Elsevier

NeuroToxicology

Volume 34, January 2013, Pages 118-127
NeuroToxicology

Manganese transport via the transferrin mechanism

https://doi.org/10.1016/j.neuro.2012.10.018Get rights and content

Abstract

Excessive manganese (Mn) uptake by brain cells, particularly in regions like the basal ganglia, can lead to toxicity. Mn2+ is transported into cells via a number of mechanisms, while Mn3+ is believed to be transported similarly to iron (Fe) via the transferrin (Tf) mechanism. Cellular Mn uptake is therefore determined by the activity of the mechanisms transporting Mn into each type of cell and by the amounts of Mn2+, Mn3+ and their complexes to which these cells are exposed; this complicates understanding the contributions of each transporter to Mn toxicity. While uptake of Fe3+ via the Tf mechanism is well understood, uptake of Mn3+ via this mechanism has not been systematically studied. The stability of the Mn3+Tf complex allowed us to form and purify this complex and label it with a fluorescent (Alexa green) tag. Using purified and labeled Mn3+Tf and biophysical tools, we have developed a novel approach to study Mn3+Tf transport independently of other Mn transport mechanisms. This approach was used to compare the uptake of Mn3+Tf into neuronal cell lines with published descriptions of Fe3+ uptake via the Tf mechanism, and to obtain quantitative information on Mn uptake via the Tf mechanism. Results confirm that in these cell lines significant Mn3+ is transported by the Tf mechanism similarly to Fe3+Tf transport; although Mn3+Tf transport is markedly slower than other Mn transport mechanisms. This novel approach may prove useful for studying Mn toxicity in other systems and cell types.

Highlights

► Mn3+ can be transported into neuronal cells via the Tf mechanism. ► This transport shows characteristics similar to those of Fe3+ transport via this same mechanism. ► The amount of this transport in the neuronal cells used was much less than transport of the same concentration of Mn2+. ► Because Mn3+ is a strong oxidizing agent, it is transport via the Tf mechanism, could also cause oxidative damage to endosomes.

Introduction

While manganese (Mn) is an essential biological element and a necessary cofactor in a number of important enzymatic reactions, excessive brain Mn accumulation particularly in the globus pallidus and striatum can lead to neurotoxicity with symptoms and signs resembling those of Parkinson's disease. The evidence indicates that in the 2+ oxidation state (Mn2+), Mn enters cells via a number of transport mechanisms, including the divalent metal transporter 1 (DMT1) (Au et al., 2008), a Mn citrate transporter (Crossgrove et al., 2003, Crossgrove and Yokel, 2004), a store activated Ca2+ channel (Yokel and Crossgrove, 2004), the ZIP8 mechanism (He et al., 2006, Liu et al., 2008), and the ZIP14 mechanism (Fujishiro et al., 2012, Girijashanker et al., 2008). In the 3+ oxidation state (Mn3+), the evidence suggests that Mn is transported via the transferrin (Tf) mechanism (Aschner and Aschner, 1990, Aschner and Gannon, 1994). Mn uptake into a specific cell type is thus determined by the activity of each type of uptake mechanism expressed in that cell type and the oxidation state of the Mn reaching the cell. Once inside the cell, most of the Mn is found in the mitochondrial and nuclear fractions (Maynard and Cotzias, 1955).

A study of Mn speciation in animal cells using X-ray absorption near edge structure (XANES) spectroscopy identified only Mn2+ and a trace amount of Mn3+ having the spectrum of the enzyme Mn superoxide dismutase (Gunter et al., 2004, Gunter et al., 2005, Gunter et al., 2006a). This is undoubtedly because Mn2+ is by far the more stable species of these two oxidation states (Latimer and Hildebrand, 1956). The concentration of free Mn3+ is not zero, but exists in a steady state in which its concentration is much lower than that of free Mn2+ and below the XANES detection limit. A number of factors influence this steady state, but generally free Mn3+ is more stable at low pH than at neutral pH (Latimer and Hildebrand, 1956). Mn3+ can also enter aqueous solution when stabilized by formation of stable complexes (Gunter et al., 2006b), including some formed with common organic anions usually found in cells and tissue, such as citrate or Tf.

The Tf system is known to transport iron (Fe) primarily into mitochondria for incorporation into hemes and other Fe-containing proteins (Sheftel et al., 2007). Fe3+ transport via the Tf mechanism involves the following steps: binding of the Fe3+ to extracellular Tf, binding of the Fe3+Tf complex to Tf receptors (TfRs) and movement of the Tf-bound TfR into clathrin-coated pits, invagination of these pits into endosomes bringing the Fe3+Tf inside the cell, movement of the endosomes into the region of the cytosol in which the mitochondrial network is found, release of Fe3+ from binding to the Tf complex by acidification of the endosomal interior, reduction of the Fe3+ to Fe2+ by Steap proteins in the endosomes, transport of the Fe2+ out of the endosomes by DMT1, and uptake of the Fe2+ by mitochondria where it can be incorporated into hemes and other iron-containing proteins (Hentze et al., 2004, Ohgami et al., 2005, Ohgami et al., 2006, Richardson and Ponka, 1997, Sheftel et al., 2007).

Mn and Fe are elements with atomic numbers 25 and 26, respectively, within the transition metal series and have many similar characteristics. Mn uptake via the Tf mechanism has usually been assumed to be analogous to that of Fe; nevertheless, there are differences in their chemistry, which could cause Mn3+ transport via Tf to differ from transport of Fe3+. First, the oxidation state in which Fe binds most strongly to Tf, Fe3+, is also its most stable oxidation state at physiological pH; in contrast, while Mn also binds Tf as Mn3+, it is most stable at physiological pH as Mn2+. Since the concentration of free blood Mn3+ is very low at physiological pH, even when Mn is present in excess, mass action would predict that Mn3+ would bind to Tf very slowly as Mn3+ transferrin (Mn3+Tf). This is supported by the observation that the procedure set up by Aisen et al. (1969) for preparing Mn3+Tf requires a week for the accumulation of the stable Mn3+Tf complex to approach completion. We have studied this slow conversion of Mn2+ to Mn3+ more closely under somewhat more physiological conditions in a XANES experiment described below. Studies of transport of Fe3+ via the Tf mechanism show that the Fe3+ is reduced to Fe2+ by Steap proteins inside endosomes and transported out of the endosomes via DMT1 (Hentze et al., 2004, Ohgami et al., 2005, Ohgami et al., 2006, Richardson and Ponka, 1997). Mn2+, like Fe2+ is transported by DMT1 (Au et al., 2008) and hence should be released from the endosomes similarly to Fe2+. Furthermore, the Tf transport mechanism for Fe is known to deliver Fe2+ to the vicinity of the mitochondrial network, where it is sequestered into mitochondria for incorporation into hemes (Sheftel et al., 2007). If it also delivers Mn2+ to mitochondria, it could contribute to mitochondrial Mn toxicity associated with deficits in energy production (Brouillet et al., 1993, Galvani et al., 1995, Gavin et al., 1992, Malecki, 2001, Malthankar et al., 2004, Roth et al., 2000, Roth et al., 2002, Zwingmann et al., 2003).

The systems transporting Mn2+ into cells are complex and difficult to functionally isolate experimentally. Furthermore, the expression of each type of transporter varies with cell type. However, we have been able to purify the Mn3+Tf complex and also to covalently bind a fluorescent label (Alexa green) to Mn3+Tf because of the stability of this complex. This has permitted us to study the transport of Mn via the Tf mechanism independently of the other cellular Mn transport mechanisms, to follow the transport of Mn3+Tf into neuronal cells and into the region of the mitochondrial network using confocal microscopy, and to confirm that Mn transport via the Tf system functions analogously to the transport of ferric iron (Fe3+) by this mechanism. Using the purified Mn3+Tf and atomic absorption spectroscopy (AA), we have also been able to measure the accumulation of Mn via Mn3+Tf independently of the other Mn transport mechanisms using atomic absorption and to compare it with the accumulation from a similar concentration of Mn2+. The primary goal of the work reported here is to introduce this novel approach to the study of transport of Mn3+ via the Tf system to those interested in its role in Mn neurotoxicity so that it may be applied to studies of Mn3+Tf transport in additional systems and cell types and to more complete studies of its uptake kinetics.

The uptake of Mn3+ via the Tf mechanism in the two neuronal cell types used in the current study is slower than that of Mn2+, but is not negligible and therefore could contribute to Mn toxicity, along with Mn transported into cells by the other transport mechanisms. However, this does not necessarily mean that transport of Mn3+ via the Tf system is so small in all cell types, and hence other cell types should be studied. Furthermore, Mn3+ is also a strong oxidizing agent, which might be expected to oxidize many components of the endosome in addition to the Steap proteins. Thus, transport of Mn via the Tf mechanism exposes an additional set of cell components to Mn, particularly within the endosomal system, which could be damaged by this transport process, but not by other processes of Mn transport into the cell.

Section snippets

Neuronal cells

Mn uptake via the Tf mechanism was studied in cultured mouse hippocampal (HT22) and striatal neurons (STHdhQ7/Q7) (Trettel et al., 2000). The latter were chosen because the striatum is an area where Mn preferentially accumulates, and one of the areas most affected by Mn toxicity (Finkelstein et al., 2007). HT22 cells were grown at 37 °C in an incubator with 5% CO2 in Gibco F-12 mixture (HAM) containing 5% FBS, 5% HS, 20 mM glucose, and Pen Strep. The STHdhQ7/Q7 cells were grown at 33 °C with 5% CO2

Conversion of Mn2+ to Mn3+ through stabilization in a Tf complex

An experimental sample containing separately added Mn2+ (0.2 mM) and Tf (2.0 mM) was incubated at 22 °C for 24 h in HEPES (10 mM) (pH 7.2) buffered saline (160 mM NaCl). The sample was then frozen and maintained at −80 °C until a XANES spectrum could be obtained (Fig. 1). The sample was analyzed with curve-fitting techniques in which its spectrum was compared with the spectra of the Mn2+ and Mn3+ model compounds shown in Fig. S2A and B in the supplementary material. These techniques are described in

Discussion

While Fe uptake via the Tf mechanism has been thoroughly studied and described in a large number of publications over the last 30 years, the transport of Mn via this mechanism has not been systematically studied. Initial evidence that Mn could be transported into cells via the Tf mechanism was published over 20 years ago (Aschner and Aschner, 1990); however, many of the characteristics of this transport have remained speculative and significant differences may exist between Fe and Mn transport

Conflict of interest

There are no conflict of interest.

Acknowledgments

The authors would like to thank Dr. William Bernhard for use of his EPR and UV–vis spectrometers and Mr. Paul Black for instructions on the use and help in operating these spectrometers. They would like to thank Dr. Marcy MacDonald for developing and permitting us to use the STHdhQ7/Q7 striatal neurons and Dr. Gail Johnson-Voll for providing us with these cells. They would like to thank Dr. Pamela Meher for providing us with the HT22 hippocampal neurons. They thank Dr. Beth Van Winkle for help

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