Mapping alterations to the endogenous elemental distribution within the lateral ventricles and choroid plexus in brain disorders using X-ray fluorescence imaging

The choroid plexus and cerebral ventricles are critical structures for the production of cerebral spinal fluid (CSF) and play an important role in regulating ion and metal transport in the brain, however many aspects of its roles in normal physiology and disease states, such as psychiatric illness, remain unknown. The choroid plexus is difficult to examine in vivo, and in situ ex vivo, and as such has typically been examined indirectly with radiolabeled tracers or ex vivo stains, making measurements of the endogenous K+, Cl-, and Ca+ distributions unreliable. In the present study, we directly examined the distribution of endogenous ions and biologically relevant transition metals in the choroid plexus and regions surrounding the ventricles (ventricle wall, cortex, corpus callosum, striatum) using X-ray fluorescence imaging (XFI). We find that the choroid plexus was rich in Cl- and Fe while K+ levels increase further from the ventricle as Cl- levels decrease, consistent with the known role of ion transporters in the choroid plexus CSF production. A polyI:C offspring displayed enlarged ventricles, elevated Cl- surrounding the ventricles, and intraventricular calcifications. These observations fit with clinical findings in patients with schizophrenia and suggest maternal treatment with polyI:C may lead to dysfunctional ion regulation in offspring. This study demonstrates the power of XFI for examining the endogenous elemental distributions of the ventricular system in healthy brain tissue as well as disease models

Computational studies of copper(II)-binding to model protein fragments, and their associated redox chemistry in an aqueous medium

Bibliography: p. 135-13

Computational and experimantal studies of the copper-induced structure within the n-terminal domain of the prion protein

Bibliography: p. 264-28

Modeling by Assembly and Molecular Dynamics Simulations of the Low Cu2+ Occupancy Form of the Mammalian Prion Protein Octarepeat Region: Gaining Insight into Cu2+-Mediated β-Cleavage

The prion protein has garnered considerable interest because of its involvement in prion disease as well as its unresolved cellular function. The octarepeat region in the flexible N-domain is capable of binding copper through multiple coordination modes. Under conditions of low pH and low Cu2+ concentration, the four octarepeats (ORs) cooperatively coordinate a single copper ion. Based on the average structure of the PHGG and GWGQ portions of a copper-free OR2 model from molecular dynamics simulations, the starting structures of the OR4 complex could be constructed by assembling the repeating structure of PHGG and GWGQ fragments. The resulting model contains a preformed site suitable for Cu2+ coordination. Molecular dynamics simulations of Cu2+ bound to the assembled OR4 model (Cu:OR4) reveal a close association of specific Trp and Gly residues with the Cu2+ center. This low Cu2+-occupancy form of prion protein is redox-active and can readily initiate cleavage of the OR region, mediated by reactive oxygen species generated by Cu+. The OR region is known to be required for β-cleavage, as are the Trp residues within the OR region. The β-cleaved form of the prion protein accumulates in amyloid fibrils. Hence, the close approach of Trp and Gly residues to the Cu2+ coordination site in the low Cu2+-occupancy form of the OR region may signal an important interaction for the initiation of prion disease

High Affinity Binding of Indium and Ruthenium Ions by Gastrins

<div><p>The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga³⁺ were fitted by a 2 site model with dissociation constants (K_d) of 3.3 x 10⁻⁷ and 1.1 x 10⁻⁶ M. Although the absorption of gastrin did not change upon the addition of In³⁺ ions, the changes in absorbance on Fe³⁺ ion binding in the presence of indium ions were fitted by a 2 site model with K_d values for In³⁺ of 6.5 x 10⁻¹⁵ and 1.7 x 10⁻⁷ M. Similar results were obtained with Ru³⁺ ions, although the K_d values for Ru³⁺ of 2.6 x 10⁻¹³ and 1.2 x 10⁻⁵ M were slightly larger than observed for In³⁺. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In³⁺ or Ru³⁺ ions, and that the affinity of the first site for In³⁺ or Ru³⁺ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.</p></div

Proposed structural models of FeIII2Ggly and RuIII2Ggly.

<p>The model for Fe^III₂Ggly (A) is based on the EXAFS data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g005" target="_blank">Fig 5B</a>, and is consistent with previous NMR and visible spectroscopic studies of Ggly and mutant peptides.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.ref008" target="_blank">8</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.ref009" target="_blank">9</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.ref012" target="_blank">12</a>] The two Fe^III ions are coordinated by the carboxylate side chains likely from glutamates 6, 7, 8, 9 and 10, with glutamate 7 acting as a ligand to both Fe^III ions. One or more oxygens also act as bridging ligands between the two Fe^III ions. The peptide backbone and non-coordinating side chains have been omitted for simplicity. The model for Ru^III₂Ggly (B) is based on the EXAFS data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g005" target="_blank">Fig 5H</a>, and differs from the model for Fe^III₂Ggly in the presence of a Ru≡Ru bond and a chloride ion ligand.</p

Ruthenium ions compete with ferric ions for the gastrin binding sites.

<p>Addition of aliquots of ruthenium chloride (black ▼) to 10 μM Gamide or Gly in the buffer described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g002" target="_blank">Fig 2</a> legend resulted in an increase in absorbance at 280 nm which was significantly less than the changes seen on addition of aliquots of ferric chloride (red π). However in the presence of 5.30 (green ⬛) or 26.48 μM (blue •) ruthenium chloride the changes in absorbance seen on addition of aliquots of ferric chloride were considerably different from the changes seen in the absence of ruthenium chloride. The points are means from three separate experiments; bars represent the SEM. The lines were constructed with the dissociation constants and maximum absorbance values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.t001" target="_blank">Table 1</a>) obtained by fitting the data to the 2 site competitive model shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g001" target="_blank">Fig 1</a> with the program BioEqs.</p

Models of metal ion binding.

<p>In the 2 site model gastrin binds two metal ions with dissociation constants K_d1M and K_d2M. In the 2 site competitive model gastrin binds two ferric ions with dissociation constants K_d1Fe and K_d2Fe, and two metal ions (M) to the same two sites with dissociation constants K_d1M and K_d2M. The dissociation constant K_d3M describes the formation of the mixed FeGastrinM complex.</p

Indium ions compete with ferric ions for the gastrin binding sites.

<p>Addition of aliquots of indium nitrate (black ▼) to 10 μM Gamide or Ggly in the buffer described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g002" target="_blank">Fig 2</a> legend resulted in little change in absorbance at 280 nm when compared to the changes seen on addition of aliquots of ferric chloride (red π). However in the presence of 3.99 (green ⬛) or 39.85 μM (blue •) indium nitrate the changes in absorbance seen on addition of aliquots of ferric chloride were considerably different from the changes seen in the absence of indium nitrate. The points are means from three separate experiments; bars represent the SEM. The lines were constructed with the dissociation constants and maximum absorbance values (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.t001" target="_blank">Table 1</a>) obtained by fitting the data to the 2 site competitive model shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140126#pone.0140126.g001" target="_blank">Fig 1</a> with the program BioEqs.</p