Expansion and mechanistic insights into de novo DEAF1 variants in DEAF1-associated neurodevelopmental disorders.

De novo deleterious and heritable biallelic mutations in the DNA binding domain (DBD) of the transcription factor deformed epidermal autoregulatory factor 1 (DEAF1) result in a phenotypic spectrum of disorders termed DEAF1-associated neurodevelopmental disorders (DAND). RNA-sequencing using hippocampal RNA from mice with conditional deletion of Deaf1 in the central nervous system indicate that loss of Deaf1 activity results in the altered expression of genes involved in neuronal function, dendritic spine maintenance, development, and activity, with reduced dendritic spines in hippocampal regions. Since DEAF1 is not a dosage-sensitive gene, we assessed the dominant negative activity of previously identified de novo variants and a heritable recessive DEAF1 variant on selected DEAF1-regulated genes in 2 different cell models. While no altered gene expression was observed in cells over-expressing the recessive heritable variant, the gene expression profiles of cells over-expressing de novo variants resulted in similar gene expression changes as observed in CRISPR-Cas9-mediated DEAF1-deleted cells. Altered expression of DEAF1-regulated genes was rescued by exogenous expression of WT-DEAF1 but not by de novo variants in cells lacking endogenous DEAF1. De novo heterozygous variants within the DBD of DEAF1 were identified in 10 individuals with a phenotypic spectrum including autism spectrum disorder, developmental delays, sleep disturbance, high pain tolerance, and mild dysmorphic features. Functional assays demonstrate these variants alter DEAF1 transcriptional activity. Taken together, this study expands the clinical phenotypic spectrum of individuals with DAND, furthers our understanding of potential roles of DEAF1 on neuronal function, and demonstrates dominant negative activity of identified de novo variants.

Estrogen genomic and membrane actions at an intersection.

Estradiol is a primary hormonal signal that synchronizes central neuronal activity changes during the female reproductive cycle. The cellular and molecular mechanisms for many of these changes are still not well understood. Exciting new findings of Roepke and colleagues indicate that estradiol regulates expression of key potassium channels as well as modulatory intracellular signaling molecules. This regulation might contribute to region-specific changes in neuronal excitability in the hypothalamic arcuate nucleus and to the integration of neuronal responses that influence homeostatic functions.

Phosphorylation state of tyrosine hydroxylase in the stalk-median eminence is decreased by progesterone in cycling female rats.

Progesterone has the capacity to suppress hypothalamic dopaminergic neuronal activity on proestrous afternoon and prolong or amplify the preovulatory prolactin surge in rats. In the present study, we examined enzyme activity and phosphorylation state of tyrosine hydroxylase (TH) in the stalk-median eminence of cycling female rats on proestrus and estrus and related these to circulating progesterone levels. Phospho-TH levels were evaluated by Western blot analysis. TH activity was determined from the rate of 3,4-dihydroxyphenylalanine (DOPA) accumulation. Phospho-TH levels at Ser-19, Ser-31, and Ser-40 were similar at 1100, 1300, and 1500 h on proestrus but declined at 1700, 1900, and 2200 h, coincident with rising serum progesterone levels. Similarly, DOPA accumulation was 30-50% lower at 1700, 1900, and 2200 h as compared with 1100-1500 h on proestrus. Ser-31 and Ser-40 phosphorylation states were increased by 1100 h on estrus to a level similar to 1100 h on proestrus, whereas DOPA accumulation was 30% greater on estrous as compared with proestrous morning. There were no significant differences among the several time points on proestrus and estrus with regard to TH protein or beta-tubulin levels. Exogenous progesterone administration (7.5 mg/kg, sc) before the preovulatory progesterone surge decreased TH activity and phospho-TH at Ser-19, Ser-31, and Ser-40, accompanied by premature increased serum prolactin. Our study suggests that decreased TH phosphorylation at Ser-19, Ser-31, and Ser-40 contributes to the decline in TH activity in the stalk-median eminence on proestrous afternoon and that progesterone may cause this initial cytoplasmic response of TH dephosphorylation.

Regulation of cytokine-inducible SH2-containing protein (CIS) by ubiquitination and Elongin B/C interaction.

Cytokine-inducible SH2-containing protein (CIS) inhibits prolactin receptor (PRLR) signaling and acts as part of an E3 ubiquitin ligase complex through interactions with Elongin B/C proteins. This study aimed to identify CIS lysine ubiquitination sites and determine roles of ubiquitination and Elongin B/C interactions on CIS protein stability and PRLR signaling inhibition. Site-directed mutations revealed that CIS can be ubiquitinated on all six lysine residues. Elongin B/C interaction box mutation had no influence on CIS ubiquitination. CIS stability was increased by mutation of lysine residues and further enhanced by co-mutation of Elongin B/C interaction domain. CIS inhibition of STAT5B phosphorylation and casein promoter activation was dependent on CIS interactions with Elongin B/C, but not on CIS ubiquitination. These data indicate CIS protein stability is regulated through multiple mechanisms, including ubiquitination and interaction with Elongin B/C proteins, whereas CIS functional inhibition of PRLR signaling is dependent on the Elongin B/C interaction.

Progesterone decreases tyrosine hydroxylase phosphorylation state and increases protein phosphatase 2A activity in the stalk-median eminence on proestrous afternoon.

The progesterone (P(4)) rise on proestrous afternoon is associated with dephosphorylation of tyrosine hydroxylase (TH) and reduced TH activity in the stalk-median eminence (SME), which contributes to the proestrous prolactin surge in rats. In the present study, we investigated the time course for P(4) effect on TH activity and phosphorylation state, as well as cAMP levels and protein phosphatase 2A (PP2A) activity and quantity, in the SME on proestrous morning and afternoon. P(4) (7.5 mg/kg, s.c.) treatment on proestrous afternoon decreased TH activity and TH phosphorylation state at Ser-31 and Ser-40 within 1 h, whereas morning administration of P(4) had no 1 h effect on TH. PP2A activity in the SME was enhanced after P(4) treatment for 1 h on proestrous afternoon without a change in PP2A catalytic subunit quantity, whereas P(4) treatment had no effect on PP2A activity or quantity on proestrous morning. cAMP levels in the SME were unchanged with 1 h P(4) treatment. At 5 h after P(4) treatment, TH activity and phosphorylation state declined coincident with an increase in plasma prolactin in both P(4)-treated morning and afternoon groups. PP2A activity in the SME was unchanged in 5 h P(4)-treated rat. Our data suggest that P(4) action on tuberoinfundibular dopaminergic (TIDA) neurons involves at least two components. A more rapid (1 h) P(4) effect engaged only on proestrous afternoon likely involves the activation of PP2A. The longer P(4) action on TIDA neurons is evident on both the morning and afternoon of proestrus and may involve a common, as yet unidentified, mechanism.

Adenosine receptor antagonists and behavioral activation in NF-kappaB p50 subunit knockout mice.

AIMS: Our previous work revealed that mice lacking the p50 subunit of transcription factor nuclear factor kappa B (NF-kappaB) (p50 KO mice) and genetically intact F2 mice have similar locomotion under basal conditions, yet p50 KO mice show greater locomotor activation after caffeine ingestion. In this report, we test whether KO mice display altered caffeine pharmacokinetics or increased caffeine-induced DA turnover relative to F2 mice, and evaluate the impact of intraperitoneal administration of specific adenosine and DA receptor antagonists on locomotor activity. MAIN METHODS: Concentrations of DA and caffeine were measured using high performance liquid chromatography. DA turnover was measured after treatment of mice with an inhibitor of tyrosine hydroxylase. Locomotor activity was measured using telemetry. KEY FINDINGS: The data reveal that 1) caffeine concentrations in blood and brain are similar in KO and F2 mice after oral or intraperitoneal administration; 2) KO mice show greater DA turnover under basal conditions, but turnover is similar in both strains after caffeine administration; 3) the specific A2AAR antagonist SCH 58261 induces greater locomotion in KO versus F2 mice; and 4) the activating effect of SCH 58261 in KO mice is prevented by prior treatment with the D2R antagonist raclopride. SIGNIFICANCE: These findings support the conclusions that 1) A2AAR has a major impact on behavioral activation of p50 KO mice, and 2) D2R mediated neurotransmission is important to this effect.

Progesterone induces dephosphorylation and inactivation of tyrosine hydroxylase in rat hypothalamic dopaminergic neurons.

After in vivo treatment, progesterone initially decreases tyrosine hydroxylase (TH) activity in the TIDA neurons, but subsequently increases TH activity with prolonged treatment. In order to explore the cellular mechanism for progesterone's effect, this study examined the acute inhibitory action of progesterone on TH activity in rat fetal hypothalamic dopaminergic neurons in vitro. Progesterone caused a rapid decrease in TH activity within 1 h, which was sustained for at least 6 h. However, the dopaminergic cells became refractory to progesterone with continuous treatment for 12 h to 10 days. Progesterone (10-100 nM) treatment suppressed TH activity in a concentration-dependent manner. The inhibitory effect of progesterone was dependent on prior exposure to estradiol. Whereas progesterone decreased TH activity, A ring-reduced metabolites of progesterone did not alter TH activity, suggesting that the response was specific to progesterone. Progesterone decreased radiolabeled phosphate incorporation into TH protein. Okadaic acid, a phosphoprotein phosphatase inhibitor, prevented the progesterone-induced suppression of TH activity and phosphate incorporation into TH, implicating dephosphorylation of TH as the cellular mechanism. In contrast, neither TH mRNA levels nor TH protein content was altered after 1 or 12 h of progesterone treatment. Progesterone decreased TH activity after pretreatment of the hypothalamic cells for 2 or 24 h with actinomycin D, an RNA synthesis inhibitor, suggesting that increased transcription does not mediate the effect. These data indicate that the acute progesterone-induced decline in TH activity is caused by dephosphorylation of TH.

Prolactin modulates hypothalamic preproenkephalin, but not proopiomelanocortin, gene expression during lactation.

The aim of this study was to examine prolactin (PRL) regulation of preproenkephalin and proopiomelanocortin (POMC) gene expression in the hypothalamus during lactation. In the first experiment, lactating rats were deprived of pups for 3, 6, 12, or 24 h. Preproenkephalin mRNA levels were decreased in the arcuate nucleus (ARC) to 60 or 53% of suckled levels and in the ventromedial nucleus to 70% of suckled levels after 12 or 24 h but were unchanged in the striatum. POMC mRNA levels in the ARC and periarcuate area were increased to 165% of suckled levels within 3 h and remained elevated two- to threefold for 24 h. Subcutaneous administration of bromocriptine to suckled dams markedly suppressed circulating PRL levels and decreased preproenkephalin mRNA signal levels to 38 and 50% of control levels in the arcuate and ventromedial nuclei, respectively. Intravenous administration of oPRL completely reversed this effect. By contrast, bromocriptine with or without administration of ovine PRL (oPRL) did not alter POMC mRNA signal levels in the ARC. Administration of oPRL to pup-deprived dams increased preproenkephalin mRNA levels in the arcuate and ventromedial nuclei and reduced POMC mRNA levels in the ARC to levels similar to suckled control levels. In conclusion, POMC neurons in the ARC appear to be refractory to PRL regulation in the presence of a suckling stimulus, and other components of the suckling stimulus may contribute to the suppression of POMC mRNA levels during lactation. By contrast, PRL provides a regulatory influence for the suckling-induced increase in preproenkephalin mRNA signal levels in arcuate and ventromedial nuclei.