Sigrid Reinhardt

A vertebrate globin expressed in the brain.

Haemoglobins and myoglobins constitute related protein families that function in oxygen transport and storage in humans and other vertebrates. Here we report the identification of a third globin type in man and mouse. This protein is predominantly expressed in the brain, and therefore we have called it neuroglobin. Mouse neuroglobin is a monomer with a high oxygen affinity (half saturation pressure, P50 ≈ 2 torr). Analogous to myoglobin, neuroglobin may increase the availability of oxygen to brain tissue. The human neuroglobin gene (NGB), located on chromosome 14q24, has a unique exon–intron structure. Neuroglobin represents a distinct protein family that diverged early in metazoan evolution, probably before the Protostomia/Deuterostomia split.

Nicotinic Receptor Function in the Mammalian Central Nervous Systema

The diversity of neuronal nicotinic receptors (nAChRs) in addition to their possible involvement in such pathological conditions as Alzheimers disease have directed our research towards the characterization of these receptors in various mammalian brain areas. Our studies have relied on electrophysiological, biochemical, and immunofluorescent techniques applied to cultured and acutely dissociated hippocampal neurons, and have been aimed at identifying the various subtypes of nAChRs expressed in the mammalian central nervous system (CNS), at defining the mechanisms by which CNS nAChR activity is modulated, and at determining the ion permeability of CNS nAChR channels. Our findings can be summarized as follows: (1) hippocampal neurons express at least three subtypes of CNS nAChRs--an alpha 7-subunit-bearing nAChR that subserves fast-inactivating, alpha-BGT-sensitive currents, which are referred to as type IA, and alpha 4 beta 2 nAChR that subserves slowly inactivating, dihydro-beta-erythroidine-sensitive currents, which are referred to as type II, and an alpha 3 beta 4 nAChR that subserves slowly inactivating, mecamylamine-sensitive currents, which are referred to as type III; (2) nicotinic agonists can activate a single type of nicotinic current in olfactory bulb neurons, that is, type IA currents; (3) alpha 7-subunit-bearing nAChR channels in the hippocampus have a brief lifetime, a high conductance, and a high Ca2+ permeability; (4) the peak amplitude of type IA currents tends to rundown with time, and this rundown can be prevented by the presence of ATP-regenerating compounds (particularly phosphocreatine) in the internal solution; (5) rectification of type IA currents is dependent on the presence of Mg2+ in the internal solution; and (6) there is an ACh-insensitive site on neuronal and nonneuronal nAChRs through which the receptor channel can be activated. These findings lay the groundwork for a better understanding of the physiological role of these receptors in synaptic transmission in the CNS.

Selective Adhesion of Cells from Different Telencephalic Regions

We asked whether specifications of different regions of the rodent and avian telencephalon during development involved the acquisition of differential adhesive properties. Cells from different regions were aggregated in a short-term aggregation assay, and their segregation was analyzed. Both neurons and precursor cells from cortex segregate from striatal cells at early, but not later, stages, whereas cells from rodent neocortex and hippocampus segregated only during later stages. Segregation was abolished when Ca2+-dependent but not Ca2+-independent adhesion molecules were selectively removed. Thus, selective adhesion appears to be a conserved mechanism that restricts cellular mixing and might serve to maintain positional information during forebrain development. A candidate for mediating the Ca2+-dependent segregation is the CD15 (Lewis(x)) carbohydrate epitope, which is selectively expressed by mammalian cortex but not striatum.

Neuronal nicotinic receptors in the locust Locusta migratoria. Cloning and expression.

We have identified five cDNA clones that encode nicotinic acetylcholine receptor (nAChR) subunits expressed in the nervous system of the locust Locusta migratoria. Four of the subunits are ligand-binding α subunits, and the other is a structural β subunit. The existence of at least one more nAChR gene, probably encoding a β subunit, is indicated. Based on Northern analysis and in situ hybridization, the five subunit genes are expressed. locα1, locα3, andlocβ1 are the most abundant subunits and are expressed in similar areas of the head ganglia and retina of the adult locust. Because Locα3 binds α-bungarotoxin with high affinity, it may form a homomeric nAChR subtype such as the mammalian α7 nAChR. Locα1 and Locβ1 may then form the predominant heteromeric nAChR in the locust brain. locα4 is mainly expressed in optic lobe ganglionic cells and locα2 in peripherally located somata of mushroom body neurons. locα3 mRNA was additionally detected in cells interspersed in the somatogastric epithelium of the locust embryo, suggesting that this isoform may also be involved in functions other than neuronal excitability. Transcription of all nAChR subunit genes begins approximately 3 days before hatching and continues throughout adult life. Electrophysiological recordings from head ganglionic neurons also indicate the existence of more than one functionally distinct nAChR subtype. Our results suggest the existence of several nAChR subtypes, at least some of them heteromeric, in this insect species.

Expression of functional α7 nicotinic acetylcholine receptor during mammalian muscle development and denervation

We have studied, on the transcriptional, protein and functional level, the expression of α7 nicotinic acetylcholine receptors (nAChR) in the course of rat muscle development, denervation and renervation. At foetal day 13, α7 nAChR expression was observed in somites and developing muscles of the back, but not yet in migrating myoblasts. Two days later, concomitant with myoblast aggregation, the α7 isoform began to be expressed in isolated myoblasts, with the highest level of expression in the frontal zone of the migrating wave. On foetal day 18, a time when the myoblasts in the upper hindleg have fused, α7 nAChR expression was most prominent in the outer layer of muscle tissue. The highest level of expression was observed in the first postnatal week, when practically all muscle cells stained positively for α7 protein. During the following weeks, α7 nAChR expression slowly decreased and practically disappeared in adult hindleg muscle. Following chronic denervation of adult Soleus muscle fibres, expression of α7 nAChR returned within 2–4 weeks.

The murine nuclear orphan receptor GCNF is expressed in the XY body of primary spermatocytes

We have studied the expression of the nuclear orphan receptor GCNF (germ cell nuclear factor) on the mRNA and protein level in pubertal and adult mouse testes. We show by Northern and Western blot analyses and by in situ hybridization that GCNF is expressed in spermatocytes and round spermatids of adult mouse testis suggesting that GCNF may be a transcriptional regulator of spermatogenesis. Since the GCNF protein is accumulated in the XY body of late pachytene spermatocytes, it may be involved in transcriptional inactivation of sex chromosomes.

The protein kinase C (PKC) substrate GAP-43 is already expressed in neural precursor cells, colocalizes with PKCη and binds calmodulin

Expression of the growth‐associated protein of 43‐kDa (GAP‐43), which is described as a postmitotic, neuron‐specific major protein kinase C (PKC) substrate, was investigated in the murine embryonic carcinoma cell line PCC7‐Mz1 which develops into a brain‐tissue‐like pattern of neuronal, fibroblast‐like and astroglial cells upon stimulation with all‐trans retinoic acid (RA). GAP‐43 expression was very low in stem cells, but increased on mRNA and protein level within the 12 h after differentiation was initiated. While the P1 promoter of the GAP‐43 gene gave rise to a 1.6‐kb mRNA and was already active at a very low level in PCC7‐Mz1 stem cells, transcription of the P2 promoter, which resulted in a 1.4‐kb mRNA, was completely blocked in stem cells but increased rapidly after RA treatment.

EX-1, a surface antigen of mouse neuronal progenitor cells and mature neurons

Using membrane fragments of PCC7-Mz1 embryonal carcinoma cells, an established in vitro model of neural differentiation (Lang et al., J. Cell Biol., 109 (1989) 2481-2493), we have raised monoclonal antibodies (mAb) against developmental stage-specific cell surface antigens. As shown by double-immunofluorescence labeling studies, employing differentiating PCC7-Mz1 cells, primary cultures of mouse cerebellum cells and cryosections of mouse brain and other tissues, rat mAb anti-mouse EX-1 recognizes a membrane protein which is exclusively expressed by cells of the neuronal cell lineage. EX-1-expressing neuronal precursor cells were identified by double labeling with antibodies directed against stem cell markers or BrdU, EX-1-expressing postmitotic neurons by labeling with antibodies directed against phenotypic markers. In the developing mouse brain, the EX-1 antigen is expressed in the neuroepithelium already at prenatal day 8, i.e. clearly before the onset of mature neuron-specific marker expression. Increasing co-expression with the latter is observed from embryonic day E10 throughout neuronal maturation, but not with markers of other cell types tested. From these studies, the EX-1 antigen is the earliest marker for the mouse brain neuronal cell lineage so far discovered.

Analysis of NO synthase expression in neuronal, astroglial and fibroblast-like derivatives differen-tiating from PCC7-Mzl embryonic carcinoma cells

We studied the expression of the NO synthase isoforms in an in vitro model of neural development using RT-PCR, Western blot and immunohistochemistry. Murine PCC7-Mz1 cells (Jostock et al., Eur. J. Cell Biol. 76, 63-76, 1998) differentiate in the presence of all-trans retinoic acid and dibutyryl cAMP along the neural pathway into neuron-like, fibroblast-like and astroglia-like cells. Undifferentiated cells showed immunofluorescent staining for neuronal-type NOS I and endothelial-type NOS III. This expression pattern was retained in those cells differentiating into neurofilament- and tau protein-positive neuronal cells. Thymocyte alloantigen (Thy1.2/CD 90.2)-positive fibroblasts, appearing around day 3, and glial fibrillary acidic protein (GFAP)-positive astroglial cells, appearing after day 6 of differentiation, stained negative for any NOS isoform. Starting at day 6 of differentiation, expression of inducible-type NOS II could be stimulated with cytokines in a subset of cells, which may represent activated astrocytes. NOS II was always undetectable in non-induced cultures. These data indicate that the ability of stem cells to express NOS I and NOS III is only retained when the cells differentiate along the neuronal lineage, while a small subpopulation of cells acquires the ability to express NOS II in response to cytokines.