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The pathogenesis of Alzheimer’s disease (AD), a slowly-developing age-related neurodegenerative disorder, is a result of the action of multiple factors including deregulation of Ca 2+ homeostasis, mitochondrial dysfunction, and dysproteostasis. Interaction of these factors in astrocytes, principal homeostatic cells in the central nervous system, is still poorly understood. Here we report that in immortalized hippocampal astrocytes from 3xTg-AD mice (3Tg-iAstro cells) bioenergetics is impaired, including reduced glycolysis and mitochondrial oxygen consumption, and increased production of reactive oxygen species. Shotgun proteomics analysis of mitochondria-ER-enriched fraction showed no alterations in the expression of mitochondrial and OxPhos proteins, while those related to the ER functions and protein synthesis were deregulated. Using ER- and mitochondria-targeted aequorin-based Ca 2+ probe we show that, in 3Tg-iAstro cells, ER was overloaded with Ca 2+ while Ca 2+ uptake by mitochondria upon ATP stimulation was reduced. This was accompanied by the increase in short distance (≈8–10 nm) contact area between mitochondria and ER, upregulation of ER-stress/unfolded protein response genes Atf4, Atf6 and Herp, and reduction of global protein synthesis rate. We suggest that familial AD mutations in 3Tg-iAstro cells induce mitochondria-ER interaction changes that deregulate astrocytic bioenergetics, Ca 2+ homeostasis and proteostasis. These factors may interact, creating a pathogenic loop compromising homeostatic and defensive functions of astroglial cells predisposing neurons to dysfunction.

Background Alzheimer’s disease (AD) is the major age-related form of dementia in which dysfunctional ubiquitin-proteasome system (UPS) and autophagy represent primary mechanisms leading to accumulation of misfolded proteins, dysfunction of astroglial cells, neuroinflammation and neurodegeneration. Alterations of the endoplasmic reticulum (ER)-mitochondria contact sites (MERCS), specifically the shortening of the distance between the organelles, was proposed as a key mechanism of cell dysfunction in AD. However, its link to the impairment of the proteolytic system in AD remains unexplored. Methods We used, as a model, hippocampal astrocytes from 3xTg-AD mice expressing either control plasmid or synthetic linkers stabilizing ER-mitochondrial interaction at 10 nm (10 nm-EML) or at 20 nm (20 nm-EML). Alternatively, astrocytes were treated with mitochondrial Ca 2+ uptake inhibitor benzethonium chloride or activator amorolfine. We used Western blot to assess protein expression and specific enzymatic activity tests for the analysis of proteasomal, autophagic and lysosomal activities. Single cell fluorescent Ca 2+ imaging, using 4mtD3cpv probe targeted to the mitochondrial matrix, was used to assess mitochondrial Ca 2+ uptake. Results Stabilization of MERCS at 20 nm (20 nm-MERCS), which promotes mitochondrial Ca 2+ uptake, rescued protein ubiquitination, UPS composition and activity. Immunoproteasome components β2i and β5i, upregulated in AD astrocytes, and INFγ, a master-regulator of UPS remodelling in inflammatory conditions, were also rescued. Autophagic markers beclin 1, LC3II and p62, and lysosome-related marker cathepsin B, all upregulated in AD astrocytes, were significantly reduced, while autophagic flux was rescued, by stabilizing 20 nm-MERCS. Furthermore, stabilization of 20 nm-MERCS fully rescued previously reported deficit of mitochondrial Ca 2+ uptake. Strikingly, application of a mitochondrial Ca 2+ uptake positive modulator, amorolfine, partially rescued pathological remodelling of UPS and autophagy, suggesting that both mitochondrial Ca 2+ -related and Ca 2+ -unrelated mechanisms play a role in the beneficial effect of 20 nm-MERCS stabilization on protein dyshomeostasis. Conclusions Our results suggest that disruption of ER-mitochondrial interaction is a key factor for AD-related dysregulation of protein degradation and provide a proof that stabilization of MERCS at a defined distance and/or pharmacological rescue of mitochondrial Ca 2+ uptake represent valuable strategies for the development of future anti-AD therapy.

Ataxia-telangiectasia (A-T) is a rare neurodegenerative disorder caused by the deficiency of the serine/threonine kinase ataxia telangiectasia mutated (ATM) protein, whose loss of function leads to altered cell cycle, apoptosis, oxidative stress balance and DNA repair after damage. The clinical manifestations are multisystemic, among them cerebellar degeneration and muscular ataxia. The molecular mechanism by which ATM loss leads to A-T is still uncertain and, currently only symptomatic treatments are available. In this study, we generated a functional skeletal muscle cell model that recapitulates A-T and highlights the role of ATM in calcium signalling and muscle contraction. To this aim, by using CRISPR/Cas9 technology, we knocked out the ATM protein in urine-derived stem cells (USCs) from healthy donors. The resulting USCs-ATM-KO maintained stemness but showed G2/S cell cycle progression and an inability to repair DNA after UV damage. Moreover, they showed increased cytosolic calcium release after ATP stimulation to the detriment of the mitochondria. The alterations of calcium homoeostasis were maintained after differentiation of USCs-ATM-KO into skeletal muscle cells (USC-SkMCs) and correlated with impaired cell contraction. Indeed, USC-SkMCs-ATM-KO contraction kinetics were dramatically accelerated compared to control cells. These results highlight the relevant function of ATM in skeletal muscle, which is not only dependent on a non-functional neuronal communication, paving the way for future studies on a muscular interpretation of A-T ataxia.

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.

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.

IP 3 receptor (IP 3 R)-mediated Ca 2+ transfer at the mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) drives mitochondrial Ca 2+ uptake and oxidative metabolism and is linked to different pathologies, including Parkinson’s disease (PD). The dependence of Ca 2+ transfer efficiency on the ER-mitochondria distance remains unexplored. Employing molecular rulers that stabilize ER-mitochondrial distances at 5 nm resolution, and using genetically encoded Ca 2+ indicators targeting the ER lumen and the sub-mitochondrial compartments, we now show that a distance of ~20 nm is optimal for Ca 2+ transfer and mitochondrial oxidative metabolism due to enrichment of IP 3 R at MERCS. In human iPSC-derived astrocytes from PD patients, 20 nm MERCS were specifically reduced, which correlated with a reduction of mitochondrial Ca 2+ uptake. Stabilization of the ER-mitochondrial interaction at 20 nm, but not at 10 nm, fully rescued mitochondrial Ca 2+ uptake in PD astrocytes. Our work determines with precision the optimal distance for Ca 2+ flux between ER and mitochondria and suggests a new paradigm for fine control over mitochondrial function. The distance-dependence analysis of ER-mitochondria Ca 2+ transfer suggests that a distance of ∼20 nm between the organelles ensures maximal efficiency of mitochondrial Ca 2+ uptake and OXPHOS and rescues Ca 2+ uptake in Parkinson’s disease mitochondria.

Alterations of endoplasmic reticulum (ER)-mitochondrial interaction have been associated with different pathological conditions, including neurodegenerative diseases, characterized by dysregulation of protein homeostasis. However, little is known about how enhanced ER-mitochondrial tethering affects cellular proteostatic machinery. Here, we transiently overexpressed synthetic ER-mitochondrial linkers (EMLs), stabilizing the ER-mitochondrial distance at ≤5 nm (denominated as 5 nm-EML) and ∼10 nm (10 nm-EML), in HeLa cells. No alterations were found in cell growth, although metabolic activity and total ATP were significantly reduced. In EML-expressing cells, global protein synthesis was significantly reduced, accompanied by a reduction of total PERK and eIF2α protein levels, but increased p-eIF2α. Unfolded protein response (UPR) markers ATF4 and ATF6 were upregulated, suggesting that enhanced ER-mitochondrial tethering deranges protein synthesis and induces a low-grade ER stress/UPR. To further investigate ER-mitochondrial tethering-induced protein dyshomeostasis, we performed shotgun mass spectrometry proteomics followed by bioinformatic analysis. Analysis of highly changed proteins and the most significantly overrepresented gene ontology (GO) terms revealed that ≤5 nm tethering preferentially affected the expression of proteins involved in RNA processing and splicing and proteasomal protein degradation, while ∼10 nm tethering preferentially affected protein translation. Both EMLs affected expression of proteins involved in mitochondrial bioenergetics and metabolism, defense against oxidative stress, ER protein homeostasis, signaling and secretion. Finally, lipidomic analysis suggests that 5 nm-EML and 10 nm-EML differentially affect lipid homeostasis. Altogether, our results suggest that enhanced ER-mitochondrial tethering leads to a profound remodeling of cellular protein homeostasis, which may play a key role in pathogenesis of Alzheimer's and other neurodegenerative diseases.

Prion diseases arise from the conformational conversion of the cellular prion protein (PrPC) into a self-replicating prion isoform (PrPSc). Although this process has been studied mostly in neurons, a growing body of evidence suggests that astrocytes express PrPC and are able to replicate and accumulate PrPSc. Currently, prion diseases remain incurable, while downregulation of PrPC represents the most promising therapy due to the reduction of the substrate for prion conversion. Here we show that the astrocyte-specific genetic ablation or pharmacological inhibition of the calcium-activated phosphatase calcineurin (CaN) reduces PrPC expression in astrocytes. Immunocytochemical analysis of cultured CaN-KO astrocytes and isolation of synaptosomal compartments from the hippocampi of astrocyte-specific CaN-KO (ACN-KO) mice suggest that PrPC is downregulated both in vitro and in vivo. The downregulation occurs without affecting the glycosylation of PrPC and without alteration of its proteasomal or lysosomal degradation. Direct assessment of the protein synthesis rate and shotgun mass spectrometry proteomics analysis suggest that the reduction of PrPC is related to the impairment of global protein synthesis in CaN-KO astrocytes. When WT-PrP and PrP-D177N, a mouse homologue of a human mutation associated with the inherited prion disease fatal familial insomnia, were expressed in astrocytes, CaN-KO astrocytes showed an aberrant localization of both WT-PrP and PrP-D177N variants with predominant localization to the Golgi apparatus, suggesting that ablation of CaN affects both WT and mutant PrP proteins. These results provide new mechanistic details in relation to the regulation of PrP expression in astrocytes, suggesting the therapeutic potential of astroglial cells.

Calcineurin (CaN), a Ca 2+ /calmodulin-activated serine/threonine phosphatase, acts as a Ca 2+ -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 Ca 2+ 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.

Alzheimer’s disease (AD) is a progressive neurodegeneration with dysfunctions in both the ubiquitin–proteasome system (UPS) and autophagy. Astroglia participation in AD is an attractive topic of research, but molecular patterns are partially defined and available in vitro models have technical limitations. Immortalized astrocytes from the hippocampus of 3xTg-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively) have been obtained as an attempt to overcome primary cell line limitations and this study aims at characterizing their proteolytic systems, focusing on UPS and autophagy. Both 26S and 20S proteasomal activities were downregulated in 3Tg-iAstro, in which a shift in catalytic subunits from constitutive 20S proteasome to immunoproteasome occurred, with consequences on immune functions. In fact, immunoproteasome is the specific complex in charge of clearing damaged proteins under inflammatory conditions. Parallelly, augmented expression and activity of the lysosomal cathepsin B, enhanced levels of lysosomal-associated membrane protein 1, beclin1, and LC3-II, together with an increased uptake of monodansylcadaverine in autophagic vacuoles, suggested autophagy activation in 3Tg-iAstro. The two proteolytic pathways were linked by p62 that accumulated in 3Tg-iAstro due to both increased synthesis and decreased degradation in the UPS defective astrocytes. Treatment with 4-phenylbutyric acid, a neuroprotective small chemical chaperone, partially restored proteasome and autophagy-mediated proteolysis in 3Tg-iAstro. Our data shed light on the impaired proteostasis in 3Tg-iAstro with proteasome inhibition and autophagic compensatory activation, providing additional validation of this AD in vitro model, and propose a new mechanism of action of 4-phenylbutyric acid in neurodegenerative disorders.