Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31–246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate α- and β-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3′ region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3′ splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.
Endothelins regulate cellular functions in the mammalian brain through the endothelin receptors A and B (EDNRA and EDNRB). In this study, we investigated the role of EDNRB on cell proliferation in the cerebellum by using the spotting lethal (sl) rat, which carries a naturally occurring deletion in the EDNRB gene. Proliferating cells in the three genotypes, wild-type (+/+), heterozygous (+/sl) and homozygous mutant (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) at postnatal day 2. The density of BrdU-positive cells (per mm(2)) in the external germinal layer of sl/sl rats (Mean +/- SEM, 977 +/- 388) was significantly reduced compared to +/+ (4915 +/- 631) and +/sl (2304 +/- 557) rats. Subsequently, we examined the effects of EDNRB mutation on neural apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the cerebella of sl/sl rats (9.3 +/- 0.5/mm(2)) was significantly more increased than +/+ rats (4 +/- 0.7). The expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were measured with standard ELISA, but were unchanged in all genotypes. These results suggest that ENDRB mediates neural proliferation and have anti-apoptotic effects in the cerebellum of the postnatal rat, and that these effects are independent of changes in the expression of BDNF and GDNF. Our findings will lead to better understanding of the morphological changes in the cerebellum of Hirschsprung's disease patients with congenital EDNRB mutation.
Astrocytes can function as antigen-presenting cells (APC) upon expression of class II major histocompatibility complex (MHC) antigens, which are induced by interferon-gamma (IFN-gamma). Previous data from this laboratory had shown that the cytokine tumor necrosis factor-alpha (TNF-alpha) enhances IFN-gamma-mediated class II antigen expression on astrocytes. We have now investigated the effect of IFN-gamma and TNF-alpha on class II MHC mRNA expression in astrocytes using Northern blot analysis. Astrocytes do not constitutively express mRNA for class II MHC. Kinetic analysis of class II MHC mRNA expression in IFN-gamma-treated cells demonstrated an 8 h time lag, which was followed by an increase over the next 16 h. Optimal expression of class II mRNA was detected after a 24 h incubation with IFN-gamma. This level of expression was further enhanced by the simultaneous addition of IFN-gamma and TNF-alpha to the astrocytes, while TNF-alpha alone had no effect on class II mRNA expression. TNF-alpha does not act by increasing the stability of IFN-gamma-induced class II mRNA, indicating its action is not at that specific level of post-transcriptional control. Furthermore, astrocyte class II mRNA expression was inhibited when cycloheximide (CHX) was added together with IFN-gamma or IFN-gamma/TNF-alpha, and when CHX was added up to 4 h after treatment with IFN-gamma or IFN-gamma/TNF-alpha. These results indicate that astrocyte class II mRNA expression is mediated by newly synthesized proteins induced by IFN-gamma and/or IFN-gamma/TNF-alpha. The expression of class II antigens on astrocytes, and cytokine modulation of their expression, may be important in the initiation and perpetuation of intracerebral immune responses.
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