The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 ...neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson's disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for α- and β-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing α- and β-globin chains, changes in genes involved in Oâ homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases.
The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 ...neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson's disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for alpha- and beta-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing alpha- and beta-globin chains, changes in genes involved in O(2) homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases.
: To develop new treatments for β‐thalassemia, it is essential to identify the genes involved in the relevant pathophysiological processes. Iron metabolism in thalassemia mice being investigated, ...focusing on the expression of a gene called hepcidin (Hamp), which is expressed in the liver and whose product (Hamp) is secreted into the bloodstream. In mice, iron overload leads to overexpression of Hamp, while Hamp‐knockout mice suffer from hemochromatosis. The aim of this study is to investigate Hamp in the mouse model of β‐thalassemia and to address the potential gene transfer of Hamp to prevent abnormal iron absorption.
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Background/Aims: HFE mutations are associated with over 80% of cases of Hereditary Hemochromatosis (HH), an iron‐overload disease in which the liver is the most frequently affected ...organ. Research on HFE has traditionally focused on its interaction with the transferrin receptor. Recent studies suggested a more complex function for this non‐classical MHC‐I protein. The aim of this study was to examine how HFE and its two most common mutations affect the expression of selected genes in a hepatocyte‐like cell line.
Methods: Gene expression was analyzed in HepG2 cells over‐expressing wild‐type and mutant HFE. The effect of HFE in iron import and oxidative stress levels was assessed. Unfolded protein response (UPR)‐activated gene expression was analyzed in peripheral blood mononuclear cells from HH patients.
Results: C282Y‐HFE down‐regulated hepcidin and enhanced calreticulin mRNA expression. Calreticulin levels correlated with intracellular iron increase and were associated with protection from oxidative stress. In C282Y
+/+
patients calreticulin mRNA levels correlated with the expression of the UPR marker BiP and showed a negative association with the number of HH clinical manifestations.
Conclusions: The data show that expression of C282Y‐HFE triggers a stress‐protective response and suggest a role for calreticulin as a modifier of the clinical expression of HH.
Studies done in non-hepatic cell lines, focusing on the interaction between HFE with TFR1 and β-2M proved insufficient to explain the discrepancies found in the clinical penetrance of hemochromatosis ...in subjects carrying the C282Y mutation. Our first goal was to investigate the role of HFE wild type (wt) and mutant proteins (C282Y and H63D) in a human hepatic cell line, focusing on the cellular localization and interaction of HFE with the expression of other iron related proteins. HFE mutant C282Y was found to be retained in the endoplasmic reticulum (ER). Thus, in addition, we investigated the effect of HFE wt and mutant proteins on Calreticulin, which is a chaperon protein that responds to ER stress and has a protective effect on oxidative damage in some cell lines. Here we report setting up a stable transfection of wt- and mutant-HFE in a hepatic cell line (HepG2) and examine the intracellular distribution of wt- and HFE mutants, their effect on iron intake independently of TFR1 and on the expression of other iron and ER stress response genes, namely Hepcidin and Calreticulin. In addition, we validated some of the novel effects of HFE on Calreticulin using peripheral blood mononuclear cells from HFE patients. The localization of the HFE variants was analyzed using KDEL and Golgin-97 as ER and the Golgi complex markers, respectively. HFE C282Y shows a high degree of overlap with the ER markers, confirming a retention of this variant in this organelle. Over-expression of the HFE wt impaired the intake of 55Fe relatively to transfected control cells (P<0.008) independently of TFR1, as demonstrated by RNAi silencing. Hamp RNA expression was decreased in cells over expressing C282Y in comparison to HFE wt cells (P<0.011). Finally over-expression of HFE wt decreases Calreticulin mRNA, whereas the C282Y had an opposite effect, compared to the control cell line. A similar result was observed in peripheral blood mononuclear cells (PMBC) of C282Y homozygous HFE patients, compared to wild type blood donors (P<0.006). Interestingly, this data suggest that synthesis of the HFE mutant C282Y triggers a protective effect on oxidative damage mediated by Calreticulin. In fact, HepG2 cells over-expressing C282Y showed lower levels of ROS than HFE wt (P<0.004). This observation might contribute to explain some of the discrepancies seen in the clinical penetrance of the disease in C282Y carrying subjects. The direct effect of the mutant HFE C282Y on mRNA expression of hepcidin also demonstrated here for the first time corroborates and provides a molecular basis for earlier reports of low hepcidin levels in HH patients and in Hfe-KO mice.
The β-thalassemias and sickle cell disease are severe congenital anemias that are caused by mutations that alter the production of the β chain of hemoglobin. Allogeneic hematopoietic stem cell (HSC) ...transplantation is curative, but this therapeutic option is not available to the majority of patients. The transfer of a functional globin gene in autologous HCSs thus represents a highly attractive alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling the expression of the globin transgene, which ideally should be erythroid specific, differentiation-stage restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, we have demonstrated that an optimised combination of proximal and distal transcriptional control elements permits lineage-specific, elevated expression of the β-globin gene, resulting in therapeutic hemoglobin production and correction of anemia in β-thalassemic mice. Several groups have now confirmed and extended these findings in various mouse models of severe hemoglobinopathies, thus generating enthusiasm for a genetic treatment based on globin gene transfer. Furthermore, globin vectors represent a general paradigm for the regulation of transgene function and the improvement of vector safety by restricting transgene expression to the differentiated progeny within a single lineage, thereby reducing the risk of activating oncogenes in hematopoietic progenitors. Here we review the principles underlying the genesis of regulated vectors for stem cell therapy
Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30% of inherited ...diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in β
0
39‐thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well‐described NMD (nonsense‐mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon–anticodon base‐pairing, inducing a ribosomal read‐through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read‐through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of β
0
39‐thalassaemia. In this context, we started the development of a cellular model of the β
0
39‐thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the β
0
39‐thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of β‐globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the β
0
39‐globin mutation causing β‐thalassaemia.
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