Role of Mitochondria-Associated ER membranes on microglia activation: implications for Alzheimer's disease.

Projektinformationen

INFLAMAM

ID Finanzhilfevereinbarung: 101106857

DOI

10.3030/101106857

Das Projekt endete am 31 Januar 2025

EK-Unterschriftsdatum 8 Mai 2023

Startdatum 1 Januar 2024

Enddatum 31 Dezember 2025

Finanziert unter

• Marie Skłodowska-Curie Actions (MSCA)

Gesamtkosten

Keine Daten

EU-Beitrag

€ 165 312,96

Koordiniert durch

AGENCIA ESTATAL CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
Spain

Periodic Reporting for period 1 - INFLAMAM (Role of Mitochondria-Associated ER membranes on microglia activation: implications for Alzheimer's disease.)

Berichtszeitraum: 2024-01-01 bis 2025-12-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Alzheimer’s disease (AD) is a common debilitating age-related neurodegenerative condition characterized by progressive cognitive decline and pre-symptomatic accumulation of amyloid-β. Over 14 million people in Europe are living with dementia (60-70% AD diagnosis), which is projected to double by 2050. Currently, there is no treatment for AD; however, the dementia research community has made progress in characterizing the clinical and pathologic features of the disease and identifying risk factors. Indeed, many genetic loci robustly associated with AD code for proteins that are preferentially or exclusively expressed in microglia, supporting a causal role of microglia as key players in disease initiation and progression. Upon activation, as a means to coordinate the immune response, the lipid organization of microglial cellular membranes is reorganized. To sustain this dynamic lipid remodeling, the cell coordinates a network of lipid pathways at unique subcellular regions, called MAMs (Mitochondria-associated ER membranes).
The primary objective of the INFLAMAM project is to elucidate the mechanisms through which MAMs regulate microglial activation and to understand the consequences of MAM and lipid homeostasis defects in the context of AD. To achieve this, the project will integrate advanced lipidomics and proteomics techniques with both pharmacological and genetic interventions targeting MAMs. This comprehensive approach aims to identify key molecular drivers that mediate the interaction between lipid metabolism and immune signaling at MAMs in microglia. By establishing the role of MAMs as crucial hubs for immunometabolic regulation and drivers of AD pathogenesis, the INFLAMAM project aspires to identify new lipid pathways that could serve as targets for pharmacological interventions. These interventions could potentially mitigate the symptoms related to microglial activity in AD, offering new avenues for therapeutic development.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

Using murine-derived and human-derived microglia-like cell lines (BV2 and HMC3) and primary cultured murine microglia upon exposure to different immune challenges (TLR4 pathway, E. coli Lipopolysaccharide LPS; TLR3 activation, via the double-stranded RNA synthetic analog polyI:C; RIG-I/MDA-5-MAVS-IFN axis, using Lipofectamine-encapsulated polyI:C; and a pro-inflammatory activation cocktail, LPS + IFN-), we characterized the activation profile detecting the levels of IL1 and nitrites. LPS and LPS + IFN- stimuli increased drastically the levels of both pro-inflammatory mediators while polyI:C showed slight upregulation. To complement these, the levels of IFN- expression were measured by qRT-PCR, and found that any of these stimulati triggered an increase.
Then, we tracked MAM activities by measuring 3H-serine into 3H-phosphatidylserine and 3H-phosphatidylethanolamine will be tracked by thin-layer chromatography. By doing so, we found an upregulation of MAM upon 24h of stimulation with LPS or LPS + IFN- while polyI:C showed a decrease. Similar results were found in primary microglia when assaying the MAM-located ACAT1 (acyl-coenzyme A:cholesterol acyltransferase 1) activity by tracking the incorporation of 3H-cholesterol into 3H-cholesteryl esters. However, ACAT1 activity was below the detection limit in BV-2 and HMC3 cell lines, probably due to low uptake of 3H-cholesterol.
Proteomics and lipidomics analysis were performed to address the molecular landscape of MAM upon microglia activation. These results – still under analysis – will pinpoint relevant lipid pathways as potential targets for intervention.
Once MAM changes triggered by inflammatory stimuli were established, we aimed to modulate MAM by exposure to exogenously added recombinant sphingomyelinase (SMase), which has been shown to induce the mobilization of cholesterol from plasma membrane towards the ER, triggering MAM upregulation. By the same token, inhibition of MAM formation was achieved with specific inhibitors of SMase activity (GW4869 and/or desipramine), which are known to prevent cholesterol delivery to ER and impede the formation of MAM domains. Our results indicated, as expected, that SMase treatment enhanced MAM activities and microglia activation profile upon inflammatory stimulation. However, SMase inhibition showed no consistent alteration on the activation profile. Analyzing the lipidomic profile of microglia upon MAM intervention will help address whether this differential effect is due to alterations in any specific lipid pathway.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

INFLAMAM combined functional characterization, image analysis, state-of-the-art omics technologies, and bioinformatics integration to help elucidate the role and relevance of MAM and lipid metabolism in microglia as a driver of disease. Our data indicates the relationship between MAM activities and modulation of microglia inflammatory profile. Upon completion, Integration of lipidomic and proteomic data by multiparametric bioinformatics analysis will be pivotal to prioritize specific lipid pathways and define strategies for future intervention approaches. These results highlight the need to integrate functional readouts with lipidomic data to ensure the efficacy of MAM-targeted strategies.