Emanuela Urso

Cuprizone neurotoxicity, copper deficiency and neurodegeneration

Cuprizone is used to obtain demyelination in mice. Cuprizone-treated mice show symptoms similar to several neurodegenerative disorders such as severe status spongiosus. Although it has a simple chemical formula, its neurotoxic mechanism is still unknown. In this work, we examined both physico-chemical properties and biological effects of cuprizone. Our results indicate that cuprizone has very complicated and misunderstood solution chemistry. Moreover, we show here the inability of cuprizone to cross neither the intestinal epithelial barrier nor the neuronal cell membrane, as well its high tolerability by cultured neurons. If added to mice diet, cuprizone does not accumulate in liver or in brain. Therefore, its neurotoxic effect is explainable only in terms of its capability to chelate copper, leading to chronic copper deficiency.

Behind the Link between Copper and Angiogenesis: Established Mechanisms and an Overview on the Role of Vascular Copper Transport Systems

Angiogenesis critically sustains the progression of both physiological and pathological processes. Copper behaves as an obligatory co-factor throughout the angiogenic signalling cascades, so much so that a deficiency causes neovascularization to abate. Moreover, the progress of several angiogenic pathologies (e.g. diabetes, cardiac hypertrophy and ischaemia) can be tracked by measuring serum copper levels, which are being increasingly investigated as a useful prognostic marker. Accordingly, the therapeutic modulation of body copper has been proven effective in rescuing the pathological angiogenic dysfunctions underlying several disease states. Vascular copper transport systems profoundly influence the activation and execution of angiogenesis, acting as multi-functional regulators of apparently discrete pro-angiogenic pathways. This review concerns the complex relationship among copper-dependent angiogenic factors, copper transporters and common pathological conditions, with an unusual accent on the multi-faceted involvement of the proteins handling vascular copper. Functions regulated by the major copper transport proteins (CTR1 importer, ATP7A efflux pump and metallo-chaperones) include the modulation of endothelial migration and vascular superoxide, known to activate angiogenesis within a narrow concentration range. The potential contribution of prion protein, a controversial regulator of copper homeostasis, is discussed, even though its angiogenic involvement seems to be mainly associated with the modulation of endothelial motility and permeability.

Real-time monitoring of copper ions-induced cytotoxicity by EIS cell chips

An important goal of biomedical research is the development of tools for high-throughput evaluation of drug effects and cytotoxicity tests. Here we demonstrate EIS cell chips able to monitor cell growth, morphology, adhesion and their changes as a consequence of treatment with drugs or toxic compounds. As a case study, we investigate the uptake of copper ions and its effect on two cell lines: B104 and HeLa cells. For further understanding, we also carried out in parallel with EIS studies, a complete characterization of cell morphology and changes induced by copper ions through complementary methodologies (including state-of-the-art AFM, viability test and Western blot). Our results reveal a strong correlation between EIS data and both MTT test and AFM characterization so our chip can be used as powerful tools in all biology lab in combination with other standard methods giving additional information that can be useful in a complete and deep investigation of a biological process. This chip can be used even alone replacing in vitro drug tests based on conventional biochemical methods, being very cheap and reusable and allowing to perform cytotoxicity tests without using any expensive reagent or equipment.

The prion protein: structural features and related toxic peptides

Prion diseases are characterized by the conversion of the physiological cellular form of the prion protein (PrPC) into an insoluble, partially protease‐resistant abnormal scrapie form (PrPSc). PrPC is normally expressed in mammalian cell and is highly conserved among species, although its role in cellular function remains elusive. The conversion of PrPC to PrPSc parallels a conformational change of the polypeptide from a predominantly α‐helical to a highly β‐sheet secondary structure. The pathogenesis and molecular basis of the consequent nerve cell loss are not understood. Limited structural information is available on aggregate formation by this protein as the possible cause of these diseases and on its toxicity. This brief overview focuses on the large amount of structure‐activity studies based on the prion fragment approach, hinging on peptides derived from the unstructured N‐terminal and globular C‐terminal domains. It is well documented that most of the fragments with regular secondary structure, with the exception of helices 1 and 3, possess a high β‐sheet propensity and tendency to form β‐sheet‐like aggregates. In this context, helix 2 plays a crucial role because it is able to adopt both misfolded and partially helical conformation. However, only a few mutants are able to display its intrinsic neurotoxicity.

RFID-based tracing systems for drugs: Technological aspects and potential exposure risks

Radio Frequency Identification (RFID) is a very promising wireless technology able to trace and track individual objects. The pharmaceutical supply chain is a challenging scenario, where an item-level traceability is crucial to guarantee transparency and safety in the drug flow. Unfortunately, there are still some barriers limiting the large-scale deployment of these innovative technologies. In order to face these challenges, multidisciplinary skills are required. A recent research project has attempted to coordinate heterogeneous activities focused on drug traceability. One of these is related to the evaluation of potential effects of exposure to electromagnetic fields on drugs. This paper aims to briefly describe both the main features of the defined framework for the item-level tracing of drugs on the whole supply chain and the most interesting results obtained by the evaluation of the potential effects of RFID systems on drugs. In particular, the potential alterations of the molecular structure of a commercial human insulin preparation have been analyzed by using investigative techniques such as Reverse Phase-High Pressure Liquid Chromatography and in vitro cell proliferation assays. The experimental results are strongly encouraging the use of RFID-based technologies for item-level tracing systems in the pharmaceutical supply chain.

Evaluation of Thermal and Nonthermal Effects of UHF RFID Exposure on Biological Drugs

The radio frequency identification (RFID) technology promises to improve several processes in the healthcare scenario, especially those related to the traceability of people and things. Unfortunately, there are still some barriers limiting the large-scale deployment of these innovative technologies in the healthcare field. Among these, the evaluation of potential thermal and nonthermal effects due to the exposure of biopharmaceutical products to electromagnetic fields is very challenging, but still slightly investigated. This paper aims to setup a controlled RF exposure environment, in order to reproduce a worst case exposure of pharmaceutical products to the electromagnetic fields generated by the UHF RFID devices placed along the supply chain. Radiated powers several times higher than recommended by current normative limits were applied (10 and 20 W). The electric field strength at the exposed sample location, used in tests, was as high as 100 V/m. Nonthermal effects were evaluated by chromatography techniques and in vitro assays. The results obtained for a particular case study, the ActrapidTM human insulin preparation, showed temperature increases lower than 0.5 °C and no significant changes in the structure and performance of the considered drug.

Monitoring prion protein expression in complex biological samples by SERS for diagnostic applications.

Surface-enhanced Raman spectroscopy (SERS) allows a new insight into the analysis of cell physiology. In this work, the difficulty of producing suitable substrates that, besides permitting the amplification of the Raman signal, do not interact with the biological material causing alteration, has been overcome by a combined method of hydrothermal green synthesis and thermal annealing. The SERS analysis of the cell membrane has been performed with special attention to the cellular prion protein PrP(C). In addition, SERS has also been used to reveal the prion protein-Cu(II) interaction in four different cell models (B104, SH-SY5Y, GN11, HeLa), expressing PrP(C) at different levels. A significant implication of the current work consists of the intriguing possibility of revealing and quantifying prion protein expression in complex biological samples by a cheap SERS-based method, replacing the expensive and time-consuming immuno-assay systems commonly employed.

Fluorimetric Analysis of Copper Transport Mechanisms in the B104 Neuroblastoma Cell Model: A Contribution from Cellular Prion Protein to Copper Supplying

Dysregulated body copper homeostasis can negatively impact neuronal functions, but full knowledge of the mechanisms underlying the cell metal distribution has not been achieved yet. The high-affinity copper transporter 1 (Ctr1) is considered the main route for cell copper entry, while the cellular prion protein (PrPC) is presumed to be involved in the same process. Anchored to the outer side of the plasma membrane, this protein has the ability to bind copper ions and undergo internalization. To provide indications about the contribution of Ctr1 and PrPC proteins in cell copper transport, we used a fluorimetric method to characterize the kinetic properties of ion internalization in a neuroblastoma cell model, overexpressing prion protein (B104). Biochemical characteristics of intake delineated in the presence of other metal ions and an excess of extracellular potassium were compatible with PrPC-mediated endocytotic transport. Accordingly, inhibition of clathrin-dependent endocytosis by hypertonic shock and enzymatic removal of surface prion protein reduced copper influx by the same extent. On the whole, experimental evidence collected in a neuron-like cell model sustains a role for PrPC in mediating copper uptake by clathrin-dependent endocytosis.

Bioenergetics profile of CD4(+) T cells in relapsing remitting multiple sclerosis subjects.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune demyelinating disease of the central nervous system. There are four clinical forms of MS, the most common of which is characterized by a relapsing remitting course (RRMS). The etiology of MS is unknown, but many studies suggested that genetic, environmental and infectious agents may contribute to the development of this disease. In experimental autoimmune encephalomyelitis (EAE), the animal model for MS, it has been shown that CD4(+) T cells play a key role in MS pathogenesis. In fact, these cells are able to cross the blood-brain barrier and cause axonal damage with neuronal death. T cell activation critically depends on mitochondrial ATP synthesis and reactive oxygen species (ROS) production. Interestingly, lots of studies linked the oxidative damage arising from mitochondrial changes to neurodegenerative disorders, such as MS. Based on these evidences, this work focused on the metabolic reprogramming of CD4(+) T cells in MS subjects, being this cell population directly implicated in pathogenesis of disease, paying attention to mitochondrial function and response to oxidative stress. Such aspects, once clarified, may open new opportunities for a therapeutic metabolic modulation of MS disorder.

Investigating potential effects of RFID systems on the molecular structure of the human insulin

The Radio Frequency Identification (RFID) is a wireless technology that is becoming more and more important as auto-identification solution for many application scenarios. The adoption of this innovative technology in the pharmaceutical sector promises to solve several problems related to tracing and tracking systems at item level. Unfortunately, there are still some barriers limiting the largescale deployment of RFID technologies. One of these is related to very interesting research topics on the evaluation of potential effects of electromagnetic fields on drugs. In detail, this work aimed to analyze the impact of UHF RFID devices, used in tracing systems, on the molecular structure and potency of a commercial human insulin preparation, ActrapidTM. In order to investigate possible induced alterations of molecular structure, the Reverse Phase-High Pressure Liquid Chromatography and the Nuclear Magnetic Resonance spectroscopy have been mainly used in the experimental protocol. To obtain some indications about drug performance, in vitro cell proliferation assays have been also conducted. The experimental results, achieved by a protocol combining an accurate structural analysis on 5 min to 24 h irradiated drug samples with functional in vitro assays, have shown that the electromagnetic field generated by UHF RFID devices does not cause significant effects on ActrapidTM insulin. These findings are strongly encouraging the use of RFID-based technologies for item-level tracing systems in the pharmaceutical supply chain.