Antipsychotic drugs counteract autophagy and mitophagy in multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by myelin damage followed by axonal and ultimately neuronal loss. The etiology and physiopathology of MS are still elusive, and no fully effective therapy is yet available. We investigated the role in MS of autophagy (physiologically, a controlled intracellular pathway regulating the degradation of cellular components) and of mitophagy (a specific form of autophagy that removes dysfunctional mitochondria). We found that the levels of autophagy and mitophagy markers are significantly increased in the biofluids of MS patients during the active phase of the disease, indicating activation of these processes. In keeping with this idea, in vitro and in vivo MS models (induced by proinflammatory cytokines, lysolecithin, and cuprizone) are associated with strongly impaired mitochondrial activity, inducing a lactic acid metabolism and prompting an increase in the autophagic flux and in mitophagy. Multiple structurally and mechanistically unrelated inhibitors of autophagy improved myelin production and normalized axonal myelination, and two such inhibitors, the widely used antipsychotic drugs haloperidol and clozapine, also significantly improved cuprizone-induced motor impairment. These data suggest that autophagy has a causal role in MS; its inhibition strongly attenuates behavioral signs in an experimental model of the disease. Therefore, haloperidol and clozapine may represent additional therapeutic tools against MS.

Classical and Innovative Evidence for Therapeutic Strategies in Retinal Dysfunctions.

The human retina is a complex anatomical structure that has no regenerative capacity. The pathogenesis of most retinopathies can be attributed to inflammation, with the activation of the inflammasome protein platform, and to the impact of oxidative stress on the regulation of apoptosis and autophagy/mitophagy in retinal cells. In recent years, new therapeutic approaches to treat retinopathies have been investigated. Experimental data suggest that the secretome of mesenchymal cells could reduce oxidative stress, autophagy, and the apoptosis of retinal cells, and in turn, the secretome of the latter could induce changes in mesenchymal cells. Other studies have evidenced that noncoding (nc)RNAs might be new targets for retinopathy treatment and novel disease biomarkers since a correlation has been found between ncRNA levels and retinopathies. A new field to explore is the interaction observed between the ocular and intestinal microbiota; indeed, recent findings have shown that the alteration of gut microbiota seems to be linked to ocular diseases, suggesting a gut-eye axis. To explore new therapeutical strategies for retinopathies, it is important to use proper models that can mimic the complexity of the retina. In this context, retinal organoids represent a good model for the study of the pathophysiology of the retina.

Various Aspects of Calcium Signaling in the Regulation of Apoptosis, Autophagy, Cell Proliferation, and Cancer.

Calcium (Ca2+) is a major second messenger in cells and is essential for the fate and survival of all higher organisms. Different Ca2+ channels, pumps, or exchangers regulate variations in the duration and levels of intracellular Ca2+, which may be transient or sustained. These changes are then decoded by an elaborate toolkit of Ca2+-sensors, which translate Ca2+ signal to intracellular operational cell machinery, thereby regulating numerous Ca2+-dependent physiological processes. Alterations to Ca2+ homoeostasis and signaling are often deleterious and are associated with certain pathological states, including cancer. Altered Ca2+ transmission has been implicated in a variety of processes fundamental for the uncontrolled proliferation and invasiveness of tumor cells and other processes important for cancer progression, such as the development of resistance to cancer therapies. Here, we review what is known about Ca2+ signaling and how this fundamental second messenger regulates life and death decisions in the context of cancer, with particular attention directed to cell proliferation, apoptosis, and autophagy. We also explore the intersections of Ca2+ and the therapeutic targeting of cancer cells, summarizing the therapeutic opportunities for Ca2+ signal modulators to improve the effectiveness of current anticancer therapies.

Endoplasmic reticulum-mitochondria Ca2+ crosstalk in the control of the tumor cell fate.

Mitochondria-associated membranes are juxtaposed between the endoplasmic reticulum and mitochondria and have been identified as a critical hub in the regulation of apoptosis and tumor growth. One key function of mitochondria-associated membranes is to provide asylum to a number of proteins with tumor suppressor and oncogenic properties. In this review, we discuss how Ca2+ flux manipulation represents the primary mechanism underlying the action of several oncogenes and tumor-suppressor genes and how these networks might be manipulated to provide novel therapies for cancer. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Loss of miR-204 expression is a key event in melanoma.

Cutaneous melanoma (CM) is a malignancy with increasing occurrence. Its microRNA repertoire has been defined in a number studies, leading to candidates for biological and clinical relevance: miR-200a/b/c, miR-203, miR-205, miR-204, miR-211, miR-23b and miR-26a/b. Our work was aimed to validate the role of these candidate miRNAs in melanoma, using additional patients cohorts and in vitro cultures. miR-26a, miR-204 and miR-211 were more expressed in normal melanocytes, while miR-23b, miR-200b/c, miR-203 and miR-205 in epidermis and keratinocytes. None of the keratinocyte-related miRNAs was associated with any known mutation or with clinical covariates in melanoma. On the other hand, the loss of miR-204 was enriched in melanomas with NRAS sole mutation (Fisher exact test, P = 0.001, Log Odds = 1.67), and less frequent than expected in those harbouring CDKN2A mutations (Fisher exact test, P = 0.001, Log Odds - 1.09). Additionally, miR-204 was associated with better prognosis in two independent melanoma cohorts and its exogenous expression led to growth impairment in melanoma cell lines. Thus, miR-204 represents a relevant mechanism in melanoma, with potential prognostic value and its loss seems to act in the CDKN2A pathway, in cooperation with NRAS.

Calcium regulates cell death in cancer: Roles of the mitochondria and mitochondria-associated membranes (MAMs).

Until 1972, the term 'apoptosis' was used to differentiate the programmed cell death that naturally occurs in organismal development from the acute tissue death referred to as necrosis. Many studies on cell death and programmed cell death have been published and most are, at least to some degree, related to cancer. Some key proteins and molecular pathways implicated in cell death have been analyzed, whereas others are still being actively researched; therefore, an increasing number of cellular compartments and organelles are being implicated in cell death and cancer. Here, we discuss the mitochondria and subdomains of the endoplasmic reticulum (ER) that interact with mitochondria, the mitochondria-associated membranes (MAMs), which have been identified as critical hubs in the regulation of cell death and tumor growth. MAMs-dependent calcium (Ca2+) release from the ER allows selective Ca2+ uptake by the mitochondria. The perturbation of Ca2+ homeostasis in cancer cells is correlated with sustained cell proliferation and the inhibition of cell death through the modulation of Ca2+ signaling. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

Reprogramming of miRNA networks in cancer and leukemia.

We studied miRNA profiles in 4419 human samples (3312 neoplastic, 1107 nonmalignant), corresponding to 50 normal tissues and 51 cancer types. The complexity of our database enabled us to perform a detailed analysis of microRNA (miRNA) activities. We inferred genetic networks from miRNA expression in normal tissues and cancer. We also built, for the first time, specialized miRNA networks for solid tumors and leukemias. Nonmalignant tissues and cancer networks displayed a change in hubs, the most connected miRNAs. hsa-miR-103/106 were downgraded in cancer, whereas hsa-miR-30 became most prominent. Cancer networks appeared as built from disjointed subnetworks, as opposed to normal tissues. A comparison of these nets allowed us to identify key miRNA cliques in cancer. We also investigated miRNA copy number alterations in 744 cancer samples, at a resolution of 150 kb. Members of miRNA families should be similarly deleted or amplified, since they repress the same cellular targets and are thus expected to have similar impacts on oncogenesis. We correctly identified hsa-miR-17/92 family as amplified and the hsa-miR-143/145 cluster as deleted. Other miRNAs, such as hsa-miR-30 and hsa-miR-204, were found to be physically altered at the DNA copy number level as well. By combining differential expression, genetic networks, and DNA copy number alterations, we confirmed, or discovered, miRNAs with comprehensive roles in cancer. Finally, we experimentally validated the miRNA network with acute lymphocytic leukemia originated in Mir155 transgenic mice. Most of miRNAs deregulated in these transgenic mice were located close to hsa-miR-155 in the cancer network.

Nutrients, herbal bioactive derivatives and commensal microbiota as tools to lower the risk of SARS-CoV-2 infection.

The SARS-CoV-2 outbreak has infected a vast population across the world, causing more than 664 million cases and 6.7 million deaths by January 2023. Vaccination has been effective in reducing the most critical aftermath of this infection, but some issues are still present regarding re-infection prevention, effectiveness against variants, vaccine hesitancy and worldwide accessibility. Moreover, although several old and new antiviral drugs have been tested, we still lack robust and specific treatment modalities. It appears of utmost importance, facing this continuously growing pandemic, to focus on alternative practices grounded on firm scientific bases. In this article, we aim to outline a rigorous scientific background and propose complementary nutritional tools useful toward containment, and ultimately control, of SARS-CoV-2 infection. In particular, we review the mechanisms of viral entry and discuss the role of polyunsaturated fatty acids derived from α-linolenic acid and other nutrients in preventing the interaction of SARS-CoV-2 with its entry gateways. In a similar way, we analyze in detail the role of herbal-derived pharmacological compounds and specific microbial strains or microbial-derived polypeptides in the prevention of SARS-CoV-2 entry. In addition, we highlight the role of probiotics, nutrients and herbal-derived compounds in stimulating the immunity response.

The role of the mTOR pathway in diabetic retinopathy.

The retina, the part of the eye, translates the light signal into an electric current that can be sent to the brain as visual information. To achieve this, the retina requires fine-tuned vascularization for its energy supply. Diabetic retinopathy (DR) causes alterations in the eye vascularization that reduce the oxygen supply with consequent retinal neurodegeneration. During DR, the mammalian target of rapamycin (mTOR) pathway seems to coordinate retinal neurodegeneration with multiple anabolic and catabolic processes, such as autophagy, oxidative stress, cell death, and the release of pro-inflammatory cytokines, which are closely related to chronic hyperglycemia. This review outlines the normal anatomy of the retina and how hyperglycemia can be involved in the neurodegeneration underlying this disease through over activation or inhibition of the mTOR pathway.

Epigenetic Regulation: A Link between Inflammation and Carcinogenesis.

Epigenetics encompasses a group of dynamic, reversible, and heritable modifications that occur within cells that are independent of gene mutations. These alterations are highly influenced by the environment, from the environment that surrounds the human being to the internal microenvironments located within tissues and cells. The ways that pigenetic modifications promote the initiation of the tumorigenic process have been widely demonstrated. Similarly, it is well known that carcinogenesis is supported and prompted by a strong proinflammatory environment. In this review, we introduce our report of a proinflammatory microenvironment that encircles the tumor core but can be responsible for the induction of epigenetic drift. At the same time, cancer cells can alter their epigenetic profile to generate a positive loop in the promotion of the inflammatory process. Therefore, an in-depth understanding of the epigenetic networks between the tumor microenvironment and cancer cells might highlight new targetable mechanisms that could prevent tumor progression.

Relevance of Autophagy and Mitophagy Dynamics and Markers in Neurodegenerative Diseases.

During the past few decades, considerable efforts have been made to discover and validate new molecular mechanisms and biomarkers of neurodegenerative diseases. Recent discoveries have demonstrated how autophagy and its specialized form mitophagy are extensively associated with the development, maintenance, and progression of several neurodegenerative diseases. These mechanisms play a pivotal role in the homeostasis of neural cells and are responsible for the clearance of intracellular aggregates and misfolded proteins and the turnover of organelles, in particular, mitochondria. In this review, we summarize recent advances describing the importance of autophagy and mitophagy in neurodegenerative diseases, with particular attention given to multiple sclerosis, Parkinson's disease, and Alzheimer's disease. We also review how elements involved in autophagy and mitophagy may represent potential biomarkers for these common neurodegenerative diseases. Finally, we examine the possibility that the modulation of autophagic and mitophagic mechanisms may be an innovative strategy for overcoming neurodegenerative conditions. A deeper knowledge of autophagic and mitophagic mechanisms could facilitate diagnosis and prognostication as well as accelerate the development of therapeutic strategies for neurodegenerative diseases.

Methods to Monitor Mitophagy and Mitochondrial Quality: Implications in Cancer, Neurodegeneration, and Cardiovascular Diseases.

Mitochondria are dynamic organelles that participate in a broad array of molecular functions within the cell. They are responsible for maintaining the appropriate energetic levels and control the cellular homeostasis throughout the generation of intermediary metabolites. Preserving a healthy and functional mitochondrial population is of fundamental importance throughout the life of the cells under pathophysiological conditions. Hence, cells have evolved fine-tuned mechanisms of quality control that help to preserve the right amount of functional mitochondria to meet the demand of the cell. The specific recycling of mitochondria by autophagy, termed mitophagy, represents the primary contributor to mitochondrial quality control. During this process, damaged or unnecessary mitochondria are recognized and selectively degraded. In the past few years, the knowledge in mitophagy has seen rapid progress, and a growing body of evidence confirms that mitophagy holds a central role in controlling cellular functions and the progression of various human diseases.In this chapter, we will discuss the pathophysiological roles of mitophagy and provide a general overview of the current methods used to monitor and quantify mitophagy. We will also outline the main established approaches to investigate the mitochondrial function, metabolism, morphology, and protein damage.

Sodium azide induced neuronal damage in vitro: evidence for non-apoptotic cell death.

The features of neuronal damage induced by the mitochondrial toxin NaN(3) were investigated in rat primary cortical neuron cultures. Cell viability (MTT colorimetric determination) and transmembrane mitochondrial potential (J-C1 fluorescence) were concentration-dependently reduced 24 h after NaN(3); neither nuclear fragmentation by DAPI, nor Annexin V positivity by flow cytometry were detected, ruling out the occurrence of apoptosis. The loss in cell viability (to 54 +/- 2%) observed 24 h after a 10-min treatment with 3 mM NaN(3) was prevented by the NMDA glutamate receptor antagonist MK801 (1 microM), by the antioxidants trolox (100 microM) and acetyl-L-carnitine (1 mM) and by the nitric oxide synthase inhibitor, L-NAME (100 microM), but not by the guanylylcyclase inhibitor ODQ, 10 microM. The mitochondrial dysfunction induced by NaN(3) provides a common platform for investigating the mechanisms of both ischemic and degenerative neuronal injury, useful for screening potential protective agents against neuronal death.

Mitochondria-associated membranes (MAMs) and inflammation.

The endoplasmic reticulum (ER) and mitochondria are tightly associated with very dynamic platforms termed mitochondria-associated membranes (MAMs). MAMs provide an excellent scaffold for crosstalk between the ER and mitochondria and play a pivotal role in different signaling pathways that allow rapid exchange of biological molecules to maintain cellular health. However, dysfunctions in the ER-mitochondria architecture are associated with pathological conditions and human diseases. Inflammation has emerged as one of the various pathways that MAMs control. Inflammasome components and other inflammatory factors promote the release of pro-inflammatory cytokines that sustain pathological conditions. In this review, we summarize the critical role of MAMs in initiating inflammation in the cellular defense against pathogenic infections and the association of MAMs with inflammation-mediated diseases.

Screen for MicroRNA and Drug Interactions in Breast Cancer Cell Lines Points to miR-126 as a Modulator of CDK4/6 and PIK3CA Inhibitors.

Background: Breast cancer (BC) represents the most common cancer in women worldwide. Due to its heterogeneous nature, breast cancer management might benefit from differential treatments toward personalized medicine. Additionally, drug resistance is a common phenomenon. We systematically investigated the effect of 14 different drugs administered on BC cell lines in combination with microRNAs (miRNA, miR). Methods: Thirty-eight miRNAs, all associated with BC by clinical and molecular parameters including progression, prognosis and subtypes, were tested for their effects on the viability of 12 different BC cell lines. Four miRNAs with the strongest impact on viability were further assayed in combination with 14 BC drugs. Mann-Whitney U-test with Bonferroni correction was used for statistical analysis. Results: In a miRNA only pre-screen we observed effects on BC cell lines' viability for 34 out of 38 candidate miRNAs. We then identified 14 miRNA/drug combinations for which the combination IC50 was lower than that of both miRNA and drug as single agents. miR-181a, paired with GSK1070916, Doxorubicin, XL765 and AMG511, was the only miRNA active on the triple negative (TNBC) MDA-MB-468 cell line. miR-126 was the only miRNA (in combination with CDK4/6 or PIK3CA inhibitors) with significant effects on cell lines from different subtypes: MCF7 (Luminal) and MDA-MB-453 (HER2+). Because of its activity on different BC subtypes, we investigated the genome wide effects of miR-126 using transcriptomics and confirmed that expression of miR-126 in BC cell lines affected cell cycle and mitosis. Conclusion: Our results show that a combination treatment with miRNAs, in particular miR-181a, miR-326, miR-9 and miR-126, enhance the activity of specific BC drugs in vitro, even on the most aggressive BC subtypes, HER2+ and TNBC. Finally, as expected from its drug interactions, based on a whole transcriptome study we could confirm a role for miR-126 in cell cycle regulation.

Ethanolic extract from Hemidesmus indicus (Linn) displays otoprotectant activities on organotypic cultures without interfering on gentamicin uptake.

The ethanolic extract from Hemidesmus indicus (Linn) (Apocynaceae) (Hie) was studied for its otoprotective effects in ex vivo rat organotypic model of gentamicin (GM) toxicity. In organ of Corti organotypic cultures (OC), GM can induce a fast dose-dependent apoptosis of hair cells (HC), both external and internal. We found that, after coadministration of GM and Hie to organotypic cultures, the extract was able to significantly counteract this toxic effect on HC, at the concentration of 25 and 50microg/ml. Interestingly, at these concentrations the extract was present in the cell medium at a concentration 1.6- and 3.3-fold lower than GM, suggesting its otoprotective activity could not merely due to an aspecific inhibition of GM entry. To support this hypothesis, we evaluated the amount of GM present in organotypic cultures after the coadministration of 1.5mg/ml GM and Hie, and found no significant reduction of GM uptake in the presence of 100microg/ml Hie. These data suggest the otoprotective action of Hie derives from specific inhibition of the apoptotic routine induced by GM treatment.

Regulation of Endoplasmic Reticulum-Mitochondria Ca2+ Transfer and Its Importance for Anti-Cancer Therapies.

Inter-organelle membrane contact sites are emerging as major sites for the regulation of intracellular Ca2+ concentration and distribution. Here, extracellular stimuli operate on a wide array of channels, pumps, and ion exchangers to redistribute intracellular Ca2+ among several compartments. The resulting highly defined spatial and temporal patterns of Ca2+ movement can be used to elicit specific cellular responses, including cell proliferation, migration, or death. Plasma membrane (PM) also can directly contact mitochondria and endoplasmic reticulum (ER) through caveolae, small invaginations of the PM that ensure inter-organelle contacts, and can contribute to the regulation of numerous cellular functions through scaffolding proteins such as caveolins. PM and ER organize specialized junctions. Here, many components of the receptor-dependent Ca2+ signals are clustered, including the ORAI1-stromal interaction molecule 1 complex. This complex constitutes a primary mechanism for Ca2+ entry into non-excitable cells, modulated by intracellular Ca2+. Several contact sites between the ER and mitochondria, termed mitochondria-associated membranes, show a very complex and specialized structure and host a wide number of proteins that regulate Ca2+ transfer. In this review, we summarize current knowledge of the particular action of several oncogenes and tumor suppressors at these specialized check points and analyze anti-cancer therapies that specifically target Ca2+ flow at the inter-organelle contacts to alter the metabolism and fate of the cancer cell.

Risk factors associated with relapse of eyelid basal cell carcinoma: results from a retrospective study of 142 patients.

Of skin cancers, 9% arise at the periocular level, constituting a significant threat due to the proximity to intracranial structures, such as the eyes, nerve endings and proximal tissues. Tumour recurrence can be frequent and represents a primary clinical challenge for the surgeon. To present a retrospective study on the treatment of eyelid tumours at a tertiary care centre in Italy over an eight-year period and, in particular, to underline the risk factors associated with tumour relapse. Among a cohort of 205 patients, a retrospective study was conducted on 142 basal cell carcinoma (BCC) patients with eyelid tumours treated with surgical excision. Relapse-free survival was assessed using univariate Kaplan-Meier and multivariate Cox regression analysis. Over an eight-year study period, we detected 23 cases of BCC recurrence, with tumour localization associating with tumour relapse, representing an independent risk factor. The extent of the area of excision was significantly associated with relapse, but not margin positivity which was associated with reduced relapse-free survival. To minimize relapse of basal cell carcinoma during patient management, relevant factors to consider before and after tumour excision include tumour localization, margin invasion, and extension of the excision, but not the surgical technique used.

Cisplatin-induced apoptosis in human promyelocytic leukemia cells.

Cis-diamminedichloroplatinum (II) cisplatin (CDDP) is an organometallic compound frequently used in anti-cancer therapy, in particular ovarian, testicular, and head and neck tumors. We found cisplatin was effective against human promyelocytic leukemia cell line HL-60, inhibiting cell cycle progression and inducing time- and concentration- dependent cell death. Presence of nuclear fragmentation, caspase-3 cleavage and annexin V positivity suggests cell death occurred by apoptosis, although DNA internucleosomal fragmentation was not detected. In addition, analysis of malondialdehyde (MDA) production and protein carbonylation indicated that cisplatin increased lipid peroxidation and oxidation of cell proteins. This occurrence was prevented by antioxidants such as N-acetylcysteine (N-aC) and glutathione (GSH), which, consistently, were also able to prevent CDDP-induced cell death. Collectively, these findings indicate that, besides growth inhibition, an increase of oxygen radicals and lipid degradation can account for a significant part of CDDP-induced apoptosis.

Mitochondrial reactive oxygen species and inflammation: Molecular mechanisms, diseases and promising therapies.

Over the last few decades, many different groups have been engaged in studies of new roles for mitochondria, particularly the coupling of alterations in the redox pathway with the inflammatory responses involved in different diseases, including Alzheimer's disease, Parkinson's disease, atherosclerosis, cerebral cavernous malformations, cystic fibrosis and cancer. Mitochondrial dysfunction is important in these pathological conditions, suggesting a pivotal role for mitochondria in the coordination of pro-inflammatory signaling from the cytosol and signaling from other subcellular organelles. In this regard, mitochondrial reactive oxygen species are emerging as perfect liaisons that can trigger the assembly and successive activation of large caspase-1- activating complexes known as inflammasomes. This review offers a glimpse into the mechanisms by which inflammasomes are activated by mitochondrial mechanisms, including reactive oxygen species production and mitochondrial Ca2+ uptake, and the roles they can play in several inflammatory pathologies.