Presence of Adp-Ribosylation Factor 1 (ARF1) in the Choroid Plexus: Impact on Copper Levels in the Cerebrospinal Fluid and Lead Exposure

Lara T Sang, Purdue University

Abstract

Copper (Cu) dyshomeostasis in the brain, especially Cu overload, has been associated with neurodegenerative disorders such as Alzheimer’s disease (AD). Brain Cu levels are partly regulated by the choroid plexus (CP), a tissue that forms a barrier between the blood and cerebrospinal fluid (CSF) and is rich in Cu-transporting proteins. Literature data have shown that ADP-ribosylation factor 1 (ARF1) plays a role in regulating cellular Cu homeostasis; yet its presence and function in the blood-CSF barrier was unknown. The main purpose of the project was to prove the presence of ARF1 in the CP and explore its possible function with regards to Cu regulation. Since the CP is known to accumulate toxic metal lead (Pb) from human and animal studies, the project also aimed to test whether Pb exposure caused CSF Cu dyshomeostasis through disrupting ARF1-mediated Cu regulatory mechanism. Using quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC), our data clearly demonstrated that ARF1 was highly enriched in the choroidal epithelial Z310 cells and expressed in CP tissues. Acute Pb exposure in mice (one ip. dose at 27 mg Pb/kg followed by tissue dissection 24 hrs later) significantly increased ARF1 expression in the CP as compared to saline-injected controls, suggesting a high responsiveness of the protein to Pb exposure. In the subsequent chronic study, mice were exposed to Pb via drinking water at 200ppm (low-dose) or 800ppm (high-dose) at libitum for 4 weeks (control group had sodium in drinking water). Atomic absorption spectrometry (AAS) analyses verified a dose-dependent Pb accumulation in the CP. Importantly, Cu concentrations in the CSF displayed a Pb dose-dependent increase as compared to controls. IHC data further revealed an altered ARF1 expression in the choroidal epithelia in chronic Pb exposed animals. Further knocking down ARF1 expression in choroidal epithelial Z310 cells using in vitro Arf1-targeting siRNA transfection approach revealed a decreased Cu level in cells, suggesting a critical role of ARF1 in cellular Cu regulation. In choroidal epithelial cells exposed to Pb in culture medium, a down-regulated ARF1 expression partly reversed Pb-induced Cu overload. Taken together, this study provides first-hand evidence to support the presence of ARF1 in the bloodCSF barrier. Further, our data demonstrate that Pb exposure causes Cu overload in the CSF, which is likely mediated by an altered expression of ARF1 in the blood-CSF barrier.

Degree

M.Sc.

Advisors

Zheng, Purdue University.

Subject Area

Aging|Cellular biology|Neurosciences|Physiology

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