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.

Figure 1

Hemoglobin α- and β-chain mRNA expression. A: Arrangements of genes in the rat α- and β-globin gene cluster (not to scale) with the respective ideogram. Adult α- and β-globin-like gene products assemble to α2β2 hemoglobin in erythroid cells. Positions were taken from the MapViewer (RGSC v3.4) at The National Center for Biotechnology Information (NCBI) website. B: mRNA levels (qPCR) of α- and β-globin gene expression in dopaminergic neurons of the SNC, cortical pyramidal neurons, and striatal projection neurons. Con and rot indicate samples from control (n = 6) and rotenone-treated (n = 6) animals, respectively. Data are means ± SEM (*, P < 0.01; **, P < 0.001). C: In situ hybridization immunohistochemistry with DIG-labeled RNA probes shows intense blue staining for Hba-a2 (a–c) and Hbb (d–f) in the cortex (Cx; a,d), dentate gyrus of the hippocampus (DG; b,e), and substantia nigra pars compacta (SNC; c,f). Cells containing hemoglobin β-chain mRNA (blue) and the neuronal marker NeuN (brown nuclei) in the (g) cortex, (h) dentate gyrus, and (i) SNC. Scale bar = 50 μm in a–i (insets are 2.5-fold magnifications).

Figure 2

Hemoglobin α-or β-chains on 40-μm rat brain sections (A–F) and 4-μm human brain sections (G–L). Hemoglobin α-chains (Hb α; A,D,G,J) and hemoglobin β-chains (Hb β; B,E,H,K) are present in neurons, as shown in the rat and human (hu) cortex (Cx), the rat substantia nigra pars compacta/substantia nigra pars reticulata (SNC/R), and the human dentate gyrus of the hippocampus (DG), visible as brown staining. C, F, I, and L show negative controls with blocked primary antibodies, IgG control, or secondary antibody only. Scale bar = 50 μm in A–L; 10 μmin insets to A,B,D,E,G,H,J,K.

Figure 3

Western blot with extracted protein from rat blood (0.25 μg and 8 ng), brain (50 μg), and primary midbrain cultures enriched for neurons (“Neuron” 5 μg) probed with whole hemoglobin antiserum. A strong band at 16 kDa is present in all lanes, which is indicative of the presence of the hemoglobin chains monomers. Rat blood shows additional bands at 32 kDa (dimer) and 64 kDa (tetramer) with higher protein concentrations. Comparable-sized bands also become visible in the brain sample after overexposure of the blot.