Human frataxin, the Friedreich ataxia deficient protein, interacts with mitochondrial respiratory chain.

Friedreich ataxia (FRDA) is a rare, inherited neurodegenerative disease caused by an expanded GAA repeat in the first intron of the FXN gene, leading to transcriptional silencing and reduced expression of frataxin. Frataxin participates in the mitochondrial assembly of FeS clusters, redox cofactors of the respiratory complexes I, II and III. To date it is still unclear how frataxin deficiency culminates in the decrease of bioenergetics efficiency in FRDA patients' cells. We previously demonstrated that in healthy cells frataxin is closely attached to the mitochondrial cristae, which contain both the FeS cluster assembly machinery and the respiratory chain complexes, whereas in FRDA patients' cells with impaired respiration the residual frataxin is largely displaced in the matrix. To gain novel insights into the function of frataxin in the mitochondrial pathophysiology, and in the upstream metabolic defects leading to FRDA disease onset and progression, here we explored the potential interaction of frataxin with the FeS cluster-containing respiratory complexes I, II and III. Using healthy cells and different FRDA cellular models we found that frataxin interacts with these three respiratory complexes. Furthermore, by EPR spectroscopy, we observed that in mitochondria from FRDA patients' cells the decreased level of frataxin specifically affects the FeS cluster content of complex I. Remarkably, we also found that the frataxin-like protein Nqo15 from T. thermophilus complex I ameliorates the mitochondrial respiratory phenotype when expressed in FRDA patient's cells. Our data point to a structural and functional interaction of frataxin with complex I and open a perspective to explore therapeutic rationales for FRDA targeted to this respiratory complex.

The proper interplay between the expression of Spo11 splice isoforms and the structure of the pseudoautosomal region promotes XY chromosomes recombination.

XY chromosome missegregation is relatively common in humans and can lead to sterility or the generation of aneuploid spermatozoa. A leading cause of XY missegregation in mammals is the lack of formation of double-strand breaks (DSBs) in the pseudoautosomal region (PAR), a defect that may occur in mice due to faulty expression of Spo11 splice isoforms. Using a knock-in (ki) mouse that expresses only the single Spo11ß splice isoform, here we demonstrate that by varying the genetic background of mice, the length of chromatin loops extending from the PAR axis and the XY recombination proficiency varies. In spermatocytes of C57Spo11ßki/- mice, in which loops are relatively short, recombination/synapsis between XY is fairly normal. In contrast, in cells of C57/129Spo11ßki/- males where PAR loops are relatively long, formation of DSBs in the PAR (more frequently the Y-PAR) and XY synapsis fails at a high rate, and mice produce sperm with sex-chromosomal aneuploidy. However, if the entire set of Spo11 splicing isoforms is expressed by a wild type allele in the C57/129 background, XY recombination and synapsis is recovered. By generating a Spo11αki mouse model, we prove that concomitant expression of SPO11ß and SPO11α isoforms, boosts DSB formation in the PAR. Based on these findings, we propose that SPO11 splice isoforms cooperate functionally in promoting recombination in the PAR, constraining XY asynapsis defects that may arise due to differences in the conformation of the PAR between mouse strains.

S100A8/A9-alarmin promotes local myeloid-derived suppressor cell activation restricting severe autoimmune arthritis.

Immune-suppressive effects of myeloid-derived suppressor cells (MDSCs) are well characterized during anti-tumor immunity. The complex mechanisms promoting MDSC development and their regulatory effects during autoimmune diseases are less understood. We demonstrate that the endogenous alarmin S100A8/A9 reprograms myeloid cells to a T cell suppressing phenotype during autoimmune arthritis. Treatment of myeloid precursors with S100-alarmins during differentiation induces MDSCs in a Toll-like receptor 4-dependent manner. Consequently, knockout of S100A8/A9 aggravates disease activity in collagen-induced arthritis due to a deficit of MDSCs in local lymph nodes, which could be corrected by adoptive transfer of S100-induced MDSCs. Blockade of MDSC function in vivo aggravates disease severity in arthritis. Therapeutic application of S100A8 induces MDSCs in vivo and suppresses the inflammatory phenotype of S100A9ko mice. Accordingly, the interplay of T cell-mediated autoimmunity with a defective innate immune regulation is crucial for autoimmune arthritis, which should be considered for future innovative therapeutic options.

SAMHD1 restricts the deoxyguanosine triphosphate pool contributing to telomere stability in telomerase-positive cells.

SAMHD1 (Sterile alpha motif and histidine/aspartic acid domain-containing protein 1) is a dNTP triphosphohydrolase crucial in the maintenance of balanced cellular dNTP pools, which support genome integrity. In SAMHD1 deficient fibroblasts isolated from Aicardi-Goutières Syndrome (AGS) patients, all four DNA precursors are increased and markedly imbalanced with the largest effect on dGTP, a key player in the modulation of telomerase processivity. Here, we present data showing that SAMHD1, by restricting the dGTP pool, contributes to telomere maintenance in hTERT-immortalized human fibroblasts from AGS patients as well as in telomerase positive cancer cell lines. Only in cells expressing telomerase, the lack of SAMHD1 causes excessive lengthening of telomeres and telomere fragility, whereas primary fibroblasts lacking both SAMHD1 and telomerase enter normally into senescence. Telomere lengthening observed in SAMHD1 deficient but telomerase proficient cells is a gradual process, in accordance with the intrinsic property of telomerase of adding only a few tens of nucleotides for each cycle. Therefore, only a prolonged exposure to high dGTP content causes telomere over-elongation. hTERT-immortalized AGS fibroblasts display also high fragility of chromosome ends, a marker of telomere replication stress. These results not only demonstrate the functional importance of dGTP cellular level but also reveal the critical role played by SAMHD1 in restraining telomerase processivity and safeguarding telomere stability.

The Interplay between Ghrelin and Microglia in Neuroinflammation: Implications for Obesity and Neurodegenerative Diseases.

Numerous studies have shown that microglia are capable of producing a wide range of chemokines to promote inflammatory processes within the central nervous system (CNS). These cells share many phenotypical and functional characteristics with macrophages, suggesting that microglia participate in innate immune responses in the brain. Neuroinflammation induces neurometabolic alterations and increases in energy consumption. Microglia may constitute an important therapeutic target in neuroinflammation. Recent research has attempted to clarify the role of Ghre signaling in microglia on the regulation of energy balance, obesity, neuroinflammation and the occurrence of neurodegenerative diseases. These studies strongly suggest that Ghre modulates microglia activity and thus affects the pathophysiology of neurodegenerative diseases. This review aims to summarize what is known from the current literature on the way in which Ghre modulates microglial activity during neuroinflammation and their impact on neurometabolic alterations in neurodegenerative diseases. Understanding the role of Ghre in microglial activation/inhibition regulation could provide promising strategies for downregulating neuroinflammation and consequently for diminishing negative neurological outcomes.

Alarming and Calming: Opposing Roles of S100A8/S100A9 Dimers and Tetramers on Monocytes.

Mechanisms keeping leukocytes distant of local inflammatory processes in a resting state despite systemic release of inflammatory triggers are a pivotal requirement for avoidance of overwhelming inflammation but are ill defined. Dimers of the alarmin S100A8/S100A9 activate Toll-like receptor-4 (TLR4) but extracellular calcium concentrations induce S100A8/S100A9-tetramers preventing TLR4-binding and limiting their inflammatory activity. So far, only antimicrobial functions of released S100A8/S100A9-tetramers (calprotectin) are described. It is demonstrated that extracellular S100A8/S100A9 tetramers significantly dampen monocyte dynamics as adhesion, migration, and traction force generation in vitro and immigration of monocytes in a cutaneous granuloma model and inflammatory activity in a model of irritant contact dermatitis in vivo. Interestingly, these effects are not mediated by the well-known binding of S100A8/S100A9-dimers to TLR-4 but specifically mediated by S100A8/S100A9-tetramer interaction with CD69. Thus, the quaternary structure of these S100-proteins determines distinct and even antagonistic effects mediated by different receptors. As S100A8/S100A9 are released primarily as dimers and subsequently associate to tetramers in the high extracellular calcium milieu, the same molecules promote inflammation locally (S100-dimer/TLR4) but simultaneously protect the wider environment from overwhelming inflammation (S100-tetramer/CD69).

Immunoregulation of Ghrelin in neurocognitive sequelae associated with COVID-19: an in silico investigation.

Some patients suffering from the new Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) develop an exaggerated inflammatory response triggered by a "cytokine storm" resulting in acute respiratory distress syndrome (ARDS) with the concomitant activation of non-specific inflammatory reactivity in the circulatory system and other organs, leading to multiorgan failure, leaky vasculature, coagulopathies and stroke. Impairment of brain functions may also occur as dysregulations in immune function resulting from neuroendocrine interactions. In this study, we explored, by bioinformatics approaches, the interaction between the multiple inflammatory agents involved in SARS-CoV-2 and Ghrelin (Ghre) together with its receptor GHSR-1A, which are described as anti-inflammatory mediators, in order to investigate what could trigger the hyper-inflammatory response in some SARS-CoV-2 patients. In our analysis, we found several interactions of Ghre and GHSR-1A with SARS-CoV-2 interacting human genes. We observed a correlation between Ghre, angiotensin-converting enzyme 2 ACE2, toll-like receptors 9 (TLR9), and Acidic chitinase (CHIA), whereas its receptor GHSR-1A interacts with chemokine receptor 3 (CXCR3), CCR3, CCR5, CCR7, coagulation factor II (thrombin) receptor-like 1 (F2RL1), vitamin D receptor (VDR), Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and DDP4 in receptor dipeptidyl peptidase-4. To our knowledge, our findings show, for the first time, that Ghre and GHSR-1A may exert an immunomodulatory function in the course of SARS-Cov-2 infection.

Analysis of monocyte cell tractions in 2.5D reveals mesoscale mechanics of podosomes during substrate-indenting cell protrusion.

Podosomes are mechanosensitive protrusive actin structures that are prominent in myeloid cells, and they have been linked to vascular extravasation. Recent studies have suggested that podosomes are hierarchically organized and have coordinated dynamics on the cell scale, which implies that the local force generation by single podosomes can be different from their global combined action. Complementary to previous studies focusing on individual podosomes, here we investigated the cell-wide force generation of podosome-bearing ER-Hoxb8 monocytes. We found that the occurrence of focal tractions accompanied by a cell-wide substrate indentation cannot be explained by summing the forces of single podosomes. Instead, our findings suggest that superimposed contraction on the cell scale gives rise to a buckling mechanism that can explain the measured cell-scale indentation. Specifically, the actomyosin network contraction causes peripheral in-plane substrate tractions, while the accumulated internal stress results in out-of-plane deformation in the central cell region via a buckling instability, producing the cell-scale indentation. Hence, we propose that contraction of the actomyosin network, which connects the podosomes, leads to a substrate indentation that acts in addition to the protrusion forces of individual podosomes. This article has an associated First Person interview with the first author of the paper.

Toward a Molecular Approach to Chronotype Assessment.

The aim of the present study was to develop a Polygenic Score-based model for molecular chronotype assessment. Questionnaire-based phenotypical chronotype assessment was used as a reference. In total, 54 extremely morning/morning (MM/M; 35 females, 39.7 ± 3.8 years) and 44 extremely evening/evening (EE/E; 20 females, 27.3 ± 7.7 years) individuals donated a buccal DNA sample for genotyping by sequencing of the entire genetic variability of 19 target genes known to be involved in circadian rhythmicity and/or sleep duration. Targeted genotyping was performed using the single primer enrichment technology and a specifically designed panel of 5526 primers. Among 2868 high-quality polymorphisms, a cross-validation approach lead to the identification of 83 chronotype predictive variants, including previously known and also novel chronotype-associated polymorphisms. A large (35 single-nucleotide polymorphisms [SNPs]) and also a small (13 SNPs) panel were obtained, both with an estimated predictive validity of approximately 80%. Potential mechanistic hypotheses for the role of some of the newly identified variants in modulating chronotype are formulated. Once validated in independent populations encompassing the whole range of chronotypes, the identified panels might become useful within the setting of both circadian public health initiatives and precision medicine.

Ghrelin peptide improves glial conditioned medium effects on neuronal differentiation of human adipose mesenchymal stem cells.

The influences of ghrelin on neural differentiation of adipose-derived mesenchymal stem cells (ASCs) were investigated in this study. The expression of typical neuronal markers, such as protein gene product 9.5 (PGP9.5) and Microtubule Associated Protein 2 (MAP2), as well as glial Fibrillary Acid Protein (GFAP) as a glial marker was evaluated in ASCs in different conditions. In particular, 2 µM ghrelin was added to control ASCs and to ASCs undergoing neural differentiation. For this purpose, ASCs were cultured in Conditioned Media obtained from Olfactory Ensheathing cells (OEC-CM) or from Schwann cells (SC-CM). Data on marker expression were gathered after 1 and 7 days of culture by fluorescence immunocytochemistry and flow cytometry. Results show that only weak effects were induced by the addition of only ghrelin. Instead, dynamic ghrelin-induced modifications were detected on the increased marker expression elicited by glial conditioned media. In fact, the combination of ghrelin and conditioned media consistently induced a further increase of PGP9.5 and MAP2 expression, especially after 7 days of treatment. The combination of ghrelin with SC-CM produced the most evident effects. Weak or no modifications were found on conditioned medium-induced GFAP increases. Observations on the ghrelin receptor indicate that its expression in control ASCs, virtually unchanged by the addition of only ghrelin, was considerably increased by CM treatment. These increases were enhanced by combining ghrelin and CM treatment, especially at 7 days. Overall, it can be assumed that ghrelin favors a neuronal rather than a glial ASC differentiation.

The displacement of frataxin from the mitochondrial cristae correlates with abnormal respiratory supercomplexes formation and bioenergetic defects in cells of Friedreich ataxia patients.

Friedreich ataxia (FRDA) is a neurodegenerative disease resulting from a severe decrease of frataxin (FXN). Most patients carry a GAA repeat expansion in both alleles of the FXN gene, whereas a small fraction of them are compound heterozygous for the expansion and a point mutation in the other allele. FXN is involved in the mitochondrial biogenesis of the FeS-clusters. Distinctive feature of FRDA patient cells is an impaired cellular respiration, likely due to a deficit of key redox cofactors working as electrons shuttles through the respiratory chain. However, a definite relationship between FXN levels, FeS-clusters assembly dysregulation and bioenergetics failure has not been established. In this work, we performed a comparative analysis of the mitochondrial phenotype of cell lines from FRDA patients, either homozygous for the expansion or compound heterozygotes for the G130V mutation. We found that, in healthy cells, FXN and two key proteins of the FeS-cluster assembly machinery are enriched in mitochondrial cristae, the dynamic subcompartment housing the respiratory chain. On the contrary, FXN widely redistributes to the matrix in FRDA cells with defects in respiratory supercomplexes assembly and altered respiratory function. We propose that this could be relevant for the early mitochondrial defects afflicting FRDA cells and that perturbation of mitochondrial morphodynamics could in turn be critical in terms of disease mechanisms.

Lipids from Insects in Cosmetics and for Personal Care Products.

Insects, the most varied group of known organisms on Earth, are arousing great interest also for the possibility to use them as a feed and food source. The mass rearing of some species, defined as "bioconverters", is spreading worldwide, thanks to their sustainability. At the end of the bioconversion process, breeders obtain eco-friendly biomolecules of high biological and economic value, including proteins and lipids, from larvae of bioconverter insects, in particular Hermetia illucens. Besides the most classical use of insect lipids as food additives, they are also used in the formulation of several products for personal care. The composition of insect lipids depends on the substrate on which the insects are reared but also on the insect species, so the cosmetic producers should consider these features to choose their insect starting point. The most abundant fatty acids detected in H. illucens are lauric, myristic, palmitic, and oleic acids, regardless of feed substrate; its fatty acids composition is favorable for soap composition, while their derivatives are used for detergent and shampoo. Here, we offer an overview of insect lipids, their extraction methods, and their application in cosmetics and personal care products.

Effects of Ghrelin on Olfactory Ensheathing Cell Viability and Neural Marker Expression.

Ghrelin (Ghre), a gut-brain peptide hormone, plays an important role in the entire olfactory system and in food behavior regulation. In the last years, it has aroused particular interest for its antioxidant, anti-inflammatory, and anti-apoptotic properties. Our previous research showed that Ghre and its receptor are expressed by peculiar glial cells of the olfactory system: Olfactory Ensheathing Cells (OECs). These cells are able to secrete different neurotrophic factors, promote axonal growth, and show stem cell characteristics. The aim of this work was to study, in an in vitro model, the effect of Ghre on both cell viability and the expression of some neural markers, such as Nestin (Ne), Glial Fibrillary Acid Protein (GFAP), Neuregulin (Neu), and ß-III-tubulin (Tuj1), in primary mouse OEC cultures. The MTT test and immunocytochemical procedures were used to highlight cell viability and marker expression, respectively. Our results demonstrate that Ghre, after 7 days of treatment, exerted a positive effect, stimulating OEC viability compared with cells without Ghre treatment. In addition, Ghre was able to modify the expression of some biomarkers, increasing Neu and Tuj1 expression, while GFAP was constant; on the contrary, the presence of positive Ne cells was drastically reduced after 7 days, and this showed a loss of stem cell characteristic and therefore the possible orientation towards an adult neural phenotype.

Prenatal music exposure influences weight, ghrelin expression, and morphology of rat hypothalamic neuron cultures.

Music plays an important role in brain physiology, in some areas related to emotions, food intake and body weight, such as the hypothalamus. There are different frequencies to which it can be tuned, today the most utilized is at 440 Hz, while in the past the 432 Hz frequency was more used to show particular effects on brain. It is known that Ghrelin, a peptide hormone, regulates food intake in the hypothalamus; in a previous paper, we reported that musical stimuli at 432 Hz modified the Ghrelin expression in the rat, increasing beneficial effects on metabolism. In this study, we used this frequency and we focused our attention on body weight, Ghrelin expression, and neuron morphology in hypothalamic cultures. To investigate the role of music, we utilized newborn pups from pregnant rats that were exposed to music stimuli at 432 Hz during the perinatal period and for the postnatal period, some for 3 days (P3) and others for 6 days (P6). Some pups were not exposed to music stimuli (controls). Our results showed that music increased the body weight of pups; in addition, enhanced Ghrelin expression in hypothalamic neurons and their axonal elongation were highlighted by immunocytochemical techniques. Moreover, we found that the positive music effect started in pups at P3 and increased at P6 compared with controls. These results suggest that the musical frequency at 432 Hz could stimulate the orexigenic Ghrelin effects influencing the increase in body weight and affecting the number of hypothalamic neurons expressing Ghrelin.

CD163 expression defines specific, IRF8-dependent, immune-modulatory macrophages in the bone marrow.

BACKGROUND: Scavenger receptor CD163 is exclusively expressed on monocytes/macrophages and is widely used as a marker for alternatively activated macrophages. However, the role of CD163 is not yet clear. OBJECTIVES: We sought to examine the function of CD163 in steady-state as well as in sterile and infectious inflammation. METHODS: Expression of CD163 was analyzed under normal and inflammatory conditions in mice. Functional relevance of CD163 was investigated in models of inflammation in wild-type and CD163-/- mice. RESULTS: We describe a subpopulation of bone marrow-resident macrophages (BMRMs) characterized by a high expression of CD163 and functionally distinct from classical bone marrow-derived macrophages. Development of CD163+ BMRMs is strictly dependent on IFN regulatory factor-8. CD163+ BMRMs show a specific transcriptome and cytokine secretion pattern demonstrating a specific immunomodulatory profile of these cells. Accordingly, CD163-/- mice show a stronger inflammation in allergic contact dermatitis, indicating a regulatory role of CD163. However, CD163-/- mice are highly susceptible to S aureus infections, demonstrating the relevance of CD163 for antimicrobial defense as well. CONCLUSIONS: Our data indicate that anti-inflammatory and immunosuppressive mechanisms are not necessarily associated with a decreased antimicrobial activity. In contrast, our data define a novel macrophage population that controls overwhelming inflammation on one hand but is also necessary for an effective control of infections on the other hand.

SAMHD1-deficient fibroblasts from Aicardi-Goutières Syndrome patients can escape senescence and accumulate mutations.

In mammalian cells, the catabolic activity of the dNTP triphosphohydrolase SAMHD1 sets the balance and concentration of the four dNTPs. Deficiency of SAMHD1 leads to unequally increased pools and marked dNTP imbalance. Imbalanced dNTP pools increase mutation frequency in cancer cells, but it is not known if the SAMHD1-induced dNTP imbalance favors accumulation of somatic mutations in non-transformed cells. Here, we have investigated how fibroblasts from Aicardi-Goutières Syndrome (AGS) patients with mutated SAMHD1 react to the constitutive pool imbalance characterized by a huge dGTP pool. We focused on the effects on dNTP pools, cell cycle progression, dynamics and fidelity of DNA replication, and efficiency of UV-induced DNA repair. AGS fibroblasts entered senescence prematurely or upregulated genes involved in G1/S transition and DNA replication. The normally growing AGS cells exhibited unchanged DNA replication dynamics and, when quiescent, faster rate of excision repair of UV-induced DNA damages. To investigate whether the lack of SAMHD1 affects DNA replication fidelity, we compared de novo mutations in AGS and WT cells by exome next-generation sequencing. Somatic variant analysis indicated a mutator phenotype suggesting that SAMHD1 is a caretaker gene whose deficiency is per se mutagenic, promoting genome instability in non-transformed cells.

Olfactory Ensheathing Cells express both Ghrelin and Ghrelin Receptor in vitro: a new hypothesis in favor of a neurotrophic effect.

Olfactory Ensheathing Cells (OECs) are glial cells able to secrete different neurotrophic growth factors and thus promote axonal growth, also acting as a mechanical support. In the olfactory system, during development, they drive the non-myelinated axons of the Olfactory Receptor Neurons (ORNs) towards the Olfactory Bulb (OB). Ghrelin (Ghre), a gut-brain peptide hormone, and its receptor (GHS-R 1a) are expressed in different parts of the central nervous system. In the last few years, this peptide has stimulated particular interest as results show it to be a neuroprotective factor with antioxidant, anti-inflammatory and anti-apoptotic properties. Our previous studies showed that OB mitral cells express Ghre, thus being able to play an important role in regulating food behavior in response to odors. In this study, we investigated the presence of Ghre and GHS-R 1a in primary mouse OECs. The expression of both Ghre and its receptor was assessed by an immunocytochemical technique, Western Blot and Polymerase Chain Reaction (PCR) analysis. Our results demonstrated that OECs are able to express both Ghre and GHS-R 1a and that these proteins are detectable after extensive passages in vitro; in addition, PCR analysis further confirmed these data. Therefore, we can hypothesize that Ghre and GHS-R 1a interact with a reinforcement function, in the peripheral olfactory circuit, providing a neurotrophic support to the synaptic interaction between ORNs and mitral cells.

Individual Radiosensitivity in Oncological Patients: Linking Adverse Normal Tissue Reactions and Genetic Features.

Introduction: Adverse effects of radiotherapy (RT) significantly affect patient's quality of life (QOL). The possibility to identify patient-related factors that are associated with individual radiosensitivity would optimize adjuvant RT treatment, limiting the severity of normal tissue reactions, and improving patient's QOL. In this study, we analyzed the relationships between genetic features and toxicity grading manifested by RT patients looking for possible biomarkers of individual radiosensitivity. Methods: Early radiation toxicity was evaluated on 143 oncological patients according to the Common Terminology Criteria for Adverse Events (CTCAE). An individual radiosensitivity (IRS) index defining four classes of radiosensitivity (highly radiosensitive, radiosensitive, normal, and radioresistant) was determined by a G2-chromosomal assay on ex vivo irradiated, patient-derived blood samples. The expression level of 15 radioresponsive genes has been measured by quantitative real-time PCR at 24 h after the first RT fraction, in blood samples of a subset of 57 patients, representing the four IRS classes. Results: By applying univariate and multivariate statistical analyses, we found that fatigue was significantly associated with IRS index. Interestingly, associations were detected between clinical radiation toxicity and gene expression (ATM, CDKN1A, FDXR, SESN1, XPC, ZMAT3, and BCL2/BAX ratio) and between IRS index and gene expression (BBC3, FDXR, GADD45A, and BCL2/BAX). Conclusions: In this prospective cohort study we found that associations exist between normal tissue reactions and genetic features in RT-treated patients. Overall, our findings can contribute to the identification of biological markers to predict RT toxicity in normal tissues.