Adelaide Maria Afonso Fernandes Borralho - iMED, University of Lisboa
Title: Relevance of neuron-microglia vesicular trafficking, stress-related microRNAS and DAMPs in Alzheimer's disease
Abstract:
Neuroinflammation is a known hallmark of Alzheimer’s Disease (AD), where microglial cells play a major role. Vesicular trafficking mediated by cell-derived exosomes have recently gain increased interest in the context of AD, where they are crucial in the interplay between neurons and glial cells. Exosomes may carry endogenous inflammatory microRNAs (miRNAs) or Damage-associated molecular patterns (DAMPs) molecules, such as high mobility group box 1 (HMGB1) and S100B, which may change the phenotype of recipient cells. Here, we used an in vitro model of AD to investigate whether: (i) AD neuron-derived secretome affects microglia phenotype; (ii) if exosomal cargo, including inflammatory-related miRNAs and DAMPs recapitulate the donor cell ; and whether (ii) exosomes released by AD neurons are able to activate the recipient microglial cell.
Human microglia cell line CHME3 was co-cultured with human neuroblastoma cells expressing amyloidprecursor protein (APP)695 Swedish mutation (SH-SY5Y APPSwe). After co-culture, the expression of miRNAs, their targets and inflammatory cytokines were evaluated by qRT-PCR. Exosomes from CHME3, SH-SY5Y and SH-SY5Y APPSwe were isolated by differential ultracentrifugation, characterized by NanoSight, and their content evaluated for miRNA profiling and HMGB1/S100B mRNA expression by qRTPCR. In parallel, exosomes from AD neurons were fluorescently labelled with PKH-67 and incubated on CHME3 cells. Microglia were then evaluated for exosome internalization and lysosome processing.
Exposure of CHME3 cells to SH-SY5Y APPSwe cells acutely increased the cellular expression of miR-155, followed by delayed upregulation of SOCS1 and miR-124, together with CEBP-α reduction. Increased levels of TNF-α, IL-1β, IL-6 and IL-10 occurred at 72 h of co-culturing. High expression of miR-124 was observed in the SH-SY5Y APPSwe cells, while miR-21 was increased in both SH-SY5Y APPSwe and CHME3 cells, and recapitulated in cell-derived exosomes. While HMGB1 mRNA expression was increased in SH-SY5Y APPSwe and not detected in their exosomes, S100B was decreased in SH-SY5Y APPSwe, when compared to SH-SY5Y, and reflected in their respective exosomes. Low mRNA levels of both DAMPs were found in CHME3 cells. Exosomes from SH-SY5Y and SH-SY5Y APPSwe showed similar concentration (~1.6 x106 cells/ml) and 2 populations with different sizes (~85 and ~135 nm). Microglia treated with SH-SY5Y APPSwe exosomes revealed an increased density of incorporated exosomes preferentially localized in lysosomes.
Collectively, communication between SH-SY5Y APPSwe and CHME3 cells determine early microglia M1 phenotype with later heterogeneous M1/M2 cell subsets. Increase of stress-related miRNAs in both cells and their derived exosomes, suggest a key role of exosomes on the delivery of miRNAs and DAMPs from donor to recipient cells. Finally, neuronal exosomes are efficiently internalized by microglial cells and directed to lysosomal processing.
Title: Relevance of neuron-microglia vesicular trafficking, stress-related microRNAS and DAMPs in Alzheimer's disease
Abstract:
Neuroinflammation is a known hallmark of Alzheimer’s Disease (AD), where microglial cells play a major role. Vesicular trafficking mediated by cell-derived exosomes have recently gain increased interest in the context of AD, where they are crucial in the interplay between neurons and glial cells. Exosomes may carry endogenous inflammatory microRNAs (miRNAs) or Damage-associated molecular patterns (DAMPs) molecules, such as high mobility group box 1 (HMGB1) and S100B, which may change the phenotype of recipient cells. Here, we used an in vitro model of AD to investigate whether: (i) AD neuron-derived secretome affects microglia phenotype; (ii) if exosomal cargo, including inflammatory-related miRNAs and DAMPs recapitulate the donor cell ; and whether (ii) exosomes released by AD neurons are able to activate the recipient microglial cell.
Human microglia cell line CHME3 was co-cultured with human neuroblastoma cells expressing amyloidprecursor protein (APP)695 Swedish mutation (SH-SY5Y APPSwe). After co-culture, the expression of miRNAs, their targets and inflammatory cytokines were evaluated by qRT-PCR. Exosomes from CHME3, SH-SY5Y and SH-SY5Y APPSwe were isolated by differential ultracentrifugation, characterized by NanoSight, and their content evaluated for miRNA profiling and HMGB1/S100B mRNA expression by qRTPCR. In parallel, exosomes from AD neurons were fluorescently labelled with PKH-67 and incubated on CHME3 cells. Microglia were then evaluated for exosome internalization and lysosome processing.
Exposure of CHME3 cells to SH-SY5Y APPSwe cells acutely increased the cellular expression of miR-155, followed by delayed upregulation of SOCS1 and miR-124, together with CEBP-α reduction. Increased levels of TNF-α, IL-1β, IL-6 and IL-10 occurred at 72 h of co-culturing. High expression of miR-124 was observed in the SH-SY5Y APPSwe cells, while miR-21 was increased in both SH-SY5Y APPSwe and CHME3 cells, and recapitulated in cell-derived exosomes. While HMGB1 mRNA expression was increased in SH-SY5Y APPSwe and not detected in their exosomes, S100B was decreased in SH-SY5Y APPSwe, when compared to SH-SY5Y, and reflected in their respective exosomes. Low mRNA levels of both DAMPs were found in CHME3 cells. Exosomes from SH-SY5Y and SH-SY5Y APPSwe showed similar concentration (~1.6 x106 cells/ml) and 2 populations with different sizes (~85 and ~135 nm). Microglia treated with SH-SY5Y APPSwe exosomes revealed an increased density of incorporated exosomes preferentially localized in lysosomes.
Collectively, communication between SH-SY5Y APPSwe and CHME3 cells determine early microglia M1 phenotype with later heterogeneous M1/M2 cell subsets. Increase of stress-related miRNAs in both cells and their derived exosomes, suggest a key role of exosomes on the delivery of miRNAs and DAMPs from donor to recipient cells. Finally, neuronal exosomes are efficiently internalized by microglial cells and directed to lysosomal processing.