S100B actions on glial and neuronal cells in the developing brain: an overview.

The S100B is a member of the S100 family of "E" helix-loop- "F" helix structure (EF) hand calcium-binding proteins expressed in diverse glial, selected neuronal, and various peripheral cells, exerting differential effects. In particular, this review compiles descriptions of the detection of S100B in different brain cells localized in specific regions during the development of humans, mice, and rats. Then, it summarizes S100B's actions on the differentiation, growth, and maturation of glial and neuronal cells in humans and rodents. Particular emphasis is placed on S100B regulation of the differentiation and maturation of astrocytes, oligodendrocytes (OL), and the stimulation of dendritic development in serotoninergic and cerebellar neurons during embryogenesis. We also summarized reports that associate morphological alterations (impaired neurite outgrowth, neuronal migration, altered radial glial cell morphology) of specific neural cell groups during neurodevelopment and functional disturbances (slower rate of weight gain, impaired spatial learning) with changes in the expression of S100B caused by different conditions and stimuli as exposure to stress, ethanol, cocaine and congenital conditions such as Down's Syndrome. Taken together, this evidence highlights the impact of the expression and early actions of S100B in astrocytes, OL, and neurons during brain development, which is reflected in the alterations in differentiation, growth, and maturation of these cells. This allows the integration of a spatiotemporal panorama of S100B actions in glial and neuronal cells in the developing brain.

Expression and estrogen regulation of G protein-coupled estrogen receptor in human glioblastoma cells.

Glioblastoma (GB) is the most frequent primary brain tumor with a very poor prognosis. Sex hormones are crucial players in the development of GBs. 17 ß-estradiol (E2) signaling is involved through its corresponding intracellular receptors [estrogen receptor α (ERα) and ß (ERß)] in GB cell proliferation and progression. E2 activates G-protein coupled estrogen receptor (GPER), leading to rapidly occurring effects, independently of gene transcription. GPER activation is involved in tumor progression in various cancer types. Currently, available data concerning the occurrence and role of GPER in GB are very limited. In the present study, it was observed that GPER was expressed in human brain tumor cell lines [U251 (astrocytoma-derived cell line), U87, LN229 and T98 (glioblastoma-derived cell line)]. Immunofluorescence assays revealed that GPER localizes in the plasma membrane, cytoplasm and nucleus. An in silico analysis identified two potential E2 response elements in the promoter region of the GPER gene. E2 increased GPER expression in the U251, U87 and LN229 cell lines. Molecular modeling data derived from in silico analysis predicted the three-dimensional conformation of GPER, and docking analysis identified potential binding sites of E2 and its specific agonist, G1. Taken together, these results indicate that GPER may be differentially expressed in human GB cell lines with E2 possibly upregulating GPER expression. The present study raises further questions about the implications of GPER-mediated E2 signaling in the biology of GBs.

EZH2 Mediates Proliferation, Migration, and Invasion Promoted by Estradiol in Human Glioblastoma Cells.

Glioblastomas (GBM) are the most frequent and aggressive brain tumors. 17ß-estradiol (E2) increases proliferation, migration, and invasion of human GBM cells; however underlying mechanisms are no fully understood. Zeste 2 Enhancer Homologous enzyme (EZH2) is a methyltransferase part of Polycomb 2 repressor complex (PRC2). In GBM, EZH2 is overexpressed and involved in the cell cycle, migration, and invasion processes. We studied the role of EZH2 in the pro-oncogenic actions of E2 in human GBM cells. EZH2 gene silencing and pharmacological inhibition of EZH2 blocked proliferation, migration, and invasion of GBM cells induced by E2. We identified in silico additional putative estrogen response elements (EREs) at the EZH2 promoter, but E2 did not modify EZH2 expression. In silico analysis also revealed that among human GBM samples, EZH2 expression was homogeneous; in contrast, the heterogeneous expression of estrogen receptors (ERs) allowed the classification of the samples into groups. Even in the GBM cluster with high expression of ERs and those of their target genes, the expression of PCR2 target genes did not change. Overall, our data suggest that in GBM cells, pro-oncogenic actions of E2 are mediated by EZH2, without changes in EZH2 expression and by mechanisms that appear to be unrelated to the transcriptional activity of ERs.

Quantitative proteomic analysis of extracellular vesicle subgroups isolated by an optimized method combining polymer-based precipitation and size exclusion chromatography.

The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies.

Insights Into the Proteomic Profiling of Extracellular Vesicles for the Identification of Early Biomarkers of Neurodegeneration.

Extracellular vesicles (EVs) are involved in the development and progression of neurodegenerative diseases, including Alzheimer's and Parkinson's disease. Moreover, EVs have the capacity to modify the physiology of neuronal circuits by transferring proteins, RNA, lipids, and metabolites. The proteomic characterization of EVs (exosomes and microvesicles) from preclinical models and patient samples has the potential to reveal new proteins and molecular networks that affect the normal physiology prior to the appearance of traditional biomarkers of neurodegeneration. Noteworthy, many of the genetic risks associated to the development of Alzheimer's and Parkinson's disease affect the crosstalk between mitochondria, endosomes, and lysosomes. Recent research has focused on determining the role of endolysosomal trafficking in the onset of neurodegenerative diseases. Proteomic studies indicate an alteration of biogenesis and molecular content of EVs as a result of endolysosomal and autophagic dysfunction. In this review, we discuss the status of EV proteomic characterization and their usefulness in discovering new biomarkers for the differential diagnosis of neurodegenerative diseases. Despite the challenges related to the failure to follow a standard isolation protocol and their implementation for a clinical setting, the analysis of EV proteomes has revealed the presence of key proteins with post-translational modifications that can be measured in peripheral fluids.

The Thyrotropin-Releasing Hormone-Degrading Ectoenzyme, a Therapeutic Target?

Thyrotropin releasing hormone (TRH: Glp-His-Pro-NH2) is a peptide mainly produced by brain neurons. In mammals, hypophysiotropic TRH neurons of the paraventricular nucleus of the hypothalamus integrate metabolic information and drive the secretion of thyrotropin from the anterior pituitary, and thus the activity of the thyroid axis. Other hypothalamic or extrahypothalamic TRH neurons have less understood functions although pharmacological studies have shown that TRH has multiple central effects, such as promoting arousal, anorexia and anxiolysis, as well as controlling gastric, cardiac and respiratory autonomic functions. Two G-protein-coupled TRH receptors (TRH-R1 and TRH-R2) transduce TRH effects in some mammals although humans lack TRH-R2. TRH effects are of short duration, in part because the peptide is hydrolyzed in blood and extracellular space by a M1 family metallopeptidase, the TRH-degrading ectoenzyme (TRH-DE), also called pyroglutamyl peptidase II. TRH-DE is enriched in various brain regions but is also expressed in peripheral tissues including the anterior pituitary and the liver, which secretes a soluble form into blood. Among the M1 metallopeptidases, TRH-DE is the only member with a very narrow specificity; its best characterized biological substrate is TRH, making it a target for the specific manipulation of TRH activity. Two other substrates of TRH-DE, Glp-Phe-Pro-NH2 and Glp-Tyr-Pro-NH2, are also present in many tissues. Analogs of TRH resistant to hydrolysis by TRH-DE have prolonged central efficiency. Structure-activity studies allowed the identification of residues critical for activity and specificity. Research with specific inhibitors has confirmed that TRH-DE controls TRH actions. TRH-DE expression by ß2-tanycytes of the median eminence of the hypothalamus allows the control of TRH flux into the hypothalamus-pituitary portal vessels and may regulate serum thyrotropin secretion. In this review we describe the critical evidences that suggest that modification of TRH-DE activity in tanycytes, and/or in other brain regions, may generate beneficial consequences in some central and metabolic disorders and identify potential drawbacks and missing information needed to test these hypotheses.

MicroRNA Expression in the Locus Coeruleus, Entorhinal Cortex, and Hippocampus at Early and Middle Stages of Braak Neurofibrillary Tangle Pathology.

The present study analyzes by RT-qPCR the expression of microRNA (miRNA)-27a-3p, miRNA-124-3p, miRNA-132-3p, and miRNA-143-3p in the locus coeruleus (LC), entorhinal cortex (EC), CA1 region of the hippocampus (CA1), and dentate gyrus (DG) of middle-aged (MA) individuals with no brain lesions and of cases at Braak and Braak stages I-II and II-IV of neurofibrillary tangle (NFT) pathology. The most affected region is the LC in which miRNA-27a-3p, miRNA-124-3p, and miRNA-143-3p show a trend to increase at stages I-II and are significantly up-regulated at stages III-IV when compared with MA. Only miRNA-143-3p is up-regulated in the EC at stages III-IV when compared with MA and with stages I-II. No modifications in the expression levels of miRNA-27a-3p, miRNA-124-3p, miRNA-132-3p, and miRNA-143-3p are found in CA1 at any stage, whereas miRNA-124-3p is significantly down-regulated in DG at stages I-II. Accompanying in situ hybridization reveals miRNA-27a-3p, miRNA-124-3p, and miRNA-143-3 localization in neurons, indicating that changes in miRNA expression are not a direct effect of changes in the numbers of neurons and glial cells. Present observations show for the first time important miRNA de-regulation in the LC at the first stages of NFT. Since the LC is the main noradrenergic input to the cerebral cortex, key regulator of mood and depression, and one of the first nuclei affected in aging and Alzheimer's disease (AD), these findings provide insights for additional study of the LC in aging and AD.

Altered mechanisms of protein synthesis in frontal cortex in Alzheimer disease and a mouse model.

Expression of the nucleolar chaperones nucleolin (NCL) and nucleophosmin (NPM1), upstream binding transcription factor (UBTF), rRNA18S, rRNA28S, and several genes encoding ribosomal proteins (RPs) is decreased in frontal cortex area 8 at advanced stages of Alzheimer's disease (AD). This is accompanied by reduced protein levels of elongation factors eEF1A and eEF2. Changes are more marked in AD cases with rapid course (rpAD), as initiation factor eIF3η is significantly down-regulated and several RP genes up-regulated in rpAD when compared with typical AD. These changes contrast with those seen in APP/PS1 transgenic mice used as a model of AD-like ß-amyloidopathy; Ncl mRNA, rRNA18S, rRNA28S and seven out of fifteen assessed RP genes are up-regulated in APP/PS1 mice aged 20 months; only eEF2 protein levels are reduced in transgenic mice. Our findings show marked altered expression of molecules linked to the protein synthesis machinery from the nucleolus to the ribosome in frontal cortex at terminal stages of AD which differs from that seen in APP/PS1 transgenic mice, thus further suggesting that molecular signals in mouse models do not apply to real human disease counterparts.

Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD.

Altered Machinery of Protein Synthesis in Alzheimer's: From the Nucleolus to the Ribosome.

Ribosomes and protein synthesis have been reported to be altered in the cerebral cortex at advanced stages of Alzheimer's disease (AD). Modifications in the hippocampus with disease progression have not been assessed. Sixty-seven cases including middle-aged (MA) and AD stages I-VI were analyzed. Nucleolar chaperones nucleolin, nucleophosmin and nucleoplasmin 3, and upstream binding transcription factor RNA polymerase I gene (UBTF) mRNAs are abnormally regulated and their protein levels reduced in AD. Histone modifications dimethylated histone H3K9 (H3K9me2) and acetylated histone H3K12 (H3K12ac) are decreased in CA1. Nuclear tau declines in CA1 and dentate gyrus (DG), and practically disappears in neurons with neurofibrillary tangles. Subunit 28 ribosomal RNA (28S rRNA) expression is altered in CA1 and DG in AD. Several genes encoding ribosomal proteins are abnormally regulated and protein levels of translation initiation factors eIF2α, eIF3η and eIF5, and elongation factor eEF2, are altered in the CA1 region in AD. These findings show alterations in the protein synthesis machinery in AD involving the nucleolus, nucleus and ribosomes in the hippocampus in AD some of them starting at first stages (I-II) preceding neuron loss. These changes may lie behind reduced numbers of dendritic branches and reduced synapses of CA1 and DG neurons which cause hippocampal atrophy.