Category Archives: Cell Cycle

On p300, enhancers and neurodevelopmental disorders.

The P300 or adenovirus E1A-associated cellular p300 transcriptional co-activator protein, is a transcriptional regulator (1, 2). It harbours an intrinsic acetyltransferase activity. Thus, by definition it affects gene transcription by inducing chromatin remodelling close to promoter sites and by providing chromatin accessibility to transcription factors and the transcriptional machinery (1). Moreover, P300 can interact with all four histone types of the nucleosome core i.e., H2A, H2B, H3 and H4 (2). Depending on the context of interactions with its co-regulators, gene transcription can either be upregulated  or downregulated, like in the case of p53 and ACTR respectively (3). By specifically interacting with the phosphorylated form of CREB, it also affects cAMP-gene regulation. Furthermore, the P300 protein has a critical role in embryonic development and neuraldevelopment. This is evident in humans, in the case of p300 mutations that cause loss of function and/or copy number alteration (reductions in copy number). These mutations cause an embryo to develop a condition called, broad thumb-hallux syndrome. Some of the phenotypic features of this syndrome, is craniofacial and limb formation abnormalities and mental retardation, highlighting the importance of p300 function during morphogenesis and neuraldevelopment in humans. The massive amount of genes under P300 control during these critical stages of embryonic development was revealed when Visel et al. (2009)(4), examined P300 binding sites by chromatin immunoprecipitation coupled to parallel massive sequencing (Chip-seq) in mouse embryos. Specifically the authors examined, P300 binding sites, in mouse embryos of embryonic age 11.5 (E11.5). This is an important stage for especially for neuraldevelopment, since in the mouse embryo, this is the stage were the neocortex epithelium initiates to expand by increasing proliferative divisions of radial glia cells (neural progenitors). Binding sites where examined in the forebrain (includes both the neocortex and the ventral telencephalon), the limbs and the midbrain. The sample size was more than 150 (!!!) embryos per tissue. In this case P300 binding was used to predict enhancer areas, since P300 was shown to associate in vitro with enhancer areas.Enhancers are DNA regions which enhance the transcription of a gene. Just for the forebrain, 2,543 P300 binding sites were identified by Chip-seq. Moreover, to examine the correspondence of binding sites to known genes, the authors performed tissue specific microarrays. In the forebrain’s case, they found that for the 885 genes which are overexpressed in the forebrain, 14 % of the identified P300 binding sites are within 101 kb from the promoter. Moreover, the enrichment for P300 binding sites was observed to increase according to the level of overexpression of forebrain-specific genes. The conclusion by the authors was that mapping P300 binding is a very accurate way to detect enhancers. In a relatively recent review, Williamson et al. (2011), provide information around enhancers and how knowing more about them might be useful to understand human disease. For example, quoting the authors and Noonan and McCallion (2010): “almost half of single nucleotide polymorphisms (SNPs) that show statistical associations with common/complex human disease and quantitative traits in genome-wide association studies (GWAS) are within noncoding regions and gene deserts and thus, potentially involve enhancers“. For the case of neurodevelopmental disorders, such as autism and schizophrenia, gaining more insights on how early imbalances in the brain structure come up though gene expression de-regulation, is critical. Achieving such progress, will help to understand how the disorder evolves and establishes in the brain. Also, this information, can hopefully lead us into finding ways to treat and perhaps prevent the disorder from evolving .

P300 Binding Sites in the mouse heart and (on the left ) conservation in rat, human, dog and elephant, by Hardison R.C., Nature Genetics (2010)

We are still a long way from home. Nevertheless, the rapid advancement of next generation sequencing technology (NGS), coupled with the parallel advancement in the computational methods created to explain sequencing and gene expression data, provide significant insights towards steps of progress.

References
1. Wikipedia>p300-CBP
2. GeneCards>p300
3. Li,Q. et al.(2002), Mol.Endo., 16(12),1819-2827.
4. Visel, A., et al.(2009), Nature 457(12), 854-858.
5. Williamson et al. (2011), Dev.Cell., 21(1), 17-19.
6. Noonan, J.P., and McCallion, A.S. (2010), Annu.Rev. Genomics Hum. Genet., 11, 1–23.
7. Hardison, R.C. (2010), Nature Genetics, 42, 734–735.

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Hippocampal Interneurons (PtI)

The information below is taken from Danglot et al. review on hippocampal interneurons, and it can serve as short introduction on the subject.

Introduction

Interneuron hereby signifies a local circuit neuron (unless specified differently), which synthesizes and releases γ-aminobutyric acid (GABA). This type of cell is critical for hippocampal function since it keeps the excitatory glutamatergic component under a yin-yan balance. Accordingly, if the GABA-ergic component is reduced, epileptiform activity develops in the hippocampus, whereas enhancing the GABA-ergic tone impairs hippocampal function. Various types of interneurons are characterized from their axonal projection pattern/target, for example oriens-lacunosum-moleculare cells target the distal apical dendrites of principal cells in the stratum lacunosum-moleculare.

Hippocampal Neurogenesis

Pyramids and other cells

Pioneer work by Bayer and Altman in the rat and of Soriano et al in mice, back in the late 70s and 80s, gave insight in the neurogenetic events taking place in the putative hippocampus. It was recognized that the hippocampal neuroepithelium consists of three distinct components, each  giving birth to different neuronal types. Accordingly, the Ammonic neuroepithelium, gives rise to the pyramidal cells and large neurons in the stratum oriens and radiatum, the Dentate neuroepithelium generates granule cells and stratum moleculare large neurons. Lastly, the Fimbrial glioepithelium generates the fimbria glial cells. There is a specific timeline in the generation of each cell type , which differs among the hippocampal areas, as also within the same area pending on the cell type. Therefore, pyramidal cells of CA3 show a peak in neurogenesis on embryonic day 17 (E17) in the rat, whereas for CA1 pyramids peak neurogenesis is seen on E19.  In mice, CA3 pyramids are generated between E14-E15 and CA1 between  E15-E16 (Soriano et al., 1986, 1989a,b). Generally there is a succession of steps before the pyramids are established in the pyramidal layer: one day folowing their genesis, they migrate in the intermediate plate , a temporary lamina, and the next day they migrate towards the hippocampal plate, taking around four days for CA1 pyramids to reach their lamina and even longer for CA3.Hence, in rat, the CA1 pyramidal layer is obvious around E20 and CA3 on E22. The Dentate Gyrus can be distinguished around E21 since around 85% of the granules are generated postnatally.

Interneurons

Both in rats and mice GABA-ergic interneurons are generated prenatally, around E13-E18 in rat and E11-E17 for mice. Again there are regional differences in the birth time of the interneurons even within one hippocampal area. Hence, like cortical neurons there is a inside-out gradient of interneuron settling in the pyramidal layer, early generated neurons populate the deep positions whereas younger  interneurons pass by them to occupy higher positions.  Moreover, interneurons of the stratum oriens and stratum radiatum (plexiform layers or dendritic layers) are formed before the ones of the stratum pyramidale. In contrast to most CA1 and CA3 interneurons generated between E12-E13, DG interneurons arise later, between E13-E14.

Hippocampal Interneuron Matrix  (or matrices? )

Early tracing studies by Altman and Bayer suggested that interneurons may originate from the roof of the telencephalon. There is still dichotomy as to where the GABA-ergic interneurons originate, but according to Danglot et al., a considerable number of interneurons arise from the subpallial telencephalon (ventral telencephalon) , migrate tangenially ( in contrast to cortical neurons that migrate radially) and populate the hippocampus, striatum and neocortex. The Medial  and  Caudal Ganglionic Eminences  supply the hippocampus with interneurons (MGE: supplies only CA areas and CGE: supplies both CA and DG). These GABA-ergic interneurons are positive for Dlx2 (Dlx1/2 are homeobox genes expressed in the subpallium and have a role in the induction of GABA-ergic interneuron fate). Dlx2 positive cells can be seen on E15.5 in stratum radiatum and on E16.5 in stratum oriens (Pleasure et al. 2000). Due to this early placement in the hippocampus, its has been postulated that interneurons serve as “lighthouses” for incoming pyramidal cells and hippocampal afferents.

References

Danglot, L., Triller, A. & Marty, S. The development of hippocampal interneurons in rodents. Hippocampus 16, 1032-1060 (2006).

Soriano, E., Cobas, A. & Fairen, A. Asynchronism in the neurogenesis of GABAergic and non-GABAergic neurons in the mouse hippocampus. Brain Res 395, 88-92 (1986).

Soriano, E., Cobas, A. & Fairen, A. Neurogenesis of glutamic acid decarboxylase immunoreactive cells in the hippocampus of the mouse. II: Area dentata. J Comp Neurol 281, 603-611 (1989).

Soriano, E., Cobas, A. & Fairen, A. Neurogenesis of glutamic acid decarboxylase immunoreactive cells in the hippocampus of the mouse. I: Regio superior and regio inferior. J Comp Neurol 281, 586-602 (1989).

Pleasure, S.J., et al. Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons. Neuron 28, 727-740 (2000).

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Glucocorticoids and Fetal Development

Figure 1. The word Gluco-corticoids is derived from the Greek word "γλυκός" for sweet and from the Latin word for cortex.

1. What are Glucocorticoids?

Glucocorticoids (from Greek “γλυκός” meaning ”sweet” and cortex) are a class of steroid hormones that are produced in the adrenal cortex. In human the main glucocorticoid (GC) is cortisol, whereas in rodents, the main glucocorticoid  is corticosterone. GC hormones are vital for the homeostatic regulation of the bodily functions, and their actions span a hierarchy of control levels. Accordingly, GCs act on the basic molecular level to control critical functions such the cell cycle, cellular metabolism, viability, synaptic plasticity and the immune response.  At the top of the hierarchical pyramid, GCs fine-tune the stress behavior and cognitive function. It is well-known and as every human has experienced, high levels of GCs caused by stressful situations impair memory. Also, GCs are very important for fetal and brain development.

2. The Glucocorticoid Receptor

GCs bind onto their receptor, the Glucocorticoid Receptor (GR), which is located in the cytoplasm of the cell. GR belongs to the superfamily of the ligand-activated nuclear transcription factors. Hence, in the absence of ligand, GR is located in the cellular cytoplasm in a complex with chaperone proteins such as HSP90, HSP60 and FKBP51 (1). Upon hormone binding, the receptor is shuttled in the nucleus (Fig.2).  There it recognizes and associates with partially pallidromic regulatory DNA areas in target genes, called Glucocorticoid Responsive Elements (i.e GREs) (2,3) . GR can either act as a transcriptional activator, by directly binding onto the promoter of the target gene. Alternatively, GR interacts with other co-factors and causes trans-repression of target genes (1, 4) .  Structure of the GR: GR shares the modular structure common among the steroid receptors. It consists of a variable N-terminal domain (NTD), a highly conserved DNA binding domain with two zinc finger motifs (DBD), a hinge region, and a C-terminal hormone binding domain (5).  The DBD is conserved throughout the members of the nuclear receptorsuperfamily and almostall vertebrate species (6). The hinge region participates in receptor–ligand binding.

Figure 2. The ligated GR can either activate or repress gene transcription. (by Holgate and Polosa, 2008) Figure 2. The ligated GR can either activate or repress gene transcription. (by Holgate and Polosa, 2008)

3. GCs and Adverse Effects on Fetal and Infant Brain Development

GC hormones are potent stimulators of organ maturation. Due to this property,  synthetic GC analogs are vastly used in prenatal medicine.   Specifically,  in pregnancies under the risk of pre-term delivery (10% of pregnancies in North America) .  GC administration is the preferred route followed, to accelerate fetal lung maturation (12,13). Moreover, other clinical conditions of the mother or the fetus make use of synthetic GC administration such as allergies, asthma, and when the fetus is suspected to suffer from  Congenital Adrenal Hyperplasia (CAH) (14). Synthetic GCs are marginally different from their endogenous equivalents and more potent agonists of the GR. Specifically, dexamethasone (DEX) one of the most widely used synthetic GC is a very potent GR agonist. DEX is less sensitive to degradation by 11β-hydroxysteroid dehydrogenase-2  (11β-HSD2) (8, 14); a key enzyme that transforms active GCs to inactive 11 keto-analogs in the fetoplacental area. Therefore, 11β-HSD2 reduces the exposure of the fetus to GCs.

Despite the benefits of DEX, accumulating data suggest a dark side of DEX.  Specifically, clinical follow-up studies have indicated that DEX can have detrimental effects on brain development. Specifically, in infants and school aged children having exposed to DEX around birth, cognitive and behavioral deficits such as reduced IQ, poor social interaction and difficulty in coping with stress have been documented. Moreover, imaging data have pointed to reduced cortical convolution and reduced head circumference of DEX exposed infants in respect to untreated controls. Even more significantly , animal studies on prenatal DEX treatment, have documented that brain development can be permanently impaired by DEX. For example, Uno et al.(1990 and 1994) demonstrated that the size of the  hippocampal structure is reduced by DEX treatment in utero in rhesus macaques (15, 16).

Importantly, the observed impairments in the brain architecture could be the result of changes in different processes. Hence, the formation of the brain can be disrupted by altered migration of neuronal cells,or by impairments in the proliferation of Neural Progenitor Cells from which neurons originate. Accordingly, a series of in vitro studies demonstrated that exposure to GCs inhibits neural progenitor and stem cell proliferation and changes the balance between proliferation and differentiation (17, 18). The anti- proliferative effects of GCs are not limited in embryonic NPCs as increased GCs due to stress, or DEX exposure cause the same effects in adult NPCs of the hippocampus. Moreover, GCs are potent inhibitors of the proliferation of tumor cell lines such as medulloblastoma, neuroblastoma and osteosarcoma cells (17, 18).

4. Conclusion

In the light of the clinical and experimental observations on GC’s  dynamics in the developing brain and NPCs, it  is very important to understand the mechanisms by which they can impair brain development. Results from this line of research could be applied for improving the current GC-based treatments and the mode of DEX use in perinatal medicine.

References

1. Lu, N.Z., et al. International Union of Pharmacology. LXV. The pharmacology and classification of the nuclear receptor superfamily: glucocorticoid, mineralocorticoid, progesterone, and androgen receptors. Pharmacol Rev 58, 782-797 (2006).

2. Strahle, U., Schmid, W. & Schutz, G. Synergistic action of the glucocorticoid receptor with transcription factors. EMBO J 7, 3389-3395 (1988).

3. Strahle, U., Klock, G. & Schutz, G. A DNA sequence of 15 base pairs is sufficient to mediate both glucocorticoid and progesterone induction of gene expression. Proc Natl Acad Sci U S A 84, 7871-7875 (1987).

4. Zanchi, N.E., et al. Glucocorticoids: Extensive physiological actions modulated through multiple mechanisms of gene regulation. Journal of Cellular Physiology 224, 311-315.

5. McMaster, A. & Ray, D.W. Modelling the glucocorticoid receptor and producing therapeutic agents with anti-inflammatory effects but reduced side-effects. Experimental Physiology 92, 299-309 (2007).

6. Stolte, E.H., van Kemenade, B.M., Savelkoul, H.F. & Flik, G. Evolution of glucocorticoid receptors with different glucocorticoid sensitivity. J Endocrinol 190, 17-28 (2006).

7. Cole, T.J., et al. Molecular genetic analysis of glucocorticoid signaling during mouse development. Steroids 60, 93-96 (1995).

8. Speirs, H.J., Seckl, J.R. & Brown, R.W. Ontogeny of glucocorticoid receptor and 11beta-hydroxysteroid dehydrogenase type-1 gene expression identifies potential critical periods of glucocorticoid susceptibility during development. J Endocrinol 181, 105-116 (2004).

9. Noorlander, C.W., De Graan, P.N., Middeldorp, J., Van Beers, J.J. & Visser, G.H. Ontogeny of hippocampal corticosteroid receptors: effects of antenatal glucocorticoids in human and mouse. J Comp Neurol 499, 924-932 (2006).

10. Pryce, C.R. Postnatal ontogeny of expression of the corticosteroid receptor genes in mammalian brains: inter-species and intra-species differences. Brain Res Rev 57, 596-605 (2008).

11. Patel, P.D., et al. Glucocorticoid and mineralocorticoid receptor mRNA expression in squirrel monkey brain. J Psychiatr Res 34, 383-392 (2000).

12. Effect of corticosteroids for fetal maturation on perinatal outcomes. NIH Consensus Development Panel on the Effect of Corticosteroids for Fetal Maturation on Perinatal Outcomes. JAMA 273, 413-418 (1995).

13. Effect of corticosteroids for fetal maturation on perinatal outcomes. NIH Consens Statement 12, 1-24 (1994).

14. Tegethoff, M., Pryce, C. & Meinlschmidt, G. Effects of intrauterine exposure to synthetic glucocorticoids on fetal, newborn, and infant hypothalamic-pituitary-adrenal axis function in humans: a systematic review. Endocr Rev 30, 753-789 (2009).

15.Uno H, Lohmiller L, Thieme C, Kemnitz JW, Engle MJ, Roecker EB et al. Brain damage induced by prenatal exposure to dexamethasone in fetal rhesus macaques. I. Hippocampus. Brain Res Dev Brain Res 1990 May 1; 53(2): 157-167.

16.Uno H, Eisele S, Sakai A, Shelton S, Baker E, DeJesus O et al. Neurotoxicity of glucocorticoids in the primate brain. Horm Behav 1994 Dec; 28(4): 336-348.

17.Glick RD, Medary I, Aronson DC, Scotto KW, Swendeman SL, La Quaglia MP. The effects of serum depletion and dexamethasone on growth and differentiation of human neuroblastoma cell lines. J Pediatr Surg 2000 Mar; 35(3): 465-472.

18. Sundberg M, Savola S, Hienola A, Korhonen L, Lindholm D. Glucocorticoid hormones decrease proliferation of embryonic neural stem cells through ubiquitin-mediated degradation of cyclin D1. J Neurosci 2006 May 17; 26(20): 5402-5410.

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Cell Cycle Regulators Related to the Glucocorticoid Receptor

Eukaryotic cell-cycle, with the relative duration of each phase

G1/S-specific cyclin-D1 (CD1) : The protein encoded by this gene belongs to the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance throughout the cell cycle. Cyclins function as regulators of CDK kinases. Different cyclins exhibit distinct expression and degradation patterns which contribute to the temporal coordination of each mitotic event. CD1 forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. This protein has been shown to interact with tumor suppressor protein Rb which also regulates the expression of CD1.

P21: p21 is a potent cyclin-dependent kinase inhibitor (CKI). The p21 (WAF1) protein binds to and inhibits the activity of cyclinCDK2 or –CDK4 complexes, and thus functions as a regulator of cell cycle progression at G1. The expression of this gene is tightly controlled by the tumor suppressor protein p53, through which this protein mediates the p53-dependent cell cycle G1 phase arrest in response to a variety of stress stimuli. This was a major discovery in the early 1990’s that revealed how cells stop dividing after being exposed to damaging agents such as radiation. In addition to growth arrest, p21 can mediate cellular senescence and one of the ways it was discovered was as a senescent cell-derived inhibitor. The p21(WAF1) protein can also interact with proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory factor, and plays a regulatory role in S phase DNA replication and DNA damage repair. This protein was reported to be specifically cleaved by CASP3-like caspases, which thus leads to a dramatic activation of CDK2, and may be instrumental in the execution of apoptosis following caspase activation. However p21 may inhibit apoptosis and does not induce cell death on its own [4].

P27 or Cyclin-dependent kinase inhibitor 1B belongs to the Cip/Kip family of cyclin dependent kinase (Cdk) inhibitor proteins. The p27 protein binds to and prevents the activation of cyclin ECDK2 or cyclin DCDK4 complexes, and thus controls the cell cycle progression at G1. It is often referred to as a cell cycle inhibitor protein because its major function is to stop or slow down the cell division cycle.  p27Kip1 binds to cyclin D either alone, or when complexed to its catalytic subunit CDK4. In doing so p27Kip1 inhibits the catalytic activity of Cdk4, which means that it prevents Cdk4 from adding phosphate residues to its principal substrate, the retinoblastoma (pRb) protein. Increased levels of the p27Kip1 protein typically cause cells to arrest in the G1 phase of the cell cycle. Likewise, p27Kip1 is able to bind other Cdk proteins when complexed to cyclin subunits such as Cyclin E/Cdk2 and Cyclin A/Cdk2.

Retinoblastoma protein (pRb): pRb prevents the cell from replicating damaged DNA by preventing its progression along the cell cycle through G1 (first gap phase) into S (synthesis phase).[7] pRb binds and inhibits transcription factors of the E2F family, which are composed of dimers of an E2F protein and a DP protein.[8] The transcription activating complexes of E2 promoter-binding–protein-dimerization partners (E2F-DP) can push a cell into S phase.[9][10][11][12][13] As long as E2F-DP is inactivated, the cell remains stalled in the G1 phase. When pRb is bound to E2F, the complex acts as a growth suppressor and prevents progression through the cell cycle [3]. The pRb-E2F/DP complex also attracts a histone deacetylase (HDAC) protein to the chromatin, reducing transcription of S phase promoting factors, further suppressing DNA synthesis.

Source: Wikipedia (links are highlighted)

This list definitely does not cover the whole range of cell-cycle modulators, which have been shown or linked with GR . Albeit, it does include some critical molecules, linked to the antiproliferative effects of glucocorticoids and I intent to gradually expand the list as I go.

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