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 confocal microscopy; iii) able to monitor sub-cellular movements by live-cell imaging. We will also study other derivatives of DCHQ family characterised by improved photochemical and biological features to map intracellular magnesium.
We plan to extend the application of the DCHQ fluorochromes to the evaluation of the total intracellular magnesium of blood cells in a case-control study performed by in vivo magnetic resonance spectroscopy aimed at assessing the magnesium tissue distribution in a pathology characterised by intestinal malabsorption (CIF).
A further approach will make use of synchrotron radiation X-ray fluorescence microscopy and x-ray microimaging which, having the unique capability to image the intracellular topography of atomic elements with a spatial resolution of hundreds of nm, will allow to assess the intracellular magnesium distribution together with the intracellular partition of the different oxidation states of the cation with an unprecedented accuracy.
The study of magnesium homeostasis will be performed on: i) different cell lines in basal conditions; ii) cell lines under specific metabolic stimuli; iii) cells adapted to grow at nonphysiological (low and high) concentration of magnesium; and iv) endothelial cells engineered for the expression of the TRPM (7 and 6) channels, which are responsible of the magnesium influx.
The feasibility of the project relies on the mutually dependent expertises of the four Research Units, which are working in the fields of biochemistry, in vivo nuclear magnetic resonance, fluorimetry, cell biology, analytical methodologies for magnesium measurements, live cell imaging, molecular biology and genetics, physics of materials and nanotechnology. Furthermore, three of the four Research Units working on this project have a long experience in the study of different aspects of cell magnesium. The past and well established scientific collaboration among these groups testifies the interdependency of the scientific competencies. In addition, the contribution of the Research Unit of CNR will be fundamental for the set up of advanced X-ray imagine techniques in the study of intracellular magnesium, exploiting the application of synchrotron radiation X-ray fluorescence microscopy to cell biology, which represents a novel scientific frontier in the imaging of the intracellular topography. We believe this project will greatly contribute to the advancement of the biological knowledge on the magnesium which, despite its relevance, it has been nicknamed “the forgotten ion”. <<<

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 magnesium by synchrotron-based X-ray fluorescence microscopy (SXRFM) and microimaging. These innovative approaches allow to evaluate directly magnesium distribution with a very small manipulation of the samples, and to map not only the intracellular distribution, but also the valence, revealing the kind of bond to which participate in the different cellular subcompartments.
To this purpose, we will take advantage of the following cell types:
1) epithelial cells adapted to grow in very low or very high magnesium;
2) human endothelial cells genetically modified to overexpress or silence TRPM-7, a magnesium transporter.
3) peripheral blood cells isolated from patients with chronic intestinal failure and healthy age-matched controls;
These various experimental models will allow to correlate intracellular magnesium with cellular functions.
Furthermore we plan to study magnesium homeostasis measuring, in different tissues, the intracellular magnesium with different spectroscopic techniques, among them 31P- MRS. In fact, the correlation between these data will allow to identify the mechanisms of regulation of magnesium concentration in different tissues as well as of the effect of magnesium decompensation. Thanks to their complementary expertises ranging from physics and biochemistry to cellular and molecular biology, and their longstanding experience in the field of magnesium research, the four research teams will be the first in Italy attempting to set up pioneering experimental protocols for molecular imaging some of which with extremely high spatial resolution (about few hundreds nm) to evaluate the intracellular distribution of magnesium. Combining results obtained by the different experimental approaches will contribute to decipher the role of this cation in diverse physiopathological conditions. Furthermore clarifying its mechanism of action and of its homeostasis at cellular and tissutal level will bring to a more accurate approach in therapy and prophilaxys. <<<

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). DCHQ probes appear to map specific cellular compartments with high sensibility. Such subcellular mapping, if confirmed by further studies included in this project, such as X-ray imaging techniques, might provide the first description of the actual cellular distribution of magnesium and identify intracellular pools with functional significance.

3) KINETIC STUDIES: use of DCHQ derivatives to study magnesium fluxes by live cell imaging. In particular, the ester derivative that can be trapped in the cytoplasm should be sensitive and specific enough to measure fluctuations in cellular magnesium induced by specific stimuli, such as mitochondrial inhibitors, mitogens or apoptosis inducers. It is fundamental to verify and better characterise such fluctuations, as data in the literature are very scanty: this could help to elucidate the mechanisms whereby magnesium affects several important biological functions including activation, proliferation and death.

4) MAGNESIUM TRANSPORT: Assessing the role of specific magnesium transporters, such as TRPM6/7 and Na/Mg antiporter and consequently magnesium homeostasis, in epithelial and endothelial cell functions. Study of the relationship between transporter expression and magnesium fluxes, by using cell lines characterised by altered expression of these transporters. Some cell lines are already available (UR Rome: HC11 high and low); others will be selected by modification of their gene expression (UR Milan: Engineered HUVEC)
5) ENDOTHELIAL DISFUNCTION: use of genetically modified endothelial cells to study the mechanisms responsible for endothelial disfunction induced by magnesium deficiency previously described by the UR of Milan.

6) X-RAY IMAGING TECHNIQUES. As for microfluorescence and microimaging techniques, we expect two-sided results: on one hand, the setup of experimental protocols and data analysis for the general issue of localising, quantifying and determining the oxidation status of elements in the cellular environment; on the other hand, the specific issue of subcellular localisation of magnesium. By using different techniques (microfluorescence, microimaging and differential absorption by means of both an X-ray microscope and a lens-less geometry), it will be possible to compare the methods and to understand their potential application to high resolution cellular imaging. Since these are novel techniques, very promising but still not diffusely utilised, it is necessary to validate the results with complementary techniques, as we plan to do within this project. Should this approach prove successful, it will boost a wider diffusion of these techniques in the scientific community. Moreover, the protocols established in this project might be applied to other issues of current interest, both in the biomedical field and in other scientific fields, such as, for example, environmental issues, study and conservation of Arts, material science and engineering, nanoscience, etc. <<<

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 glucose transport requires Mg (Romani,2000; Barbagallo,2001); therefore hypomagnesemia has a role in promoting hyperglycemia. Mg is also involved in neuronal damage since it antagonizes glutamate receptors. Indeed, several reports support the neuroprotective effects of Mg (Turkyilmaz, 2002; Lin, 2004). Furthermore, magnetic resonance spectroscopy has demonstrated a significant reduction of cytosolic Mg in the brain of patients with neurological diseases (Lodi, 2001). Many of the aforementioned diseases share an endothelial dysfunction as an early pathogenetic event. The endothelium is, therefore, a challenging model to study. Because of their strategical location at the interface between blood and vessels, endothelial cells are readily exposed to various signals (cytokines, metabolites, ions, free radicals, shear stress), some of which may promote maladaptative functional changes. Among others, abnormal Mg status has been reported to be important in the modulating endothelial behaviour. Indeed, low Mg inhibits endothelial growth and migration, induces endothelial-monocyte interactions, and stimulates the synthesis of pro-coagulant molecules and pro-inflammatory cytokines (Maier 2004, 2007). It has also been demonstrated that Mg deficiency renders endothelial cells more sensitive to oxidative stress by impairing the enzymatic systems involved in scavenging free radicals.
In spite of the wealth of data correlating Mg metabolism to cellular dysfuctions and to several pathologic conditions, our knowledge of its cellular homeostasis is still scattered and incomplete. Though the concentration gradient across the cell is not very steep, both biochemical and molecular data point to the existence of specific control mechanisms in the membrane that regulate Mg influx and efflux, as well as the possibility that Mg is compartmentalised within the cell. In particular, Mg efflux is regulated by a Na/Mg antiport, while its influx is regulated by channels of the Transient Receptor Potential Melastatin (TRPM) family, namely TRPM-6 and -7, which share the unique functional duality of being ion channels and kinases (Cahalan, 2001; Takezawa,2004; Wolf,2004). Interestingly, cultured cells rendered TRPM7-deficient of the TRPM7 gene undergo growth arrest and die after a few days in culture (Nadler,2001; Schmitz,2003). The role of TRPM-6 in Mg homeostasis is demonstrated by the fact that mutations in the TRPM6 gene cause an autosomal recessive disease characterized by hypomagnesemia with secondary hypocalcemia (Schlingmann,2002; Chubanov,2004).
Although i) clinical studies demonstrate the pathogenic role of an altered Mg metabolism in several diseases, and ii) the existence of Mg transporters has been studied at the molecular level, direct evidence for the association between physiopathological events and alterations in fluxes and/or compartmentalisation of magnesium is still missing.
Most of the studies concerning TRPM-modified cells were based on ion current measurements by patch-clamp electrodes, while efflux data were inferred from total intra or extracellular Mg measurements by atomic absorption spectroscopy of cellular extracts. On the other hand, in vivo assessment of dynamic changes in magnesium is hampered by the lack of suitable fluorescent indicators. This is due to the fact that commercially available Mg fluorescent tracers (Fura, Indo, Quin and, more recently, Fluo) derive from calcium probes and usually retain a significant affinity for calcium (Kd around 100 microM). Subsequently, these probes were modified to improve their selectivity towards Mg, obtaining Mag-Fura and Mag-Fluo, which display a Kd for Mg in the mM range. However, both Mag-Fura and Mag-Fluo retain a significant affinity for calcium (Kd around 20 microM) and can sense pH, which hinders interpretation of results (Schachter,1990; Hurley,1992).
In the last decades, the attention has been mainly focused on the study of the regulation of calcium cellular homeostasis and rapid signalling.
When measuring changes in cytosolic Mg, some issues should be considered: 1) basal Mg concentration is much higher than calcium concentration (sub-mM vs. sub-microM, respectively); 2) Mg modifications are much smaller than those of calcium (1 or 2-fold vs. 10-fold or more, respectively); 3) Mg modifications are generally much slower than those of calcium. This implies that an appropriate fluorescent probe should be very sensitive and selective for Mg, and should be well tolerated by the cell to allow long-term observations. In addition, Mg shows a high affinity for different molecules, among which ATP4- and polyamines. Moreover, alterations of intracellular Mg stores, which occur without modifications of free or bound Mg, have been observed (Fatolahi,2000; Wolf 1998; Di Francesco and Wolf,1998). Apart from adenine nucleotide-bound Mg which can be measured by magnetic resonance, at the moment it is not possible to identify the different Mg pools and to correlate their alterations with their biological function. The ideal probe for Mg should be highly specific, sensitive, stable and lack cytotoxicity. Recently, a new class of Mg tracers have been proposed to measure free intracellular Mg (Komatsu, 2004; Komatsu, 2005). These molecules have been used to detect rapid and significant intracellular movements which are indicative of an active mobilisation of Mg pools that does not depend on ATP hydrolysis (Kubota, 2005), in contrast to what previously thought. The significance of these movements is still waiting for an interpretation to link them to cellular functions such as apoptosis or growth. Lately, new fluorescent probes derived from benzochromene have been utilized in two-photon confocal microscopy. These molecules seem very interesting since i) they allow the discrimination of free and bound Mg; and ii) they allow the use of thick tissue samples (Kim et al.2007). This is a relevant issue since, by analyzing samples like these, it will be possible to understand the involvement of Mg in pathophysiologic conditions.
The photochemical properties of several derivatives based on diaza-18-crown-6 appended with two 8-hydroxyquinoline (DCHQ), which show a high affinity for Mg, have characterised (Prodi, 1998a and b). Some of us (teams in Roma and Bologna) are now collaborating to apply DCHQ and the Cl-substituted derivative (DCHQ2) to the measurement of intracellular Mg. These probes proved to be very specific for Mg, with a Kd=44 microM and to possess a significantly lower interference with calcium than classical indicators. The molecules are well tolerated by the cells and allow the detection of rapid intracellular Mg transients in response to stimuli interfering with mitochondrial activities (Farruggia, 2006). Thus, DCHQ probes seem to possess all the requisites to remedy the lack of knowledge in cellular magnesium homeostasis, and represent a promising tool to decipher the role of this cation in diverse physiopathological conditions. Preliminary data indicate that DCHQ1 could be useful to quantitate cellular Mg, thus creating an alternative to atomic absorption spectroscopy. The advantage is that it will be possible to operate on fewer cells, which renders such an approach of relevance for clinical pathology (Farruggia et al. submitted).
Another promising approach is represented by the use of novel experimental protocols for molecular imaging with extremely high spatial resolution (about 100 nm). In particular, high brilliance x-ray sources, as the third generation synchrotron radiation large facilities, have allowed the access to high value innovative methodologies in many scientific fields, and in particular in biomedical science. Thanks to the enormous progress in x-ray optics and to the x-ray short wavelength, it is now possible to achieve spatial resolution at the level of nanometer. The high penetrating power of x-rays gives useful information on the bulk of the samples and not only on their surface, and x-ray fluorescence techniques allow to quantitatively determine elemental concentration, even in traces. The combination of beam focusing techniques with spectroscopic analysis permitted the development of methods like microfluorescence, which gives the spatial distribution of a specific element with very high spatial resolution (down to 100 nm). These kinds of measurements can be carried out either with direct microfluorescence experiments, or with measurement of the differential absorption at two incident beam energies, one just below and one just above the absorption edge for that given element. Microfluorescence and microimaging have been used to study the intracellular distribution of Cu (Yang, 2005) and of essential elements in cardiomyocytes (Palmer, 2006) and in aquatic protist cells (Twining, 2003). A recent work reached the record in x-ray microfluorescence spatial resolution, studying at 90 nm the Iron storage within Dopamine neurovesicles (Ortega, 2007).
Therefore, new fluorescent indicators in association with an x-ray approach will provide innovative research tools that might open up new horizons for the study of this cation at the cellular level, and contribute to understanding the role of Mg in important physiopathological conditions.

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