Research ReportChronic expression of H-ferritin in dopaminergic midbrain neurons results in an age-related expansion of the labile iron pool and subsequent neurodegeneration: implications for Parkinson's disease
Introduction
Iron is essential for life due to its requirement in several basic cellular functions. Conversely, iron is potentially toxic since it can participate in redox reactions that lead to generation of reactive oxygen species (ROS). Cells therefore strive to maintain a transient pool of readily available iron, known as the labile iron pool (LIP), within a narrow range, providing enough iron for cellular function while limiting any excess that can participate in toxic reactions (Eisenstein, 2000, Schneider and Leibold, 2000). Excess intracellular iron not utilized for metabolic purposes is normally stored in the cytosol within the iron-binding protein ferritin. Fully assembled ferritin consists of 24 subunits of H- and L-subunits that form nanocages capable of storing up to 4500 molecules of iron. The H-subunit harbors ferroxidase activity used to catalyse oxidation of ferrous to ferric iron for entry into the ferritin cavity. The L-subunit catalyzes ferrihydrite formation promoting iron storage. Thus, ferritin stores iron in a nontoxic, bioavailable form (Arosio and Levi, 2002). Iron may be mobilized from ferritin by oxidative stress, following localized protein unfolding, or ferritin degradation within lysosomes. Ferritin itself can also undergo degradation by the proteasome following either iron depletion or ferritin oxidation (Galaris and Pantopoulos, 2008).
In Parkinson's disease (PD), iron levels in the SN, the brain region that undergoes preferential neurodegeneration, are reported to be elevated (Riederer, 1992, Youdim et al., 1993, Gerlach, 1997, Bush,, 2000, Schenck and Zimmerman, 2004). This appears to occur within the dopaminergic neurons that are preferentially affected in this condition (Carmona, 2008, Oakley, 2007, Ortega, 2007). Iron can interact with hydrogen peroxide, a major by-product of dopamine oxidation, leading to production of highly reactive hydroxyl radicals via the Fenton reaction (Comporti, 2002). This in turn may contribute to subsequent dopaminergic neurodegeneration.
Previously, we created transgenic mouse lines in which ferritin levels were selectively elevated within DA SN neurons resulting in an increase in ferritin-bound iron within these cells (Kaur et al., 2003). In young animals, this was found to afford protection against neurodegeneration associated with two widely used animal models of the disease, systemic 1methyl-4-phenyl 2,3,6 tetrahydropyridine (MPTP) (Kaur et al., 2003) and paraquat administration (McCormack et al., 2005). This suggested a direct involvement of iron-catalyzed oxidative stress in subsequent neurodegenerative events associated with these agents. However, prolonged ferritin elevation within the dopaminergic SN neurons was found to lead to a selective age-related neurodegeneration of these cells and to exacerbate MPTP neurotoxicity in older animals (Kaur et al., 2007). Age-related neurodegeneration has also been described to occur in iron regulatory protein 2-deficient (IRP2−/−) mice; this neurodegeneration has been suggested to be attributable to increased ferritin levels within degenerating neuronal populations in these animals (LaVaute et al., 2001). IRP1+/−, IRP2−/− mice were found to have an even more severe age-related neurodegeneration accompanied by still higher levels of ferritin accumulation (Smith et al., 2004). It has been speculated that iron maintained primarily in a ferritin-bound state may result in a LIP deficiency which could impact on cellular function by reducing the amount of easily available iron needed for the synthesis of important iron-sulfur containing enzymes including those of the mitochondria (Rouault, 2006). In this study, we assessed whether age-related dopaminergic SN neurodegeneration in our transgenic ferritin mice was due to subsequent deficiencies in the LIP in the affected cells as a consequence of prolonged ferritin elevation.
Section snippets
Results
In order to assess whether iron availability in our ferritin transgenic mice results in subsequent age-related neurodegeneration, we examined the LIP within DA SN neurons from ferritin-expressing transgenics versus wildtype mice with increasing age in isolated dopaminergic striatal synaptosomes utilizing a novel magnetic bead isolation protocol recently reported by our laboratory (Chinta, 2007, Mallajosyula, 2008). This method allows selective isolation of terminals emanating from dopaminergic
Discussion
In this present study, we demonstrate that while DA LIP levels are reduced in younger ferritin transgenics (verifying previously published results, Kaur et al., 2003), they become elevated with age. Iron within the young ferritin transgenic brains may be kept in a more non-reactive form due to a relatively high ferritin-to-iron ratio compared to age-matched controls however, with age, increasing brain iron levels may lead to increases in ferritin-bound iron levels (Kaur et al., 2007). It has
Animals
Construction and characterization of the ferritin transgenics used in this study have been previously described (Kaur et al., 2003). Animals were bred in-house and housed according to standard animal care protocols, fed Harlan Teklad 7912 irradiated chow ad libitum, kept on a 12-h light/dark cycle, and maintained in a pathogen-free environment in the Buck Institute Vivarium. All animal experiments were approved by local IACUC review and conducted according to current NIH policies on the use of
Acknowledgment
This work was funded by National Institutes of Health (NIH) R01 NS041264 (JKA).
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Authors equally contributed to this study.