Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer's disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction

Astroglial calcium signaling and homeostasis in tuberous sclerosis complex

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder characterized by the development of benign tumors in various organs, including the brain, and is often accompanied by epilepsy, neurodevelopmental comorbidities including intellectual disability and autism. A key hallmark of TSC is the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling pathway, which induces alterations in cortical development and metabolic processes in astrocytes, among other cellular functions. These changes could modulate seizure susceptibility, contributing to the progression of epilepsy and its associated comorbidities. Epilepsy is characterized by dysregulation of calcium (Ca 2+) channels and intracellular Ca 2+ dynamics. These factors contribute to hyperexcitability, disrupted synaptogenesis, and altered synchronization of neuronal networks, all of which contribute to seizure activity. This study investigates the intricate interplay between altered Ca 2+ dynamics, mTOR pathway dysregulation, and cellular metabolism in astrocytes. The transcriptional profile of TSC patients revealed significant alterations in pathways associated with cellular respiration, ER and mitochondria, and Ca 2+ regulation. TSC astrocytes exhibited lack of responsiveness to various stimuli, compromised oxygen consumption rate and reserve respiratory capacity underscoring their reduced capacity to react to environmental changes or cellular stress. Furthermore, our study revealed significant reduction of store operated calcium entry (SOCE) along with strong decrease of basal mitochondrial Ca 2+ concentration and Ca 2+ influx in TSC astrocytes. In addition, we observed alteration in mitochondrial membrane potential, characterized by increased depolarization in TSC astrocytes. Lastly, we provide initial evidence of structural abnormalities in mitochondria within TSC patient-derived astrocytes, suggesting a potential link between disrupted Ca 2+ signaling and mitochondrial dysfunction. Our findings underscore the complexity of the relationship between Ca 2+ signaling, mitochondria dynamics, apoptosis, and mTOR hyperactivation. Further exploration is required to shed light on the pathophysiology of TSC and on TSC associated neuropsychiatric disorders offering further potential avenues for therapeutic development

Ca2+ related mechanisms of cellular dysfunction in neurodegeneration

Calcium is a pleiotropic intracellular messenger regulating mitochondrial bioenergetics and proteostasis, both altered in Alzheimer’s disease (AD). In my PhD project I have studied the cause-effect relationships between calcium dishonesties and cellular dysfunction in astrocytes. In this context I investigated the discrepancy, described in AD astrocytes, between deficient mitochondrial calcium uptake and increased ER-mitochondria interaction, which in principle should promote ER-mitochondrial calcium transfer. To solve this issue, I investigated the correlation between ER-mitochondria distances and efficiency of calcium transfer: I designed a palette of ER-mitochondria linkers, discovering that ER-mitochondria calcium transfer is strongly inhibited at 10nm distances, and is strongly potentiated at 20nm, explaining why increased ER-mitochondria interaction at 8-10nm, in AD astrocytes, results in reduced mitochondrial calcium uptake. Another aspect of my PhD thesis is the investigation of Calcineurin (CaN) role in both physiology of astrocytes and AD related astrocytes dysfunction. In astrocytes, eIF2α-mediated protein synthesis and proteasomal degradation are under CaN control. While, in and AD mice model, deletion of astroglial CaN displayed a protective effect, reducing memory impairment, aβ plaques deposition and tau hyper-phosphorylation. These results indicate that CaN, under pathological conditions can drive pathology progression. I demonstrated that ER-mitochondria interaction and calcineurin represents promising therapeutic targets to mitigate AD related astroglial pathophysiology, given the importance of astrocytes for brain functions further studies are warranted to elucidate mechanisms of these dysfunctions

Calcineurin Signalling in Astrocytes: From Pathology to Physiology and Control of Neuronal Functions

Calcineurin (CaN), a Ca2+/calmodulin-activated serine/threonine phosphatase, acts as a Ca2+-sensitive switch regulating cellular functions through protein dephosphorylation and activation of gene transcription. In astrocytes, the principal homeostatic cells in the CNS, over-activation of CaN is known to drive pathological transcriptional remodelling, associated with neuroinflammation in diseases such as Alzheimer's disease, epilepsy and brain trauma. Recent reports suggest that, in physiological conditions, the activity of CaN in astrocytes is transcription-independent and is required for maintenance of basal protein synthesis rate and activation of astrocytic Na+/K+ pump thereby contributing to neuronal functions such as neuronal excitability and memory formation. In this contribution we overview the role of Ca2+ and CaN signalling in astroglial pathophysiology focusing on the emerging physiological role of CaN in astrocytes. We propose a model for the context-dependent switch of CaN activity from the post-transcriptional regulation of cell proteostasis in healthy astrocytes to the CaN-dependent transcriptional activation in neuroinflammation-associated diseases

Immortalized hippocampal astrocytes from 3xTg-AD mice, a new model to study disease-related astrocytic dysfunction: a comparative review

Alzheimer's disease (AD) is characterized by complex etiology, long-lasting pathogenesis, and celltype-specific alterations. Currently, there is no cure for AD, emphasizing the urgent need for a comprehensive understanding of cell-specific pathology. Astrocytes, principal homeostatic cells of the central nervous system, are key players in the pathogenesis of neurodegenerative diseases, including AD. Cellular models greatly facilitate the investigation of cell-specific pathological alterations and the dissection of molecular mechanisms and pathways. Tumor-derived and immortalized astrocytic cell lines, alongside the emerging technology of adult induced pluripotent stem cells, are widely used to study cellular dysfunction in AD. Surprisingly, no stable cell lines were available from genetic mouse AD models. Recently, we established immortalized hippocampal astroglial cell lines from amyloid-beta precursor protein/presenilin-1/Tau triple-transgenic (3xTg)-AD mice (denominated as wild type (WT)and 3Tg-iAstro cells) using retrovirus-mediated transduction of simian virus 40 large T-antigen and propagation without clonal selection, thereby maintaining natural heterogeneity of primary cultures. Several groups have successfully used 3Tg-iAstro cells for single-cell and omics approaches to study astrocytic AD-related alterations of calcium signaling, mitochondrial dysfunctions, disproteostasis, altered homeostatic and signaling support to neurons, and blood-brain barrier models. Here we provide a comparative overview of the most used models to study astrocytes in vitro, such as primary culture, tumor-derived cell lines, immortalized astroglial cell lines, and induced pluripotent stem cell-derived astrocytes. We conclude that immortalized WT- and 3Tg-iAstro cells provide a noncompetitive but complementary, low-cost, easy-to-handle, and versatile cellular model for dissection of astrocyte-specific AD-related alterations and preclinical drug discovery

The endoplasmic reticulum stress and unfolded protein response in Alzheimer's disease: A calcium dyshomeostasis perspective

Protein misfolding is prominent in early cellular pathology of Alzheimer's disease (AD), implicating pathophysiological significance of endoplasmic reticulum stress/unfolded protein response (ER stress/UPR) and highlighting it as a target for drug development. Experimental data from animal AD models and observations on human specimens are, however, inconsistent. ER stress and associated UPR are readily observed in in vitro AD cellular models and in some AD model animals. In the human brain, components and markers of ER stress as well as UPR transducers are observed at Braak stages III-VI associated with severe neuropathology and neuronal death. The picture, however, is further complicated by the brain region- and cell type-specificity of the ADrelated pathology. Terms 'disturbed' or 'non-canonical' ER stress/UPR were used to describe the discrepancies between experimental data and the classic ER stress/UPR cascade. Here we discuss possible 'disturbing' or 'interfering' factors which may modify ER stress/UPR in the early AD pathogenesis. We focus on the dysregulation of the ER Ca2+ homeostasis, store-operated Ca2+ entry, and the interaction between the ER and mitochondria. We suggest that a detailed study of the CNS cell type-specific alterations of Ca2+ homeostasis in early AD may deepen our understanding of AD-related dysproteostasis

Ca²⁺ handling at the mitochondria-ER contact sites in neurodegeneration

Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are morpho-functional units, formed at the loci of close apposition of the ER-forming endomembrane and outer mitochondrial membrane (OMM). These sites contribute to fundamental cellular processes including lipid biosynthesis, autophagy, apoptosis, ER-stress and calcium (Ca2+) signalling. At MERCS, Ca2+ ions are transferred from the ER directly to mitochondria through a core protein complex composed of inositol-1,4,5 trisphosphate receptor (InsP3R), voltage-gated anion channel 1 (VDAC1), mitochondrial calcium uniporter (MCU) and adaptor protein glucose-regulated protein 75 (Grp75); this complex is regulated by several associated proteins. Deregulation of ER-mitochondria Ca2+ transfer contributes to pathogenesis of neurodegenerative and other diseases. The efficacy of Ca2+ transfer between ER and mitochondria depends on the protein composition of MERCS, which controls ER-mitochondria interaction regulating, for example, the transversal distance between ER membrane and OMM and the extension of the longitudinal interface between ER and mitochondria. These parameters are altered in neurodegeneration. Here we overview the ER and mitochondrial Ca2+ homeostasis, the composition of ER-mitochondrial Ca2+ transfer machinery and alterations of the ER-mitochondria Ca2+ transfer in three major neurodegenerative diseases: motor neurone diseases, Parkinson disease and Alzheimer's disease.</p

Ca

From PubMed via Jisc Publications RouterHistory: received 2021-07-07, revised 2021-08-03, accepted 2021-08-03Publication status: aheadofprintMitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are morpho-functional units, formed at the loci of close apposition of the ER-forming endomembrane and outer mitochondrial membrane (OMM). These sites contribute to fundamental cellular processes including lipid biosynthesis, autophagy, apoptosis, ER-stress and calcium (Ca ) signalling. At MERCS, Ca ions are transferred from the ER directly to mitochondria through a core protein complex composed of inositol-1,4,5 trisphosphate receptor (InsP R), voltage-gated anion channel 1 (VDAC1), mitochondrial calcium uniporter (MCU) and adaptor protein glucose-regulated protein 75 (Grp75); this complex is regulated by several associated proteins. Deregulation of ER-mitochondria Ca transfer contributes to pathogenesis of neurodegenerative and other diseases. The efficacy of Ca transfer between ER and mitochondria depends on the protein composition of MERCS, which controls ER-mitochondria interaction regulating, for example, the transversal distance between ER membrane and OMM and the extension of the longitudinal interface between ER and mitochondria. These parameters are altered in neurodegeneration. Here we overview the ER and mitochondrial Ca homeostasis, the composition of ER-mitochondrial Ca transfer machinery and alterations of the ER-mitochondria Ca transfer in three major neurodegenerative diseases: motor neurone diseases, Parkinson disease and Alzheimer's disease. [Abstract copyright: Copyright © 2021 Elsevier Ltd. All rights reserved.

Quantification of the Chemical Chaperone 4-Phenylbutyric Acid (4-PBA) in Cell Culture Media via LC-HRMS: Applications in Fields of Neurodegeneration and Cancer

In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography–High Resolution Mass Spectrometry (LC-HRMS) method to quantify 4-PBA in NeuroBasal-A and Dulbecco’s Modified Eagle widely used cell culture media. Samples were injected on a Luna® 3 µm PFP(2) 100 Å (100 × 2.0 mm) column maintained at 40 °C. Water and methanol both with 0.1% formic acid served as mobile phases in a step gradient mode. The mass acquisition was performed by selected ion monitoring (SIM) in negative mode for a total run time of 10.5 min at a flow rate of 0.300 mL/min. The analogue 4-(4-Nitrophenyl)-Butyric Acid served as internal standard. Validation parameters were verified according to FDA and EMA guidelines. The quantification ranges from 0.38–24 µM. Inter and intraday RSDs (Relative Standard Deviations) were within 15%. The developed LC-HRMS method allowed the estimation of 4-PBA absorption and adsorption kinetics in vitro in two experimental systems: (i) 4-PBA improvement of protein synthesis in an Alzheimer’s disease astrocytic cell model; and (ii) 4-PBA reduction of endoplasmic reticulum stress in thapsigargin-treated melanoma cell lines