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The phenotypic transformation of astrocytes in Alzheimer's disease (AD) is still not well understood. Recent analyses based on single-nucleus RNA sequencing of postmortem Alzheimer's disease (AD) samples are limited by the low number of sequenced astrocytes, small cohort sizes, and low number of differentially expressed genes detected. To optimize the detection of astrocytic genes, we employed a novel strategy consisting of the localization of pre-determined astrocyte and neuronal gene clusters in publicly available whole-brain transcriptomes. Specifically, we used cortical transcriptomes from 766 individuals, including cognitively normal subjects (Controls), and people diagnosed with mild cognitive impairment (MCI) or dementia due to AD. Samples came from three independent cohorts organized by the Mount Sinai Hospital, the Mayo Clinic, and the Religious Order Study/Memory and Aging Project (ROSMAP). Astrocyte- and neuron-specific gene clusters were generated from human brain cell-type specific RNAseq data using hierarchical clustering and cell-type enrichment scoring. Genes from each cluster were manually annotated according to cell-type specific functional Categories. Gene Set Variation Analysis (GSVA) and Principal Component Analysis (PCA) were used to establish changes in these functional categories among clinical cohorts. We highlight three novel findings of the study. First, individuals with the same clinical diagnosis were molecularly heterogeneous. Particularly in the Mayo Clinic and ROSMAP cohorts, over 50% of Controls presented down-regulation of genes encoding synaptic proteins typical of AD, whereas 30% of patients diagnosed with dementia due to AD presented Control-like transcriptomic profiles. Second, down-regulation of neuronal genes related to synaptic proteins coincided, in astrocytes, with up-regulation of genes related to perisynaptic astrocytic processes (PAP) and down-regulation of genes encoding endolysosomal and mitochondrial proteins. Third, down-regulation of astrocytic mitochondrial genes inversely correlated with the disease stages defined by Braak and CERAD scoring. Finally, we interpreted these changes as maladaptive or adaptive from the point of view of astrocyte biology in a model of the phenotypical transformation of astrocytes in AD. The main prediction is that early malfunction of the astrocytic endolysosomal system, associated with progressive mitochondrial dysfunction, contribute to Alzheimer's disease. If this prediction is correct, therapies preventing organelle dysfunction in astrocytes may be beneficial in preclinical and clinical AD.

Abstract Background 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 Ca2+ signaling is the basis of their excitability. Because APOE4 modifies membrane-lipid composition, and lipids regulate Ca2+ channels, we determined whether APOE4 dysregulates Ca2+signaling in astrocytes. Methods Ca2+ signals were recorded in astrocytes in hippocampal slices from APOE3 and APOE4 gene targeted replacement male and female mice using Ca2+ imaging. Mechanistic analyses were performed in immortalized astrocytes. Ca2+ fluxes were examined with pharmacological tools and Ca2+ probes. APOE3 and APOE4 expression was manipulated with GFP-APOE vectors and APOE siRNA. Lipidomics of lysosomal and whole-membranes were also performed. Results We found potentiation of ATP-elicited Ca2+responses in APOE4 versus APOE3 astrocytes in male, but not female, mice. The immortalized astrocytes modeled the male response, and showed that Ca2+ hyperactivity associated with APOE4 is caused by dysregulation of Ca2+ 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 Ca2+ fluxes by extracellular lipids, and present distinct lipid composition in lysosomal and plasma membranes. Conclusions Immortalized APOE4 versus APOE3 astrocytes present: increased Ca2+ excitability due to lysosome dysregulation, altered membrane lipidomes and intracellular cholesterol distribution, and impaired modulation of Ca2+ responses upon changes in extracellular lipids. Ca2+ 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.