Date of Award

4-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physics

First Advisor

Yulia Pushkar

Committee Chair

Yulia Pushkar

Committee Member 1

Stephen Durbin

Committee Member 2

Henray J. Melosh

Committee Member 3

Ken Ritchie

Abstract

Copper is an essential element in the brain playing several critical roles ranging from neurotransmitter synthesis to ATP production. As Cu is typically present in micromolar concentrations and has a spatially capricious distribution in the brain, determining concentrations has historically been challenging. X-ray fluorescence microscopy (XRF) offers excellent spatial resolution (down to 30~nm) and detection limits (sub parts per million), making it an excellent tool for analyzing metal distributions in the brain. Using XRF, it is demonstrated that Cu-rich aggregates with concentrations in the hundreds of millimolar are present in the subventricular zone of rats and mice. As the subventricular zone is a neurogenic niche in the adult brain, the hippocampal dentate gyrus and rostral migratory stream were also examined to see if these aggregates were present. It is shown that rats, but not mice, have Cu accumulations in the rostral migratory stream and dentate gyrus, which may be related to the fact that the rat hippocampus produces more neurons and new neurons are integrated more readily into neural circuits. In addition, these aggregates are found in glial fibrillary acidic protein-positive cells and a lack of co-localization with bromodeoxyuridine suggests they are specifically in type-B progenitor cells in the SVZ. Co-localization experiments with lysosomal associated membrane protein 1 and ubiquitin suggest that Cu aggregates are not simply awaiting autophagy but likely serve some purpose. (XANES) spectra recorded directly from Cu aggregates in tissue show that Cu is present as a CuxSy multimetallic cluster and has the oxidation state Cu+. Consistent with being bound as a cuprous-thiolate cluster, Cu aggregates consistently co-localize with sulfur with a molar ratio of [S]/[Cu] = 1.66 ± 0.07. By co-localizing XRF maps and DAB staining, it is shown that Cu and Fe form exclusive aggregates in the subventricular zone that exhibit peroxidase or pseudoperoxidase activity.

Towards determining the role of Cu aggregates, the metal distribution in the subventricular zone was examined under several conditions and insults. The subventricular zone is shown to selectively accumulate Cu with age owing in part to the increase of Cu concentrations within aggregates. Metallothionein is a sulfur-rich protein known to be critical in Cu homeostasis in astrocytes cells. Metallothionein(1,2) knockout mice exhibit lower subventricular Cu concentrations owed in part to the fact that there are about one third as many aggregates in the SVZ and remaining aggregates contain less Cu. As another metallothionein isoform – metallothionein 3 – is present in the SVZ, it is likely that it fulfills the role of metallothionein(1,2). While assignment is metallothionein as the binding protein is consistent with literature, its exact role of the protein in aggregate formation remains unclear; metallothioneins may simply facilitate Cu uptake into the cell. Exposure to Mn, which is known to disrupt brain barriers, did not affect Cu concentrations for rats in either the subventricular zone or rostral migratory stream. Furthermore, the cuprizone mouse model, which reproduces demyelination as observed in multiple sclerosis through dietary Cu chelation, is examined. While X-ray diffraction mapping shows that myelin-rich areas show diminished myelin there is no corresponding decrease in Cu in these regions, suggesting that Cu starvation may not be the mechanism behind demyelination. Dietary Cu chelation by cuprizone did not affect metal concentrations in the subventricular zone.

Taken together, the results demonstrate that periventricular Cu accumulations are formed by a robust mechanism and likely serve some role. While the mechanism remains unclear, it is plausible that the aggregates somehow play a role in the neurogenic activity in the regions they are present.

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