Challenges of proving a causal role of somatic mutations in the aging process.

Aging is accompanied by the progressive accumulation of permanent changes to the genomic sequence, termed somatic mutations. Small mutations, including single-base substitutions and insertions/deletions, are key determinants of the malignant transformations leading to cancer, but their role as initiators of other age-related phenotypes is controversial. Here, we present recent advances in the study of somatic mutagenesis in aging tissues and posit that the current uncertainty about its causal effects in the aging process is due to technological and methodological weaknesses. We highlight classical and novel experimental systems, including premature aging syndromes, that could be used to model the increase of somatic mutation burden and understand its functional role. It is important that studies are designed to take into account the biological context and peculiarities of each tissue and that the downstream impact of somatic mutation accumulation is measured by methods able to resolve subtle cellular changes.

The PI3K/Akt/mTOR pathway in polycystic kidney disease: A complex interaction with polycystins and primary cilium.

Over-activation of the PI3K/Akt/mTOR network is a well-known pathogenic event that leads to hyper-proliferation. Pharmacological targeting of this pathway has been developed for the treatment of multiple diseases, including cancer. In polycystic kidney disease (PKD), the mTOR cascade promotes cyst growth by boosting proliferation, size and metabolism of kidney tubule epithelial cells. Therefore, mTOR inhibition has been tested in pre-clinical and clinical studies, but only the former showed positive results. This review reports recent discoveries describing the activity and molecular mechanisms of mTOR activation in tubule epithelial cells and cyst formation and discusses the evidence of an upstream regulation of mTOR by the PI3K/Akt axis. In particular, the complex interconnections of the PI3K/Akt/mTOR network with the principal signaling routes involved in the suppression of cyst formation are dissected. These interactions include the antagonism and the reciprocal negative regulation between mTOR complex 1 and the proteins whose deletion causes Autosomal Dominant PKD, the polycystins. In addition, the emerging role of phopshoinositides, membrane components modulated by PI3K, will be presented in the context of primary cilium signaling, cell polarization and protection from cyst formation. Overall, studies demonstrate that the activity of various members of the PI3K/Akt/mTOR network goes beyond the classical transduction of mitogenic signals and can impact several aspects of kidney tubule homeostasis and morphogenesis. These properties might be useful to guide the establishment of more effective treatment protocols to be tested in clinical trials.

Whole genome DNA sequencing provides an atlas of somatic mutagenesis in healthy human cells and identifies a tumor-prone cell type.

BACKGROUND: The lifelong accumulation of somatic mutations underlies age-related phenotypes and cancer. Mutagenic forces are thought to shape the genome of aging cells in a tissue-specific way. Whole genome analyses of somatic mutation patterns, based on both types and genomic distribution of variants, can shed light on specific processes active in different human tissues and their effect on the transition to cancer. RESULTS: To analyze somatic mutation patterns, we compile a comprehensive genetic atlas of somatic mutations in healthy human cells. High-confidence variants are obtained from newly generated and publicly available whole genome DNA sequencing data from single non-cancer cells, clonally expanded in vitro. To enable a well-controlled comparison of different cell types, we obtain single genome data (92% mean coverage) from multi-organ biopsies from the same donors. These data show multiple cell types that are protected from mutagens and display a stereotyped mutation profile, despite their origin from different tissues. Conversely, the same tissue harbors cells with distinct mutation profiles associated to different differentiation states. Analyses of mutation rate in the coding and non-coding portions of the genome identify a cell type bearing a unique mutation pattern characterized by mutation enrichment in active chromatin, regulatory, and transcribed regions. CONCLUSIONS: Our analysis of normal cells from healthy donors identifies a somatic mutation landscape that enhances the risk of tumor transformation in a specific cell population from the kidney proximal tubule. This unique pattern is characterized by high rate of mutation accumulation during adult life and specific targeting of expressed genes and regulatory regions.

Healthy skeletal muscle aging: The role of satellite cells, somatic mutations and exercise.

Satellite cells (SCs) form the resident stem cell population in the skeletal muscle tissue. While their function in mediating tissue regeneration after injury is well described, their role in the undamaged-, aging-, and exercising muscle is only starting to be unraveled. Although direct evidence linking the loss of SC function to the onset of age-related loss of muscle mass and function (i.e., sarcopenia) is currently lacking, satellite cells are increasingly seen as an important component for the decline of tissue function seen with aging. This is evident from the pertinent role of SCs in maintaining homeostasis, and in mediating remodeling- and repair-responses, in the skeletal muscle. This narrative review focuses on human studies, but includes cellular and animal models, to describe the role of SCs in different physiological scenarios relevant for human aging. The intrinsic and extrinsic mechanisms underlying age-induced alterations in the SC pool are discussed, with particular emphasis on the genomic modifications that accumulate in human SCs during a lifetime (i.e., somatic mutation-burden). Finally, the role of exercise as a potential countermeasure to age-induced SC alterations is explored in the different scenarios covered.

Somatic mutagenesis in satellite cells associates with human skeletal muscle aging.

Human aging is associated with a decline in skeletal muscle (SkM) function and a reduction in the number and activity of satellite cells (SCs), the resident stem cells. To study the connection between SC aging and muscle impairment, we analyze the whole genome of single SC clones of the leg muscle vastus lateralis from healthy individuals of different ages (21-78 years). We find an accumulation rate of 13 somatic mutations per genome per year, consistent with proliferation of SCs in the healthy adult muscle. SkM-expressed genes are protected from mutations, but aging results in an increase in mutations in exons and promoters, targeting genes involved in SC activity and muscle function. In agreement with SC mutations affecting the whole tissue, we detect a missense mutation in a SC propagating to the muscle. Our results suggest somatic mutagenesis in SCs as a driving force in the age-related decline of SkM function.

Phosphoinositide 3-Kinase Gamma Inhibition Protects From Anthracycline Cardiotoxicity and Reduces Tumor Growth.

BACKGROUND: Anthracyclines, such as doxorubicin (DOX), are potent anticancer agents for the treatment of solid tumors and hematologic malignancies. However, their clinical use is hampered by cardiotoxicity. This study sought to investigate the role of phosphoinositide 3-kinase γ (PI3Kγ) in DOX-induced cardiotoxicity and the potential cardioprotective and anticancer effects of PI3Kγ inhibition. METHODS: Mice expressing a kinase-inactive PI3Kγ or receiving PI3Kγ-selective inhibitors were subjected to chronic DOX treatment. Cardiac function was analyzed by echocardiography, and DOX-mediated signaling was assessed in whole hearts or isolated cardiomyocytes. The dual cardioprotective and antitumor action of PI3Kγ inhibition was assessed in mouse mammary tumor models. RESULTS: PI3Kγ kinase-dead mice showed preserved cardiac function after chronic low-dose DOX treatment and were protected against DOX-induced cardiotoxicity. The beneficial effects of PI3Kγ inhibition were causally linked to enhanced autophagic disposal of DOX-damaged mitochondria. Consistently, either pharmacological or genetic blockade of autophagy in vivo abrogated the resistance of PI3Kγ kinase-dead mice to DOX cardiotoxicity. Mechanistically, PI3Kγ was triggered in DOX-treated hearts, downstream of Toll-like receptor 9, by the mitochondrial DNA released by injured organelles and contained in autolysosomes. This autolysosomal PI3Kγ/Akt/mTOR/Ulk1 signaling provided maladaptive feedback inhibition of autophagy. PI3Kγ blockade in models of mammary gland tumors prevented DOX-induced cardiac dysfunction and concomitantly synergized with the antitumor action of DOX by unleashing anticancer immunity. CONCLUSIONS: Blockade of PI3Kγ may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function.

Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.

Hiperparatiroidismo neonatal: una rara afección de pronóstico infausto / Neonatal hyperparathyroidism: a rare condition of untimely prognosis

El hiperparatiroidismo neonatal severo es una entidad grave y poco frecuente causada por la inactivación del receptor sensible al calcio. Las manifestaciones clínicas suelen ser precoces. Incluyen: dificultad respiratoria debido a la hipotonía y deformidades de la caja torácica, huesos con mineralización baja y múltiples fracturas. La tasa de mortalidad es elevada. La mayoría de los pacientes mueren durante el periodo neonatal. Se presenta el caso de un recién nacido que presenta signos clínicos y analíticos sospechosos de hiperparatiroidismo neonatal severo. Finalmente tras recuperación espontánea se realiza el diagnóstico de hiperparatiroidismo transitorio secundario a hipoparatiroidismo materno desconocido (AU)

The severe neonatal hyperparathyroidism is a serious institution, and rare caused by the inactivation of receiver sensitive to calcium. The clinical manifestations are usually early. Include: difficulty breathing due to the hypotonia and deformities of the chest, bones with low mineralization, and multiple fractures. The mortality rate is high. Most patients die by during the neonatal period. We present the case of a newborn with clinical signs and analytical suspects of severe neonatal hyperparathyroidism. Finally after spontaneous recovery is performed the diagnosis of hyperparathyroidism secondary to transient hypoparathyroidism unknown maternal (AU)

Rare progerin-expressing preadipocytes and adipocytes contribute to tissue depletion over time.

Accumulation of progerin is believed to underlie the pathophysiology of Hutchinson-Gilford progeria syndrome, a disease characterized by clinical features suggestive of premature aging, including loss of subcutaneous white adipose tissue (sWAT). Although progerin has been found in cells and tissues from apparently healthy individuals, its significance has been debated given its low expression levels and rare occurrence. Here we demonstrate that sustained progerin expression in a small fraction of preadipocytes and adipocytes of mouse sWAT (between 4.4% and 6.7% of the sWAT cells) results in significant tissue pathology over time, including fibrosis and lipoatrophy. Analysis of sWAT from mice of various ages showed senescence, persistent DNA damage and cell death that preceded macrophage infiltration, and systemic inflammation. Our findings suggest that continuous progerin expression in a small cell fraction of a tissue contributes to aging-associated diseases, the adipose tissue being particularly sensitive.

Rac signal adaptation controls neutrophil mobilization from the bone marrow.

Mobilization of neutrophils from the bone marrow determines neutrophil blood counts and thus is medically important. Balanced neutrophil mobilization from the bone marrow depends on the retention-promoting chemokine CXCL12 and its receptor CXCR4 and the egression-promoting chemokine CXCL2 and its receptor CXCR2. Both pathways activate the small guanosine triphosphatase Rac, leaving the role of this signaling event in neutrophil retention and egression ambiguous. On the assumption that active Rac determines persistent directional cell migration, we generated a mathematical model to link chemokine-mediated Rac modulation to neutrophil egression time. Our computer simulation indicated that, in the bone marrow, where the retention signal predominated, egression time strictly depended on the time it took Rac to return to its basal activity (namely, adaptation). This prediction was validated in mice lacking the Rac inhibitor ArhGAP15. Neutrophils in these mice showed prolonged Rac adaptation and cell-autonomous retention in the bone marrow. Our model thus demonstrates that mobilization in the presence of two spatially defined opposing chemotactic cues strictly depends on inhibitors shaping the time course of signal adaptation. Furthermore, our findings might help to find new modes of intervention to treat conditions characterized by excessively low or high circulating neutrophils.

Phosphoinositide 3-Kinase-C2α Regulates Polycystin-2 Ciliary Entry and Protects against Kidney Cyst Formation.

Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2α (PI3K-C2α) in renal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2α resides at the recycling endosome compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2α controlled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2α was sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K-C2α in mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2α is required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease.

Phosphoinositide 3-Kinase γ Restrains Neurotoxic Effects of Microglia After Focal Brain Ischemia.

Phosphoinositide 3-kinase γ (PI3Kγ) is linked to neuroinflammation and phagocytosis. This study was conducted to elucidate conjectural differences of lipid kinase-dependent and kinase-independent functions of PI3Kγ in the evolvement of brain damage induced by focal cerebral ischemia/reperfusion. Therefore, PI3Kγ wild-type, knockout, and kinase-dead mice were subjected to middle cerebral artery occlusion followed by reperfusion. Tissue damage and cellular composition were assessed by immunohistochemical stainings. In addition, microglial cells derived from respective mouse genotypes were used for analysis of PI3Kγ effects on phagocytic activity, matrix metalloproteinase-9 release, and cAMP content under conditions of oxygen/glucose deprivation and recovery. Brain infarction was more pronounced in PI3Kγ-knockout mice compared to wild-type and kinase-dead mice 48 h after reperfusion. Immunohistochemical analyses revealed a reduced amount of galectin-3/MAC-2-positive microglial cells indicating that activated phagocytosis was reduced in ischemic brains of knockout mice. Cell culture studies disclosed enhanced metalloproteinase-9 secretion in supernatants derived from microglia of PI3Kγ-deficient mice after 2-h oxygen/glucose deprivation and 48-h recovery. Furthermore, PI3Kγ-deficient microglial cells showed a failed phagocytic activation throughout the observed recovery period. Lastly, PI3Kγ-deficient microglia exhibited strongly increased cAMP levels in comparison with wild-type microglia or cells expressing kinase-dead PI3Kγ after oxygen/glucose deprivation and recovery. Our data suggest PI3Kγ kinase activity-independent control of cAMP phosphodiesterase as a crucial mediator of microglial cAMP regulation, MMP-9 expression, and phagocytic activity following focal brain ischemia/recirculation. The suppressive effect of PI3Kγ on cAMP levels appears critical for the restriction of ischemia-induced immune cell functions and in turn tissue damage.

PI3K-C2α: One enzyme for two products coupling vesicle trafficking and signal transduction.

The spatial restriction of phosphorylated phosphoinositides generated downstream activated membrane receptors is critical for proper cell response to environmental cues. The α isoform of class II PI3Ks, PI3K-C2α, has emerged as a modulator of receptor localization, acting both in the control of receptor endocytosis and resensitization. This unexpectedly versatile enzyme was found to differentially produce two distinct 3-phosphorylated phosphoinositides and to selectively control distinct steps of vesicular traffic such as endocytosis and recycling. This review focuses on the latest discoveries regarding PI3K-C2α function in vesicle trafficking and its impact on cell biology and mammalian embryonic development.

Morgana acts as an oncosuppressor in chronic myeloid leukemia.

We recently described morgana as an essential protein able to regulate centrosome duplication and genomic stability, by inhibiting ROCK. Here we show that morgana (+/-) mice spontaneously develop a lethal myeloproliferative disease resembling human atypical chronic myeloid leukemia (aCML), preceded by ROCK hyperactivation, centrosome amplification, and cytogenetic abnormalities in the bone marrow (BM). Moreover, we found that morgana is underexpressed in the BM of patients affected by atypical CML, a disorder of poorly understood molecular basis, characterized by nonrecurrent cytogenetic abnormalities. Morgana is also underexpressed in the BM of a portion of patients affected by Philadelphia-positive CML (Ph(+) CML) caused by the BCR-ABL oncogene, and in this condition, morgana underexpression predicts a worse response to imatinib, the standard treatment for Ph(+) CML. Thus, morgana acts as an oncosuppressor with different modalities: (1) Morgana underexpression induces centrosome amplification and cytogenetic abnormalities, and (2) in Ph(+) CML, it synergizes with BCR-ABL signaling, reducing the efficacy of imatinib treatment. Importantly, ROCK inhibition in the BM of patients underexpressing morgana restored the efficacy of imatinib to induce apoptosis, suggesting that ROCK inhibitors, combined with imatinib treatment, can overcome suboptimal responses in patients in which morgana is underexpressed.

The Hedgehog pathway effector smoothened exhibits signaling competency in the absence of ciliary accumulation.

Misactivation of the seven-transmembrane protein Smoothened (Smo) is frequently associated with basal cell carcinoma and medulloblastoma. Cellular exposure to secreted Hedgehog (Hh) protein or oncogenic mutations in Hh pathway components induces Smo accumulation in the primary cilium, an antenna-like organelle with mostly unknown cellular functions. Despite the data supporting an indispensable role of the primary cilium in Smo activation, the mechanistic underpinnings of this dependency remain unclear. Using a cell-membrane-impermeable Smo antagonist (IHR-1), we demonstrate that Smo supplied with a synthetic agonist or activated with oncogenic mutations can signal without ciliary accumulation. Similarly, cells with compromised ciliary Smo trafficking due to loss of the phosphatidylinositol-4-phosphate 3-kinase (PI3K)-C2α retain transcriptional response to an exogenously supplied Smo agonist. These observations suggest that assembly of a Smo-signaling complex in the primary cilium is not a prerequisite for Hh pathway activation driven by Smo agonists or oncogenic Smo molecules.

Risk factors for subclavian vein thrombosis in cancer patients with total parenteral nutrition.

BACKGROUND: There are few reports on total parenteral nutrition (TPN) and its possible prothrombotic effect. The purpose of this study was to identify risk factors for subclavian vein thrombosis (SVT) in patients receiving TPN. METHOD: Cancer patients with indwelling subclavian catheters and TPN were followed in a cohort study. Doppler ultrasound examination was performed 8 and 30 days after catheter placement. RESULTS: One hundred twenty-one patients were included, with a mean of 61 (± 11.8) years of age. We detected 36 SVT events at day 8 (29.8%) and 47 (38.8%) at day 30 after central catheter placement. Mean length of subclavian catheterization was 17.2 (± 8.2) days. Fifty-three point three percent of patients receiving ≥ 3,050 mOsm TPN in 24 hours developed SVT (relative risk (RR) = 2.01, 95% CI, 1.14 - 3.57; P = 0.016) at day 8 and 60% (RR = 1.67, 95% CI, 1.30 - 2.71; P = 0.038) at day 30 post-catheter placement. Protein administration of > 97.5 g/day was shown to be a risk factor for early thrombosis with a mean of 16.88 days for the development of SVT (95% CI, 10 - 23.7) versus 27.8 days (95% CI, 25.8 - 29.9) in the group with nutritional protein content < 97.5 g/day (P = 0.000). CONCLUSION: High-osmolarity and high-protein nutrition formulas were shown to be risk factors for SVT in cancer patients receiving TPN.

Deficiency of cannabinoid receptor of type 2 worsens renal functional and structural abnormalities in streptozotocin-induced diabetic mice.

A functionally active endocannabinoid system is present within the kidney. The cannabinoid receptor type 2 (CB2) is expressed by both inflammatory cells and podocytes, and its activation has beneficial effects in experimental diabetic nephropathy. To further explore the role of CB2 in diabetic nephropathy, we studied renal functional and structural abnormalities in streptozotocin-induced diabetic CB2 knockout mice. In diabetic mice, deletion of the CB2 receptor albuminuria, the downregulation of podocin and nephrin, mesangial expansion, overexpression of extracellular matrix components, monocyte infiltration, and reduced renal function were all exacerbated. To investigate the relative contributions of podocytes and monocytes to the phenotype of diabetic knockout mice, bone marrow transplantation experiments were performed. The lack of CB2 on bone marrow-derived cells was shown to be important in driving the enhanced glomerular monocyte accrual found in diabetic knockout mice. Absence of CB2 on resident glomerular cells had a major role in worsening diabetic nephropathy, both functional and structural abnormalities, likely by enhanced MCP-1 and CB1 signaling. Studies in cultured podocytes demonstrated that CB2 expression is not altered by a high glucose milieu but is downregulated by mechanical stretch, mimicking glomerular capillary hypertension. Thus, CB2 deletion worsens diabetic nephropathy, independent of bone marrow-derived cells.