X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder.

Biometals such as zinc, iron, copper and calcium play key roles in diverse physiological processes in the brain, but can be toxic in excess. A hallmark of neurodegeneration is a failure of homeostatic mechanisms controlling the concentration and distribution of these elements, resulting in overload, deficiency or mislocalization. A major roadblock to understanding the impact of altered biometal homeostasis in neurodegenerative disease is the lack of rapid, specific and sensitive techniques capable of providing quantitative subcellular information on biometal homeostasis in situ. Recent advances in X-ray fluorescence detectors have provided an opportunity to rapidly measure biometal content at subcellular resolution in cell populations using X-ray Fluorescence Microscopy (XFM). We applied this approach to investigate subcellular biometal homeostasis in a cerebellar cell line isolated from a natural mouse model of a childhood neurodegenerative disorder, the CLN6 form of neuronal ceroid lipofuscinosis, commonly known as Batten disease. Despite no global changes to whole cell concentrations of zinc or calcium, XFM revealed significant subcellular mislocalization of these important biological second messengers in cerebellar Cln6nclf (CbCln6nclf ) cells. XFM revealed that nuclear-to-cytoplasmic trafficking of zinc was severely perturbed in diseased cells and the subcellular distribution of calcium was drastically altered in CbCln6nclf cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6nclf cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6nclf cells. Importantly, a zinc-complex, ZnII(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6nclf cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. ZnII(atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in CbCln6nclf cells. This study highlights the complementarities of bulk and single cell analysis of metal content for understanding disease states. We demonstrate the utility and broad applicability of XFM for subcellular analysis of perturbed biometal metabolism and mechanism of action studies for novel therapeutics to target neurodegeneration.

Carboxymethylated cage amines: coordination and lactamization.

Depending upon the position and degree of substitution, carboxymethyl derivatives of cage amines of the "sarcophagine" (3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane) type vary considerably in the stability of their lactamized forms. For 1,8-diamino-3-carboxymethylsarcophagine, L(1), only indirect evidence for some involvement of a lactamized form of its Ni(II) complex has been obtained. Crystal structure determinations for [Cu(H(2)L(1))](NO(3))(3.5)Cl(0.5) x 2.5H(2)O and [Ni(HL(1))]Cl(3) x 3H(2)O show distorted octahedral coordination of all six endocyclic N-donor atoms in both cases. For related diaminosarcophagine derivatives with either two (1,8; L(2)) or three (1,1,8; L(3)) carboxymethyl substituents on the exocyclic N atoms, crystallographic studies have shown a dilactam form for the ligands in their Ni(II) and Cu(II) complexes which is of almost identical conformation to that of the diprotonated "free" ligand in [H(2)L(3)][ZnCl(4)] x 6H(2)O. The lactamized ligands appear to strongly favor square planar four-coordination, and the Co(II) complex of L(2) shows a remarkable lack of reactivity toward oxygen. Kinetic studies indicate that the hydrolytic stability of the lactam rings is comparable to that of uncoordinated analogues.

Carboxymethylation of cage amines: control of alkylation by metal ion coordination.

Reactions between chloroacetate and both "free" and coordinated forms of the cage amine diaminosarcophagine (1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane) result in the introduction of between one and four pendent carboxymethyl substituents on the nitrogen atoms of the cage. While at least the first two steps in the reaction of the free ligand have been found to involve only the secondary nitrogen centers, in both the Cu(II) and Co(III) complexes alkylation occurs only at the primary (1,8) centers, the greater ease of achieving a higher degree of alkylation of the Cu(II) complex being attributed to the lower charge of this species causing a lesser reduction of the nucleophilicity of the uncoordinated primary nitrogen atoms. All the new ligands have been characterized by X-ray structure determinations of their Cu(II) or Co(III) complexes. In some cases, this has shown that the methods used to isolate the crystalline complexes result in lactamization of the ligand.