MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling.

Rett syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. Although postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of microRNAs (miRNAs) in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 short hairpin RNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and three-dimensional (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in the wild-type mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.

Cytidylyltransferase-binding protein is identical to transcytosis-associated protein (TAP/p115) and enhances the lipid activation of cytidylyltransferase.

We previously identified a protein from rat liver that binds CTP:phosphocholine cytidylyltransferase (CT). We have now purified this protein (cytidylyltransferase-binding protein (CTBP)) from rat liver. The purification involved precipitation at pH 5 and extraction of the precipitate with buffer, followed by sequential chromatography on DEAE-Sepharose and butyl-agarose. Final purification was accomplished by either preparative electrophoresis or hydroxylapatite chromatography. Amino acid sequences from six peptides derived from pure CTBP matched sequences in transcytosis-associated protein (TAP) with 98% identity. Thus, CTBP was positively identified to be TAP. Purified CTBP increased the activity of purified CT measured with phosphatidylcholine (PC)/oleic acid. In the absence of PC/oleic acid, CTBP did not stimulate CT activity. Dilution of CT to reduce the Triton X-100 concentration produced a loss of CT activity. The lost activity was recovered by the addition of CTBP plus PC/oleic acid to the assay, but not by the addition of either PC/oleic acid or CTBP alone. Removal of CTBP from purified preparations by immunoprecipitation with CTBP antibodies eliminated the activation of CT. Both CT and CTBP were shown to bind to PC/oleic acid liposomes. The formation of complexes between CT and CTBP in the absence of PC/oleic acid liposomes could not be demonstrated. These results suggest that CTBP functions to modify the interaction of CT with PC/oleic acid liposomes, resulting in an increase in the catalytic activity perhaps by the formation of a ternary complex between CT, CTBP, and lipid. Overall, these results suggest that CTBP (TAP) may function to coordinate the biosynthesis of phosphatidylcholine with vesicle transport.

HIV prevention among Zambian adolescents: developing a value utilization/norm change model.

Peer-led interventions are effective in reducing risk for HIV among adolescents. A pre-intervention study was conducted to determine how to successfully carry out a possible future intervention to reduce HIV risk among adolescents in urban Zambia. Ethnographic and sexual data were collected on 276 males and females both attending and not attending secondary school during a 14-month period in 1992-1993. Additionally, several focus groups were conducted. This paper reviews the cultural background of Zambian adolescents and presents an overview of the study results. Among the findings, it was learned that most of the male and female adolescents (average age of 17) are sexually active, very few routinely use condoms, less than half of sexually active adolescents have ever used a condom, AIDS is omnipresent in Zambia, the threat of HIV infection is a very real concern for most of the adolescents, there is a strong desire to protect themselves from HIV infection during sex (but condoms are often seen as ineffective and other forms of safer sex are not discussed), nearly all of the sexually active females and some of the males have received money or gifts for sex, and some of the out-of-school females are engaging in very risky sex (e.g., unprotected anal intercourse, and anilingus) with adult men. The ethnographic data, including a brief trial risk reduction workshop, suggests that the core values and social norms of the adolescents may shape behavioral change. A value utilization/norm change (VUNC) model is developed, which is intended to provide a conceptual framework for understanding how to utilize selected core values of the adolescents to strengthen or alter norms within the social networks in order to elicit desired HIV risk reduction.

Fatty acids promote the formation of complexes between choline-phosphate cytidylyltransferase and cytidylyltransferase binding protein.

We previously identified a 112-kDa protein (CTBP) that binds CTP:choline-phosphate cytidylyltransferase (CT) (D.A. Feldman and P.A. Weinhold, 1993, J. Biol. Chem. 268, 3127-3135). In this study we show that fatty acids promote the binding of cytidylyltransferase to CTBP. Gel filtration chromatography separated CTBP from CT in liver cytosol. CTBP was eluted slightly slower than the thyroglobulin standard. The addition of oleate to cytosol followed by incubation at 37 degrees C resulted in the formation of aggregates containing both CT and CTBP. The aggregates eluted in the void volume of the gel filtration column, sedimented to the bottom of glycerol density gradients, and precipitated at concentrations of polyethylene glycol lower than required to precipitate CT and CTBP in untreated cytosol. Immunoprecipitation by CT antibodies of both CT and CTBP in aggregate preparations provided further evidence for the formation of CT-CTBP complexes. A smaller CT-CTBP complex was isolated by glycerol density centrifugation from cytosol incubated with oleate at 4 degrees C. Overall, these results suggest that oleate promotes the binding of CT to CTBP. The results suggest that a dissociable complex is formed at 4 degrees C in the presence of oleate which is polymerized by incubation at 37 degrees C to a large aggregate that is more resistant to dissociation. Complex formation at 37 and 4 degrees C was dose dependent at oleate concentrations between 50 and 200 microM. Complex formation at 4 degrees C was specifically promoted by long-chain, unsaturated fatty acids. These results suggest that CTBP may be involved in the fatty acid-induced translocation of cytidylyltransferase.

Regulation of CTP: phosphocholine cytidylyltransferase in HepG2 cells: effect of choline depletion on phosphorylation, translocation and phosphatidylcholine levels.

We studied the effect of choline depletion on the biosynthesis of phosphatidylcholine (PC) and the distribution and phosphorylation of cytidylyltransferase (CT) in HepG2 cells. Phosphocholine concentrations decreased within 24 h of choline depletion to values less than 2% of controls. The incorporation of [3H]glycerol into PC was reduced in choline-depleted (CD) cells. The apparent turnover of PC was similar in CD and choline-supplemented (CS) cells (T1/2 = 20 h). The methylation pathway for PC synthesis increased nearly 10-fold in CD cells. Cell growth was similar in CD and CS cells. Over 95% of CT activity in CS cells was in the soluble pool. Choline depletion resulted in a progressive decrease in CT activity and immunodetected enzyme in the soluble pool and a corresponding increase in membrane CT over a 48-h period. Choline supplementation of CD cells caused a rapid release of membrane CT (complete release by 3 h). Two phosphorylated forms of CT were identified. One form contained a higher level of phosphorylation (HPCT) than the other form (LPCT). HPCT migrated slightly slower than LPCT on SDS gels. CD cells contained only LPCT in both soluble and membrane pools. CS cells contained only HPCT. During choline depletion PC content decreased nearly 20% but CT binding did not occur until LPCT was generated in cytosol. Conversely, choline supplementation released LPCT into cytosol and HPCT was formed only after the release. We conclude that both the induction of binding sites, perhaps by depletion of PC and dephosphorylation of HPCT to LPCT, are required for CT translocation to membranes. The release of CT from membranes is initiated by changes in membrane binding sites followed by trapping of the CT in the soluble pool by phosphorylation of LPCT to HPCT.

Identification of a protein complex between choline-phosphate cytidylyltransferase and a 112-kDa protein in rat liver.

Antisera raised against purified cytidylyltransferase (CT) immunoprecipitated CT activity from liver cytosol and detected the M(r) 45,000 subunit of CT on Western blots. Antisera detected a M(r) 112,000 protein on Western blots of liver cytosol. This protein was not detected in purified CT and was not detected by preimmune serum. The 112-kDa antibodies, isolated by affinity chromatography, did not immunoprecipitate CT activity. Antiserum raised against an N-terminal sequence of CT and antibodies raised against an internal sequence of CT immunoprecipitated CT activity but did not detect the 112-kDa protein. These results showed that the 112-kDa protein was not a form of CT. We concluded that the antiserum probably contained anti-idiotypic antibodies that recognized CT binding sites on the 112-kDa protein. Purified CT that was conjugated to horseradish peroxidase bound to crude 112-kDa protein immobilized on nitrocellulose. The binding was competitively reduced by purified CT and by affinity-purified antibodies to the 112-kDa protein. CT and 112-kDa protein coeluted from DEAE-Sepharose. When the putative 112-kDa protein-CT complex was chromatographed on a second DEAE-Sepharose column or on a Bio-Gel A-1.5m column, CT activity and 112-kDa protein were eluted together. Chromatography of the complex on hydroxylapatite dissociated most of the complex, producing CT free of the 112-kDa protein. We conclude that the 112-kDa protein is a CT-binding protein. The formation and/or dissociation of this complex may be important in the regulation of CT.

Microsomal CTP:choline phosphate cytidylyltransferase: kinetic mechanism of fatty acid stimulation.

Fatty acids are known to cause an increase in the incorporation of radioactive choline into phosphatidylcholine. A coincident increase in membrane cytidylyltransferase activity is well documented. The purpose of the present studies was to determine the direct effects of oleic acid on the kinetic properties of membrane cytidylyltransferase. An examination of the reaction characteristics of membrane cytidylyltransferase revealed that membranes from adult rat lung contained high CTPase activity. This activity prevented the determination of reaction velocities at low CTP concentrations. The CTPase activity was blocked by the addition of ADP or ATP to the reaction. The addition of 6.0 mM ADP to the assay mixture enabled us to determine the effect of oleate on the CTP Km. Oleate (122 microM) caused a significant decrease in CTP Km for microsomal cytidylyltransferase (0.99 mM to 0.33 mM) and H-Form cytidylyltransferase (1.04 mM to 0.27 mM). Oleate did not decrease the CTP Km for L-Form cytidylyltransferase. Oleate had no effect on the choline phosphate Km in microsomal, H-Form or L-Form cytidylyltransferase. Oleate also increased the Vmax for cytidylyltransferase. The increase was dependent upon the concentration of oleate with a maximal increase of 50-60% at 100-130 microM oleate. We conclude that oleate has a direct stimulatory effect on cytidylyltransferase when it is in the active form (membrane bound or H-Form lipoprotein complex). We suggest that the kinetic effects operate synergistically with other regulatory mechanisms such as translocation or conversion of inactive to active species. The direct effect of oleate on the cytidylyltransferase may be an important regulatory mechanism when CTP concentrations are limiting.

Control of phosphatidylcholine synthesis in Hep G2 cells. Effect of fatty acids on the activity and immunoreactive content of choline phosphate cytidylyltransferase.

We examined the effect of fatty acids on phosphatidylcholine synthesis and cytidylyltransferase activity in Hep G2 cells. Treatment of Hep G2 cells with oleic acid caused an increase in the incorporation of [methyl-14C]choline into phosphatidylcholine and a corresponding decrease in radioactivity in choline phosphate using a pulse-chase procedure. This result is consistent with a fatty acid-induced increase in the cytidylyl-transferase step in the choline pathway. We measured cytidylyltransferase activity in membrane fractions and in cytosol (100,000 x g supernatant or soluble enzyme released by digitonin). The activity increased in both membrane and cytosol. Thus, an increase in total activity occurred. Cytidylyltransferase protein determined by Western blot immunoassay increased after oleic acid treatment. Immunotitration of cytidylyltransferase protein also indicated that an increase in enzyme protein resulted from oleic acid treatment. Cycloheximide did not prevent the oleic acid-induced increase in cytidylyltransferase activity. The increase in enzyme activity was apparent when we measured the activity in the presence or absence of lipid activators. Separation of cytosolic cytidylyltransferase into H- and L-forms showed that the increase in cytosolic activity was due to an increase in H-form. The amount of L-form did not change. We interpret these results to suggest that fatty acid treatment of Hep G2 cells promoted the formation of active cytidylyltransferase (H-form) from a preexisting inactive form. The increased activity was distributed between membranes and the lipoprotein form in cytosol (H-form).

Dexamethasone increases the activity but not the amount of choline-phosphate cytidylyltransferase in fetal rat lung.

The activity of choline-phosphate cytidylyltransferase is increased by glucocorticoids in late gestation fetal lung in association with increased phosphatidylcholine biosynthesis. Previous indirect data had suggested that the stimulatory effect of the hormone was due to activation of existing enzyme rather than synthesis of new cytidylyltransferase protein. Using a rabbit antibody raised against purified rat liver choline-phosphate cytidylyltransferase, we have now quantitated the amount of the enzyme in fetal rat lung explants cultured with and without dexamethasone. Our results show that the hormone increased the activity of the enzyme but not the amount of cytidylyltransferase protein. Thus the stimulatory effect of dexamethasone on cytidylyltransferase is due to activation of existing enzyme rather than induction of enzyme synthesis.

CTP:phosphocholine cytidylyltransferase in rat lung: relationship between cytosolic and membrane forms.

The purpose of these studies was to determine the properties of the membrane-bound cytidylyltransferase in adult lung and to assess the relationship between the microsomal enzyme and the two forms of cytidylyltransferase in cytosol. Microsomes, isolated by glycerol density centrifugation, contained significantly less cytidylyltransferase than microsomes isolated by differential centrifugation (11.6 +/- 3.2 vs. 30 +/- 11 nmol/min per g lung). The released activity was recovered as H-form cytidylyltransferase. Cytidylyltransferase activity was not removed from microsomes by washing of the microsomal pellet with homogenizing buffer. Triton X 100 extracted all of the cytidylyltransferase from microsomes. The extracted activity was similar to H-form. Chlorpromazine dissociated microsomal enzyme to L-form. Chlorpromazine has been shown previously to dissociate H-form to L-form. These results suggested that microsomal cytidylyltransferase existed in a form similar if not identical to cytosolic H-form. In vitro translocation experiments demonstrated that the L-form of cytidylyltransferase was the species which binds to microsomal membranes. Triton X 100 extraction of microsomes from translocations experiments removed the bound enzyme activity. Glycerol density fractionation indicated that the activity in the Triton extract was H-form cytidylyltransferase. We concluded that the active lipoprotein form of cytidylyltransferase (H-form) is the membrane-associated form of cytidylyltransferase in adult lung; that it is formed after the L-form binds to microsomal membranes and that cytosolic H-form is released from the membrane.

Characterization of cytosolic forms of CTP: choline-phosphate cytidylyltransferase in lung, isolated alveolar type II cells, A549 cell and Hep G2 cells.

The subcellular forms of cytidylyltransferase (EC 2.7.7.15) in rat lung, rat liver, Hep G2 cells, A549 cells and alveolar Type II cells from adult rats were separated by glycerol density centrifugation. Cytosol prepared from lung, Hep G2 cells, A549 cells and alveolar Type II cells contained two forms of the enzyme. These species were identical to the L-Form and H-Form isolated previously from lung cytosol by gel filtration. Liver cytosol contained only the L-Form. Rapid treatment of Hep G2 cells with digitonin released all of the cytoplasmic cytidylyltransferase activity. The released activity was present in both H-Form and L-Form. The molecular weight of L-Form was determined from sedimentation coefficients and Stokes radius values to be 97,690 +/- 10,175. Thus, the L-Form appears to be a dimer of the Mr 45,000 catalytic subunit. The f/f degrees value of 1.5 indicated that the protein molecule has an axial ratio of 10, assuming a prolate ellipsoid shape. The estimated molecular weight of the H-Form was 284,000 +/- 25,000. The H-Form was dissociated into L-Form by incubation of cytosol at 37 degrees C. Triton X-100 (0.1%) and chlorpromazine (1.0 mM) also dissociated the H-Form into L-Form. Western blot analysis indicated that both forms contained the catalytic subunit. An increase in Mr 45,000 subunit coincided with the increase in cytidylyltransferase activity in L-Form, which resulted from the dissociated of H-Form. The L-Form was dependent on phospholipid for activity. The H-Form was active without lipid. Phosphatidylinositol was present in the H-Form isolated from Hep G2 cells. The phosphatidylinositol dispersed when the H-Form was dissociated into L-Form. Phosphatidylinositol and phosphatidylglycerol cause L-Form to aggregate into a form similar to H-Form. Phosphatidylcholine/oleic acid (1:1 molar ratio) and oleic acid also aggregated the L-Form. Phosphatidylcholine did not produce aggregation. We conclude that the H-Form is the active form of cytidylyltransferase in cytoplasm. The H-Form appears to be a lipoprotein consisting of an apoprotein (L-Form dimer of the Mr 45,000 subunit) complexed with lipids. A change in the relative distribution of H-Form and L-Form in cytosol would alter the cellular activity and thus may be important in the regulation of phosphatidylcholine synthesis.

Gay youth and AIDS.

Gay male teenagers face considerable adversity during their "coming out" process due to the AIDS epidemic. They must decide whether to be tested for HIV-1 infection, whether to postpone sexual activity, how to select a partner, and which kinds of sexual practices to engage in. Gay youth often make such decisions based upon misinformation and faulty premises. This paper reviews what is known about gay youth and AIDS, and assesses their possible risk for HIV-1 infection. It is recommended that school and community-based health education programs be developed to teach gay and bisexual youth about safe sex. Moreover, research is needed into sociocultural variations among gay youth in order to develop appropriate and effective intervention strategies for AIDS risk reduction in this diverse population.

CTP:phosphorylcholine cytidylyltransferase from rat liver. Isolation and characterization of the catalytic subunit.

We reported previously the purification of CTP:phosphorylcholine cytidylyltransferase from rat liver (Weinhold, P. A., Rounsifer, M. E., and Feldman, D. A. (1986) J. Biol. Chem. 261, 5104-5110). The purified enzyme appeared to contain equal amounts of two nonidentical proteins, with Mr of about 38,000 and 45,000. We have now separated and purified these proteins. Polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate indicated that each protein was homogeneous. The 45,000 protein contained the catalytic activity. Analysis by gel filtration chromatography and glycerol gradient centrifugation indicated that the 38,000 and 45,000 proteins in the purified cytidylyltransferase were independently associated with Triton X-100 micelles. The apparent Mr of the complexes suggested that a tetramer of each protein was bound to one Triton X-100 micelle. The isolated 45,000 catalytic protein had the same lipid requirement and kinetic properties as the purified cytidylyltransferase containing both proteins. Enzyme activity was stimulated to maximal values by phosphatidylcholine vesicles containing 9 mol % of either oleic acid, phosphatidylinositol, or phosphatidylglycerol. The amino acid compositions of the isolated 38,000 and 45,000 proteins were distinctly different. Overall, the results suggested that a tetramer of the 45,000 protein possessed nearly optimal catalytic activity. A functional role of the 38,000 protein as part of a cytidylyltransferase enzyme complex could not be documented. However, the need for stabilizing concentrations of Triton X-100 in the purified enzyme preparation may have prevented the association of the two proteins.

Public awareness of AIDS in Rwanda.

AIDS is a rapidly growing epidemic in Kigali, Rwanda. To understand the level of public awareness of AIDS in that city, 33 informants (15 men and 18 women) were interviewed during September, 1985. Most (66.7%) said that they first heard of the disease only within the previous eight months. About half (46.9%) could not mention one or more AIDS symptoms. Younger informants and women reported less knowledge of AIDS symptoms. While nearly everyone recognized AIDS as a stigmatized disease, most informants apparently did not know why it is stigmatized. Only about one-third of the informants (34.4%) could correctly state the mode of AIDS transmission. People who are at greatest risk for the disease, unmarried men and women, were least likely to know how it is transmitted. Half (50.0%) of those informants who responded to the question of the origins of AIDS said that it began in 'America.' While many informants are frightened by the disease, no one has yet changed their sexual behavior as a response to the epidemic. All informants agreed that more information about AIDS should be made available in Rwanda. Preventive measures against the spread of AIDS are urgently needed in central Africa.

The purification and characterization of CTP:phosphorylcholine cytidylyltransferase from rat liver.

We have purified CTP:phosphorylcholine cytidylyltransferase from rat liver cytosol 2180-fold to a specific activity of 12,250 nmol/min/mg of protein. The purified enzyme was stable at -70 degrees C in the presence of Triton X-100 and 0.2 M phosphate. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide electrophoresis. Separation by sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the purified enzyme contained subunits with Mr of 39,000 and 48,000. Gel filtration analysis indicated that the native enzyme was a tetramer containing two 39,000 and two 48,000 subunits. The purified enzyme appeared to bind to Triton X-100 micelles, one molecule of tetramer/micelle. Maximal activity was obtained with 100 microM phosphatidylcholine-oleic acid vesicles (8-10-fold stimulation). Phosphatidylglycerol produced a 4-5-fold increase in activity at 10 microM. The pH optimum and true Km values for CTP and phosphorylcholine were similar to those reported previously for crude preparations of cytidylyltransferase. The overall behavior of cytidylyltransferase during purification and subsequent analysis suggested that it has hydrophobic properties similar to those exhibited by membrane proteins.

The stimulation and binding of CTP: phosphorylcholine cytidylyltransferase by phosphatidylcholine-oleic acid vesicles.

The activity of the low molecular weight form of cytidylyltransferase from fetal lung cytosol and adult liver cytosol was stimulated more by phosphatidylcholine-oleic acid (1:1 molar ratio) vesicles than by phosphatidylglycerol vesicles. Phosphatidylcholine alone did not stimulate the activity, while oleic acid alone produced only slight stimulation. Vesicles prepared from phosphatidylinositol, phosphatidylglycerol-cholesterol (2:1) and phosphatidylglycerol-phosphatidylcholine (1:1) all stimulated the activity to the same extent. Phosphatidylcholine-oleic acid vesicles (molar ratio 2:1) produced less stimulation than 1:1 vesicles. Phosphatidylcholine-palmitic acid vesicles (2:1) were about 50% as active as the corresponding phosphatidylcholine-oleic acid vesicles. All vesicles were in the size range of small unilamellar vesicles as judged by Sephacryl S-1000 chromatography. Stimulation also occurred when phosphatidylcholine vesicles and oleic acid were added separately to the assay. The stimulation by phospholipid vesicles was correlated with the ability of the vesicles to bind cytidylyltransferase, determined by sucrose density centrifugation of the enzyme-vesicles mixtures. We conclude that the stimulation of soluble cytidylyltransferase occurs through binding of the enzyme to anionic membrane surfaces. Suitable anionic membranes can be prepared either from anionic phospholipids, or by the addition of anionic lipids (unesterified fatty acids or phosphatidylglycerol) to phosphatidylcholine.