Zinc is an essential micro-nutrient that participates in catalytic and structural functions touching nearly every metabolic process in the cell. While alterations in neuronal Zn2+ distribution have been associated with multiple disease states including Alzheimer’s Disease (AD), ALS, schizophrenia, and depression, the molecular mechanism of Zn2+ homeostasis in neurons is largely unexplored. The Zinc and Iron-reg- ulated transport Proteins (ZIP) mediate the entrance of first row transition metals into the cytoplasm. hZIP12, one of the fourteen human ZIPs, is a Zn2+ uptake transporter expressed in the brain. hZIP12 cSNPs (coding single nucleotide polymorphisms) have been associated with AD and abnormalities in the caudate nucleus. Importantly, our previous structural functional studies of the homologous hZIP4 enables postulation of the likely functional alteration introduced by observed cSNP. This proposal is based on the hypothesis that studies of hZIP12 and variants associated with neurodegenerative diseases will serve as an entrance point to build testable mechanistic models of neuronal Zn2+ homeostasis. Aim one tests the hypothesis that hZIP12 mutations associated with neuronal diseases impact transport activity and/or sur- face expression. hZIP12-mediated Zn2+ transport will be measured in neuronal SH-SY5Y cells expressing wild type and mutated proteins. Membrane targeting and dimerization studies will characterize predicted structural effects of observed cSNPs. The structural and functional consequence of three types of vari- ants will be examined: 1) one cSNP linked to AD, 2) two cSNPs associated with morphological alterations of the caudate nucleus; and 3) two cSNPs hypothesized to alter metal selectivity. Aim two tests the hy- pothesis that hZIP12 dysfunction modifies cellular Zn2+ pools and metal distribution. Cytosolic Zn2+ levels, as well as transmembrane transporter expression, will be assessed under early steady state and non- toxic Zn2+ levels. To this end, targeted as well as unbiased global approaches will be employed: 1) size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) profiling of the proteome will be combined with mass spectrometry (LC-MS/MS) to identify Zn2+ binding proteins. 2) The expression of compensatory genes participating in the response will be evaluated via unbiased approaches (RNA-Seq) as well as directed Q-PCR of genes known to be involved in Zn2+ homeostasis/distribution. Results from these studies will initiate the definition of molecular and subcellular mechanisms of Zn2+ homeostasis in the brain, provide a detailed description of hZIP12 function in neuronal Zn2+ subcellular distribution, and elucidate the role of hZIP12 in relevant neurodegenerative diseases.

Project Narrative Worcester Polytechnic Institute PI: Robert Dempski The proposed research is relevant to public health because the discovery of how the human ZIP12 transporter impacts neuronal transition metal homeostasis will increase understanding of the role of this protein in diseases such as schizophrenia, Alzheimer’s disease, and brain development abnormalities. The proposed research is relevant to the part of NIH’s mission that pertains to fostering innovative research strategies to advance the Nation’s capacity to protect and improve health.