NMIHBA results from hypomorphic PRUNE1 variants that lack short-chain exopolyphosphatase activity.

Neurodevelopmental disorder with microcephaly, hypotonia and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G > A (p.Arg128Gln), c.520G > T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.

Supt20 is required for development of the axial skeleton.

Somitogenesis and subsequent axial skeletal development is regulated by the interaction of pathways that determine the periodicity of somite formation, rostrocaudal somite polarity and segment identity. Here we use a hypomorphic mutant mouse line to demonstrate that Supt20 (Suppressor of Ty20) is required for development of the axial skeleton. Supt20 hypomorphs display fusions of the ribs and vertebrae at lower thoracic levels along with anterior homeotic transformation of L1 to T14. These defects are preceded by reduction of the rostral somite and posterior shifts in Hox gene expression. While cycling of Notch target genes in the posterior presomitic mesoderm (PSM) appeared normal, expression of Lfng was reduced. In the anterior PSM, Mesp2 expression levels and cycling were unaffected; yet, expression of downstream targets such as Lfng, Ripply2, Mesp1 and Dll3 in the prospective rostral somite was reduced accompanied by expansion of caudal somite markers such as EphrinB2 and Hes7. Supt20 interacts with the Gcn5-containing SAGA histone acetylation complex. Gcn5 hypomorphic mutant embryos show similar defects in axial skeletal development preceded by posterior shift of Hoxc8 and Hoxc9 gene expression. We demonstrate that Gcn5 and Supt20 hypomorphs show similar defects in rostral-caudal somite patterning potentially suggesting shared mechanisms.

Hectd1 is required for development of the junctional zone of the placenta.

The placenta plays a critical role in the growth and survival of the fetus. Here we demonstrate that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain E3 ubiquitin ligase, Hectd1, is essential for development of the mouse placenta. Hectd1 is widely expressed during placentation with enrichment in trophoblast giant cells (TGCs) and other trophoblast-derived cell subtypes in the junctional and labyrinth zones of the placenta. Disruption of Hectd1 results in mid-gestation lethality and intrauterine growth restriction (IUGR). Variable defects in the gross structure of the mutant placenta are found including alterations in diameter, thickness and lamination. The number and nuclear size of TGCs is reduced. Examination of subtype specific markers reveals altered TGC development with decreased expression of Placental lactogen-1 and -2 (Pl1 and Pl2) and increased expression of Proliferin (Plf). Reduced numbers of spongiotrophoblasts and glycogen trophoblasts were also found at the junctional zone of the Hectd1 mutant placenta. Finally, there was an increase in immature uterine natural killer (uNK) cells in the maternal decidua of the Hectd1 mutant placenta. Proliferation and apoptosis are differentially altered in the layers of the placenta with an increase in both apoptosis and proliferation in the maternal decidua, a decrease in proliferation and increase in apoptosis in the labyrinth layer and both unchanged in the junctional zone. Together these data demonstrate that Hectd1 is required for development of multiple cell types within the junctional zone of the placenta.

Sigma2 (sigma2) receptors as a target for cocaine action in the rat striatum.

Studies from our laboratory have shown that agonists at sigma1 and sigma2 receptors inhibit N-methyl-D-aspartate (NMDA)-stimulated dopamine release from motor and limbic areas of rat brain. In the current study, we examined the effects of cocaine on N-methyl-D-aspartate (NMDA)-stimulated [3H]dopamine release in rat striatal slices. Cocaine inhibited N-methyl-D-aspartate-stimulated [3H]dopamine release in a concentration-dependent manner with a Ki of approximately 10 microM, under conditions in which the dopamine transporter (DAT) was blocked by 10 microM nomifensine. The inhibition seen by cocaine was reversed by the selective sigma2 antagonist 1'-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-1-butyl]-spiro[isobenzofuran-1(3H), 4'piperidine] (Lu28-179). Inhibition of release by cocaine and (+)pentazocine, under conditions in which sigma1 receptors were blocked, was also reversed by the conventional PKC inhibitor 3-[1-[3-(dimethylamino)propyl-1H-indole-3-yl]-1-H-pyrpole-2-5'-dione. These results suggest that cocaine or other agonists, acting through the sigma2 receptor, require an intact conventional PKC (cPKC), most likely PKCalpha or PKCgamma in order to inhibit dopamine release.

Dysphagia and disrupted cranial nerve development in a mouse model of DiGeorge (22q11) deletion syndrome.

We assessed feeding-related developmental anomalies in the LgDel mouse model of chromosome 22q11 deletion syndrome (22q11DS), a common developmental disorder that frequently includes perinatal dysphagia--debilitating feeding, swallowing and nutrition difficulties from birth onward--within its phenotypic spectrum. LgDel pups gain significantly less weight during the first postnatal weeks, and have several signs of respiratory infections due to food aspiration. Most 22q11 genes are expressed in anlagen of craniofacial and brainstem regions critical for feeding and swallowing, and diminished expression in LgDel embryos apparently compromises development of these regions. Palate and jaw anomalies indicate divergent oro-facial morphogenesis. Altered expression and patterning of hindbrain transcriptional regulators, especially those related to retinoic acid (RA) signaling, prefigures these disruptions. Subsequently, gene expression, axon growth and sensory ganglion formation in the trigeminal (V), glossopharyngeal (IX) or vagus (X) cranial nerves (CNs) that innervate targets essential for feeding, swallowing and digestion are disrupted. Posterior CN IX and X ganglia anomalies primarily reflect diminished dosage of the 22q11DS candidate gene Tbx1. Genetic modification of RA signaling in LgDel embryos rescues the anterior CN V phenotype and returns expression levels or pattern of RA-sensitive genes to those in wild-type embryos. Thus, diminished 22q11 gene dosage, including but not limited to Tbx1, disrupts oro-facial and CN development by modifying RA-modulated anterior-posterior hindbrain differentiation. These disruptions likely contribute to dysphagia in infants and young children with 22q11DS.

A comparison of the binding profiles of dextromethorphan, memantine, fluoxetine and amitriptyline: treatment of involuntary emotional expression disorder.

We compared the binding profiles of medications potentially useful in the treatment of involuntary emotional expression disorder at twenty-six binding sites in rat brain tissue membranes. Sites were chosen based on likelihood of being target sites for the mechanism of action of the agents in treating the disorder or their likelihood in producing side effects experienced by patients treated with psychoactive agents. We used radioligand binding assays employing the most selective labeled ligands available for sites of interest. Concentrations of labeled ligand were used at or below the K(i) value of the ligand for the target site. Compounds were initially screened at 1 muM. For compounds that competed for greater than 20-30% of specific binding at target sites of interest, full concentration curves were constructed. Dextromethorphan, amitriptyline and fluoxetine competed for binding to sigma(1) receptors and to serotonin transporters with high to moderate affinity. Of the target sites tested, these are the most likely to contribute to the therapeutic benefit of the various agents. In addition, all three drugs showed some activity at alpha(2) and 5-HT(1B/D) sites. Of the drugs tested, dextromethorphan bound to the fewest sites unlikely to be target sites. Although the mechanism of action of dextromethorphan or any drug that has been used in the treatment of involuntary emotional expression disorder is currently unknown, our data support that the affinity of the drug for sigma(1) receptors is consistent with its possible action through this receptor type in controlling symptoms of the disorder.

Regulation of de novo phosphatidylinositol synthesis.

Mechanisms that function to regulate the rate of de novo phosphatidylinositol (PtdIns) synthesis in mammalian cells have not been elucidated. In this study, we characterize the effect of phorbol ester treatment on de novo PtdIns synthesis in C3A human hepatoma cells. Incubation of cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) initially (1-6 h) results in a decrease in precursor incorporation into PtdIns; however, at later times (18-24 h), a marked increase is observed. TPA-induced glucose uptake from the medium is not required for observation of the stimulation of PtdIns synthesis, because the effect is apparent in glucose-free medium. Inhibition of the activation of arachidonic acid substantially blocks the synthesis of PtdIns but has no effect on the synthesis of phosphatidylcholine (PtdCho). Increasing the concentration of cellular phosphatidic acid by blocking its conversion to diacylglycerol, on the other hand, enhances the synthesis of PtdIns and inhibits the synthesis of PtdCho. The TPA-induced stimulation of PtdIns synthesis is not the result of the concomitant TPA-induced G1 arrest, because G1 arrest induced by mevastatin has no effect on PtdIns synthesis. Inhibition of protein kinase C activity blocks the stimulatory action of TPA on de novo synthesis of PtdIns but has no effect on TPA-induced inhibition. Potential sites of enzymatic regulation are discussed.

Sigma1 receptor agonist-mediated regulation of N-methyl-D-aspartate-stimulated [3H]dopamine release is dependent upon protein kinase C.

We have previously shown that sigma1 receptor agonists inhibit N-methyl-D-aspartate (NMDA)-stimulated [3H]dopamine from slices of rat striatum in a concentration-related manner and that the inhibition is reversed by sigma1 receptor-selective and nonsubtype-selective sigma receptor antagonists. Based on previous evidence from our laboratory as well as other laboratories, we hypothesized that sigma1 receptors might use a protein kinase C (PKC) signaling pathway to modulate stimulated dopamine release. We tested several inhibitors of PKC isozymes, as well as a phospholipase C inhibitor for their effects on sigma1 receptor agonist-mediated regulation of [3H]dopamine release. Although none of the inhibitors tested affected the ability of NMDA to stimulate [3H]dopamine release, they all abolished regulation by the sigma1 receptor agonist (+)-pentazocine in a concentration-related manner. We also found that prior exposure to 1 microM phorbol 2-myristate 13-acetate for 30 min abolished regulation by (+)-pentazocine. We concluded that an intact PKC system was required for sigma1 agonist-mediated regulation of NMDA-stimulated [3H]dopamine release from rat striatal slices. Based on the pharmacological profile of the PKC inhibitors tested, as well as reports in the literature on PKC

Steroids modulate N-methyl-D-aspartate-stimulated [3H] dopamine release from rat striatum via sigma receptors.

Steroids have been proposed as endogenous ligands at sigma receptors. In the current study, we examined the ability of steroids to regulate N-methyl-d-aspartate (NMDA)-stimulated [3H]dopamine release from slices of rat striatal tissue. We found that both progesterone and pregnenolone inhibit [3H]dopamine release in a concentration-dependent manner similarly to prototypical agonists, such as (+)-pentazocine. The inhibition seen by both progesterone and pregnenolone exhibits IC50 values consistent with reported Ki values for these steroids obtained in binding studies, and was fully reversed by both the sigma1 antagonist 1-(cyclopropylmethyl)-4-2'-4"flurophenyl)-2'oxoethyl)piperidine HBr (DuP734) and the sigma2 antagonist 1'-[4-[1-(4-fluorophenyl)-1-H-indol-3-yl]-1-butyl]spiro[iso-benzofuran-1(3H), 4'piperidine] (Lu28-179). Lastly, to determine whether a protein kinase C (PKC) signaling system might be involved in the inhibition of NMDA-stimulated [3H]dopamine release, we tested the PKCbeta-selective inhibitor 5,21:12,17-dimetheno-18H-dibenzo[i,o]pyrrolo[3,4 - 1][1,8]diacyclohexadecine-18,20(19H)-dione,8-[(dimethylamino)methyl]-6,7,8,9,10,11-hexahydro-monomethanesulfonate (9Cl) (LY379196) against both progesterone and pregnenolone. We found that LY379196 at 30 nM reversed the inhibition of release by both progesterone and pregnenolone. These findings support steroids as candidates for endogenous ligands at sigma receptors.

Modulation of bradykinin-induced calcium changes in SH-SY5Y cells by neurosteroids and sigma receptor ligands via a shared mechanism.

In this study we investigated the effects of sigma receptor ligands and neurosteroids on bradykinin-induced intracellular calcium concentration ([Ca2+]i) changes in SH-SY5Y neuroblastoma cells. [Ca2+]i levels in cells loaded with fura-2 were monitored with dual-wavelength ratiometric fluorescence measurement. Submicromolar concentrations of bradykinin elicited [Ca2+]i responses with a fast rise followed by a slow decline in these cells. Preincubation of low micromolar concentrations of the neurosteroids pregnenolone, dehydroepiandrosterone (DHEA), or the prototypic sigma (sigma) receptor agonist (+)pentazocine potentiated bradykinin-induced [Ca2+]i changes in SH-SY5Y cells. The sigma receptor antagonist haloperidol blocked the enhancing effects on [Ca2+]i by (+)pentazocine or pregnenolone. Progesterone did not significantly affect the basal [Ca2+]i level or bradykinin-induced [Ca2+]i changes in these cells. However, coincubation of progesterone with (+)pentazocine, pregnenolone, or DHEA reversed their potentiating effects. The antagonistic effects of haloperidol and progesterone on the potentiating effects of (+)pentazocine and pregnenolone suggested that these ligands might act through a common mechanism. We further showed that progesterone, pregnenolone, and DHEA competed for [3H]+pentazocine binding in SH-SY5Y cells with Ki values of 0.13 +/- 0.03 microM, 0.98 +/- 0.34 microM, and 5.2 +/- 1.4 microM, respectively. Thus, the modulation of bradykinin-induced [Ca2+]i changes by neurosteroids in these cells is likely due to their actions on sigma receptors.