Iron Metabolism in Parkinson's Disease: DMT1 (-IRE) and TfR2 are regulated by microRNAs
Asci, Roberta (2012) Iron Metabolism in Parkinson's Disease: DMT1 (-IRE) and TfR2 are regulated by microRNAs. [Tesi di dottorato] (Inedito)
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Parkinson’s Disease is one of the most frequent human neurodegenerations. Motor symptoms of Parkinson’s disease are the consequence of the destruction of nervous cells in the substantia nigra (SN). For many years it has been believed that the degeneration of nervous cells in SN in Parkinson’s disease is related to an important increase in the concentration of iron. This excess of iron, which may initiate Fenton reaction, may be the cause of the oxidative stress leading to the death of nervous cells in PD. However, the mechanisms involved in iron accumulation remain unclear. Transferrin Receptor type 2 (TfR2) is a protein expressed on cell membrane end involved in the cellular iron uptake. It is not ubiquitously expressed like Transferrin Receptor type 1 but it is especially expressed in several tissues such as liver and recently, it has been discovered to be strongly involved in neuronal iron uptake in neurodegenerative disease. This suggests its potential involvement in the iron overload in the Substantia Nigra, a commonly observed phenomenon in Parkinson’s Disease. The mRNA encoding for TfR2 does not contain the Iron Responsive Element (IRE) in its 3’ UTR, so its regulation is surely non mediated by Iron Regulative Proteins (IRPs). Another gene/protein strongly involved in iron metabolism but not regulated by intracellular iron concentrations via IRPs is DMT1 -IRE. It is a metal transporter so it could be involved in PD iron excess. The aim of this thesis was to evaluate the expression of both the previously mentioned gene/proteins in a commonly used in vitro model of PD composed of SH-SY5Y treated with MPP+ (1-methyl-4-phenylpyridinium), since they are not regulated by IRPs, and to answer the question: “Is supposable a regulation microRNA-mediated?”. The expression of DMT1 –IRE and TfR2 was evaluated at both mRNA and protein level in treated cells after 0, 24 and 48 hours from treatment with MPP+. For both genes/proteins an increase in the expression was found in treated cells. MicroRNA-221 was selected among two potential candidate microRNAs, restricted from a list of them resulted by bioinformatics prediction of microRNAs targeting the 3’UTR of TfR2. Using luciferase assay we demonstrated the real ability of microRNA 221 in TfR2 3’UTR binding. The function of microRNA-221 was verified in the chosen PD in vitro model. A similar experimental plan was used to verify the action of microRNA-Let-7d on the regulation of DMT1 –IRE isoform in the same PD cellular model. In this thesis is demonstrated that SH-SY5Y cells treated with MPP+ show an increase in the expression of TfR2 and DMT1 -IRE respect to control cells (after 24 and 48 hours from treatment); at the same time, a significant reduction in the expression of microRNA-221 and of microRNA-Let-7d occurs. The over-expression of microRNA-221 in treated cells causes a decrease in the expression of TfR2, while when cells are transfected with a vector containing microRNA-221 mutated in the binding site for the seed region, this microRNA loses the ability to bind the 3’UTR of TfR2 and its expression is completely restored. On the other hand, the over-expression of microRNA-Let-7d in treated cells causes a decrease in the expression of DMT1 -IRE, while cells transfected with a vector containing a mutated form of microRNA-Let-7d that loses the ability to bind the 3’UTR of TfR2, show a TfR2 expression restored and comparable with the expression in untreated cells. These results suggest that microRNA-221 is involved in the iron metabolism fine regulation, acting specifically on TfR2 expression in the used Parkinson’s Disease in vitro model and that microRNA-Let-7d is able to regulate the expression of DMT1 –IRE, a transporter that could be involved in the iron over-load in PD. Further studies could show the way forward to the therapeutic targeting of this pathway which could be directed to the down-regulation of the expression of both TfR2 and DMT1 -IRE in the dopaminergic neurons of the Substantia Nigra in PD. This findings could be important to obviate the need for the systemic ferrochelant therapy to which patients are often subjected as adjuvant treatment together with the specific therapy for Parkinson’s Disease.
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