The Role of Iron in the Pathogenesis of Parkinsonism in the Drosophila Model

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. It is characterized by the progressive degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc). Although the etiology of PD remains incompletely understood, emerging evidence suggests that iron homeostasis dysregulation may be involved. A pathological hallmark of PD is the formation of Lewy bodies, intra-cytoplasmic inclusions that are major composed of alpha-synuclein (alpha-syn). alpha-synuclein is encoded by the SNCA gene. It is generally believed that alpha-synuclein aggregation is a main pathogenic feature and the cause of PD. Previous in vitro studies have provided direct evidence showing that iron could interact with alpha-synuclein and facilitate its aggregation. Nevertheless, the exact role of iron in the pathogenesis of PD is still inconclusive, and so far no studies have proved the interaction between iron and alpha-synuclein in vivo..; Here, based on a Drosophila model, we tested the hypothesis that the interaction between iron and alpha-synuclein accumulation accelerates the pathogenesis of PD, and that restoring brain iron homeostasis provides neuroprotective effects against PD. In our present studies, two groups of Drosophila, including w1118 control and mutant alpha-synuclein A53T Drosophila were cultured under normal- (normal medium) and high-iron diet (medium added with 30mM ferric ammonium citrate (FAC)) for up to 30 days. During chronic iron treatment, startle-induced negative geotaxis assay was conducted every ten days to test the locomotor ability in the flies. After that, whole-mount immunostaining was used to assess dopaminergic neuronal survival. These flies were also collected and subjected to the quantification of brain iron content for the characterization of the brain iron content status. Furthermore, quantitative real-time PCR and western-blot analysis were conducted to investigate the amount of various alpha-synuclein conformations.;In the first part, we observed that alpha-synuclein A53T fly exhibited age-related increase of brain iron content compared with age-matched control. These were accompanied by shorter life-span, locomotor dysfunction, and TH-positive neuronal loss in PPM1/2 and PPM3 cluster. Meanwhile, we have demonstrated that neuronal toxicity and motor deficits were associated with increased proteinase K resistant, insoluble alpha-synuclein rather than the total amount of protein level. The insoluble alpha-synuclein was regarded as alpha-synuclein aggregation.;In the second part, we found that in alpha-synuclein A53T fly, excessive iron uptake aggravated locomotion defects and led to specific TH positive neuronal loss in cluster PPM3 after 30 days of iron treatment. Moreover, the excessive iron-induced neurological toxicity and motor dysfunction were also associated with increased alpha-synuclein aggregation. Overall, these two sets of results suggest that abnormal up-regulation of brain iron content may be associated with alpha-synuclein, and contribute to the pathogenesis of PD through alpha-synuclein aggregation-dependent mechanisms.;In the third part, we further explored the potential neuroprotective effect of restoring brain iron homeostasis in PD. We made use of genetic modification to manipulate iron-transport protein DMT1 expression, in turn to identify the protective effect of decreasing brain iron content in alpha-synuclein Drosophila model. Our present results proved that inactivation of Malvolio in alpha-synuclein A53T fly can suppress the increase of brain iron contents, and can also prolong life span, partially ameliorate locomotion deficits, and attenuate TH positive neuronal loss in alpha-synuclein A53T fly. In addition, these beneficial effects might occur through the inhibition of alpha-synuclein aggregation in alpha-synuclein A53T fly. Consequently, this result implicates that reducing brain iron by inactivation of iron up-take protein DMT1 can inhibit alpha-synuclein aggregation and provide beneficial effect on DA neuronal survival in PD model.;In conclusion, we demonstrated that : (1) abnormal up-regulation of brain iron content may be associated with alpha-synuclein and contributes to the pathogenesis of PD through alpha-synuclein aggregation-dependent mechanisms; (2) iron uptake protein DMT1 may serve as a potential therapeutic target for alleviating aberrant iron accumulation and retards the progression of neurodegeneration in PD.