Chronic exposure to the divalent heavy metals, lead, manganese (Mn) and chromium, has all been linked to development of severe and irreversible neurological disorders as well as increased risk to develop Parkinson disease. Although the mechanisms by which these metals induce neuronal cell death are not well defined, neurotoxicity is regulated by a number of factors, one of which is the transport of these metals across the blood brain barrier and their subsequent uptake within targeted neuron. Once inside the neuron these heavy metals provoke a series of biochemical and molecular events leading to cell death induced by apoptosis and/or necrosis. In the case of Mn, chronic high level exposure provokes a syndrome resembling Parkinson?s disease, which at the latter stages consists of severe extrapyramidal dysfunction. Recent studies have indicated that Mn toxicity is integrally linked to transport and disposition of iron. Mn is predominantly taken up into cells via the same transporter responsible for iron uptake, i.e. the divalent metal transporter, DMT1. This transporter has a very broad specificity and is responsible for the cellular uptake of other divalent cations as well, including Cd+2, Zn+2, Co+2, Ni+2, Cu+2 and Pb+2. One of the two forms of DMT1, the form containing the iron-response element (+IRE) in the 3?-noncoding region of the message, is negatively regulated by iron status such that if iron levels are low, DMT1 expression is elevated resulting in increased transport and the potential for enhanced metal toxicity. The other form (-IRE) presumably is not regulated in this fashion. Recent studies have demonstrated that the two isoforms of DMT1 (+/-IRE) are distributed in different subcellular compartments with the -IRE species selectively present in the nucleus of neuronal and neuronal-like cells. However, the function and structure of this nuclear-based form of DMT1 have not been determined. Accordingly, we propose to examine the function of both the +IRE and -IRE forms of DMT1 and their contribution to the cellular uptake and subcellular distribution of Mn and their role in supporting heavy metal neurotoxicity. We have a unique ability to study the function of DMT1 in regulating Mn and other heavy metal toxicity since Dr. Michael Garrick, a coinvestigator in this grant, has an animal model, the Belgrade rat, with a mutation in DMT1 making it functionally inactive. Accordingly, the following studies are proposed: 1) determine the contribution of the +/-IRE forms of DMT1 in supporting iron and Mn transport and toxicity, 2) characterize factors regulating expression of +/-IRE forms of DMT1, 3) determine the structural features promoting nuclear localization of the -IRE form of DMT1, 4) determine the function of nuclear DMT1 and its contribution to Mn-induced toxicity, and 5) localization of the +/-IRE isoforms in rat brain.
