FSP1 is a predictive biomarker of osteosarcoma cells' susceptibility to ferroptotic cell death and a potential therapeutic target.

Human osteosarcoma (OS) is a relatively rare malignancy preferentially affecting long body bones which prognosis is often poor also due to the lack of effective therapies. Clinical management of this cancer basically relies on surgical removal of primary tumor coupled with radio/chemotherapy. Unfortunately, most osteosarcoma cells are resistant to conventional therapy, with the undergoing epithelial-mesenchymal transition (EMT) giving rise to gene expression reprogramming, thus increasing cancer cell invasiveness and metastatic potential. Alternative clinical approaches are thus urgently needed. In this context, the recently described ferroptotic cell death represents an attractive new strategy to efficiently kill cancer cells, since most chemoresistant and mesenchymal-shaped tumors display high susceptibility to pro-ferroptotic compounds. However, cancer cells have also evolved anti-ferroptotic strategies, which somehow sustain their survival upon ferroptosis induction. Indeed, here we show that osteosarcoma cell lines display heterogeneous sensitivity to ferroptosis execution, correlating with the mesenchymal phenotype, which is consistently affected by the expression of the well-known anti-ferroptotic factor ferroptosis suppressor protein 1 (FSP1). Interestingly, inhibiting the activity or expression of FSP1 restores cancer cell sensitivity to ferroptosis. Moreover, we also found that: i) AKRs might also contribute to resistance; ii) NRF2 enhances FSP1 expression upon ferroptosis induction; while iii) p53 contributes to the regulation of FSP1 basal expression in OS cells.In conclusion, FSP1 expression can potentially be used as a valuable predictive marker of OS sensitivity to ferroptosis and as a new potential therapeutic target.

Doxorubicin loaded octacalcium phosphate particles as controlled release drug delivery systems: Physico-chemical characterization, in vitro drug release and evaluation of cell death pathway.

Mastering new and efficient ways to obtain successful drug delivery systems (DDS) with controlled release became a paramount quest in the scientific community. Increase of malignant bone tumors and the necessity to optimize an approach of localized drug delivery require research to be even more intensified. Octacalcium phosphate (OCP), with a number of advantages over current counterparts is extensively used in bone engineering. The aim of the present research was to synthesize bioactive and biocompatible doxorubicin (DOX) containing OCP particles. DOX-OCP was successfully obtained in situ in an exhaustive range of added drug (1-20 wt%, theoretical loading). Based on XRD, above 10 wt% of DOX, OCP formation was inhibited and the obtained product was low crystalline α-TCP. In-vitro drug release was performed in pH 7.4 and 6.0. In both pH environments DOX had a continuous release over six weeks. However, the initial drug burst for pH 7.4, in the first 24 h, ranged from 15.9 ± 1.3 % to 33.5 ± 12 % and for pH 6.0 23.7 ± 1.5 % to 36.2 ± 12 %.The DOX-OCP exhibited an inhibitory effect on viability of osteosarcoma cell lines MG63, U2OS and HOS. In contrast, MC3T3-E1 cells (IC50 > 0.062 µM) displayed increased viability and proliferation from 3rd to 7th day. Testing of the DDS on ferroptotic markers (CHAC1, ACSL4 and PTGS2) showed that OCP-DOX does not induce ferroptotic cell death. Moreover, the evaluation of protein levels of cleaved PARP, by western blotting analysis, corroborated that apoptosis is the main pathway of programmed cell death in osteosarcoma cells induced by DOX-OCP.

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.

Wild-type and mutant p53 in cancer-related ferroptosis. A matter of stress management?

Cancer cells within tumor masses are chronically exposed to stress caused by nutrient deprivation, oxygen limitation, and high metabolic demand. They also accumulate hundreds of mutations, potentially generating aberrant proteins that can induce proteotoxic stress. Finally, cancer cells are exposed to various damages during chemotherapy. In a growing tumor, transformed cells eventually adapt to these conditions, eluding the death-inducing outcomes of signaling cascades triggered by chronic stress. One such extreme outcome is ferroptosis, a form of iron-dependent non-apoptotic cell death mediated by lipid peroxidation. Not surprisingly, the tumor suppressor p53 is involved in this process, with evidence suggesting that it acts as a pro-ferroptotic factor and that its ferroptosis-inducing activity may be relevant for tumor suppression. Missense alterations of the TP53 gene are extremely frequent in human cancers and give rise to mutant p53 proteins (mutp53) that lose tumor suppressive function and can acquire powerful oncogenic activities. This suggests that p53 mutation provides a selective advantage during tumor progression, raising interesting questions on the impact of p53 mutant proteins in modulating the ferroptotic process. Here, we explore the role of p53 and its cancer-related mutants in ferroptosis, using a perspective centered on the resistance/sensitivity of cancer cells to exogenous and endogenous stress conditions that can trigger ferroptotic cell death. We speculate that an accurate molecular understanding of this particular axis may improve cancer treatment options.

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 AD-related 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.

Identification of novel aza-analogs of TN-16 as disrupters of microtubule dynamics through a multicomponent reaction.

Despite novel biological targets emerging at an impressive rate for anticancer therapy, antitubulin drugs remain the backbone of numerous oncological protocols and their efficacy has been demonstrated in a wide variety of adult and pediatric cancers. In the present contribution, we set to develop analogs of a potent but neglected antitubulin agent, TN-16, originally discovered via modification of tenuazonic acid (3-acetyl-5-sec-butyltetramic acid). To this extent, we developed a novel multicomponent reaction to prepare TN-16, and then we applied the same reaction for the synthesis of aza-analogs. In brief, we prepared a library of 62 novel compounds, and three of these retained nanomolar potencies. TN-16 and the active analogs are cytotoxic on cancer cell lines and, as expected from antitubulin agents, induce G2/M cell cycle arrest. These agents lead to a disruption of the microtubules and an increase in α-tubulin acetylation and affect in vitro polymerization, although they have a lesser effect in cellular tubulin polymerization assays.

The Gut-Ex-Vivo System (GEVS) Is a Dynamic and Versatile Tool for the Study of DNBS-Induced IBD in BALB/C and C57BL/6 Mice, Highlighting the Protective Role of Probiotics.

BACKGROUND: IBD is a spectrum of pathologies characterized by dysregulated immune activation leading to uncontrolled response against the intestine, thus resulting in chronic gut inflammation and tissue damage. Due to its complexity, the molecular mechanisms responsible for disease onset and progression are still elusive, thus requiring intense research effort. In this context, the development of models replicating the etiopathology of IBD and allowing the testing of new potential therapies is critical. METHODS: Colon from C57BL/6 or BALB/c mice was cultivated in a Gut-Ex-Vivo System (GEVS), exposed for 5 h to DNBS 1.5 or 2.5 mg/mL, in presence or absence of two probiotic formulations (P1 = Bifidobacterium breve BR03 (DSM16604) and B632 (DSM24706); P2 = Lacticaseibacillus rhamnosus LR04 (DSM16605), Lactiplantibacillus plantarum LP14 (DSM33401) and Lacticaseibacillus paracasei LPC09), and the main hallmarks of IBD were evaluated. RESULTS: Gene expression analysis revealed the following DNBS-induced effects: (i) compromised tight junction organization, responsible for tissue permeability dysregulation; (ii) induction of ER stress, and (iii) tissue inflammation in colon of C57BL/6 mice. Moreover, the concomitant DNBS-induced apoptosis and ferroptosis pathways were evident in colon from both BALB/c and C57BL/6 mice. Finally, the co-administration of probiotics completely prevented the detrimental effects of DNBS. CONCLUSIONS: Overall, we have provided results demonstrating that GEVS is a consistent, reliable, and cost-effective system for modeling DNBS-induced IBD, useful for studying the onset and progression of human disease at the molecular level, while also reducing animal suffering. Moreover, we have confirmed the beneficial effect of probiotics administration in promoting the remission of IBD.

Calreticulin and PDIA3, two markers of endoplasmic reticulum stress, are associated with metabolic alterations and insulin resistance in pediatric obesity: A pilot study.

Our aim was to evaluate the markers of endoplasmic reticulum (ER) stress among children and adolescents with obesity in relation to metabolic alterations. Calreticulin (CALR) and PDIA3 circulating levels were assessed on 52 pediatric subjects-26 patients with obesity and 26 normal weight controls (4-18 years)-enrolled in a pilot study. Clinical and metabolic evaluations were performed (BMI-SDS, insulin, and glucose at fasting and during an oral glucose tolerance test, lipid profile, blood pressure), and metabolic syndrome was detected. PDIA3 was higher (p < 0.02) and CALR slightly higher in children with obesity than in controls. PDIA3 was related positively to the Tanner stages. Both PDIA3 and CALR were positively associated with insulin resistance, cholesterol, and triglycerides and the number of criteria identifying metabolic syndrome and negatively with fasting and post-challenge insulin sensitivity. Our preliminary findings suggest the existence of a link between ER stress and metabolic changes behind obesity complications even at the pediatric age. CALR and PDIA3 could be early markers of insulin resistance and dyslipidemia-related ER stress useful to stratify patients at high risk of further complications.

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.

A Metabologenomic approach reveals alterations in the gut microbiota of a mouse model of Alzheimer's disease.

The key role played by host-microbiota interactions on human health, disease onset and progression, and on host response to treatments has increasingly emerged in the latest decades. Indeed, dysbiosis has been associated to several human diseases such as obesity, diabetes, cancer and also neurodegenerative disease, such as Parkinson, Huntington and Alzheimer's disease (AD), although whether causative, consequence or merely an epiphenomenon is still under investigation. In the present study, we performed a metabologenomic analysis of stool samples from a mouse model of AD, the 3xTgAD. We found a significant change in the microbiota of AD mice compared to WT, with a longitudinal divergence of the F/B ratio, a parameter suggesting a gut dysbiosis. Moreover, AD mice showed a significant decrease of some amino acids, while data integration revealed a dysregulated production of desaminotyrosine (DAT) and dihydro-3-coumaric acid. Collectively, our data show a dysregulated gut microbiota associated to the onset and progression of AD, also indicating that a dysbiosis can occur prior to significant clinical signs, evidenced by early SCFA alterations, compatible with gut inflammation.

Calcineurin Controls Cellular Prion Protein Expression in Mouse Astrocytes.

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.

A Gut-Ex-Vivo System to Study Gut Inflammation Associated to Inflammatory Bowel Disease (IBD).

Inflammatory bowel disease (IBD) is a complex, chronic, and dysregulated inflammatory condition which etiology is still largely unknown. Its prognosis and disease progression are highly variable and unpredictable. IBD comprises several heterogeneous inflammatory conditions ranging from Ulcerative Colitis (UC) to Crohn's Disease (CD). Importantly, a definite, well-established, and effective clinical treatment for these pathologies is still lacking. The urgent need for treatment is further supported by the notion that patients affected by UC or CD are also at risk of developing cancer. Therefore, a deeper understanding of the molecular mechanisms at the basis of IBD development and progression is strictly required to design new and efficient therapeutic regimens. Although the development of animal models has undoubtedly facilitated the study of IBD, such in vivo approaches are often expensive and time-consuming. Here we propose an organ ex vivo culture (Gut-Ex-Vivo system, GEVS) based on colon from Balb/c mice cultivated in a dynamic condition, able to model the biochemical and morphological features of the mouse models exposed to DNBS (5-12 days), in 5 h. Indeed, upon DNBS exposure, we observed a dose-dependent: (i) up-regulation of the stress-related protein transglutaminase 2 (TG2); (ii) increased intestinal permeability associated with deregulated tight junction protein expression; (iii) increased expression of pro-inflammatory cytokines, such as TNFα, IFNγ, IL1ß, IL6, IL17A, and IL15; (iv) down-regulation of the anti-inflammatory IL10; and (v) induction of Endoplasmic Reticulum stress (ER stress), all markers of IBD. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of IBD, in a time- and cost-effective manner.

High Levels of TRIM5α Are Associated with Xenophagy in HIV-1-Infected Long-Term Nonprogressors.

Autophagy is a lysosomal-dependent degradative mechanism essential in maintaining cellular homeostasis, but it is also considered an ancient form of innate eukaryotic fighting against invading microorganisms. Mounting evidence has shown that HIV-1 is a critical target of autophagy that plays a role in HIV-1 replication and disease progression. In a special subset of HIV-1-infected patients that spontaneously and durably maintain extremely low viral replication, namely, long-term nonprogressors (LTNP), the resistance to HIV-1-induced pathogenesis is accompanied, in vivo, by a significant increase in the autophagic activity in peripheral blood mononuclear cells. Recently, a new player in the battle of autophagy against HIV-1 has been identified, namely, tripartite motif protein 5α (TRIM5α). In vitro data demonstrated that TRIM5α directly recognizes HIV-1 and targets it for autophagic destruction, thus protecting cells against HIV-1 infection. In this paper, we analyzed the involvement of this factor in the control of HIV-1 infection through autophagy, in vivo, in LTNP. The results obtained showed significantly higher levels of TRIM5α expression in cells from LTNP with respect to HIV-1-infected normal progressor patients. Interestingly, the colocalization of TRIM5α and HIV-1 proteins in autophagic vacuoles in LTNP cells suggested the participation of TRIM5α in the autophagy containment of HIV-1 in LTNP. Altogether, our results point to a protective role of TRIM5α in the successful control of the chronic viral infection in HIV-1-controllers through the autophagy mechanism. In our opinion, these findings could be relevant in fighting against HIV-1 disease, because autophagy inducers might be employed in combination with antiretroviral drugs.

Probiotics Supplements Reduce ER Stress and Gut Inflammation Associated with Gliadin Intake in a Mouse Model of Gluten Sensitivity.

Exposure to gluten, a protein present in wheat rye and barley, is the major inducer for human Celiac Disease (CD), a chronic autoimmune enteropathy. CD occurs in about 1% worldwide population, in genetically predisposed individuals bearing human leukocyte antigen (HLA) DQ2/DQ8. Gut epithelial cell stress and the innate immune activation are responsible for the breaking oral tolerance to gliadin, a gluten component. To date, the only treatment available for CD is a long-term gluten-free diet. Several studies have shown that an altered composition of the intestinal microbiota (dysbiosis) could play a key role in the pathogenesis of CD through the modulation of intestinal permeability and the regulation of the immune system. Here, we show that gliadin induces a chronic endoplasmic reticulum (ER) stress condition in the small intestine of a gluten-sensitive mouse model and that the coadministration of probiotics efficiently attenuates both the unfolded protein response (UPR) and gut inflammation. Moreover, the composition of probiotics formulations might differ in their activity at molecular level, especially toward the three axes of the UPR. Therefore, probiotics administration might potentially represent a new valuable strategy to treat gluten-sensitive patients, such as those affected by CD.

Inhibition of the Histone Methyltransferase EZH2 Enhances Protumor Monocyte Recruitment in Human Mesothelioma Spheroids.

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a long latency period and dismal prognosis. Recently, tazemetostat (EPZ-6438), an inhibitor of the histone methyltransferase EZH2, has entered clinical trials due to the antiproliferative effects reported on MPM cells. However, the direct and indirect effects of epigenetic reprogramming on the tumor microenvironment are hitherto unexplored. To investigate the impact of tumor-associated macrophages (TAMs) on MPM cell responsiveness to tazemetostat, we developed a three-dimensional MPM spheroid model that recapitulates in vitro, both monocytes' recruitment in tumors and their functional differentiation toward a TAM-like phenotype (Mo-TAMs). Along with an increased expression of genes for monocyte chemoattractants, inhibitory immune checkpoints, immunosuppressive and M2-like molecules, Mo-TAMs promote tumor cell proliferation and spreading. Prolonged treatment of MPM spheroids with tazemetostat enhances both the recruitment of Mo-TAMs and the expression of their protumor phenotype. Therefore, Mo-TAMs profoundly suppress the antiproliferative effects due to EZH2 inhibition in MPM cells. Overall, our findings indicate that TAMs are a driving force for MPM growth, progression, and resistance to tazemetostat; therefore, strategies of TAM depletion might be evaluated to improve the therapeutic efficacy of pharmacological inhibition of EZH2.

Gut-Ex-Vivo system as a model to study gluten response in celiac disease.

Celiac disease (CD) is a complex immune-mediated chronic disease characterized by a consistent inflammation of the gastrointestinal tract induced by gluten intake in genetically predisposed individuals. Although initiated by the interaction between digestion-derived gliadin, a gluten component, peptides, and the intestinal epithelium, the disorder is highly complex and involving other components of the intestine, such as the immune system. Therefore, conventional model systems, mainly based on two- or three-dimension cell cultures and co-cultures, cannot fully recapitulate such a complex disease. The development of mouse models has facilitated the study of different interacting cell types involved in the disorder, together with the impact of environmental factors. However, such in vivo models are often expensive and time consuming. Here we propose an organ ex vivo culture (gut-ex-vivo system) based on small intestines from gluten-sensitive mice cultivated in a dynamic condition, able to fully recapitulate the biochemical and morphological features of the mouse model exposed to gliadin (4 weeks), in 16 h. Indeed, upon gliadin exposure, we observed: i) a down-regulation of cystic fibrosis transmembrane regulator (CFTR) and an up-regulation of transglutaminase 2 (TG2) at both mRNA and protein levels; ii) increased intestinal permeability associated with deregulated tight junction protein expression; iii) induction and production of pro-inflammatory cytokines such as interleukin (IL)-15, IL-17 and interferon gamma (IFNγ); and iv) consistent alteration of intestinal epithelium/villi morphology. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of CD, test new or repurposed molecules to accelerate the search for new treatments, and to study the impact of the microbiome and derived metabolites, in a time- and cost- effective manner.

Proteomic analysis links alterations of bioenergetics, mitochondria-ER interactions and proteostasis in hippocampal astrocytes from 3xTg-AD mice.

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 Ca2+ 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 Ca2+ probe we show that, in 3Tg-iAstro cells, ER was overloaded with Ca2+ while Ca2+ 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, Ca2+ homeostasis and proteostasis. These factors may interact, creating a pathogenic loop compromising homeostatic and defensive functions of astroglial cells predisposing neurons to dysfunction.

Ferroptosis: a new unexpected chance to treat metastatic melanoma?

Human skin melanoma is one of the most aggressive and difficult to treat human malignancies, with an increasing incidence over the years. While the resection of the early diagnosed primary tumor remains the best clinical approach, advanced/metastatic melanoma still remains with a poor prognosis. Indeed, although enormous progress in the therapeutic treatment of human tumors has been made in recent years, patients affected by metastatic melanoma are still poorly affected by these clinical advances. Therefore, new valuable therapeutic approaches are urgently needed, to design and define effective treatments to consistently increase the overall survival rate of patients affected by this malignancy. In this review we summarize the main signaling pathways studied to kill human skin melanoma, and introduce the ferroptotic cell death as a new pathway to be explored to eradicate this tumor.

Haptoglobin Phenotypes Are Associated with the Postload Glucose and Insulin Levels in Pediatric Obesity.

PURPOSE: Haptoglobin (Hp) is a protein involved in the acute-phase reaction of inflammation. Humans have three major phenotypes (Hp1-1, Hp1-2, and Hp2-2). Several studies have shown altered Hp regulation in adults with obesity and metabolic alterations. The Hp2-2 phenotype is associated with a high cardiovascular risk. Our aim was to investigate if Hp levels and the Hp2-2 phenotype are associated with glucose metabolism in pediatric obesity. METHODS: We retrospectively studied 192 participants (92 males and 100 females), aged 4-18 years. Clinical and biochemical data were collected. The Hp phenotype (Hp1-1, Hp1-2, and Hp2-2) was identified through Western immunoblot. RESULTS: Subjects carrying Hp1-1, Hp1-2, and Hp2-2 phenotypes were 13.6, 50.8, and 35.6%, respectively. Hp serum, fasting glucose, and insulin levels, as well as HOMA-IR, were similar among groups. Postload glucose and insulin levels (as insulin AUC) were progressively higher from the Hp1-1 to Hp2-2 phenotype. CONCLUSION: To our knowledge, this is the first study on Hp phenotypes conducted in a pediatric population with obesity. We showed that the presence of Hp2 allele is associated with a worse response of glucose load in terms of both glucose and insulin levels. Thus, the Hp2-2 phenotype could predispose in pediatrics, at the same degree of obesity, to a worse glycemic and insulinemic compensation.