RICERCA ITALIANA     

ADVANCED TOOLS OF MOLECULAR IMAGING AND GENETIC ENGINEERING TO STUDY DISTRIBUTION AND DYNAMICS OF CELL MAGNESIUM: NOVEL APPROACHES TO LINK MAGNESIUM HOMEOSTASIS AND CELL FUNCTIONS.

Università Cattolica del Sacro Cuore

Abstract

Magnesium is an essential cofactor of numerous enzymatic activities being crucial for vital cellular functions such as DNA and protein synthesis. Many important pathologies are associated to a tissue and/or systemic magnesium deficit, and in some cardiovascular diseases and particularly in cerebral trauma magnesium supplementation is currently used as therapeutic aid.
Albeit a wealth of scientific and clinical data highlights the key role of this cation for most of the cellular functions, cell magnesium homeostasis still waits to be elucidated. The major lack of knowledge pertains both the transport mechanisms and the functional meaning of the intracellular movements and compartmentalization. This is mainly due to a poor molecular characterisation of magnesium transporters, as well as to the paucity of methodological approaches suitable to map intracellular magnesium with a high degree of specificity and sensitivity.
The present project is aimed at developing new approaches for the study of magnesium intracellular distribution combining advanced molecular imaging techniques with molecular biology and genetic engineering methods. In particular the project will focus on the use of a new fluorochrome family (DCHQ) derived from 8-hydroxyquinoline, which showed to have a high affinity and specificity to magnesium and was found to be: i) capable of assessing the intracellular magnesium content, ii) suitable to map magnesium intracellular distribution by two photon >>>

Principal Investigator

Federica Wolf Università Cattolica del Sacro Cuore

Research Objectives

Our attempt is to identify and characterize the intracellular magnesium pools (free or bound, cytosolic or localized in subcellular compartments). To this purpose, because of the physical and chemical properties of magnesium (e.g. high concentrations, binding to different species, hydrated ion form), the development of sophisticated techniques of cellular imaging is required.
The aim of this project is to contribute to a deeper knowledge on the distribution and dynamics of intracellular magnesium in order to correlate the homeostasis of this metal to its multiple biological functions. Until now, studies on the homeostasis of intracellular Mg have been jeopardized by the lack of suitable tracers. We therefore propose to develop and utilize novel techniques of molecular imaging like:
i) new fluorescent probes with a high affinity and specificity for magnesium, to be used in cytofluorimetry and live cell imaging. The high sensitivity of fluorimetric methods allows to analyze very small samples, unlike classic methods, as atomic absorption spectroscopy. Furthermore, cytofluorimetry and microphotometry allows to work on unhomogeneous samples if selective counterstaining are available, allowing to identify the different cells without purification steps. This could lead to develop methodologies which could clarify several aspects of cellular biology of magnesium but also ameliorate diagnostic applications.
ii) an unprecedented protocol to map intracellular >>>

First Results

Expected results from this project will contribute to elucidate various aspects of magnesium homeostasis at the cellular level, by means of molecular imaging techniques in combination with genetic engineering. In particular:

1) QUANTITATIVE MEASUREMENTS OF MAGNESIUM: validity and advantages provided by DCHQ fluorescent probes. The high specificity and selectivity of these molecules suggests that they might be used as an alternative to atomic absorption spectrometry (AAS) to quantify cellular magnesium in biological samples, especially if only little amounts of starting material are available. In particular, this possibility will be assessed in a control-case study for benign chronic intestinal failure (CIF); the quantification of magnesium contents of circulating blood cells by DCHQ1 will be compared with measurements obtained by AAS and correlated with measurements of ionised magnesium in muscle and brain by nuclear magnetic resonance. If the validity of the technique we propose in this project is confirmed, it will provide an important applicative tool in diagnostic procedures in the field of clinical biochemistry. Basically, magnesium content in blood cells could be measured in samples as small as a few mL, by a simple and rapid protocol.

2) SUBCELLULAR DISTRIBUTION: use of DCHQ derivatives to measure magnesium distribution in subcellular compartments by confocal imaging (two-photon source, possibly combined with FLIM and FRAP techniques) >>>

Timescale

24 months

National and international background

Magnesium (Mg) is an important intracellular divalent cation, which plays a key role in a wide range of biochemical reactions (it acts as a cofactor in as many as 300 enzymatic reactions) and of essential biological functions (e.g., nerve impulse, muscular contraction, DNA and protein synthesis, energy metabolism). Many serious human pathologies, such as cardiovascular diseases, diabetes, metabolic syndrome and several neurological disorders, are characterised by a deficiency in Mg, either tissue-specific or systemic. Alterations of cellular Mg metabolism have been demonstrated in hypertensive subjects and proposed to contribute to the pathogenesis of primary hypertension (Kosch, 2001). In addition, Mg is reduced in smooth muscle cells from spontaneously hypertensive rats and a Mg-supplemented diet attenuates the development of hypertension in this experimental model (Touyz, 1999). In humans, the consumption of dairy products, which retain substantial amounts of K, Ca and Mg, may be associated with reduced blood pressure and risk of stroke (Massey, 2001). In addition, oral Mg therapy has been associated with a significant improvement of endothelial function in patients with coronary artery disease and with a decrease of plasma concentrations of triglycerides, VLDL and apo-B (Schechter, 2000). Also diabetes frequently associates with Mg deficiency (Valk,1999), because of the increased Mg secretion due to hyperglycemia (Djurhuus, 2000). It is noteworthy that insulin-mediated >>>