Brain specific human genes, NELL1 and NELL2, are predominantly expressed in neuroblastoma and other embryonal neuroepithelial tumors.

NELL1 and NELL2 encode cysteine-rich amino acid sequences including six epidermal growth factor-like motifs, which contain signal peptides at the N-terminals. The deduced amino acid sequences of both genes are 55% identical and their cysteine stretch structures are conserved. NELL1 is expressed in the brain and kidney, whereas NELL2 is expressed specifically in the brain. The cell lineage expressing NELLs in the nervous system was investigated in established cell lines and central nervous system tumor tissues obtained from patients by Northern blot and reverse transcriptase-polymerase chain reaction analyses. NELL1 and NELL2 were predominantly expressed in neuroblastoma cell lines and little expressed in glioblastoma cell lines. NELL1 and NELL2 were also expressed in central neurocytoma, medulloblastoma, and some astrocytic tumors. Immunohistochemical analysis revealed that NELL2 protein was localized in the cytoplasm of neurons. These results suggest that NELL2 is predominantly expressed in the neuronal cell lineage in the human nervous system. NELL1 is expressed mainly in tumors in the neuronal cell lineage.

ENH, containing PDZ and LIM domains, heart/skeletal muscle-specific protein, associates with cytoskeletal proteins through the PDZ domain.

The Enigma homologue protein (ENH), containing an N-terminal PDZ domain and three C-terminal LIM domains, is a heart and skeletal muscle-specific protein that has been shown to preferentially interact with protein kinase C beta (PKCbeta) through the LIM domains (Kuroda et al., J. Biol. Chem. 271, 31029-31032, 1996). We here demonstrate that ENH is colocalized with a cytoskeletal protein alpha-actinin in the Z-disk region of rat neonatal cardiomyocytes. Pull-down assays using the glutathione-S-transferase-fusion system also showed the interaction of the PDZ domain of ENH with actin and alpha-actinin. Furthermore, by combined use of the in silico and conventional cDNA cloning methods, we have isolated three ENH-related clones from a mouse heart-derived cDNA library: mENH1 (591 amino acid residues) corresponding to rat ENH, mENH2 (337 residues), and mENH3 (239 residues); the latter two containing only a single PDZ domain. Deciphering their cDNA sequences, these mENH1-3 mRNAs appear to be generated from a single mENH gene by alternative splicing. Northern blot analyses using human cancer cells and mouse embryos have shown expression of each mENH mRNA to vary considerably among the cell types and during the developmental stage. Together with a recent finding that PKCbeta is markedly activated in the cardiac hypertrophic signaling, these results suggest that ENH1 plays an important role in the heart development by scaffolding PKCbeta to the Z-disk region and that ENH2 and ENH3 negatively modulate the scaffolding activity of ENH1.

Immunocytochemical localization of a neuron-specific thrombospondin-1-like protein, NELL2: light and electron microscopic studies in the rat brain.

We have studied the cellular and intracellular localization of NELL2, a neural thrombospondin-1-like protein. NELL2 protein was detected as doublet bands of 140 and 90 kDa with the use of the specific antibodies raised against the C-terminal region of NELL2 and was recognized only in the brain but not in the peripheral tissues. Within the brain, NELL2 was abundantly present in the hippocampus and cerebral cortex, found moderately in the olfactory bulb and hypothalamus, and at a low level in the thalamus, cerebellum, and medulla. Immunocytochemically, NELL2 was seen only in neurons but not in glial cells or in the white matter. NELL2-immunoreactive cells were distributed throughout the brain with the highest density in the hippocampus and cerebral cortex. NELL2 was mainly found in the cell bodies of neurons and the immunoreactivity was often seen as dots in the perikarya. The distribution of NELL2 immunoreactivity did not completely correspond to that of any subtypes of protein kinase C (PKC). Under electron microscopy, NELL2 protein was associated with the endoplasmic reticulum (ER), especially with rough ER. NELL2 immunoreactivity was found in the restricted parts of the ER and found commonly inside the ER. These results suggest that NELL2 protein is synthesized by neurons and may be secreted from the neurons involved in certain neuronal functions.

Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2.

Two closely related genes coding for NELL proteins (NELL1 and NELL2) have been cloned by the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C betaI (PKCbetaI) as bait. The rat NELL proteins show about 55% identity with each other and contain several protein motifs assigned to a secretion signal peptide, an NH(2)-terminal thrombospondin-1 (TSP-1)-like module, five von Willebrand factor C domains, and six epidermal growth factor-like domains; the NELL proteins share many protein motifs with TSP-1. The NELL proteins expressed in COS-7 cells are homotrimeric glycoproteins and possess heparin-binding activity. Furthermore, while NELL1 and NELL2 show distinct subcellular localization in cytoplasm, they both are partially secreted into the culture medium of COS-7 cells. Although the NELL1 mRNA is faintly expressed in adult neural cells, the NELL2 mRNA is expressed abundantly, particularly in the pyramidal cells of rat hippocampus, showing neuronal high plasticity. During mouse embryogenesis, expression of the NELL2 mRNA is initiated 7-11 days postcoitum, simultaneously with neural plate formation. These results strongly suggest that the NELL2 protein, similar to but not identical with TSP-1, is involved in the growth and differentiation of neural cells. Additionally, the NELL1 and NELL2 mRNAs were found to be expressed abundantly in Burkitt's lymphoma Raji cells and colorectal adenocarcinoma SW480 cells, respectively. Thus, it is likely that the NELL proteins also participate in the growth, differentiation, and oncogenesis of cancer cell lines.

Immunohistochemical localization of serotonin transporter in normal and colchicine treated rat brain.

Distribution of serotonin transporter (SET) was examined immunohistochemically in the rat brain using two specific polyclonal antibodies raised against oligopeptides corresponding with 15 amino acids of carboxyl terminus and 14 amino acids of amino terminus of rat SET. The distribution and density of SET immunoreactive varicose fibers were quite similar to those of serotonin immunoreactive fibers, however no neuronal cell bodies in the brainstem raphe nuclei was stained in normal rat brain. Electron microscopic study showed that SET immunoreactivity was predominantly localized in the presynaptic terminals. After intraventricular infusion of colchicine, neuronal perikarya of dorsal, median, and pontine raphe nuclei became visible. These results suggest that SET is likely present at the synaptic terminals of serotonergic neurons and such localization may be in good agreement with its pharmacological action which includes reuptake of serotonin at presynaptic nerve terminals.