CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca2+ signalling molecules, but its role in the neuroendocrine system is unknown. Here we show that adult CD38 knockout (CD38-/-) female and male mice show marked defects in maternal nurturing and social behaviour, respectively, with higher locomotor activity. Consistently, the plasma level of oxytocin (OT), but not vasopressin, was strongly decreased in CD38-/- mice. Replacement of OT by subcutaneous injection or lentiviral-vector-mediated delivery of human CD38 in the hypothalamus rescued social memory and maternal care in CD38-/- mice. Depolarization-induced OT secretion and Ca2+ elevation in oxytocinergic neurohypophysial axon terminals were disrupted in CD38-/- mice; this was mimicked by CD38 metabolite antagonists in CD38+/+ mice. These results reveal that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.

34 These authors contributed equally to the work.Key Words: CD38, oxytocin, mutation, polymorphism, autism, high-functioning autism Author information Correspondence and requests for materials should be addressed to H. Higashida (haruhiro@med.kanazawa-u.ac.jp). 3 ABSTRACTThe neurobiological basis of autism spectrum disorder (ASD) remains poorly understood.Given the role of CD38 in social recognition through oxytocin (OT) release, we hypothesized that CD38 may play a role in the etiology of ASD. Here, we first examined the immunohistochemical expression of CD38 in the hypothalamus of post-mortem brains of non-ASD subjects and found that CD38 was colocalized with OT in secretory neurons.In studies of the association between CD38 and autism, we analyzed 10 single nucleotide polymorphisms (SNPs) and mutations of CD38 by re-sequencing DNAs mainly from a case-control study in Japan, and Caucasian cases mainly recruited to the Autism Genetic Resource Exchange (AGRE). The SNPs of CD38, rs6449197 (p<0.040) and rs3796863 (p<0.005) showed significant associations with a subset of ASD (IQ>70; designated as high-functioning autism (HFA)) in the U.S. 104 AGRE family trios, but not with Japanese 188 HFA subjects. A mutation that caused tryptophan to replace arginine at amino acid residue 140 (R140W; (rs1800561, 4693C>T)) was found in 0.6%-4.6% of the Japanese population and was associated with ASD in the smaller case-control study. The SNP was clustered in pedigrees in which the fathers and brothers of T-allele-carrier probands had ASD or ASD traits. In this cohort OT plasma levels were lower in subjects with the T allele than in those without. One proband with the T allele who was taking nasal OT spray showed relief of symptoms. The two variant CD38 poloymorphysms tested may be of interest with regard of the pathophysiology of ASD.4

Cyclic ADP-ribose (cADP-ribose) 1 is synthesized from ␤-NAD ϩ , an abundant intracellular substrate, by ADP-ribosyl cyclase in sea urchin eggs and in mammalian cells (1, 2). Pharmacological studies suggest that cADP-ribose is an endogenous modulator of ryanodine-sensitive Ca 2ϩ release channels (3-10). If cADP-ribose acts as an intracellular second messenger, ADPribosyl cyclase, as an effector enzyme, should be activated or inhibited in response to stimulation by hormones or neurotransmitters, which should simultaneously be associated with a transient decrease in the intracellular NAD ϩ concentration ([NAD ϩ ] i ) and an increase in cADP-ribose concentration (11).ADP-ribosyl cyclase seems to be present in both cytosolic and membrane-bound forms (1, 2, 12). The mammalian membranebound form of ADP-ribosyl cyclase has been identified as a cellsurface antigen, CD38 (13-19) and .Recently, it has been shown that the formation of cADPribose is regulated by nitric oxide or cGMP (21-23) and that nitric oxide or cGMP is increased by stimulation with agonists (24, 25). These findings suggest the hypothesis that the regulation of the cADP-ribose level is located far downstream in the signal transduction cascade from receptors (11). An alternative hypothesis is that the cADP-ribose formation is regulated by ADP-ribosyl cyclase through the direct action of G proteins activated by receptors within the surface membrane, as already shown for the formation of cyclic AMP, inositol 1,4,5-trisphosphate, and diacylglycerol (26 -28). To test this hypothesis, we used NG108-15 neuroblastoma ϫ glioma hybrid cells (29), in which signal transduction from receptors to effectors has been extensively characterized (29,30). In particular, in NGPM1-27 cells (31), which overexpress muscarinic acetylcholine receptors (mAChRs), it has been shown that intracellular NAD ϩ or NAD ϩ metabolites are involved in signal transduction from m1 mAChRs to K ϩ channels (32,33). In this context, such neuronal cell lines have advantages for analyzing receptor-ADP-ribosyl cyclase coupling in detail.For measurement of ADP-ribosyl cyclase, high pressure liquid chromatography (HPLC) is commonly used to separate cADP-ribose-related compounds (1,2,8,14,15,17,19,34,35). However, since it takes 30 -60 min to process one sample, it is essential to develop a much more rapid method that can allow processing of multiple samples at once. There are two papers that describe ADP-ribosyl cyclase assay by TLC (21,36), in which NAD ϩ migrates faster than cADP-ribose. The methods used in those reports seem to be affected by large amounts of radiolabeled substrates. We here developed a TLC method that overcomes this problem and allows separation of cADP-ribose in up to 19 samples within 40 -50 min. Our TLC method was first tested on COS-7 cells overexpressing human CD38 and was shown to be applicable for measuring ADP-ribosyl cyclase activity. We demonstrate that crude cell membranes of NG108-15 cells possess ADP-ribosyl cyclase activity and that such activity is activated or inhibi...

beta-NAD(+) is as abundant as ATP in neuronal cells. beta-NAD(+) functions not only as a coenzyme but also as a substrate. beta-NAD(+)-utilizing enzymes are involved in signal transduction. We focus on ADP-ribosyl cyclase/CD38 which synthesizes cyclic ADP-ribose (cADPR), a universal Ca(2+) mobilizer from intracellular stores, from beta-NAD(+). cADPR acts through activation/modulation of ryanodine receptor Ca(2+) releasing Ca(2+) channels. cADPR synthesis in neuronal cells is stimulated or modulated via different pathways and various factors. Subtype-specific coupling of various neurotransmitter receptors with ADP-ribosyl cyclase confirms the involvement of the enzyme in signal transduction in neurons and glial cells. Moreover, cADPR/CD38 is critical in oxytocin release from the hypothalamic cell dendrites and nerve terminals in the posterior pituitary. Therefore, it is possible that pharmacological manipulation of intracellular cADPR levels through ADP-ribosyl cyclase activity or synthetic cADPR analogues may provide new therapeutic opportunities for treatment of neurodevelopmental disorders.

Hepatoma cell lines can produce a massive amount of chemokines in response to various stimuli including hepatitis viruses and their products. However, it remains elusive on the types of chemokine receptor(s) expressed in the hepatoma tissues and its roles in hepatoma development. To clarify these points, we examined the chemokine receptor expression in six human hepatoma cell lines. All of the hepatoma cell lines constitutively and exclusively expressed CCR1 mRNA and its protein on their cell surface. CCR1 expression was also detected on hepatoma cells and to a lesser degree, on endothelial cells in hepatoma tissues but not in normal liver tissues. Furthermore, CCL3 expression was detected in hepatoma cells, endothelial cells, and to a lesser degree, fibroblast-like cells in hepatoma tissue, whereas only occasional vascular endothelial cells and inflammatory cells in normal liver tissues were weakly positive for CCL3. Moreover, the forskolin-mediated increases in intracellular cAMP concentrations were inhibited by the ligands for CCR1, CCL3, CCL4, and CCL5, suggesting that the expressed CCR1 was functional.