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Neurons express hemoglobin alpha- and beta-chains in rat and human brains.

ABSTRACT: Hemoglobin is the oxygen carrier in vertebrate blood erythrocytes. Here we report that hemoglobin chains are expressed in mammalian brain neurons and are regulated by a mitochondrial toxin. Transcriptome analyses of laser-capture microdissected nigral dopaminergic neurons in rats and striatal neurons in mice revealed the presence of hemoglobin alpha, adult chain 2 (Hba-a2) and hemoglobin beta (Hbb) transcripts, whereas other erythroid markers were not detected. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed the expression of Hba-a2 and Hbb in nigral dopaminergic neurons, striatal gamma-aminobutyric acid (GABA)ergic neurons, and cortical pyramidal neurons in rats. Combined in situ hybridization histochemistry and immunohistochemistry with the neuronal marker neuronal nuclear antigen (NeuN) in rat brain further confirmed the presence of hemoglobin mRNAs in neurons. Immunohistochemistry identified hemoglobin alpha- and beta-chains in both rat and human brains, and hemoglobin proteins were detected by Western blotting in whole rat brain tissue as well as in cultures of mesencephalic neurons, further excluding the possibility of blood contamination. Systemic administration of the mitochondrial inhibitor rotenone (2 mg/kg/d, 7d, s.c.) induced a marked decrease in Hba-a2 and Hbb but not neuroglobin or cytoglobin mRNA in transcriptome analyses of nigral dopaminergic neurons. Quantitative RT-PCR confirmed the transcriptional downregulation of Hba-a2 and Hbb in nigral, striatal, and cortical neurons. Thus, hemoglobin chains are expressed in neurons and are regulated by treatments that affect mitochondria, opening up the possibility that they may play a novel role in neuronal function and response to injury.

SUBMITTER: Richter F

PROVIDER: S-EPMC3123135 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Neurons express hemoglobin alpha- and beta-chains in rat and human brains.

Richter Franziska F Meurers Bernhard H BH Zhu Chunni C Medvedeva Vera P VP Chesselet Marie-Françoise MF

The Journal of comparative neurology 20090801 5

Hemoglobin is the oxygen carrier in vertebrate blood erythrocytes. Here we report that hemoglobin chains are expressed in mammalian brain neurons and are regulated by a mitochondrial toxin. Transcriptome analyses of laser-capture microdissected nigral dopaminergic neurons in rats and striatal neurons in mice revealed the presence of hemoglobin alpha, adult chain 2 (Hba-a2) and hemoglobin beta (Hbb) transcripts, whereas other erythroid markers were not detected. Quantitative reverse transcriptase ...[more]

PMID: 19479992

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Project description:In an effort to reduce the number of degrees of freedom necessary to describe a polypeptide chain we analyze the statistical behavior of polypeptide chains when represented as C alpha chains, C alpha chains with C beta atoms attached, and C alpha chains with rotational ellipsoids as models of side chains. A statistical analysis on a restricted data set of 75 unrelated protein structures is performed. The comparison of the database distributions with those obtained by model calculation on very short polypeptide stretches allows the dissection of local versus nonlocal features of the distributions. The database distribution of the bend angles of polypeptide chains of pseudo bonded C alpha atoms spans a restricted range of values and shows a bimodal structure. On the other hand, the torsion angles of the C alpha chain may assume almost all possible values. The distribution is bimodal, but with a much broader probability distribution than for bend angles. The C alpha - C beta vectors may be taken as representative of the orientation of the lateral chain, as the direction of the bond is close to the direction of the vector joining C alpha to the ad hoc defined center of the "steric mass" of the side chain. Interestingly, both the bend angle defined by C alpha i-C alpha i+1-C beta i+1 and the torsional angle offset of the pseudo-dihedral C alpha i-C alpha i+1-C alpha i+2-C beta i+2 with respect to C alpha i-C alpha i+1-C alpha i+2-C alpha i+3 span a limited range of values. The latter results show that it is possible to give a more realistic representation of polypeptide chains without introducing additional degrees of freedom, i.e., by just adding to the C alpha chain a C beta with given side-chain properties. However, a more realistic description of side chains may be attained by modeling side chains as rotational ellipsoids that have roughly the same orientation and steric hindrance. To this end, we define the steric mass of an atom as proportional to its van der Waals volume and we calculate the side-chain inertia ellipsoid assuming that the steric mass of each atom is uniformly distributed within its van der Waals volume. Finally, we define the rotational ellipsoid representing the side chain as the uniform density ellipsoid possessing the same rotationally averaged inertia tensor of the side chain. The statistics of ellipsoid parameters support the possibility of representing a side chain via an ellipsoid, independently of the local conformation. To make this description useful for molecular modeling we describe ellipsoid-ellipsoid interactions via a Lennard-Jones potential that preserves the repulsive core of the interacting ellipsoids and takes into account their mutual orientation. Tests are performed for two different forms of the interaction potential on a set of high-resolution protein structures. Results are encouraging, in view of the drastic simplifications that were introduced.

Project description:Hemoglobin (Hb) is a major protein involved in transport of oxygen (O2). It consists of Hb-α and Hb-β subunits, which are normally expressed by cells of erythroid lineage. However, till recently, it was not known whether non-erythroid cells like vaginal cells synthesize Hb and whether it has any functional significance. Therefore, we designed the following objectives: (1) to establish in-vitro culture system of human primary vaginal epithelial cells (hPVECs), (2) to determine whether Hb-α and Hb-β proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-α and Hb-β proteins (4) to decipher the significance of the Hb-α and Hb-β expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-α in hPVECs. To accomplish these studies, we applied a battery of assays such as RT-PCR, qRT-PCR, Flow cytometry, western blot, and immunofluorescence, Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The results revealed the expression of Hb-α and Hb-β at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10μg/ml for 6 hrs) and these results are comparable with the expression induced by LPS in human vaginal epithelial cell line (VK2/E6E7). These cells constitutively produced low levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines. Also, the response of phosphorylated (p65)-NF-κB to LPS was upregulated with increased expression of IL-6, Toll-like receptor-4 (TLR4) and human beta defensin-1 (hBD-1) in hPVECs and VK2/E6E7 cells. However, Bay 11-7082 treatment (5μM for 24 hrs) could neutralize the effect of LPS-induced p65-NF-κB activity and represses the production`of Hb-α and Hb-β. The results of EMSA revealed the presence of putative binding sites of NF-κB in the human Hb-α promoter region (nt-115 to -106). ChIP analysis confirmed the binding of NF-κB to Hb-α promoter. In conclusion, the present findings revealed for the first time that hPVECs synthesized Hb-α and Hb-β and the expression is comparable with the expression of VK2/E6E7 cells. The identification of NF-κB regulatory sequences in Hb-α promoter, whose activation is associated with immune response of hPVECs, indicating Hb-α and Hb-β may act as an endogenous antimicrobial defense protein against vaginal inflammation/infections.

Dataset Information

Neurons express hemoglobin alpha- and beta-chains in rat and human brains.

Publications

Neurons express hemoglobin alpha- and beta-chains in rat and human brains.

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