Functional characterization of the S41Y (C2755A) polymorphism of tryptophan hydroxylase 2.

Human TPH2 (hTPH2) catalyzes the rate-limiting step in CNS serotonin biosynthesis. We characterized a single-nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased Vmax with unchanged Km values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37 °C) for the S41Y enzyme (as compared to the wild-type enzyme). The S41Y polymorphism decreased cyclic AMP-dependent protein kinase A-mediated phosphorylation ~ 50% relative to wild-type hTPH2, suggesting that the S41Y mutation may disrupt the post-translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y-transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression. We report the functional implications of a polymorphic human tryptophan hydroxylase-2 gene associated with depression and bipolar disorder. The polymorphic enzyme (serine-41 converted to tyrosine) has increased activity, but decreased enzyme stability and serotonin production. Moreover, cyclic AMP-dependent protein kinase (PKA)-mediated phosphorylation of the mutant enzyme is decreased suggesting modified regulation of the S41Y variant leading to altered serotonin.

TPH2 in the ventral tegmental area of the male rat brain.

This study surveyed the distribution of tryptophan hydroxylase 2 (TPH2) mRNA, protein, and enzymatic activity throughout the male Sprague-Dawley rat brain. TPH2 is the genetic isoform of TPH that catalyzes the rate-limiting step in serotonin biosynthesis within the central nervous system. Although cell bodies of serotonergic neurons are located mainly in the raphe, serotonin-containing axons innervate many regions of the brain. In the present study, we assessed the levels of mRNA, protein expression, and enzyme activity of TPH2 in the rat raphe, ventral tegmental area (VTA), substantia nigra, hippocampus, cerebellum, dorsal striatum, nucleus accumbens, amygdala, and medial prefrontal cortex to more fully understand the distribution of this enzyme throughout the central nervous system. The pineal gland was used as a control tissue that expresses TPH1 (the peripheral enzyme), but not TPH2. As expected, the raphe showed the highest brain TPH2 activity and protein expression. In the contrast to other reports, however, the VTA followed the raphe as the region with the second-highest amount of TPH2 activity, mRNA and protein expression. There were significantly lower TPH activities and levels of TPH2 protein in the other regions. In addition, TPH2 immunocytochemistry demonstrated the presence of TPH-positive cell bodies within the VTA. The results of this study indicate that TPH2 and serotonergic signaling may play an important role in the mesolimbic/mesocortical reward pathway.

Robust activation of the human but not mouse telomerase gene during the induction of pluripotency.

Pluripotent stem cells (PSCs) express telomerase and have unlimited proliferative potential. To study telomerase activation during reprogramming, 3 classes of embryonic stem cell (ESC)-like clones were isolated from mouse fibroblasts containing a transgenic hTERT reporter. Class I expressed few pluripotency markers, whereas class II contained many, but not Oct4, Nanog, and Sox2. Neither class of cells differentiated efficiently. Class III cells, the fully reprogrammed induced PSCs (iPSCs), expressed all pluripotency markers, formed teratomas indistinguishable from those of mESCs, and underwent efficient osteogenic differentiation in vitro. Interestingly, whereas the endogenous mTERT gene expression was only moderately increased during reprogramming, the hTERT promoter was strongly activated in class II cells and was further elevated in class III cells. Treatment of class II cells with chemical inhibitors of MEKs and glycogen synthase kinase 3 resulted in their further reprogramming into class III cells, accompanied by a strong activation of hTERT promoter. In reprogrammed human cells, the endogenous telomerase level, although variable among different clones, was dramatically elevated. Only in cells with the highest telomerase were telomeres restored to the lengths in hESCs. Our data, for the first time, demonstrated that the hTERT promoter was strongly activated in discrete steps, revealing a critical difference in human and mouse cell reprogramming. Because telomere elongation is crucial for self-renewal of hPSCs and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPSCs.

SelSA, selenium analogs of SAHA as potent histone deacetylase inhibitors.

Cancer treatment and therapy has moved from conventional chemotherapeutics to more mechanism-based targeted approach. Disturbances in the balance of histone acetyltransferase (HAT) and deacetylase (HDAC) leads to a change in cell morphology, cell cycle, differentiation, and carcinogenesis. In particular, HDAC plays an important role in carcinogenesis and therefore it has been a target for cancer therapy. Structurally diverse group of HDAC inhibitors are known. The broadest class of HDAC inhibitor belongs to hydroxamic acid derivatives that have been shown to inhibit both class I and II HDACs. Suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA), which chelate the zinc ions, fall into this group. In particular, SAHA, second generation HDAC inhibitor, is in several cancer clinical trials including solid tumors and hematological malignancy, advanced refractory leukemia, metastatic head and neck cancers, and advanced cancers. To our knowledge, selenium-containing HDAC inhibitors are not reported in the literature. In order to find novel HDAC inhibitors, two selenium based-compounds modeled after SAHA were synthesized. We have compared two selenium-containing compounds; namely, SelSA-1 and SelSA-2 for their inhibitory HDAC activities against SAHA. Both, SelSA-1 and SelSA-2 were potent HDAC inhibitors; SelSA-2 having IC50 values of 8.9 nM whereas SAHA showed HDAC IC(50) values of 196 nM. These results provided novel selenium-containing potent HDAC inhibitors.

Reptin52 expression during in vitro neural differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) give rise to all somatic cell types, including neural cells such as astrocytes, oligodendrocytes and neurons. Commitment of hESC to a neural fate can be achieved via selection and expansion of developing neural stem cells, which, grown into non-adhering colonies called neurospheres, express nestin, a neurofilament marker. Analysis of hESC and hESC-derived neural stem cell nuclear extracts revealed an increased expression of Reptin52 in neurosphere nuclei. The increase in Reptin52 was evident throughout directed neuronal differentiation as assessed by western blotting, quantitative RT-PCR and immunocytochemistry. Reptin52 serves a pivotal regulatory role in nuclear activities such as transcription regulation and histone modification. In that regard, co-immunoprecipitation experiments showed that binding partners of Reptin52 (Pontin52, beta-catenin and ATF-2) associate with this regulatory protein in hESC-derived neuronal precursors. Moreover, expression of two of these proteins (beta-catenin - the end product of the Wnt signaling pathway - and ATF-2) is coordinately regulated with Reptin52.

Propagation of undifferentiated human embryonic stem cells with nano-liposomal ceramide.

Human embryonic stem (hES) cells, located on the periphery of the colonies, express the neuroectodermal markers nestin and Tuj1, suggesting a prematurely differentiated subgroup of cells. Here, we report that ceramide, a bioactive sphingolipid, selectively eliminates hES cells differentially expressing nestin and Tuj1. In contrast, undifferentiated cells are resistant to the apoptotic effects of ceramide. Ceramide-resistant hES cells express higher levels of the messenger RNA for ceramide-metabolizing enzymes that convert ceramide into pro-mitogenic metabolites. Based on these findings, we conducted long-term studies to determine whether liposomal ceramide can be used to maintain undifferentiated hES cells free of feeder cells. We continuously cultured hES cells on matrigel for 4 months with liposomal ceramide in a feeder cell-free system. Human ES cells treated with liposomal ceramide maintained their pluripotent state as determined by in vivo and in vitro differentiation studies and contained no chromosomal abnormalities. In conclusion, our findings suggest that exposure to ceramide provides a viable strategy to prevent premature hES cell differentiation and to maintain pluripotent stem cell populations in the absence of feeder cells.

Human embryonic and mesenchymal stem cells express different nuclear proteomes.

Human embryonic stem cells (hESCs) are characterized by their immortality and pluripotency. Human mesenchymal stem cells (hMSC), on the other hand, have limited self-renewal and differentiation capabilities. The underlying molecular differences that account for this characteristic self-renewal and plasticity are, however, poorly understood. This study reports a nuclear proteomic analysis of human embryonic and bone marrow-derived mesenchymal stem cells. Our proteomic screen highlighted a 5-fold difference in the expression of Reptin52. We show, using two-dimensional difference gel electrophoresis (2-DIGE), western analysis, and quantitative reverse transcriptase polymerase chain reaction, that Reptin52 is more abundantly expressed in hESC than hMSC. Moreover, we observed differential expression of Pontin52 and beta-catenin-proteins known to interact with Reptin52. This difference in the expression of Reptin52 and Pontin52 (known regulators of beta-catenin) further supports a role for Wnt signaling in stem cell self-renewal and proliferation.

2-D DIGE identification of differentially expressed heterogeneous nuclear ribonucleoproteins and transcription factors during neural differentiation of human embryonic stem cells.

Neural stem cells (NSC) are progenitors that can give rise to all neural lineages. They are found in specific niches of fetal and adult brains and grow in vitro as non-adherent colonies, the neurospheres. These cells express the intermediate filament nestin, commonly considered an NSC marker. NSC can be derived as neurospheres from human embryonic stem cells (hESC). The mechanisms of cellular programming that hESC undergo during differentiation remain obscure. To investigate the commitment process of hESC during directed neural differentiation, we compared the nuclear proteomes of hESC and hESC-derived neurospheres. We used 2-D DIGE to conduct a quantitative comparison of hESC and NSC nuclear proteins and detected 1521 protein spots matched across three gels. Statistical analysis (ANOVA n = 3 with false discovery correction) revealed that only 2.1% of the densitometric signal was significantly changed. The ranges of average ratios varied from 1.2- to 11-fold at a statistically significant p-value <0.05. MS/MS identified 15 regulated proteins previously shown to be involved in chromatin remodeling, mRNA processing and gene expression regulation. Notably, three members of the heterogeneous nuclear ribonucleoprotein family (AUF-1, and FBP-1 and FBP-2) register a 54, 70 and 99% increased expression, highlighting them as potential markers for NSC in vitro derivation. By contrast, Cpsf-6 virtually disappears with differentiation with an 11-fold drop in NSC, highlighting this protein as a novel marker for undifferentiated ESC.

Enhanced nuclear proteomics.

Nuclear proteomics provides an opportunity to examine protein effectors that contribute to cellular phenotype. Both the quality and sensitivity of gel-based nuclear proteomics are limited, however, by the over-representation of histones in the protein mixture. These highly charged proteins overshadow rare species and interfere with IEF. A nuclear isolation and protein extraction procedure, tested on human embryonic stem cells, is reported that effectively isolates intact nuclei and then depletes the sample of histones by taking advantage of their ability to form an insoluble complex with DNA at lower pH (even under denaturing conditions). Ubiquitous histones and abundant nuclear actin, are depleted up to 99 +/- 0.02 and 42 +/- 5%, respectively. This technique greatly improves electrofocusing efficacy and nearly doubles the number of detected protein spots. This approach to nuclear protein isolation for 2-D PAGE opens the door to better investigation of nuclear protein dynamics.

Nuclear proteomics and directed differentiation of embryonic stem cells.

During the past decade, regenerative medicine has been the subject of intense interest due, in large part, to our growing knowledge of embryonic stem (ES) cell biology. ES cells give rise to cell lineages from the three primordial germ layers--endoderm, mesoderm, and ectoderm. This process needs to be channeled if these cells are to be differentiated efficiently and used subsequently for therapeutic purposes. Indeed, an important area of investigation involves directed differentiation to influence the lineage commitment of these pluripotent cells in vitro. Various strategies involving timely growth factor supplementation, cell co-cultures, and gene transfection are used to drive lineage specific emergence. The underlying goal is to control directly the center of gene expression and cellular programming--the nucleus. Gene expression is enabled, managed, and sustained by the collective actions and interactions of proteins found in the nucleus--the nuclear proteome--in response to extracellular signaling. Nuclear proteomics can inventory these nuclear proteins in differentiating cells and decipher their dynamics during cellular phenotypic commitment. This review details what is currently known about nuclear effectors of stem cell differentiation and describes emerging techniques in the discovery of nuclear proteomics that will illuminate new transcription factors and modulators of gene expression.

Functional domains of human tryptophan hydroxylase 2 (hTPH2).

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.

Serotonin neurons derived from rhesus monkey embryonic stem cells: similarities to CNS serotonin neurons.

We sought an in vitro primate model for serotonin neurons. Rhesus monkey embryonic stem (ES) cell colonies were isolated and differentiated into embryoid bodies (EBs), then transferred to serum-free medium with 1% insulin-transferrin-selenium for 7 days to induce neural precursor cell (NPC) formation. NPCs were cultured in medium with 1% N-2 neural supplement and human fibroblast growth factor 2 (FGF2, 10 ng/ml) for 7 days to stimulate cell proliferation. Lastly, NPCs were dispersed into single cells and cultured without FGF2 for another 7 days to obtain terminal differentiation. Terminal cells were characterized for neuronal and serotonergic markers. Over 95% of the NPCs were immunopositive for nestin and Musashi1. Terminally differentiated cells appeared in both small and large morphologies. Most (>95%) of the mature cells (both small and large) were immunopositive for neuron-specific nuclear protein (NeuN), synaptophysin, microtubule-associated protein (MAP2C), Tau-1, neurofilament 160 (NF-160), beta-tubulin (TujIII), tryptophan hydroxylase (TPH), serotonin, the serotonin reuptake transporter (SERT), estrogen receptor-beta (ERbeta), and progestin receptor (PR), but not estrogen receptor-alpha (ERalpha). Less than 2-3% of cells were positive for tyrosine hydroxylase (TH). Reverse transcriptase polymerase chain reaction (RT-PCR) detected mRNA transcripts for TPH-1, TPH-2, SERT, 5-HT1A-autoreceptor, ERbeta, and PR in the differentiated population. A low level of expression of ERalpha mRNA was also detected. Quantitative RT-PCR indicated that the relative abundance of TPH-2 mRNA was greater than TPH-1 mRNA. Serotonin as measured by ELISA increased 3-fold in the mature stage compared to the selection and expansion stages. In summary, a remarkably high percentage of cells derived from monkey ES cells exhibited neuronal plus serotonergic markers as well as nuclear steroid receptors similar to primate CNS serotonin neurons, suggesting that these cells may serve as a useful primate model for serotonergic neurons.