Tet3 mediates stable glucocorticoid-induced alterations in DNA methylation and Dnmt3a/Dkk1 expression in neural progenitors.

Developmental exposure to excess glucocorticoids (GCs) has harmful neurodevelopmental effects, which include persistent alterations in the differentiation potential of embryonic neural stem cells (NSCs). The mechanisms, however, are largely unknown. Here, we investigated the effects of dexamethasone (Dex, a synthetic GC analog) by MeDIP-like genome-wide analysis of differentially methylated DNA regions (DMRs) in NSCs isolated from embryonic rat cortices. We found that Dex-induced genome-wide DNA hypomethylation in the NSCs in vitro. Similarly, in utero exposure to Dex resulted in global DNA hypomethylation in the cerebral cortex of 3-day-old mouse pups. Dex-exposed NSCs displayed stable changes in the expression of the DNA methyltransferase Dnmt3a, and Dkk1, an essential factor for neuronal differentiation. These alterations were dependent on Tet3 upregulation. In conclusion, we propose that GCs elicit strong and persistent effects on DNA methylation in NSCs with Tet3 playing an essential role in the regulation of Dnmt3a and Dkk1. Noteworthy is the occurrence of similar changes in Dnmt3a and Dkk1 gene expression after exposure to excess GC in vivo.

Recent evolutionary origin within the primate lineage of two pseudogenes with similarity to members of the transforming growth factor-beta superfamily.

Using a search engine called Motifer, we searched the public database of the human genome for genes matching a consensus pattern of cysteine residues derived from members of the transforming growth factor-beta (TGF-beta) superfamily. We identified two genes (named MDF451 and MDF628) that display sequence similarity to members of the TGF-beta superfamily in the arrangement of six conserved cysteine residues. Phylogenetic analyses revealed that MDF451 and MDF628 constitute a distinct subgroup within the TGF-beta superfamily, distantly related to the GDNF subfamily of ligands. Both genes could be identified in several primate species in addition to human, including chimpanzee, gorilla, guereza, and green and gray monkey, but not in rodents or other non-primate mammals, and appear not to be present in the genomes of mouse, rat or zebrafish. RNAs for MDF451 and MDF628 were expressed at low levels within distinct regions of the human central nervous system, including adult cerebellum, adult spinal cord and fetal brain. Despite expression at the RNA level, both genes presented a transcribed upstream stop codon that would prevent translation of the TGF-beta-like reading frame. The coding potential of alternative reading frames was not immediately apparent. The two genes may represent TGF-beta-like pseudogenes that have recently appeared in evolution in a common ancestor of the primate lineage by duplication from a GDNF/TGF-beta-like ancestral gene.

Novel, testis-specific mRNA transcripts encoding N-terminally truncated choline acetyltransferase.

Previous studies reported the presence of choline acetyltransferase (ChAT) mRNA and protein in the mammalian testis. We have now found that none of the ChAT mRNAs produced in the testis is capable of encoding a full-length ChAT protein. Two ChAT cDNAs were isolated from an adult rat testis cDNA library encoding N-terminally truncated ChAT proteins of 450 and 414 amino acids (aa), respectively, the former containing a novel N-terminal extension of 69 residues. Rapid Amplification of cDNA Ends (RACE) analysis revealed a complex pattern of 5' untranslated mRNA termini generated from the ChAT gene locus in the testis, all representing truncated versions of the ChAT enzyme. Two of these proteins were produced in transfected fibroblasts and found to lack ChAT activity. Neither did they show binding to the ChAT substrates, acetyl CoA and choline, in a competition assay. These results indicate that mammalian testis lacks a bona fide ChAT enzyme but expresses truncated ChAT proteins with a possible unique function to the testis.

Cell type-specific regulation of choline acetyltransferase gene expression. Role of the neuron-restrictive silencer element and cholinergic-specific enhancer sequences.

This study demonstrates the presence of positive and negative regulatory elements within a 2336-base pair-long region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells. A 21-base pair-long neuron-restrictive silencer element (NRSE) was identified in the proximal part of this region. This element was recognized by the neuron-restrictive silencer factor (NRSF), previously shown to regulate expression of other neuron-specific genes. The ChAT NRSE was inactive in both cholinergic and non-cholinergic neuronal cells, but repressed expression from a heterologous promoter in non-neuronal cells. Specific deletion of this element allowed ChAT gene promoter activity in non-neuronal cells, and overexpression of NRSF repressed ChAT gene promoter activity in cholinergic cells. The distal part of the ChAT gene promoter showed cholinergic-specific enhancing activity, which stimulated promoter activity in cholinergic cells, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. Thus, at least two kinds of regulatory elements cooperate to direct ChAT gene expression to cholinergic neurons, namely a neuron-restrictive silencer element and a cholinergic-specific enhancer.

Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons.

Engineering cells to secrete growth factors.

The neutrotrophins stimulate survival and differentiation of a range of target neurons. A wealth of evidence suggests that central cholinergic neurons depend on nerve growth factor (NGF) for trophic support. Grafts of NGF-producing cells rescue axotomized basal forebrain cholinergic neurons and reduce cholinergic cell death in the medial septum. Skeletal muscle cells, immortalized from embryonic day 15 (E15) rat embryos for transplantation purposes, were transfected with a human NGF construct and individual clones tested for NGF production by a biological assay using embryonic sympathetic ganglia. Clone RM22 showed a consistent ability to produce human recombinant NGF in high concentration; RM22 cells were grafted to the rat brain, following fimbria-fornix lesions, in order to examine the influence of these cells on basal forebrain cholinergic neurons. The results suggest that implantation of genetically modified cells, engineered by the introduction of expression plasmids or viral constructs to produce NGF or other neurotrophins may have therapeutic applications in rescuing damaged central cholinergic neurons in senile dementia of the Alzheimer type as well as in providing trophic support for chromaffin tissue grafts in Parkinson's disease. Moreover, the use of genetically engineered cells may be used to study the effects of administering tailor-made neurotrophins with novel activity profiles.

Role of variable beta-hairpin loop in determining biological specificities in neurotrophin family.

The neurotrophins are members of a family of structurally and functionally related neurotrophic factors that control the development and maintenance of vertebrate neurons. The crystal structure of nerve growth factor (NGF), the prototypic member of this family, contains three pairs of anti-parallel beta-strands connected by beta-hairpin loops, which contain most of the variable residues among the four neurotrophin proteins. Recently, amino acid residues in these variable loop regions have been implicated in the interaction between NGF and its signal-transducing receptor TrkA. In NGF, residues 40-49 (variable region II) span a very flexible and solvent-accessible beta-hairpin loop that is highly variable between different neurotrophins. To investigate the role of this domain in determining biological specificities in the neurotrophin family, we constructed a series of chimeric molecules by exchanging this variable region among three neurotrophins (i.e. NGF, neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4)) that bind and activate three different Trk receptors (i.e. TrkA, -C, and -B, respectively). The chimeric molecules were analyzed for their ability to activate different Trk receptor subtypes and to promote the survival of subpopulations of peripheral neurons expressing specific types of Trk mRNAs. Exchange of the 40-49 variable loop region between NGF and NT-3 resulted in molecules capable of activating both TrkA and TrkC receptors and of rescuing neurons containing TrkA and TrkC mRNAs, indicating that this loop plays an important role in determining biological specificities in these two neurotrophins. Furthermore, variable region II from NT-4 conferred the ability to differentiate nnr5 PC12-TrkB cells to a chimeric NT-3 molecule that was originally incapable of eliciting a response in these cells. In contrast, exchanges between NGF and NT-4 did not suffice to generate molecules with broader biological specificities, suggesting that other regions in these molecules are also required. Our results support the evolutionary relationships between the three polypeptides deduced from structural comparisons.

Beta-nerve growth factor influences the expression of androgen-binding protein messenger ribonucleic acid in the rat testis.

The effect of infusion of nerve growth factor (NGF) into the rat testis on the expression of androgen-binding protein (ABP) mRNA was studied. A major 1.7-kb and a minor 3.7-kb ABP mRNA were present at all stages of the seminiferous epithelium with maximal levels at stages VIII-XI and the lowest levels at stages IV-VI. Infusion of 15 ng/h of NGF with a mini-osmotic pump for 14 days resulted in a 2-fold increase of ABP mRNA as revealed by Northern blots, whereas the mRNA level of another Sertoli cell protein, urokinase-type plasminogen activator, remained unchanged. Image analysis of autoradiograms obtained by in situ hybridization of sections from treated testes showed a similar increase in APB mRNA compared to noninfused or PBS-infused testes. However, at the cellular level the labeling intensity for ABP mRNA over Sertoli cells of different stages of the seminiferous epithelium was the same in NGF-infused and control testes. This suggests that the increase of ABP mRNA in NGF-infused testes was caused by prolongation of stages VII-VIII with maximal ABP mRNA expression; the suggestion is supported by an increase of 30 percent in frequency of these stages in histological sections from NGF-infused testes.

Stage- and cell-specific expression of cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat seminiferous epithelium.

Expression of mRNAs in the rat testis encoding cyclic AMP (cAMP)-dependent protein kinases (PKAs) was studied. A microdissection method was used to isolate 10 pools of seminiferous tubules representing various stages of the cycle of the seminiferous epithelium in combination with Northern blots and in situ hybridization. The results showed a differential expression of the four isoforms of the regulatory subunits (PKA-R) at various stages of the cycle. RI alpha mRNA was detected at approximately the same levels at all stages while expression of RI beta mRNA was low at stages XIII-III, started to increase at stages IV-V, and reached a maximum at stages VIII-XI. The level of RII alpha mRNA was low at stages II-VI, increased markedly at stage VIIa,b, and reached maximal levels at stages VIIc,d and VIII, followed by a reduced expression at later stages, RII beta mRNA levels increased significantly at stage VI with maximal levels at stages VII and VIII. In situ hybridization of sections from the adult rat testis revealed RI alpha mRNA in the layers of pachytene spermatocytes and round spermatids of all stages. RI beta mRNA was detected over late pachytene spermatocytes and round spermatids of stages VII-XIII. RII alpha mRNA was seen in the layers of round spermatids of stages VII-VIII and elongating spermatids of later stages while RII beta mRNA was detected only in the round spermatid region of stages VII-VIII and in some tubules of stages I-VI. These data show that mRNAs encoding PKA-R are expressed in a stage-specific manner in differentiating male germ cells with different patterns of expression for each subunit; this suggests specific roles for these protein kinases at different times of spermatogenesis.

Expression of beta-nerve growth factor and its receptor in rat seminiferous epithelium: specific function at the onset of meiosis.

beta-Nerve growth factor (NGF) is expressed in spermatogenic cells and has testosterone-downregulated low-affinity receptors on Sertoli cells suggesting a paracrine role in the regulation of spermatogenesis. An analysis of the stage-specific expression of NGF and its low affinity receptor during the cycle of the seminiferous epithelium in the rat revealed NGF mRNA and protein at all stages of the cycle. Tyrosine kinase receptor (trk) mRNA encoding an essential component of the high-affinity NGF receptor was also present at all stages. In contrast, expression of low affinity NGF receptor mRNA was only found in stages VIIcd and VIII of the cycle, the sites of onset of meiosis. The low-affinity NGF receptor protein was present in the plasma membrane of the apical Sertoli cell processes as well as in the basal plasma membrane of these cells at stages VIIcd to XI. NGF was shown to stimulate in vitro DNA synthesis of seminiferous tubule segments with preleptotene spermatocytes at the onset of meiosis while other segments remained nonresponsive. We conclude that NGF is a meiotic growth factor that acts through Sertoli cells.

Developmental and regional expression of basic fibroblast growth factor mRNA in the rat central nervous system.

A cDNA clone encoding rat basic fibroblast growth factor (bFGF) was used as a hybridization probe to determine the level of bFGF mRNA during rat brain development as well as in different adult rat brain regions. In the rat brain, a 3.7kb bFGF mRNA was detected together with lower levels of two minor bFGF mRNA species of 1.8kb and 1.5kb, respectively. The 3.7kb bFGF mRNA was detected in the rat brain already at embryonic day 16, the earliest time point tested. The embryonic brain contained 1.5 to 2 times higher levels of the 3.7kb bFGF mRNA than the adult brain. The amount of the 3.7kb bFGF mRNA in the adult rat brain was approximately 50 times higher than the level of beta-nerve growth factor mRNA in the rat brain. bFGF mRNA was found in all 12 brain regions tested in the adult rat brain with the highest level in colliculi, cerebral cortex, thalamus, and olfactory bulb. The lowest levels were found in pons and medulla oblongata. All three bFGF mRNA species showed the same regional distribution in the brain. In contrast to nerve growth factor mRNA, the level of bFGF mRNA in the neonatal hippocampus was slightly decreased 10 days after a cholinergic denervation by transection of the fimbria-fornix.