Altres ajuts: This research was mainly funded by grants TV3-20141430, TV3-20141432 and TV3-20141431 from La Marató de Televisió de Catalunya (TV3) to EG, AG and JV respectively, co-financed by FEDER funds from European Union to AG. Co-financed by European Regional Development Fund to MDG.

The apolipoprotein E (APOE) gene exists in three isoforms in humans: APOE2, APOE3 and APOE4. APOE4 causes structural and functional alterations in normal brains, and is the strongest genetic risk factor of the sporadic form of Alzheimer's disease (LOAD). Research on APOE4 has mainly focused on the neuronal damage caused by defective cholesterol transport and exacerbated amyloid-β and Tau pathology. The impact of APOE4 on non-neuronal cell functions has been overlooked. Astrocytes, the main producers of ApoE in the healthy brain, are building blocks of neural circuits, and Ca 2+ signaling is the basis of their excitability. Because APOE4 modifies membrane-lipid composition, and lipids regulate Ca 2+ channels, we determined whether APOE4 dysregulates Ca 2+ signaling in astrocytes. Ca 2+ signals were recorded in astrocytes in hippocampal slices from APOE3 and APOE4 gene targeted replacement male and female mice using Ca 2+ imaging. Mechanistic analyses were performed in immortalized astrocytes. Ca 2+ fluxes were examined with pharmacological tools and Ca 2+ probes. APOE3 and APOE4 expression was manipulated with GFP- APOE vectors and APOE siRNA. Lipidomics of lysosomal and whole-membranes were also performed. We found potentiation of ATP-elicited Ca 2+ responses in APOE4 versus APOE3 astrocytes in male, but not female, mice. The immortalized astrocytes modeled the male response, and showed that Ca 2+ hyperactivity associated with APOE4 is caused by dysregulation of Ca 2+ handling in lysosomal-enriched acidic stores, and is reversed by the expression of APOE3, but not of APOE4, pointing to loss of function due to APOE4 malfunction. Moreover, immortalized APOE4 astrocytes are refractory to control of Ca 2+ fluxes by extracellular lipids, and present distinct lipid composition in lysosomal and plasma membranes. Immortalized APOE4 versus APOE3 astrocytes present: increased Ca 2+ excitability due to lysosome dysregulation, altered membrane lipidomes and intracellular cholesterol distribution, and impaired modulation of Ca 2+ responses upon changes in extracellular lipids. Ca 2+ hyperactivity associated with APOE4 is found in astrocytes from male, but not female, targeted replacement mice. The study suggests that, independently of Aβ and Tau pathologies, altered astrocyte excitability might contribute to neural-circuit hyperactivity depending on APOE allele, sex and lipids, and supports lysosome-targeted therapies to rescue APOE4 phenotypes in LOAD.