Lista bibliografica

Questa lista bibliografica contiene il secondo aggiornamento della bibliografia della Lisy e deriva dall’assemblaggio da parte della redazione (M. Serdoz ed E. Panfili) dei contributi parziali stilati dai gruppi regionali

Iron Metabolism in Parkinson's Disease: DMT1 (-IRE) and TfR2 are regulated by microRNAs

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

Regulation of divalent metal transporter 1 (DMT1) non-IRE isoform by the microRNA Let-7d in erythroid cells

BACKGROUND: Divalent metal transporter 1 (DMT1) is a widely expressed metal-iron transporter gene encoding four variant mRNA transcripts, differing for alternative promoter at 5' (DMT1 1A and 1B) and alternative splicing at 3' UTR, differing by a specific sequence either containing or lacking an iron regulatory element (+IRE and -IRE, respectively). DMT1-IRE might be the major DMT1 isoform expressed in erythroid cells, although its regulation pathways are still unknown. DESIGN AND METHODS: The microRNA (miRNA) Let-7d (miR-Let-7d) was selected by the analysis of four miRNAs, predicted to target the DMT1-IRE gene in CD34(+) hematopoietic progenitor cells, in K562 and in HEL cells induced to erythroid differentiation. Using a luciferase reporter assay we demonstrated the inhibition of DMT1-IRE by miR-Let-7d in K562 and HEL cells. The function of miR-Let-7d in erythroid cells was evaluated by the flow cytometry analysis of erythroid differentiation markers, by benzidine staining and by iron flame atomic absorption for the evaluation of iron concentration in the endosomes from K562 cells over-expressing miR-Let-7d. RESULTS: We show that in erythroid cells, DMT1-IRE expression is under the regulation of miR-Let-7d. DMT1-IRE and miR-Let-7d are inversely correlated with CD34(+) cells, K562 and HEL cells during erythroid differentiation. Moreover, overexpression of miR-Let-7d decreases the expression of DMT1-IRE at the mRNA and protein levels in K562 and HEL cells. MiR-Let-7d impairs erythroid differentiation of K562 cells by accumulation of iron in the endosomes. CONCLUSIONS: Overall, these data suggest that miR-Let-7d participates in the finely tuned regulation of iron metabolism by targeting DMT1-IRE isoform in erythroid cells

Molecular analysis of 42 patients with congenital dyserythropoietic anemia type II: new mutations in the SEC23B gene and a search for a genotype-phenotype relationship

BACKGROUND: The most frequent form of congenital dyserythropoietic anemia is the type II form. Recently it was shown that the vast majority of patients with congenital dyserythropoietic anemia type II carry mutations in the SEC23B gene. Here we established the molecular basis of 42 cases of congenital dyserythropoietic anemia type II and attempted to define a genotype-phenotype relationship. DESIGN AND METHODS: SEC23B gene sequencing analysis was performed to assess the diversity and incidence of each mutation in 42 patients with congenital dyserythropoietic anemia type II (25 described exclusively in this work), from the Italian and the French Registries, and the relationship of these mutations with the clinical presentation. To this purpose, we divided the patients into two groups: (i) patients with two missense mutations and (ii) patients with one nonsense and one missense mutation. RESULTS: We found 22 mutations of uneven frequency, including seven novel mutations. Compound heterozygosity for a missense and a nonsense mutation tended to produce a more severe clinical presentation, a lower reticulocyte count, a higher serum ferritin level, and, in some cases, more pronounced transfusion needs, than homozygosity or compound heterozygosity for two missense mutations. Homozygosity or compound heterozygosity for two nonsense mutations was never found. CONCLUSIONS: This study allowed us to determine the most frequent mutations in patients with congenital dyserythropoietic anemia type II. Correlations between the mutations and various biological parameters suggested that the association of one missense mutation and one nonsense mutation was significantly more deleterious that the association of two missense mutations. However, there was an overlap between the two categories

Mutational spectrum in congenital dyserythropoietic anemia type II: Identification of 19 novel variants in SEC23B gene.

SEC23B gene encodes an essential component of the coat protein complex II (COPII)-coated vesicles. Mutations in this gene cause the vast majority the congenital dyserythropoietic anemia Type II (CDA II), a rare disorder resulting from impaired erythropoiesis. Here, we investigated 28 CDA II patients from 21 unrelated families enrolled in the CDA II International Registry. Overall, we found 19 novel variants [c.2270 A>C p.H757P; c.214922 A>G; c.110911 G>A; c.387(delG) p.L129LfsX26; c.1858 A>G p.M620V; c.1832 G>C p.R611P; c.1735 T>A p.Y579N; c.1254 T>G p.I418M; c.1015 C>T p.R339X; c.1603 C>T p.R535X; c.1654 C>T p.L552F; c.1307 C>T p.S436L; c.27913 A>G; c. 2150(delC) p.A717VfsX7; c.1733 T>C p.L578P; c.110915 G>A; c.221131 A>G; c.367 C>T p.R123X; c.1857_1859delCAT; p.I619del] in the homozygous or the compound heterozygous state. Homozygosity or compound heterozygosity for two nonsense mutations was never found. In four cases the sequencing analysis has failed to find two mutations. To discuss the putative functional consequences of missense mutations, computational analysis and sequence alignment were performed. Our data underscore the high allelic heterogeneity of CDA II, as the most of SEC23B variations are inherited as private mutations. In this mutation update, we also provided a tool to improve and facilitate the molecular diagnosis of CDA II by defining the frequency of mutations in each exon

Missense mutations in the ABCB6 transporter cause dominant familial pseudohyperkalemia

Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by increased serum [K(+)] in whole blood stored at or below room temperature, without additional hematological abnormalities. Functional gene mapping and sequencing analysis of the candidate genes within the 2q35-q36 critical interval identified-in 20 affected individuals among three multigenerational FP families-two novel heterozygous missense mutations in the ABCB6 gene that cosegregated with disease phenotype. The two genomic substitutions altered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that, in erythrocyte membranes, bears the Langereis blood group antigen system. The ABCB6 R375Q mutation did not alter the levels of mRNA or protein, or protein localization in mature erythrocytes or erythroid precursor cells, but it is predicted to modestly alter protein structure. ABCB6 mRNA and protein levels increase during in vitro erythroid differentiation of CD34(+) erythroid precursors and the erythroleukemia cell lines HEL and K562. These data suggest that the two missense mutations in residue 375 of the ABCB6 polypeptide found in affected individuals of families with chromosome 2-linked FP could contribute to the red cell K(+) leak characteristic of this condition