Neuron-specific expression of neuroglobin in mammals

Abstract Neuroglobin, a vertebrate oxygen-binding protein, is expressed in many regions of the adult brain. We examined the cell type-specific expression of neuroglobin in neurons and astroglial cells in primary cultures of fetal hippocampal cells and sections of the adult mouse brain using neuroglobin-specific polyclonal antibodies and cell type-specific markers NeuN and GFAP to differentiate between neurons and glial cells. Neuroglobin is exclusively expressed in neurons, but not in astroglial cells. Accordingly, neuroglobin was detected in two neuroblastoma cell lines (N2a, SH-SY5Y) and the pheochromocytoma cell line PC-12, but not in glioblastoma cell lines (DKMG, GAMG) or other, non-neural cells (HeLa, Vero). Analysis of the neuroglobin genomic sequence from man and mouse identifies sequence motifs with similarity to the neuron-restrictive silencer element, possibly explaining a neuron-specific expression of neuroglobin. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Globin; Neuroglobin; Oxygen-binding; Glial cells; Gene expression Neuroglobin (Ngb) is a recently discovered vertebrate heme protein We have recently analyzed the expression pattern of Ngb mRNA and protein within different regions of the murine brain, revealing regionally different Ngb expression with high levels in some brain regions, including the hippocampus, the thalamus and the hypothalamus, the preposital hypoglossal and the lateral vestibular nucleus For primary hippocampal cell culture, eight to ten hippocampi from 17-to 18-day-old rat fetuses (SpragueDawley) were prepared according to Ref. [1]. Cells were plated in a concentration of 2 £ 10 5 /ml on glass coverslips (1.2 cm Ø) precoated with poly-L-lysine (Sigma-Aldrich, Seelze, Germany) and cultured in Dulbecco's modified Eagle's medium/Nut Mix F-12 (Invitrogen), 2 mM Lglutamin (PAA Laboratories, Cölbe, Germany) and penicillin/streptomycin (50 units/ml and 50 mg/ml; PAA Laboratories) with 10% fetal calf serum (FCS) for the first 24 h. This procedure allows a limited growth of astroglial cells. Further cultivation was done in medium plus 2% B-27 Supplement (Invitrogen) for up to 14 days in a humidified atmosphere of 37 8C and 5% CO 2 . Cell lines were cultured in Dulbecco's MEM (Invitrogen) with 2 mM L-glutamin (PAA Laboratories) and penicillin/streptomycin (50 units