Nicoletta Marchesi

Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates SQSTM1/p62 mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.

Autophagy is Modulated in Human Neuroblastoma Cells Through Direct Exposition to Low Frequency Electromagnetic Fields

In neurogenerative diseases, comprising Alzheimer’s (AD), functional alteration in autophagy is considered one of the pathological hallmarks and a promising therapeutic target. Epidemiological investigations on the possible causes undergoing these diseases have suggested that electromagnetic fields (EMF) exposition can contribute to their etiology. On the other hand, EMF have therapeutic implications in reactivating neuronal functionality. To partly clarify this dualism, the effect of low‐frequency EMF (LF‐EMF) on the modulation of autophagy was investigated in human neuroblastoma SH‐SY5Y cells, which were also subsequently exposed to Aβ peptides, key players in AD. The results primarily point that LF‐EMF induce a significant reduction of microRNA 30a (miR‐30a) expression with a concomitant increase of Beclin1 transcript (BECN1) and its corresponding protein. Furthermore, LF‐EMF counteract the induced miR‐30a up‐regulation in the same cells transfected with miR‐30a mimic precursor molecules and, on the other side, rescue Beclin1 expression after BECN1 siRNA treatment. The expression of autophagy‐related markers (ATG7 and LC3B‐II) as well as the dynamics of autophagosome formation were also visualized after LF‐EMF exposition. Finally, different protocols of repeated LF‐EMF treatments were assayed to contrast the effects of Aβ peptides in vitro administration. Overall, this research demonstrates, for the first time, that specific LF‐EMF treatments can modulate in vitro the expression of a microRNA sequence, which in turn affects autophagy via Beclin1 expression. Taking into account the pivotal role of autophagy in the clearance of protein aggregates within the cells, our results indicate a potential cytoprotective effect exerted by LF‐EMF in neurodegenerative diseases such as AD. J. Cell. Physiol. 229: 1776–1786, 2014.

Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonucleotides induces autophagy-dependent cell death in glioma cells.

Malignant gliomas are the most common and lethal primary central nervous system neoplasms. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrPC) may exert neuro- and cyto-protective functions: PrPC overexpression protects cultured neurons and also tumor cell lines exposed to various pro-apoptotic stimuli while, on the contrary, PrPC silencing sensitizes Adriamycin-resistant human breast carcinoma cells to TRAIL-mediated cell death. In order to determine if PrPC is involved in the resistance of glial tumors to cell death, the effects of cellular prion protein downregulation by antisense approach were investigated in different human malignant glioma cell lines. PrPC downregulation induced profound morphological changes and significant cell death. In addition, a significant tumor volume reduction was noted after PrPC silencing in a EGFP-GL261 glioma murine model. Investigations of the molecular effects induced by PrPC silencing were carried out on T98G human glioma cells by analysing autophagic as well as typical apoptotic markers (nuclear morphology, caspase-3/7, p53 and PARP-1). The results indicated that apoptosis was not induced after PrPC downregulation while, on the contrary, electron microscopy analysis, and an accumulation of GFP-LC3-II in autophagosomal membranes of GFP-LC3 transfected cells, indicated a predominant activation of autophagy. PrPC silencing also led to induction of LC3-II, increase in Beclin-1 and a concomitant decrease in p62, Bcl-2 and in the phosphorylation of 4E-BP1, a target of mTOR autophagy signaling. In conclusion, our results show for the first time that interfering with the cellular prion protein expression could modulate autophagy-dependent cell death pathways in glial tumor cells.

Gold nanostars coated with neutral and charged polyethylene glycols: A comparative study of in-vitro biocompatibility and of their interaction with SH-SY5Y neuroblastoma cells.

Gold nanostars (GNS) have been coated with four different polyethylene glycols (PEGs) equipped with a -SH function for grafting on the gold surface. These PEGs have different chain lengths with average MW=2000, 3000, 5000 and average number of -O-CH2-CH2 - units 44, 66, and 111, respectively. Two are neutral and two are terminated with -COOH and -NH2 functions, thus bearing negative and positive charges at physiological pH, thanks to the formation of carboxylate and ammonium groups. The negative charge of the GNS coated with PEG carboxylate has also been exploited to further coat the GNS with the PAH (polyallylamine hydrochloride) cationic polymer. Vitality tests have been carried out on SH-SY5Y cells treated with the five differently coated GNS for 4, 24, and 48 h, at Au concentrations ranging from 1.25 to 100 μg/mL. The same tests have been repeated with the pure PEGs and PAH. Excellent biocompatibility was found for all PEGs, independently on charge and chain length, both for coated GNS and for the pure polymers. On the contrary, poor biocompatibility was found for PAH overcoated GNS and for pure PAH, although the latter only at high concentrations. Exploiting the two-photon luminescence of GNS, we have found by confocal laser scanning microscopy that when GNS are coated with PEGs they do not enter SH-SY5Y cells, while when overcoated with PAH they massively penetrate into the cytoplasm. This causes cell death by dramatically changing cell morphology, as demonstrated also by atomic force microscopy.

Pulsed electromagnetic field (PEMF) prevents pro-oxidant effects of H2O2 in SK-N-BE(2) human neuroblastoma cells

Abstract Purpose The redox milieu, together with reactive oxygen species (ROS) accumulation, may play a role in mediating some biological effects of extremely-low-frequency electromagnetic fields (ELF-EMF). Some of us have recently reported that a pulsed EMF (PEMF) improves the antioxidant response of a drug-sensitive human neuroblastoma SH-SY5Y cell line to pro-oxidants. Since drug resistance may affect cell sensitivity to redox-based treatments, we wanted to verify whether drug-resistant human neuroblastoma SK-N-BE(2) cells respond to a PEMF in a similar fashion. Materials and methods SK-N-BE(2) cells were exposed to repeated 2 mT, 75 Hz PEMF (15 min each, repeated 3 times over 5 days), and ROS production, Mn-dependent superoxide dismutase (MnSOD)-based antioxidant protection and viability were assessed after 10 min or 30 min 1 mM hydrogen peroxide. Sham controls were kept at the same time in identical cell culture incubators. Results The PEMF increased the MnSOD-based antioxidant protection and reduced the ROS production in response to a pro-oxidant challenge. Conclusions Our work might lay foundation for the development of non-invasive PEMF-based approaches aimed at elevating endogenous antioxidant properties in cellular or tissue models.

Novel approaches to the post-myocardial infarction/heart failure neural remodeling

The review aims to discuss the role of nerve growth factor (NGF) as a potential novel biomarker in post-myocardial infarction (MI) and in heart failure (HF), with a specific focus on neural remodeling and sprouting processes occurring after tissue damage. Many experimental data show that MI induces nerve sprouting, leading to increased sympathetic outflow and higher risk of ventricular arrhythmias and sudden cardiac death. In this framework, cardiac and circulating NGF might be an indicator of the innervation process and neural remodeling: it dramatically increases after MI, while it declines along with advanced HF and ventricular dysfunction. The bimodal behavior of NGF in acute and chronic settings leads to the speculation that NGF modulation may be a pharmacological target for intervention in different stages of the ischemic heart disease. Specifically, a fascinating possibility is to support or to inhibit NGF receptors, in order to prevent negative cardiac remodeling after MI and consequent ventricular dysfunction.

Microbiota and metabolic diseases

The microbiota is a complex ecosystem of microorganisms consisting of bacteria, viruses, protozoa, and fungi, living in different districts of the human body, such as the gastro-enteric tube, skin, mouth, respiratory system, and the vagina. Over 70% of the microbiota lives in the gastrointestinal tract in a mutually beneficial relationship with its host. The microbiota plays a major role in many metabolic functions, including modulation of glucose and lipid homeostasis, regulation of satiety, production of energy and vitamins. It exerts a role in the regulation of several biochemical and physiological mechanisms through the production of metabolites and substances. In addition, the microbiota has important anti-carcinogenetic and anti-inflammatory actions. There is growing evidence that any modification in the microbiota composition can lead to several diseases, including metabolic diseases, such as obesity and diabetes, and cardiovascular diseases. This is because alterations in the microbiota composition can cause insulin resistance, inflammation, vascular, and metabolic disorders. The causes of the microbiota alterations and the mechanisms by which microbiota modifications can act on the development of metabolic and cardiovascular diseases have been reported. Current and future preventive and therapeutic strategies to prevent these diseases by an adequate modulation of the microbiota have been also discussed.

Tunable coating of gold nanostars: tailoring robust SERS labels for cell imaging

Surface modification of noble metal nanoparticles with mixed molecular monolayers is one of the most powerful tools in nanotechnology, and is used to impart and tune new complex surface properties. In imaging techniques based on surface enhanced Raman spectroscopy (SERS), precise and controllable surface modifications are needed to carefully design reproducible, robust and adjustable SERS nanoprobes. We report here the attainment of SERS labels based on gold nanostars (GNSs) coated with a mixed monolayer composed of a poly ethylene glycol (PEG) thiol (neutral or negatively charged) that ensure stability in biological environments, and of a signalling unit 7-Mercapto-4-methylcoumarin as a Raman reporter molecule. The composition of the coating mixture is precisely controlled using an original method, allowing the modulation of the SERS intensity and ensuring overall nanoprobe stability. The further addition of a positively charged layer of poly (allylamine hydrocloride) on the surface of negatively charged SERS labels does not change the SERS response, but it promotes the penetration of GNSs in SH-SY5Y neuroblastoma cells. As an example of an application of such an approach, we demonstrate here the internalization of these new labels by means of visualization of cell morphology obtained with SERS mapping.

Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38MAPK, and JNK Kinase Pathways

RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD). HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38MAPK, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuRs role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.

Synthesis of reduced-size gold nanostars and internalization in SH-SY5Y cells

The synthesis of large pentatwinned five-branched gold nanostars (GNS) has been modified so to obtain overall dimensions shrunk to 60% and a lower branches aspect ratio, leading to a dramatic blue shift of their two near-infrared (NIR) localized surface plasmon resonances (LSPR) absorptions but still maintaining one LSPR in the biotransparent NIR range. The interactions of polyethylene glycol (PEG) coated large and shrunk GNS with SH-SY5Y cells revealed that the large ones (DCI - diameter of the circumference in which GNS can be inscribed=76nm) are internalized more efficiently than the shrunk ones (DCI=46nm), correlating with a decreased cells surviving fraction.