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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 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. Methods: 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.

Mfn2 is a mitochondrial fusion protein with bioenergetic functions implicated in the pathophysiology of neuronal and metabolic disorders. Understanding the bioenergetic mechanism of Mfn2 may aid in designing therapeutic approaches for these disorders. Here we show using endoplasmic reticulum (ER) or mitochondria‐targeted Mfn2 that Mfn2 stimulation of the mitochondrial metabolism requires its localization in the ER, which is independent of its fusion function. ER‐located Mfn2 interacts with mitochondrial Mfn1/2 to tether the ER and mitochondria together, allowing Ca2+ transfer from the ER to mitochondria to enhance mitochondrial bioenergetics. The physiological relevance of these findings is shown during neurite outgrowth, when there is an increase in Mfn2‐dependent ER‐mitochondria contact that is necessary for correct neuronal arbor growth. Reduced neuritic growth in Mfn2 KO neurons is recovered by the expression of ER‐targeted Mfn2 or an artificial ER‐mitochondria tether, indicating that manipulation of ER‐mitochondria contacts could be used to treat pathologic conditions involving Mfn2.

Astrocytic excitability relies on cytosolic calcium increases as a key mechanism, whereby astrocytes contribute to synaptic transmission and hence learning and memory. While it is a cornerstone of neurosciences that experiences are remembered, because transmitters activate gene expression in neurons, long-term adaptive astrocyte plasticity has not been described. Here, we investigated whether the transcription factor CREB mediates adaptive plasticity-like phenomena in astrocytes. We found that activation of CREB-dependent transcription reduced the calcium responses induced by ATP, noradrenaline, or endothelin-1. As to the mechanism, expression of VP16-CREB, a constitutively active CREB mutant, had no effect on basal cytosolic calcium levels, extracellular calcium entry, or calcium mobilization from lysosomal-related acidic stores. Rather, VP16-CREB upregulated sigma-1 receptor expression thereby increasing the release of calcium from the endoplasmic reticulum and its uptake by mitochondria. Sigma-1 receptor was also upregulated in vivo upon VP16-CREB expression in astrocytes. We conclude that CREB decreases astrocyte responsiveness by increasing calcium signalling at the endoplasmic reticulum-mitochondria interface, which might be an astrocyte-based form of long-term depression.

BackgroundOur understanding of the impact of astrocytes in Alzheimer’s disease (AD) is hindered by the lack of astrocyte-specific omics data from patients diagnosed with dementia due to AD. Studies aiming to profile human AD astrocytes—including single-nucleus RNA sequencing—were limited by the low number of differentially expressed genes detected, and by the small size of cohorts. We improved on prior studies with a novel systems-biology-based approach.Methods Human astrocytic and neuronal gene clusters were generated from RNA sequencing data from isolated healthy human brain cells using a cell-type enrichment score and clustering. The cell-specific gene clusters were localized in 766 subjects from three AD whole-brain transcriptomes generated by the Mount Sinai Hospital, the Mayo Clinic, and the Religious Order Study/Memory and Aging Project (ROSMAP), which also contains subjects with mild cognitive impairment (MCI). Gene clusters were organized into functional categories and subcategories using manual curation. Functional changes among subject groups were determined by gene set variation analysis (GSVA) and principal component analysis (PCA).Results Hierarchical clustering of transcriptomic data revealed molecular heterogeneity in individuals with the same clinical diagnosis. Particularly in the Mayo Clinic and ROSMAP cohorts, over 50% of Controls presented massive down-regulation of genes encoding for synaptic proteins, as widely documented in AD, suggesting that these subjects might have been at a preclinical stage at the time of death. Conversely, approximately 30% of AD patients showed preservation of neuronal genes as if they were non demented subjects, suggesting that they were resilient to AD pathology (present, according to CERAD and Braak scoring), but developed dementia due to comorbidities. The astrocytic gene profiles in AD patients presenting down-regulation of neuronal genes were termed ‘AD astrocytes’. According to GSVA and PCA, AD astrocytes showed down-regulation of genes encoding for mitochondrial and endolysosomal proteins, and up-regulation of genes related to perisynaptic astrocytic processes (PAP), and survival and stress responses.Discussion Astrocytes undergo a profound transcriptional change in a remarkable percentage of Control, MCI and AD subjects, affecting organelles and astrocyte-neuron interactions. We argue that therapies preventing organelle dysfunction in astrocytes may protect neural circuits in preclinical and clinical AD.