGAGGAGGTGATGCCCCA

CCTGCTCCACGGCCTTGCTC

IL-1β

GGGCCTCAAGGAAAAGAATC

TTCTGCTTGAGAGGTGCTGA

IL-6

ATGAACTCCTTCTCCACAAGC

GTTTTCTGCCAGTGCCTGTTTG

iNOS

TCCGAGGCAAACAGCACATTCA

IRAK1

CTGGAAGGCAGAAAAGTTGG

MHC class II

AGGGATTGCGCAAAAGCA

SOCS1

TCCGTTCGCACGCCGATTAC

TCAAATCTGGAAGGGGAAGG

TNF-α

AACCTCCTCTCTGCCATC

ATGTTCGTCCTCCTCACA

RAGE

TTCACGAAGTTCCAAACAGGT

GTTCTAGGACGACTGGGGTG

S100B

GAGAGAGGGTGACAAGCACAA

GGCCATAAACTCCTGGAAGTC

HMGB1

CTCAGAGAGGTGGAAGACCATGT

GGGATGTAGGTTTTCATTTCTCTTTC

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Gene

GGGTTGGGGGTGTGGTGATGT TGTGACTCACGGCTGAACAC

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Arg1, Arginase 1; CEBP-α, CCAAT/Enhancer-Binding Protein alpha; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; IL-10, Interleukin-10; IL-1β, Interleukin-1beta; IL-6, Interleukin-6; iNOS, inducible Nitric Oxide Synthase; IRAK1, Interleukin-1 Receptor-Associated Kinase 1; MHC class II, Major Histocompatibility Complex class II; SOCS1, Suppressor of Cytokine

EP

Signalling 1; TNF-α, Tumour Necrosis Factor-alpha; RAGE, Receptor for Advanced glycation

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End-products; S100B, S100 calcium-binding protein; HMGB1, High mobility group box 1.

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miRNA

Target sequence (5’-3’)

hsa-miR-124-3p

UAAGGCACGCGGUGAAUGCC

has-miR-125b-5p

UCCCUGAGACCCUAACUUGUGA

has-miR-21-5p

UAGCUUAUCAGACUGAUGUUGA

hsa-miR-146a-5p

UGAGAACUGAAUUCCAUGGGUU

mmu-miR-155-5p

UUAAUGCUAAUUGUGAUAGGGGU

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microRNA expression in qRealTime-PCR

hsa – Human (Homo sapiens) origin; Small nuclear RNA U6 was used as a reference gene (human, mouse, rat); UniSp6 - RNA spike-in control was

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used to monitor PCR efficiency.

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Fig. 1. Transfection of neuroblastoma SH-SY5Y cells with the APPswe gene lead to upregulated expression of inflammatory markers, and of mature and immature APP695, together with increased secretion of APP-α (sAPPα) and Aβ1-40, but without clear changes on cell viability. Human neuroblastoma SH-SY5Y (SH) and SH-SY5Y APPSwe (SHSwe) cells were differentiated using 10 µM retinoic acid for 7 days, as described in Materials and methods section. A) S100B, HMGB1, RAGE and TNF-α gene expression levels were evaluated by qRealTime-PCR after 24 h incubation, as indicated in Materials and Methods section. B) Expression of mature and immature APP695 in cell lysates and of sAPPα in the media was assessed by Western blot, while cell supernatant Aβ1-40 was detected by ELISA. C) Nexin® assay analysis was used to distinguish cell populations: viable cells (annexin V-PE and 7-AAD negative), early-apoptotic cells (annexin V-PE positive and 7-AAD negative), cells in late stages of apoptosis or dead (annexin V-PE and 7-AAD positive), and necrotic cells/cellular debris (annexin V-PE negative and 7-AAD positive). Results are mean ± SEM fold change vs. SH cells A), or simply mean ± SEM B) and C) from 6 independent experiments. *p < 0.05 and **p < 0.01 vs. SH cells. 32

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Fig. 2. Interaction of CHME3 microglia with SH-SY5Y APPswe cells acutely prevents sAPPα and Aβ1-40 accumulation in the co-culture media, and later determines an increased number of senescent microglia. Human neuroblastoma SH-SY5Y (SH) and SH-SY5Y APPSwe (SHSwe) cells were differentiated using 10 µM retinoic acid for 7 days, as described in Materials and methods section. After coculturing CHME3 microglia with neuroblastoma cells for 24 h, A) expression of mature and immature APP695 in cell lysates and of sAPPα in the media was assessed by Western blot, while B) Aβ1-40 in cell supernatants was detected by ELISA, as indicated in Materials and Methods section. C) Senescence-associated β-galactosidase (SA-βgal) activity in microglia was assessed by a commercial kit to indicate senescent cells. Results are mean ± SEM, from at least 3 independent experiments. *p < 0.05 and **p < 0.01 vs SH cells.

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Fig. 3. CHME3 microglia stably express inflammatory and anti-inflammatoryrelated mediators upon interaction with SH-SY5Y APPswe cells. Human neuroblastoma SH-SY5Y (SH) and SH-SY5Y APPSwe (SHSwe) cells were differentiated using 10 µM retinoic acid for 7 days, as described in Materials and methods section. CHME3 microglial gene expression of A) pro-inflammatory iNOS, IL-1β, MHC class II, TNF-α and IL-6, as well as of B) anti-inflammatory arginase1 and IL-10 markers, were assessed by qRealTime-PCR after 24 h, 48 h and 72 h of co-incubation periods, as indicated in Materials and Methods section. Results are mean ± SEM fold change vs. basal CHME3 microglial expression, from at least 3 independent experiments. *p < 0.05 and **p < 0.01 vs. SH cells.

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Fig. 4. Human CHME3 microglial expression of inflammatory-associated miRNAs and their targets are upregulated upon interaction with SH-SY5Y APPswe cells. Human neuroblastoma SH-SY5Y (SH) and SH-SY5Y APPSwe (SHSwe) cells were differentiated using 10 µM retinoic acid for 7 days, as described in Materials and methods section. A) Expression of CHME3 microglial miR-155, miR-146 and miR-124, together with their respective targets SOCS1, IRAK1 and C/MBP-α B), was assessed by qRealTime-PCR after 24 h, 48 h and 72 h of co-incubation periods, as indicated in Materials and Methods section. Results are mean ± SEM fold change vs. basal CHME3 microglial expression, from at least 3 independent experiments. *p < 0.05 and **p < 0.01 vs. SH cells.

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Fig. 5. Morphology, size and particle concentration of exosomes derived from human neuroblastoma SH-SY5Y and SH-SY5Y APPswe cells are similar. Human neuroblastoma SH-SY5Y (SH) and SH-SY5Y APPSwe (SHSwe) cells were differentiated using 10 µM retinoic acid for 7 days, and exosomes isolated at day 9 by differential ultracentrifugation, as described in Materials and methods section. A) Representative images obtained by transmission electron microscopy (TEM) of exosomes released by SH and SHSwe cells. Scale bar equals 200 µm. B) Size distribution and volume concentration (particles/ml) by Nanoparticle Tracking Analysis using the NanoSight. C) Average exosome concentration (particles/ml). D) Average exosome diameter size in nanometers (nm). Results are mean ± SEM from at least four independent experiments.

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Fig. 6. SH-SY5Y APPSwe cells overexpress inflamma-miRNAs and release exosomes enriched in such miRNAs. Human neuroblastoma SH-SY5Y (SH) and SH-SY5Y APPSwe (SHSwe) cells were differentiated using 10 µM retinoic acid for 7 days, and exosomes isolated at day 9 by differential ultracentrifugation, as described in Materials and methods section. MiRNA expression (miR-124, miR-155, miR-146a, miR-21 and miR-125b) in cells and exosomes was evaluated by qRealTime-PCR after 24 h incubation. Results are mean ± SEM fold change vs. SH cells or SH-derived exosomes from 6 independent experiments. *p < 0.05 and **p < 0.01 vs. SH cells.

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Figure 7 – Uptake of exosomes derived from SH-SY5YSwe cells by CHME3 microglia co-localize with lysosomes. Human neuroblastoma SH-SY5Y (SH) and SH-SY5Y APPSwe (SHSwe) cells were differentiated using 10 µM retinoic acid for 7 days, and exosomes isolated at day 9 by differential ultracentrifugation, as described in Materials and methods section. A) SH and SHswe-derived exosomes were labelled with the fluorescent PKH67 probe (green) prior to incubation and revealed to be similarly internalized by CHME3 microglia after 24 h incubation B), as well as to co-localize with lysosomes identified by LysoTrackerTM (red). Cell nuclei were stained with Hoechst 33258 dye (blue) C). Decreased lysosomal intensity is observed in CHME3 microglia after the 24 h recovery period following the 24 h incubation with exosomes from SHswe cells D). Results are mean ± SEM, from at least 4 independent experiments. *p < 0.05 and **p < 0.01 vs. CHME3 microglia at 24 h after removing the media with SH-derived exosomes (solid lines), and at 24 h of exosome-microglia co-culturing (dashed lines).

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Figure 8 – Uptake of exosomes derived from SH-SY5YSwe cells by CHME3 microglia induce cell death by both apoptosis and necrosis. Human neuroblastoma SH-SY5Y (SH) and SH-SY5Y APPSwe (SHSwe) cells were differentiated using 10 µM retinoic acid for 7 days, and exosomes isolated at day 9 by differential ultracentrifugation, as described in Materials and methods section. CHME3 microglia viability was evaluated following a period of 24 h incubation with SH- or SHswe-derived exosomes A), or following an additional period of 24 h incubation after the renewal of extracellular media to allow microglia recovering B). Nexin® assay analysis was used 39

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to distinguish cell populations: viable cells (annexin V-PE and 7-AAD negative), earlyapoptotic cells (annexin V-PE positive and 7-AAD negative), cells in late stages of apoptosis or dead (annexin V-PE and 7-AAD positive), and necrotic cells/cellular debris (annexin V-PE negative and 7-AAD positive). Representative cytograms are shown and graph results are mean ± SEM from at least 4 independent experiments. *p < 0.05 vs. CHME3 microglia incubated with SH-derived exosomes.

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Figure 9 – Transfer of exosomes from SH-SY5Y APPswe cells into CHME3 microglia lead to upregulated inflammatory-associated marker and determine S100B mRNA and miR-21 dissemination via microglia-secreted exosomes. Human neuroblastoma SH-SY5Y (SH) and SH-SY5Y APPSwe (SHSwe) cells were differentiated using 10 µM retinoic acid for 7 days, and exosomes isolated at day 9 by differential ultracentrifugation, as described in Materials and methods section. CHME3 microglia were incubated for 24 h with SH - or SHswe-derived exosomes, followed by an additional period of 24 h incubation after the renewal of extracellular media to allow microglia recovering. Microglia and exosomes isolated from the extracellular media were used to assess the gene expression of S100B, HMGB1 and TNF-α, as well as 41

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miR-21, by qRealTime-PCR after 24 h incubation A) or after an additional 24 h period incubation with renewed media B). Results are mean ± SEM fold change vs. CHME3 microglia incubated with SH-derived exosomes or their respective exosomes from at least 4 independent experiments. n.d., non-detected; *p < 0.05 and **p < 0.01 vs. CHME3 microglia incubated with SH-derived exosomes or respective exosomes.

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SHSwe cells show increased inflammatory-associated markers

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CHME3 microglia activation occurs by early exposure to SHSwe cells

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SHSwe cells induce delayed upregulation of pro-resolving markers in CHME3 microglia

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Exosomes from SHSwe cells recapitulate their inflamma-miRNA profile

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• Exosomes from SHSwe cells rise S100B/HMGB1/TNF-α mRNA and miR-21 in CHME3 microglia