Parkinson's disease iron deposition caused by nitric oxide-induced loss of β-amyloid precursor protein

Abstract

Elevation of both neuronal iron and nitric oxide (NO) in the substantia nigra are associated with Parkinson's disease (PD) pathogenesis. We reported previously that the Alzheimer-associated β-amyloid precursor protein (APP) facilitates neuronal iron export. Here we report markedly decreased APP expression in dopaminergic neurons of human PD nigra and that APP(-/-) mice develop iron-dependent nigral cell loss. Conversely, APP-overexpressing mice are protected in the MPTP PD model. NO suppresses APP translation in mouse MPTP models, explaining how elevated NO causes iron-dependent neurodegeneration in PD.

Keywords: APP; iron; nitric oxide.

Figure 1.

Decreased APP expression in PD nigra. A–G, Assay data from postmortem brains of controls and PD patients. A, B, F, G, Homogenized human brain for biochemistry. C–E, Sectioned human SN for LA-ICPMS. A, Western blot for APP levels in SN. B, Iron and APP levels in SN. C, Representative sample area of human SN used for LA-ICPMS analysis. D, Representative LA-ICPMS images of APP (green) and TH (red) content in control and PD sections. Scale bar, 100 μm. E, LA-ICPMS quantification of APP and iron content in TH⁺ pixels in sectioned SN of control and PD subjects. F, Western blot for APP levels in the cortex. G, Iron and APP levels in the cortex. H, Nigral APP content in controls and mice administered l-DOPA for 5 months (n = 4 each). I, SH-SY5Y cells, transfected with luciferase reporter for APP (IRE-containing) UTR (Rogers et al., 2002), were treated with and without MPP⁺ (0–200 μm) for 24 h and then assayed for luminescence (reporting APP translation). Independent experiments indicated in the columns. J, Toxicity assay [cell counting kit (CCK)] performed in parallel to I. K, C57BL/6 mice were injected with MPTP (40 mg/kg) and killed at varying intervals indicated (n = 10 each). SN was microdissected and analyzed for iron content and APP levels. Data are means ± SE. *p < 0.05, **p < 0.01, ***p < 0.001 compared with untreated/WT controls. n is shown in the graph columns.

Figure 2.

APP protects against nigral neuron loss. A, Iron content in the cortex (Ctx), hippocampus (HC), and SN from WT and APP^−/− mice. B, Dopamine (DA) content in the adrenal gland (AG), brainstem (BS), and caudate–putamen (CPu) from WT and APP^−/− mice. C–E, Reduced motor performance of APP^−/− mice in open-field measures of distance moved (C), velocity (D), and time in movement (E). SNc, Substantia nigra pars compacta. F, Impaired cognitive performance of APP^−/− mice in the Y-maze. H, Improved pole test performance in APP^−/− mice. J–L, WT and APP^−/− mice were aged to 6 months before analysis. A subgroup of APP^−/− mice were treated with DFP from 3 months of age. SN neuron number was estimated by stereology, with representative images (scale bar, 250 μm) shown in I–K and counts displayed in L. Cont, Control. M, Nigral neuron counts of APP overexpressing (Tg2576) and WT mice treated with and without MPTP and killed at 21 d. Data are means ± SE. *p < 0.05, **p < 0.01, ***p < 0.001 compared with untreated, WT controls. ^p < 0.05, ^^^p < 0.001 as indicated. n is shown in the graph columns.

Figure 3.

NO lowers APP translation. A, Pearson's correlation between nigral iron and APP content in control and PD brains. B, Biotinylated APP mRNA and APPmut mRNA was used to pull down IRP1 from SY5Y cell homogenate. IRP1 binding was increased in the presence of the NO donor DETA-NONOate. C, Iron and APP levels in M17 control cells and cells treated for 24 h with the NO donor DETA-NONOate (100 μm). D, Iron and APP levels in SN of control mice and mice treated with 7-NI over 21 d. E, SH-SY5Y cells, transfected with luciferase reporter for APP (IRE-containing) UTR (Rogers et al., 2002), were treated with or without MPP⁺ (100 μm) and with or without l-NAME (100 μm) for 24 h and then assayed for luminescence (reporting APP translation). Independent experiments indicated in the columns. F, WT mice treated with and without MPTP were compared with mice cotreated with 7-NI over 21 d. Nigral APP levels in SN from different treatment groups were measured by Western blot. Data are means ± SE. *p < 0.05, ***p < 0.001 compared with untreated/WT controls. ^p < 0.05, ^^^p < 0.001 as indicated. n is shown in the graph columns.

Figure 4.

Inhibition of APP translation by NO causes iron elevation in PD. A, B, WT and APP knock-out mice treated with and without MPTP were compared with mice cotreated with 7-NI. A, Nigral iron content. B, Nigral neuron count (stereology). C, SN neuron counts of WT mice administered MPTP and then treated with or without selegiline. D, SN of control and PD patients were assayed for TfR1, DMT1 + IRE, and DMT1 − IRE. E, F, Pearson's correlations between nigral iron and DMT1 + IRE (E) and TfR1 (F) in control and PD brains. G, Model: Elevated NO in PD depresses APP translation by promoting IRP binding to the IRE on 5′UTR. Iron accumulates as a result of reduced APP-mediated iron export. Drugs that inhibit NO elevation (7-NI/l-NAME) or remove excess iron (DFP) confer neuroprotection. Data are means ± SE. *p < 0.05, ***p < 0.001 compared with untreated/WT controls. ^p < 0.05, ^^^p < 0.001 as indicated. n is shown in the graph columns.