| Name | Simona |
| Surname | Paladino |
| Institution | University of Naples – Federico II |
| simona.paladino@unina.it | |
| Address | Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy |
Trisomy of chromosome 21 (TS21) is the most common autosomal aneuploidy compatible with postnatal survival with a prevalence of 1 in 700 newborns. Its phenotype is highly complex with constant features, such as mental retardation, dysmorphic traits and hypotonia, and variable features including heart defects, susceptibility to Alzheimer's disease (AD), type 2 diabetes, obesity and immune disorders. Overexpression of genes on chromosome-21 (Hsa21) is responsible for the pathogenesis of Down syndrome (DS) phenotypic features either in a direct or in an indirect manner since many Hsa21 genes can affect the expression of other genes mapping to different chromosomes. Many of these genes are involved in mitochondrial function and energy conversion, and play a central role in the mitochondrial dysfunction and chronic oxidative stress, consistently observed in DS subjects.Recent studies highlight the deep interconnections between mitochondrial dysfunction and DS phenotype. In this short review we first provide a basic overview of mitochondrial phenotype in DS cells and tissues. We then discuss how specific Hsa21 genes may be involved in determining the disruption of mitochondrial DS phenotype and biogenesis. Finally we briefly focus on drugs that affect mitochondrial function and mitochondrial network suggesting possible therapeutic approaches to improve and/or prevent some aspects of the DS phenotype.
Recently, a new form of autosomal recessive early-onset parkinsonism (PARK20), due to mutations in the gene encoding the phosphoinositide phosphatase, Synaptojanin 1 (Synj1), has been reported. Several genes responsible for hereditary forms of Parkinson's disease are implicated in distinct steps of the endolysosomal pathway. However, the nature and the degree of endocytic membrane trafficking impairment in early-onset parkinsonism remains elusive. Here, we show that depletion of Synj1 causes drastic alterations of early endosomes, which become enlarged and more numerous, while it does not affect the morphology of late endosomes both in non-neuronal and neuronal cells. Moreover, Synj1 loss impairs the recycling of transferrin, while it does not alter the trafficking of the epidermal growth factor receptor. The ectopic expression of Synj1 restores the functions of early endosomes, and rescues these trafficking defects in depleted cells. Importantly, the same alterations of early endosomal compartments and trafficking defects occur in fibroblasts of PARK20 patients. Our data indicate that Synj1 plays a crucial role in regulating the homeostasis and functions of early endosomal compartments in different cell types, and highlight defective cellular pathways in PARK20. In addition, they strengthen the link between endosomal trafficking and Parkinson's disease.
Oxidative stress is a hallmark of Alzheimer's Disease (AD) and promotes tau phosphorylation. Since Thioredoxin Interacting protein (TXNIP), the inhibitor of the anti-oxidant system of Thioredoxin, is up regulated in the hippocampus of AD patients, we investigated whether TXNIP plays a role in promoting tau phosphorylation and whether Verapamil, an inhibitor of TXNIP expression, prevents TXNIP downstream effects.
Mucopolysaccharidosis (MPS) IIIB is an inherited lysosomal storage disease caused by the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The defective lysosomal clearance of undigested HS results in dysfunction of multiple tissues and organs. We recently demonstrated that the murine model of MPS IIIB develops cardiac disease, valvular abnormalities, and ultimately heart failure. To address the molecular mechanisms governing cardiac dysfunctions in MPS IIIB, we generated a model of the disease by silencing NAGLU gene expression in H9C2 rat cardiomyoblasts. NAGLU-depleted H9C2 exhibited accumulation of abnormal lysosomes and a hypertrophic phenotype. Furthermore, we found the specific activation of the epidermal growth factor receptor (EGFR), and increased phosphorylation levels of extracellular signal-regulated kinases (ERKs) in NAGLU-depleted H9C2. The inhibition of either EGFR or ERKs, using the selective inhibitors AG1478 and PD98059, resulted in the reduction of both lysosomal aberration and hypertrophy in NAGLU-depleted H9C2. We also found increased phosphorylation of c-Src and a reduction of the hypertrophic response in NAGLU-depleted H9C2 transfected with a dominant-negative c-Src. However, c-Src phosphorylation remained unaffected by AG1478 treatment, posing c-Src upstream EGFR activation. Finally, heparin-binding EGF-like growth factor (HB-EGF) protein was found overexpressed in our MPS IIIB cellular model, and its silencing reduced the hypertrophic response. These results indicate that both c-Src and HB-EGF contribute to the hypertrophic phenotype of NAGLU-depleted cardiomyoblasts by synergistically activating EGFR and subsequent signaling, thus suggesting that EGFR pathway inhibition could represent an effective therapeutic approach for MPS IIIB cardiac disease.
Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal properties of their membrane organization is of fundamental importance. Here, by using direct stochastic optical reconstruction microscopy coupled to pair correlation analysis (pc-STORM), we were able to visualize and measure the size of these clusters. Specifically, we show that they are non-randomly distributed and have an average size of 67 nm. We also demonstrated that polarized MDCK and non-polarized CHO cells have similar cluster distribution and size, but different sensitivity to cholesterol depletion. Finally, we derived a model that allowed a quantitative characterization of the cluster organization of GPI-APs at the apical surface of polarized MDCK cells for the first time. Experimental FRET (fluorescence resonance energy transfer)/FLIM (fluorescence-lifetime imaging microscopy) data were correlated to the theoretical predictions of the model.
Neuroglobin (Ngb) is expressed in the central and peripheral nervous system, cerebrospinal fluid, retina, and endocrine tissues where it is involved in binding O2 and other gasotransmitters. Several studies have highlighted its endogenous neuroprotective function. Huntington's disease (HD), a dominant hereditary disease, is characterized by the gradual loss of neurons in discrete areas of the central nervous system. We analyzed the expression of Ngb in the brain tissue of a mouse model of HD, in order to define the role of Ngb with respect to individual cell type vulnerability in HD and to gender and age of mice. Our results showed different expressions of Ngb among neurons of a specific region and between different brain regions. We evidenced a decreased intensity of Ngb at 13 weeks of age, compared to 7 weeks of age. The double immunofluorescence and fluorescence resonance energy transfer (FRET) experiments showed that the co-localization between Ngb and huntingtin at the subcellular level was not close enough to account for a direct interaction. We also observed a different expression of Ngb in the striatum, depending on the sex and age of animals. These findings provide the first experimental evidence for an adaptive response of Ngb in HD, suggesting that Ngb may exert neuroprotective effects in HD beyond its role in reducing sensitivity to oxidative stress.
We disclose herein the first example of stable monodispersed hybrid nanoparticles (termed MelaSil-NPs) made up of eumelanin biopolymer intimately integrated into a silica nanoscaffold matrix and endowed with high antioxidant and cytoprotective effects associated with a specific subcellular localization. MelaSil-NPs have been fabricated by an optimized sol-gel methodology involving ammonia-induced oxidative polymerization of a covalent conjugate of the eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA) with 3-aminopropyltriethoxysilanes (APTS). They displayed a round-shaped (ca. 50-80 nm) morphology, exhibited the typical electron paramagnetic resonance signal of eumelanin biopolymers, and proved effective in promoting decomposition of hydrogen peroxide under physiologically relevant conditions. When administered to human ovarian cancer cells (A2780) or cervical cancer cells (HeLa), MelaSil-NPs were rapidly internalized and colocalized with lysosomes and exerted efficient protecting effects against hydrogen peroxide-induced oxidative stress and cytotoxicity.
Autophagy is a physiological process, important for recycling of macromolecules and maintenance of cellular homeostasis. Defective autophagy is associated with tumorigenesis and has a causative role in chemotherapy resistance in leukemia and in solid cancers. Here, we report that autophagy is regulated by the lysine-specific demethylase LSD1/KDM1A, an epigenetic marker whose overexpression is a feature of malignant neoplasia with an instrumental role in cancer development. In the present study, we determine that two different LSD1 inhibitors (TCP and SP2509) as well as selective ablation of LSD1 expression promote autophagy in neuroblastoma cells. At a mechanistic level, we show that LSD1 binds to the promoter region of Sestrin2 (SESN2), a critical regulator of mTORC1 activity. Pharmacological inhibition of LSD1 triggers SESN2 expression that hampers mTORC1 activity, leading to enhanced autophagy. SESN2 overexpression suffices to promote autophagy in neuroblastoma cells, while loss of SESN2 expression reduces autophagy induced by LSD1 inhibition. Our findings elucidate a mechanism whereby LSD1 controls autophagy in neuroblastoma cells through SESN2 transcription regulation, and we suggest that pharmacological targeting of LSD1 may have effective therapeutic relevance in the control of autophagy in neuroblastoma.Oncogene advance online publication, 7 August 2017; doi:10.1038/onc.2017.267.
High Mobility Group A1 (HMGA1) is an architectural chromatin protein whose overexpression is a feature of malignant neoplasias with a causal role in cancer initiation and progression. HMGA1 promotes tumor growth by several mechanisms, including increase of cell proliferation and survival, impairment of DNA repair and induction of chromosome instability. Autophagy is a self-degradative process that, by providing energy sources and removing damaged organelles and misfolded proteins, allows cell survival under stress conditions. On the other hand, hyper-activated autophagy can lead to non-apoptotic programmed cell death. Autophagy deregulation is a common feature of cancer cells in which has a complex role, showing either an oncogenic or tumor suppressor activity, depending on cellular context and tumor stage. Here, we report that depletion of HMGA1 perturbs autophagy by different mechanisms. HMGA1-knockdown increases autophagosome formation by constraining the activity of the mTOR pathway, a major regulator of autophagy, and transcriptionally upregulating the autophagy-initiating kinase Unc-51-like kinase 1 (ULK1). Consistently, functional experiments demonstrate that HMGA1 binds ULK1 promoter region and negatively regulates its transcription. On the other hand, the increase in autophagosomes is not associated to a proportionate increase in their maturation. Overall, the effects of HMGA1 depletion on autophagy are associated to a decrease in cell proliferation and ultimately impact on cancer cells viability. Importantly, silencing of ULK1 prevents the effects of HMGA1-knockdown on cellular proliferation, viability and autophagic activity, highlighting how these effects are, at least in part, mediated by ULK1. Interestingly, this phenomenon is not restricted to skin cancer cells, as similar results have been observed also in HeLa cells silenced for HMGA1. Taken together, these results clearly indicate HMGA1 as a key regulator of the autophagic pathway in cancer cells, thus suggesting a novel mechanism through which HMGA1 can contribute to cancer progression.Cell Death and Differentiation advance online publication, 4 August 2017; doi:10.1038/cdd.2017.117.