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Brain-derived neurotrophic factor (BDNF) and its receptors tropomyosin kinase receptor B (TrkB) and the p75 neurotrophin receptor (p75) are the primary regulators of dendritic growth in the CNS. After being bound by BDNF, TrkB and p75 are endocytosed into endosomes and continue signaling within the cell soma, dendrites, and axons. We studied the functional role of BDNF axonal signaling in cortical neurons derived from different transgenic mice using compartmentalized cultures in microfluidic devices. We found that axonal BDNF increased dendritic growth from the neuronal cell body in a cAMP response element-binding protein (CREB)-dependent manner. These effects were dependent on axonal TrkB but not p75 activity. Dynein-dependent BDNF-TrkB-containing endosome transport was required for long-distance induction of dendritic growth. Axonal signaling endosomes increased CREB and mTOR kinase activity in the cell body, and this increase in the activity of both proteins was required for general protein translation and the expression of Arc, a plasticity-associated gene, indicating a role for BDNF-TrkB axonal signaling endosomes in coordinating the transcription and translation of genes whose products contribute to learning and memory regulation.

Memories are thought to be stored in ensembles of neurons across multiple brain regions. However, whether and how these ensembles are coordinated at the time of learning remains largely unknown. Here, we combined CREB-mediated memory allocation with transsynaptic retrograde tracing to demonstrate that the allocation of aversive memories to a group of neurons in one brain region directly affects the allocation of interconnected neurons in upstream brain regions in a behavioral- and brain region-specific manner in mice. Our analysis suggests that this cross-regional recruitment of presynaptic neurons is initiated by downstream memory neurons through a retrograde mechanism. Together with statistical modeling, our results indicate that in addition to the anterograde flow of information between brain regions, the establishment of interconnected, brain-wide memory traces relies on a retrograde mechanism that coordinates memory ensembles at the time of learning.

Protein kinases are central regulators of numerous cellular processes including growth, cell cycle progression, apoptosis and metabolism by phosphorylation of a plethora of protein substrates. Mitochondria are cell organelles which take the center stage in energy production and are involved in signalling and promoting cell death, among other functions. Deregulated kinase and mitochondrial activities have been shown to participate in the progression of several diseases including cancer. In recent years, kinases have become key targets for therapeutic intervention which is underlined by currently more than 60 FDA-approved kinase inhibitor drugs. Kinases are subject of intensive research and inhibitors are among the most effective drugs in cancer therapy. Interdependencies of kinase functions and mitochondrial activities have been described leading to the question of how perturbation of their activities affects mitochondria allocated functions. In this thesis, the effect of several small molecules targeting kinase functions was analysed using the Oroboros O2ks, the gold-standard for high-resolution respirometry. First, melanoma drugs of key MAPK pathway kinase inhibitors (BRAF: Vemurafenib, PLX8394; MEK: Trametinib; BRAF and MEK Combination: Dabrafenib and Trametinib) were analysed to describe their effects on mitochondrial respiration in the melanoma cell line A375 in a time and dose-dependent manner. Second, mitochondria and kinase profiles of the small bioactive metabolic regulator Spermidine were determined using the colon cancer cell line SW620 and non-cancer human embryonic kidney cell line HEK293T. Third, protein conformation reporters were generated to track dynamic changes of key kinases involved in central signalling cascades. Overall, no strong direct impact of these small molecules on mitochondrial function was revealed. Kinase profiling of Spermidine regarding key cellular signalling kinases (MEK, ERK, CREB, AMPK and Akt) showed no major impact on their ph, Masterarbeit Universität Innsbruck 2022

Dopamine levels in the central nervous system change under pathological conditions such as Parkinson's disease, Huntington's disease, and addiction. Under those pathological conditions, astrocytes become reactive astrocytes characterized by morphological changes and the release of inflammatory cytokines involved in pathogenesis. However, it remains unclear whether dopamine regulates astrocytic morphology and functions. Elucidating these issues will help us to understand the pathogenesis of neurodegenerative diseases caused by abnormal dopamine signaling. In this study, we investigated the effects of dopamine on IL-6 expression and process formation in rat primary cultured astrocytes and acute hippocampal slices. Dopamine increased IL-6 expression in a concentration-dependent manner, and this was accompanied by CREB phosphorylation. The effects of a low dopamine concentration (1 mu M) were inhibited by a D1-like receptor antagonist, whereas the effects of a high dopamine concentration (100 mu M) were inhibited by a beta-antagonist and enhanced by a D2-like receptor antagonist. Furthermore, dopamine (100 mu M) promoted process formation, which was inhibited by a beta-antag-onist and enhanced by both an alpha-antagonist and a D2-like receptor antagonist. In acute hippocampal slices, both a D1-like receptor agonist and beta-agonist changed astrocytic morphology. Together, these results indicate that dopamine promotes IL-6 expression and process formation via D1-like receptors and beta-adrenoceptors. Further-more, bidirectional regulation exists; namely, the effects of D1-like receptors and beta-adrenoceptors were nega-tively regulated by D2-like receptors and alpha(2)-adrenoceptors.

Intracranial mesenchymal tumors with FET-CREB fusions are a recently described group of neoplasms in children and young adults characterized by fusion of a FET family gene (usually EWSR1, but rarely FUS) to a CREB family transcription factor (ATF1, CREB1, or CREM), and have been variously termed intracranial angiomatoid fibrous histiocytoma or intracranial myxoid mesenchymal tumor. The clinical outcomes, histologic features, and genomic landscape are not well defined. Here, we studied 20 patients with intracranial mesenchymal tumors proven to harbor FET-CREB fusion by next-generation sequencing (NGS). The 16 female and four male patients had a median age of 14 years (range 4-70). Tumors were uniformly extra-axial or intraventricular and located at the cerebral convexities (n = 7), falx (2), lateral ventricles (4), tentorium (2), cerebellopontine angle (4), and spinal cord (1). NGS demonstrated that eight tumors harbored EWSR1-ATF1 fusion, seven had EWSR1-CREB1, four had EWSR1-CREM, and one had FUS-CREM. Tumors were uniformly well circumscribed and typically contrast enhancing with solid and cystic growth. Tumors with EWSR1-CREB1 fusions more often featured stellate/spindle cell morphology, mucin-rich stroma, and hemangioma-like vasculature compared to tumors with EWSR1-ATF1 fusions that most often featured sheets of epithelioid cells with mucin-poor collagenous stroma. These tumors demonstrated polyphenotypic immunoprofiles with frequent positivity for desmin, EMA, CD99, MUC4, and synaptophysin, but absence of SSTR2A, myogenin, and HMB45 expression. There was a propensity for local recurrence with a median progression-free survival of 12 months and a median overall survival of greater than 60 months, with three patients succumbing to disease (all with EWSR1-ATF1 fusions). In combination with prior case series, this study provides further insight into intracranial mesenchymal tumors with FET-CREB fusion, which represent a distinct group of CN

Inducing regeneration in injured spinal cord represents one of modern medicine's greatest challenges. Research from a variety of model organisms indicates that Hedgehog (Hh) signaling may be a useful target to drive regeneration. However, the mechanisms of Hh signaling-mediated tissue regeneration remain unclear. Here, we examined Hh signaling during post-amputation tail regeneration in Xenopus laevis larvae. We found that while Smoothened (Smo) activity is essential for proper spinal cord and skeletal muscle regeneration, transcriptional activity of the canonical Hh effector Gli is repressed immediately following amputation, and inhibition of Gli1/2 expression or transcriptional activity has minimal effects on regeneration. In contrast, we demonstrate that protein kinase A is necessary for regeneration of both muscle and spinal cord, in concert with and independent of Smo, respectively, and that its downstream effector CREB is activated in spinal cord following amputation in a Smo-dependent manner. Our findings indicate that non-canonical mechanisms of Hh signaling are necessary for spinal cord and muscle regeneration.

[Background]: DICER1 plays a central role in microRNA biogenesis and functions as a tumor suppressor in thyroid cancer, which is the most frequent endocrine malignancy with a rapidly increasing incidence. Thyroid cancer progression is associated with loss of cell differentiation and reduced expression of thyroid differentiation genes and response to thyrotropin (TSH). Here we investigated whether a molecular link exists between DICER1 and thyroid differentiation pathways. [Methods]: We used bioinformatic tools to search for transcription factor binding sites in the DICER1 promoter. DICER1, NKX2-1, PAX8, and CREB expression levels were evaluated by gene and protein expression in vitro and by interrogation of The Cancer Genome Atlas (TCGA) thyroid cancer data. Transcription factor binding and activity were assayed by chromatin immunoprecipitation, band-shift analysis, and promoter–reporter gene activity. Gene-silencing and overexpression approaches were used to elucidate the functional link between DICER1 and differentiation. [Results]: We identified binding sites for NKX2-1 and CREB within the DICER1 promoter and found that both transcription factors are functional in thyroid cells. TSH induced DICER1 expression in differentiated thyroid cells, at least in part, through the cAMP/PKA/CREB pathway. TCGA analysis revealed a significant positive correlation between CREB and DICER1 expression in human thyroid tumors. NKX2-1 overexpression increased DICER1 promoter activity and expression in vitro, and this was significantly greater in the presence of CREB and/or PAX8. Gain- and loss-of-function assays revealed that DICER1 regulates NKX2-1 expression in thyroid tumor cells and vice versa, thus establishing a positive feedback loop between both proteins. We also found a positive correlation between NKX2-1 and DICER1 expression in human thyroid tumors. DICER1 silencing decreased PAX8 expression and, importantly, the expression and activity of the sodium iodide symporter, whi

[ES] La adolescencia es un período de maduración del cerebro, en el que tienen lugar cambios plásticos y dinámicos, que hacen que el cerebro adolescente se especialice para llevar a cabo funciones superiores. Algunas áreas en las que se producen estos cambios estructurales son el hipocampo y la corteza prefrontal. La correcta maduración del cerebro es importante para llevar a cabo funciones como la memoria y el aprendizaje. Diferentes estudios demuestran la implicación de la autofagia en los procesos de maduración sináptica. La autofagia es un mecanismo celular que elimina material residual o componentes no funcionales y, su principal regulador es mTOR (mammalian target of rapamycin). En el sistema nervioso central, la autofagia es importante para el mantenimiento de las funciones neuronales y, las alteraciones en este proceso participan en enfermedades neurodegenerativas e inflamatorias. Inhibidores de mTOR, como la rapamicina, se han utilizado como tratamiento puesto que disminuyen la inflamación y la progresión de la enfermedad. El alcohol es un compuesto neurotóxico y, se ha demostrado que su abuso durante la adolescencia produce neuroinflamación, alteraciones sinápticas y de mielina, daño neural y, cambios en la memoria y aprendizaje. Hay que destacar, que la autofagia tiene lugar en diferentes regiones cerebrales (ej. corteza prefrontal, hipocampo), donde participa durante la remodelación cerebral hasta llegar al cerebro adulto (Pascual et al.,2019). Estudios del laboratorio de Patología Celular del CIPF han sido pioneros en demostrar que el abuso de alcohol en forma de atracón o “binge drinking” durante la adolescencia altera los procesos de autofagia que se asocian con cambios en los niveles de proteínas sinápticas estructurales excitatorias, como PSD-95 y SHANK3 (Montesinos et al.,2018). Considerando la participación de la autofagia en la plasticidad sináptica y el papel del factor de transcripción CREB (cAMP-responsive element-binding protein) en mecanismo, [EN] Adolescence is an important brain maturation period in which plastic and dynamic processes take place, allowing the adolescent brain to specialize to perform more complex functions. Structural remodeling occurs in a variety of brain regions, including the hippocampus and the prefrontal cortex. The correct brain maturation is critical for a correct development of learning and memory processes. Increasing evidence demonstrates the role of the autophagy machinery in the synaptic pruning. Autophagy is a cellular mechanism that removes residual or dysfunctional components and its main regulator is mTOR (mammalian target of rapamycin). In the central nervous system, autophagy is critical for maintaining several neuronal functions, and dysfunction of this pathway take part in several inflammatory and neurodegenerative diseases. Thus, modulators of mTOR, such as rapamycin, have been used as treatment by reducing the inflammation and ameliorating the progression of the disease. Alcohol is a neurotoxic compound and it has been demonstrated that its abuse in adolescence induces neuroinflammation, synaptic and myelin alterations, neural damage and changes in memory and learning. Indeed, autophagy takes place in different brain regions (e.g., prefrontal cortex, hippocampus), where it participates during brain remodeling until adulthood (Pascual et al., 2019). Studies from the Cell Pathology laboratory of CIPF have been pioneered in proving evidence that alcohol abuse in the form of binge drinking in adolescence alters the autophagy mechanisms associated with changes in the levels of excitatory scaffolding synaptic proteins, PSD-95 and SHANK3 (Montesinos et al., 2018). Taking into consideration the involvement of the autophagy pathway in the synaptic plasticity as well as the role of the transcriptional factor CREB (cAMP-responsive element-binding protein) in mechanisms involved in cognitive processes, the hypothesis of this study is to evaluate whether binge alcohol drinkin

Organ ischemia reperfusion injury (IRI), associated with acute hepatocyte death, remains an unresolved problem in clinical orthotopic liver transplantation (OLT). Autophagy, an intracellular self-digesting progress, is responsible for cell reprograming required to regain post-stress homeostasis. Methods: Here, we analyzed the cytoprotective mechanism of pituitary adenylate cyclase-activating polypeptide (PACAP)-promoted hepatocellular autophagy in a clinically relevant mouse model of extended hepatic cold storage (4 °C UW solution for 20 h) followed by syngeneic OLT. Results: In contrast to 41.7% of liver graft failure by day 7 post-transplant in control group, PACAP treatment significantly improved graft survival (91.7% by day 14), and promoted autophagy-associated regeneration programs in OLT. In parallel in vitro studies, PACAP-enhanced autophagy ameliorated cellular damage (LDH/ALT levels), and diminished necrosis in H2O2-stressed primary hepatocytes. Interestingly, PACAP not only induced nuclear cAMP response element-binding protein (CREB), but also triggered reprogramming factor Kruppel-like factor 4 (KLF4) expression in IR-stressed OLT. Indeed, CREB inhibition attenuated hepatic autophagy and recreated hepatocellular injury in otherwise PACAP-protected livers. Furthermore, CREB inhibition suppressed PACAP-induced KLF4 expression, whereas KLF4 blockade abolished PACAP-promoted autophagy and neutralized PACAP-mediated hepatoprotection both in vivo and in vitro. Conclusion: Current study documents the essential neural regulation of PACAP-promoted autophagy in hepatocellular homeostasis in OLT, which provides the emerging therapeutic principle to combat hepatic IRI in OLT.

Although banned from production for decades, PCBs remain a significant risk to human health. A primary target of concern is the developing brain. Epidemiological studies link PCB exposures in utero or during infancy to increased risk of neuropsychiatric deficits in children. Nonclinical studies of legacy congeners found in PCB mixtures synthesized prior to the ban on PCB production suggest that non-dioxin-like (NDL) congeners are predominantly responsible for the developmental neurotoxicity associated with PCB exposures. Mechanistic studies suggest that NDL PCBs alter neurodevelopment via ryanodine receptor-dependent effects on dendritic arborization. Lightly chlorinated congeners, which were not present in the industrial mixtures synthesized prior to the ban on PCB production, have emerged as contemporary environmental contaminants, but there is a paucity of data regarding their potential developmental neurotoxicity. PCB 11, a prevalent contemporary congener, is found in the serum of children and their mothers, as well as in the serum of pregnant women at increased risk for having a child diagnosed with a neurodevelopmental disorder (NDD). Recent data demonstrates that PCB 11 modulates neuronal morphogenesis via mechanisms that are convergent with and divergent from those implicated in the developmental neurotoxicity of legacy NDL PCBs. This review summarizes these data and discusses their relevance to adverse neurodevelopmental outcomes in humans.

Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Alphaherpesvirinae Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, US3. The functions of US3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV US3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV US3 and identified chicken CREB (chCREB) as a substrate of MDV US3. We show that wild-type MDV US3, but not kinase-dead US3 (US3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using US3 deletion and US3 kinase-dead recombinant MDV, we identified US3-responsive MDV genes during infection and found that the majority of US3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some US3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which is also regulated by US3. Furthermore, biochemical studies demonstrated that MDV US3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, in vitro kinase studies revealed that Meq is a US3 substrate. MDV US3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression.IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. US3 encoded by alphaherpesviruses is a multifunctional kinas

Objective: Pharmacoresistance and the lack of disease-modifying actions of current antiseizure drugs persist as major challenges in the treatment of epilepsy. Experimental models of chemoconvulsant-induced status epilepticus remain the models of choice to discover potential antiepileptogenic drugs, but doubts remain as to the extent to which they model human pathophysiology. The aim of the present study was to compare the molecular landscape of the intra-amygdala kainic acid model of status epilepticus in mice with findings in resected brain tissue from patients with drug-resistant temporal lobe epilepsy (TLE). Methods: Status epilepticus was induced via intra-amygdala microinjection of kainic acid in C57BL/6 mice, and gene expression was analyzed via microarrays in hippocampal tissue at acute and chronic time-points. Results were compared to reference datasets in the intraperitoneal pilocarpine and intrahippocampal kainic acid model and to human resected brain tissue (hippocampus and cortex) from patients with drug-resistant TLE. Results: Intra-amygdala kainic acid injection in mice triggered extensive dysregulation of gene expression that was ~3-fold greater shortly after status epilepticus (2729 genes) when compared to epilepsy (412). Comparison to samples from patients with TLE revealed a particularly high correlation of gene dysregulation during established epilepsy. Pathway analysis found suppression of calcium signaling to be highly conserved across different models of epilepsy and patients. cAMP response element-binding protein (CREB) was predicted as one of the main upstream transcription factors regulating gene expression during acute and chronic phases, and inhibition of CREB reduced seizure severity in the intra-amygdala kainic acid model. Significance: Our findings suggest the intra-amygdala kainic acid model faithfully replicates key molecular features of human drug-resistant TLE and provides potential rational target approaches for disease-modificatio

Overnutrition and metabolic disorders impair cognitive functions through molecular mechanisms still poorly understood. In mice fed with a high fat diet (HFD) we analysed the expression of synaptic plasticity‐related genes and the activation of cAMP response elementbinding protein (CREB)‐brain‐derived neurotrophic factor (BDNF)‐tropomyosin receptor kinase B (TrkB) signalling. We found that a HFD inhibited both CREB phosphorylation and the expression of a set of CREB target genes in the hippocampus. The intranasal administration of neural stem cell (NSC)‐derived exosomes (exo‐NSC) epigenetically restored the transcription of Bdnf, nNOS, Sirt1, Egr3, and RelA genes by inducing the recruitment of CREB on their regulatory sequences. Finally, exo‐NSC administration rescued both BDNF signalling and memory in HFD mice. Collectively, our findings highlight novel mechanisms underlying HFD‐related memory impairment and provide evidence of the potential therapeutic effect of exo‐NSC against metabolic disease‐related cognitive decline

submitted by Georg Zegg, University of Innsbruck, Diplomarbeit, 2019, (VLID)3414372

Radiotherapy is a highly effective tool for the treatment of brain cancer. However, radiation also causes detrimental effects in the healthy tissue, leading to neurocognitive sequelae that compromise the quality of life of brain cancer patients. Despite the recognition of this serious complication, no satisfactory solutions exist at present. Here we investigated the effects of intranasal administration of human mesenchymal stem cells (hMSCs) as a neuroprotective strategy for cranial radiation in mice. Our results demonstrated that intranasally delivered hMSCs promote radiation-induced brain injury repair, improving neurological function. This intervention confers protection against inflammation, oxidative stress, and neuronal loss. hMSC administration reduces persistent activation of damage-induced c-AMP response element-binding signaling in irradiated brains. Furthermore, hMSC treatment did not compromise the survival of glioma-bearing mice. Our findings encourage the therapeutic use of hMSCs as a non-invasive approach to prevent neurological complications of radiotherapy, improving the quality of life of brain tumor patients.

The Golgi organelle duplicates its protein and lipid content to segregate evenly between two daughter cells after mitosis. However, how Golgi biogenesis is regulated during interphase remains largely unknown. Here we show that messenger RNA (mRNA) expression of GOLPH3 and GOLGA2, two genes encoding Golgi proteins, is induced specifically in G1 phase, suggesting a link between cell cycle regulation and Golgi growth. We have examined the role of E2F transcription factors, critical regulators of G1 to S progression of the cell cycle, in the expression of Golgi proteins during interphase. We show that promoter activity for GOLPH3, a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the GOLPH3 promoter region abrogates E2F1-mediated induction of a GOLPH3 luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the GOLPH3 promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of GOLPH3 with small interfering RNA (siRNA) delays the G1 to S transition in synchronized U2OS cells. Taken together, our results reveal a link between cell cycle regulation and Golgi function, and suggest that E2F-mediated regulation of Golgi genes is required for the timely progression of the cell cycle.

Radiotherapy is a highly effective tool for the treatment of brain cancer. However, radiation also causes detrimental effects in the healthy tissue, leading to neurocognitive sequelae that compromise the quality of life of brain cancer patients. Despite the recognition of this serious complication, no satisfactory solutions exist at present. Here we investigated the effects of intranasal administration of human mesenchymal stem cells (hMSCs) as a neuroprotective strategy for cranial radiation in mice. Our results demonstrated that intranasally delivered hMSCs promote radiation-induced brain injury repair, improving neurological function. This intervention confers protection against inflammation, oxidative stress, and neuronal loss. hMSC administration reduces persistent activation of damage-induced c-AMP response element-binding signaling in irradiated brains. Furthermore, hMSC treatment did not compromise the survival of glioma-bearing mice. Our findings encourage the therapeutic use of hMSCs as a non-invasive approach to prevent neurological complications of radiotherapy, improving the quality of life of brain tumor patients.

Synaptic plasticity, the change in number, position, and strength of synapses, requires the synthesis of new cellular components that contribute to changes in synaptic composition, and it has long-been appreciated that there is an important role for de novo transcription in the maintenance of long-term potentiation (LTP). As plasticity-inducing signals are received at synapses that can be hundreds of microns away from the cell’s nucleus, which contains its transcriptional machinery, a signal must be faithfully communicated from synapse to nucleus. CREB-Regulated Transcription Coactivator 1 (CRTC1) acts as a retrograde signaling molecule that travels from stimulated synapses to the nucleus, where it alters gene expression through interactions with bZIP transcription factors such as CREB. CRTC1 has been shown to be necessary for the maintenance of long-term potentiation in the hippocampus, and undergoes dramatic and complex post-translational modifications that correlate with its nuclear transport following synaptic activity. There is little known, however, about the transcriptional targets of CRTC1 after plasticity-induction, and whether it may play a role in modulating specific programs of gene expression during different types of long-term plasticity. To address this question, I first investigated the nuclear translocation of CRTC1 in response to different plasticity-induction protocols. Next, I optimized a Chromatin Immunoprecipitation-sequencing (ChIP-seq) protocol to investigate the genomic targets of CRTC1 following dihydroxyphenylglycine (DHPG)-induced long-term depression (LTD) in CA1 cells of the hippocampus, and found that CRTC1-containing protein complexes occupy loci including transcriptional start sites, promoters, enhancers, gene bodies, and intergenic regions. The genes associated with these loci include, but are not limited to, immediate-early genes, as well as other important neuronal genes. Finally, I conducted ChIP-seq and RNA-seq on a wider set of

Mice lacking a functional Biogenesis of Lysosome-related Organelles Complex 1 (BLOC-1), such as those of the pallid line, display cognitive and behavioural impairments reminiscent of those presented by individuals with intellectual and developmental disabilities. Although disturbances in the sleep/wake cycle are commonly lamented by these individuals, the underlying mechanisms, including the possible role of the circadian timing system, are still unknown. In this paper, we have explored sleep/circadian malfunctions and underlying mechanisms in BLOC-1-deficient pallid mice. These mutants exhibited less sleep behaviour in the beginning of the resting phase than wild-type mice with a more broken sleeping pattern in normal light-dark conditions. Furthermore, the strength of the activity rhythms in the mutants were reduced with significantly more fragmentation and lower precision than in age-matched controls. These symptoms were accompanied by an abnormal preference for the open arm in the elevated plus maze in the day and poor performance in the novel object recognition at night. At the level of the central circadian clock (the suprachiasmatic nucleus, SCN), loss of BLOC-1 caused subtle morphological changes including a larger SCN and increased expression of the relative levels of the clock gene Per2 product during the day but did not affect the neuronal activity rhythms. In the hippocampus, the pallid mice presented with anomalies in the cytoarchitecture of the Dentate Gyrus granule cells, but not in CA1 pyramidal neurones, along with altered PER2 protein levels as well as reduced pCREB/tCREB ratio during the day. Our findings suggest that lack of BLOC-1 in mice disrupts the sleep/wake cycle and performance in behavioural tests associated with specific alterations in cytoarchitecture and protein expression.

The cAMP response element (CRE) binding protein (CREB) is central in the transcription regulation by cAMP, and the CREB-regulated transcriptional coactivators (CRTCs) play critical roles in CREB-mediated transcription activation. Upon stimulation, CRTCs translocate into the nucleus and complex with CREB on CRE promoters to activate target gene transcription. Their physiological importance is underscored by their function in energy balance, long-term memory, longevity and other processes. The CREB binding domain on CRTCs has been mapped, which interacts with the CREB basic leucine zipper domain that also mediates interaction with CRE-containing DNA. We report here crystal structures of a complex containing the CRTC2 CREB binding domain, the CREB basic leucine zipper domain and a CRE-containing DNA. The structures revealed that CRTC and CREB form a 2:2 complex on CRE-containing DNA, and CRTC interacts with both CREB and DNA through highly conserved residues. Structure-guided functional studies revealed that both interactions are crucial for the complex assembly and CREB stabilization on DNA. Interestingly, we found that the CRTC–DNA interaction confers selectivity toward the intrinsic DNA shape, which may play a role in selective transcription activation of the CRE genes. © 2018

BackgroundWe recently identified the human leukocyte immunoglobulin-like receptor B2 (LILRB2) and its mouse ortholog-paired Ig-like receptor (PirB) as receptors for several angiopoietin-like proteins (Angptls). We also demonstrated that PirB is important for the development of acute myeloid leukemia (AML), but exactly how an inhibitory receptor such as PirB can support cancer development is intriguing.ResultsHere, we showed that the activation of Ca (2+)/calmodulin-dependent protein kinases (CAMKs) is coupled with PirB signaling in AML cells. High expression of CAMKs is associated with a poor overall survival probability in patients with AML. Knockdown of CAMKI or CAMKIV decreased human acute leukemia development in vitro and in vivo. Mouse AML cells that are defective in PirB signaling had decreased activation of CAMKs, and the forced expression of CAMK partially rescued the PirB-defective phenotype in the MLL-AF9 AML mouse model. The inhibition of CAMK kinase activity or deletion of CAMKIV significantly slowed AML development and decreased the AML stem cell activity. We also found that CAMKIV acts through the phosphorylation of one of its well-known target (CREB) in AML cells.ConclusionCAMKs are essential for the growth of human and mouse AML. The inhibition of CAMK signaling may become an effective strategy for treating leukemia.

Synaptic vesicle and active zone proteins are required for synaptogenesis. The molecular mechanisms for coordinated synthesis of these proteins are not understood. Using forward genetic screens, we identified the conserved THO nuclear export complex (THOC) as an important regulator of presynapse development in C. elegans dopaminergic neurons. In THOC mutants, synaptic messenger RNAs are retained in the nucleus, resulting in dramatic decrease of synaptic protein expression, near complete loss of synapses, and compromised dopamine function. CRE binding protein (CREB) interacts with THOC to mark synaptic transcripts for efficient nuclear export. Deletion of Thoc5, a THOC subunit, in mouse dopaminergic neurons causes severe defects in synapse maintenance and subsequent neuronal death in the substantia nigra compacta. These cellular defects lead to abrogated dopamine release, ataxia, and animal death. Together, our results argue that nuclear export mechanisms can select specific mRNAs and be a rate-limiting step for neuronal differentiation and survival.

Shank is a post-synaptic scaffolding protein that has many binding partners. Shank mutations and copy number variations (CNVs) are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. It is not known which Shank binding partners are responsible for these defects. Here we show that the C. elegans SHN-1/Shank binds L-type calcium channels and that increased and decreased shn-1 gene dosage alter L-channel current and activity-induced expression of a CRH-1/CREB transcriptional target (gem-4 Copine), which parallels the effects of human Shank copy number variations (CNVs) on Autism spectrum disorders and schizophrenia. These results suggest that an important function of Shank proteins is to regulate L-channel current and activity induced gene expression.

Background: PDE4 cyclic nucleotide phosphodiesterases regulate 3′, 5′ cAMP abundance in the CNS and thereby regulate PKA activity and phosphorylation of CREB, which has been implicated in learning and memory, depression and other functions. The PDE4 isoform PDE4B1 also interacts with the DISC1 protein, implicated in neural development and behavioral disorders. The cellular functions of PDE4B1 have been investigated extensively, but its function(s) in the intact organism remained unexplored. Results: To specifically disrupt PDE4B1, we developed mice that express a PDE4B1-D564A transgene in the hippocampus and forebrain. The transgenic mice showed enhanced phosphorylation of CREB and ERK1/2 in hippocampus. Hippocampal neurogenesis was increased in the transgenic mice. Hippocampal electrophysiological studies showed increased baseline synaptic transmission and enhanced LTP in male transgenic mice. Behaviorally, male transgenic mice showed increased activity in prolonged open field testing, but neither male nor female transgenic mice showed detectable anxiety-like behavior or antidepressant effects in the elevated plus-maze, tail-suspension or forced-swim tests. Neither sex showed any significant differences in associative fear conditioning or showed any demonstrable abnormalities in pre-pulse inhibition. Conclusions: These data support the use of an isoform-selective approach to the study of PDE4B1 function in the CNS and suggest a probable role of PDE4B1 in synaptic plasticity and behavior. They also provide additional rationale and a refined approach to the development of small-molecule PDE4B1-selective inhibitors, which have potential functions in disorders of cognition, memory, mood and affect.

Background: PDE4 cyclic nucleotide phosphodiesterases regulate 3′, 5′ cAMP abundance in the CNS and thereby regulate PKA activity and phosphorylation of CREB, which has been implicated in learning and memory, depression and other functions. The PDE4 isoform PDE4B1 also interacts with the DISC1 protein, implicated in neural development and behavioral disorders. The cellular functions of PDE4B1 have been investigated extensively, but its function(s) in the intact organism remained unexplored. Results: To specifically disrupt PDE4B1, we developed mice that express a PDE4B1-D564A transgene in the hippocampus and forebrain. The transgenic mice showed enhanced phosphorylation of CREB and ERK1/2 in hippocampus. Hippocampal neurogenesis was increased in the transgenic mice. Hippocampal electrophysiological studies showed increased baseline synaptic transmission and enhanced LTP in male transgenic mice. Behaviorally, male transgenic mice showed increased activity in prolonged open field testing, but neither male nor female transgenic mice showed detectable anxiety-like behavior or antidepressant effects in the elevated plus-maze, tail-suspension or forced-swim tests. Neither sex showed any significant differences in associative fear conditioning or showed any demonstrable abnormalities in pre-pulse inhibition. Conclusions: These data support the use of an isoform-selective approach to the study of PDE4B1 function in the CNS and suggest a probable role of PDE4B1 in synaptic plasticity and behavior. They also provide additional rationale and a refined approach to the development of small-molecule PDE4B1-selective inhibitors, which have potential functions in disorders of cognition, memory, mood and affect.

Little is knownabout the functions of downstreamregulatory element antagonist modulator (DREAM) inembryonic stem cells (ESCs). However, DREAM interacts with cAMP response element-binding protein (CREB) in a Ca2+-dependent manner, preventing CREB binding protein (CBP) recruitment. Furthermore, CREB and CBP are involved in maintaining ESC self-renewal and pluripotency. However, a previous knockout study revealed the protective function of DREAMdepletion in brain aging degeneration and that aging is accompanied by a progressive decline in stem cells (SCs) function. Interestingly, we found that DREAM is expressed in different cell types, including human ESCs (hESCs), human adipose-derived stromal cells (hASCs), human bone marrow-derived stromal cells (hBMSCs), and human newborn foreskin fibroblasts (hFFs), and that transitory inhibition of DREAMin hESCs reduces their pluripotency, increasing differentiation.We stipulate that these changes are partly mediated by increased CREB transcriptional activity. Overall, our data indicates that DREAMacts in the regulation of hESC pluripotency and could be a target to promote or prevent differentiation in embryonic cells.

The starvation-inducible coactivator cAMP response element binding protein (CREB)-cAMP-regulated transcription coactivator (Crtc) has been shown to promote starvation resistance in Drosophila by up-regulating CREB target gene expression in neurons, although the underlying mechanism is unclear. We found that Crtc and its binding partner CREB enhance energy homeostasis by stimulating the expression of short neuropeptide F (sNPF), an ortholog of mammalian neuropeptide Y, which we show here is a direct target of CREB and Crtc. Neuronal sNPF was found to promote energy homeostasis via gut enterocyte sNPF receptors, which appear to maintain gut epithelial integrity. Loss of Crtc-sNPF signaling disrupted epithelial tight junctions, allowing resident gut flora to promote chronic increases in antimicrobial peptide (AMP) gene expression that compromised energy balance. Growth on germ-free food reduced AMP gene expression and rescued starvation sensitivity in Crtc mutant flies. Overexpression of Crtc or sNPF in neurons of wild-type flies dampens the gut immune response and enhances starvation resistance. Our results reveal a previously unidentified tolerance defense strategy involving a brain-gut pathway that maintains homeostasis through its effects on epithelial integrity.

Ocular Albinism (OA) is a disease characterized by hypopigmentation of the eyes and misrouting of the optic nerves. It is caused by mutations in the OA1 gene, which encodes a G-protein coupled receptor localized in the retinal pigment epithelium. Previously, microarrays carried out in the Farber lab comparing mRNAs from E15 control and Oa1-/- mouse eyes showed that doublecortin and Creb-binding protein (CBP) were down-regulated in Oa1-/- mice. To further explore if these proteins are involved in the Oa1 signaling cascade, I conducted a series of immunohistochemistry experiments to determine the localization of doublecortin, phosphorylated Creb (pCreb) and CBP in control and Oa1-/- retina/RPE. In addition to confirming their down-regulation in Oa1-/- retinas, my results suggest that doublecortin, pCreb and CBP are indeed involved in the Oa1 signaling cascade and may be related to the misrouting of the optic nerves in Oa1-/- mice and OA patients.

Background: Historically, traditional Chinese medicine has been widely used to treat stroke. Based on the theory of Chinese medicine and the modern pharmacological knowledge of herbal medicines, we have designed a neuroprotective formula called Post-Stroke Rehabilitation (PSR), comprising seven herbs - Astragalus membranaceus (Fisch.) Bunge, Salvia miltiorrhiza Bunge, Paeonia lactiflora Pall., Cassia obtusifolia L., Ligusticum chuanxiong Hort., Angelica sinensis (Oliv.) Diels, and Glycyrrhiza uralensis Fisch. We aim to examine the neuroprotective activity of PSR in vitro and in vivo, and to explore the underlying molecular mechanisms, to better understand its therapeutic effect and to further optimize its efficacy. Methods: PSR extract or vehicle was applied to primary rat neurons to examine their survival effects against N-methyl-D-aspartate (NMDA)- elicited excitotoxicity. Whole-cell patch-clamp recording was conducted to examine the NMDA-induced current in the presence of PSR. ERK- and CREB-activation were revealed by western blot analysis. Furthermore, PSR was tested for CRE promoter activation in neurons transfected with a luciferase reporter. The protective effect of PSR was then studied in the rat middle cerebral artery occlusion (MCAO) model. MCAO rats were either treated with PSR extract or vehicle, and their neurobehavioral deficit and cerebral infarct were evaluated. Statistical differences were analyzed by ANOVA or t-test. Results: PSR prominently reduced the death of cultured neurons caused by NMDA excitotoxicity in a dose-dependent manner, indicating its neuroprotective property. Furthermore, PSR significantly reduced NMDA-evoked current reversibly and activated phosphorylation of ERK and CREB with distinct time courses, with the latter's kinetics slower. PSR also triggered CRE-promoter activity as revealed by the increased expression of luciferase reporter in transfected neurons. PSR effectively reduced cerebral infarct and deficit in neurological beh

Functional activation of the neuronal K+-Cl- co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor beta 2 (TGF-beta 2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-beta 2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl- extrusion. We also identify the signaling pathway from TGF-beta 2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-beta 2-mediated KCC2 trafficking and functional activation. TGF-beta 2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-beta 2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-beta 2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission.

Introduction: Traumatic brain injury is a major cause of mortality and morbidity. We have previously shown that the injectable glucagon-like peptide-1 (GLP-1) analog, liraglutide, significantly improved the outcome in mice after severe traumatic brain injury (TBI). In this study we are interested in the effects of oral treatment of a different class of GLP-1 based therapy, dipeptidyl peptidase IV (DPP-IV) inhibition on mice after TBI. DPP-IV inhibitors reduce the degradation of endogenous GLP-1 and extend circulation of this protective peptide in the bloodstream. This class has yet to be investigated as a potential therapy for TBI. Methods: Mice were administrated once-daily 50 mg/kg of sitagliptin in a Nutella® ball or Nutella® alone throughout the study, beginning 2 days before severe trauma was induced with a stereotactic cryo-lesion. At 2 days post trauma, lesion size was determined. Brains were isolated for immunoblotting for assessment of selected biomarkers for pathology and protection. Results: Sitagliptin treatment reduced lesion size at day 2 post-injury by ~28% (p < 0.05). Calpain-driven necrotic tone was reduced ~2-fold in sitagliptin-treated brains (p < 0.001) and activation of the protective cAMP-response element binding protein (CREB) system was significantly more pronounced (~1.5-fold, p < 0.05). The CREB-regulated, mitochondrial antioxidant protein manganese superoxide dismutase (MnSOD) was increased in sitagliptin-treated mice (p < 0.05). Conversely, apoptotic tone (alpha-spectrin fragmentation, Bcl-2 levels) and the neuroinflammatory markers IL-6, and Iba-1 were not affected by treatment. Conclusions: This study shows, for the first time, that DPP-IV inhibition ameliorates both anatomical and biochemical consequences of TBI and activates CREB in the brain. Moreover, this work supports previous studies suggesting that the effect of GLP-1 analogs in models of brain damage relates to GLP-1 receptor stimulation in a dose-dependent manner.

Mitochondrial dysfunction has been linked to several neurodegenerative diseases such as Parkinson's disease (PD). Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha) is a master gene for mitochondrial biogenesis and has been shown to be neuroprotective in models of PD. In this work we have studied the mechanisms by which peroxisome proliferator-activated receptor-gamma (PPAR gamma) selective agonist N-(2-benzoylphenyl)-O-[2-(methyl-2-pyridinylamino)ethyl]-L-tyrosine hydrate (GW1929) acts on human dopaminergic neurons in culture. Data showed that GW1929 increased the viability of human dopaminergic neurons and protected them against oxidative stress induced by H2O2 and the mitochondrial toxin Rotenone. The enhanced resilience of the neurons was attributed to increased levels of mitochondrial antioxidants and of PGC-1 alpha. GW1929 treatment further increased cell respiration, mitochondrial biogenesis and sirtuin-1 (SIRT1) expression in the human dopaminergic neurons. Phosphorylation of cAMP responsive element-binding protein (CREB) was also robustly increased in GW1929-treated cells. Together these results show that the PPAR gamma agonist GW1929 influences CREB signaling and PGC-1 alpha activities in the human dopaminergic neurons contributing to an increased cell viability. This supports the view that drugs acting on the PPAR gamma-PGC-1 alpha signaling in neurons may have beneficial effects in PD and possible also in other brain disorders. (c) 2015 Elsevier Ltd. All rights reserved.

Adult neurogenesis plays increasingly recognized roles in brain homeostasis and repair and is profoundly affected by energy balance and nutrients. We found that the expression of Hes-1 (hairy and enhancer of split 1) is modulated in neural stem and progenitor cells (NSCs) by extracellular glucose through the coordinated action of CREB (cyclic AMP responsive element binding protein) and Sirt-1 (Sirtuin 1), two cellular nutrient sensors. Excess glucose reduced CREB-activated Hes-1 expression and results in impaired cell proliferation. CREB-deficient NSCs expanded poorly in vitro and did not respond to glucose availability. Elevated glucose also promoted Sirt-1-dependent repression of the Hes-1 promoter. Conversely, in low glucose, CREB replaced Sirt-1 on the chromatin associated with the Hes-1 promoter enhancing Hes-1 expression and cell proliferation. Thus, the glucose-regulated antagonism between CREB and Sirt-1 for Hes-1 transcription participates in the metabolic regulation of neurogenesis.

Little is known about the functions of downstream regulatory element antagonist modulator (DREAM) in embryonic stem cells (ESCs). However, DREAM interacts with cAMP response element-binding protein (CREB) in a Ca-dependent manner, preventing CREB binding protein (CBP) recruitment. Furthermore, CREB and CBP are involved in maintaining ESC self-renewal and pluripotency. However, a previous knockout study revealed the protective function of DREAM depletion in brain aging degeneration and that aging is accompanied by a progressive decline in stem cells (SCs) function. Interestingly, we found that DREAM is expressed in different cell types, including human ESCs (hESCs), human adipose-derived stromal cells (hASCs), human bone marrow-derived stromal cells (hBMSCs), and human newborn foreskin fibroblasts (hFFs), and that transitory inhibition of DREAM in hESCs reduces their pluripotency, increasing differentiation. We stipulate that these changes are partly mediated by increased CREB transcriptional activity. Overall, our data indicates that DREAM acts in the regulation of hESC pluripotency and could be a target to promote or prevent differentiation in embryonic cells.

Robust sleep/wake rhythms are important for health and cognitive function. Unfortunately, many people are living in an environment where their circadian system is challenged by inappropriate meal- or work-times. Here we scheduled food access to the sleep time and examined the impact on learning and memory in mice. Under these conditions, we demonstrate that the molecular clock in the master pacemaker, the suprachiasmatic nucleus (SCN), is unaltered while the molecular clock in the hippocampus is synchronized by the timing of food availability. This chronic circadian misalignment causes reduced hippocampal long term potentiation and total CREB expression. Importantly this mis-timed feeding resulted in dramatic deficits in hippocampal-dependent learning and memory. Our findings suggest that the timing of meals have far-reaching effects on hippocampal physiology and learned behaviour.

Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1) in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules. We identify a nuclear localization signal within CRTC1, and characterize three conserved serine residues whose dephosphorylation is required for nuclear import. Domain analysis reveals that the amino-terminal third of CRTC1 contains all of the signals required for regulated nucleocytoplasmic trafficking. We fuse this region to Dendra2 to generate a reporter construct and perform live-cell imaging coupled with local uncaging of glutamate and photoconversion to characterize the dynamics of stimulus-induced retrograde transport and nuclear accumulation.

Glioblastoma (GBM) is both the most common and malignant type of brain tumour with a median survival of 7.4 months from the date of diagnosis; even following current treatment regimens of resection, radiation and chemotherapy, highlighting the need for a greater understanding of the molecular mechanisms driving tumour biology. The Cyclic-AMP Response Element Binding (CREB) protein is a kinase-inducible transcription factor which sits at a hub of pro-oncogenic signalling pathways. CREB is a critical regulator of normal brain development where it regulates proliferation, differentiation and survival. The phosphorylated form of CREB (pCREB) is overexpressed in several types of cancer where it regulates various aspects of tumour biology but little is known about the role of CREB in GBM. As such, this thesis hypothesises that pCREB is an oncogene in GBM where it is activated by multiple cancer signalling pathways and is essential for the maintenance of tumour biology. To investigate the pathways responsible for CREB activation in GBM and the potential of pCREB to serve as a prognostic biomarker, a combination of immunohistochemistry (IHC) and bioinformatics analysis were undertaken. pCREB was co-expressed in cells along with either pMAPK or pAKT in GBM specimens, implicating the PI3K/AKT and MEK/MAPK pathways in differentially activating CREB. Signalling through a MAPK-CREB axis was associated with cell proliferation whilst an AKT-CREB signalling axis was implicated in invasion. pCREB was not prognostic for survival in GBM however was associated with poor prognosis when analysed in the context of MEK/MAPK signalling. Investigation of the role of CREB in vitro confirmed the role of CREB in regulating proliferation. Knockdown of CREB resulted in slower cell cycle progression and this occurred through the transcriptional regulation of the cell cycle factor Cyclin D1. In addition, CREB was selectively regulated the activity of the PI3K/AKT pathway via a feedback mechanism wh

Sonic hedgehog (Shh) is a morphogenic protein that operates through the Gli transcription factor-dependent canonical pathway to orchestrate normal development of many tissues. Because aberrant levels of Gli activity lead to a wide spectrum of diseases ranging from neurodevelopmental defects to cancer, understanding the regulatory mechanisms of Shh canonical pathway is paramount. During early stages of spinal cord development, Shh specifies neural progenitors through the canonical signaling. Despite persistence of Shh as spinal cord development progresses, Gli activity is switched off by unknown mechanisms. In this study we find that Shh inverts its action on Gli during development. Strikingly, Shh decreases Gli signaling in the embryonic spinal cord by an electrical activity- and cAMP-dependent protein kinase-mediated pathway. The inhibition of Gli activity by Shh operates at multiple levels. Shh promotes cytosolic over nuclear localization of Gli2, induces Gli2 and Gli3 processing into repressor forms, and activates cAMP-responsive element binding protein that in turn represses gli1 transcription. The regulatory mechanisms identified in this study likely operate with different spatiotemporal resolution and ensure effective down-regulation of the canonical Shh signaling as spinal cord development progresses. The developmentally regulated intercalation of electrical activity in the Shh pathway may represent a paradigm for switching from canonical to noncanonical roles of developmental cues during neuronal differentiation and maturation.

Dietary restriction (DR, in the form of reduced calorie intake or alternate fasting with overall normal energy supply) elicits cell protective responses in nearly all tissues and organs including brain, and extends lifespan in a fashion that is conserved from the simplest model organisms to mammals and non-human primates. Importantly, studies on DR promise to reveal novel strategies to prolong healthspan and prevent age-related disorders in human beings. The present review focuses on the neuroprotective actions of DR as demonstrated by accumulating experimental and encouraging albeit still limited clinical and epidemiological data. Following an overview of the most relevant evidence for the benefit of DR on neurodegenerative disorders and brain aging and damage in animals and human beings, the article will address the major mechanisms currently believed to participate in these effects, at a tissue (antiinflammation, enhanced adult neurogenesis and neuronal plasticity) and cellular (autophagy and mitochondrial biogenesis) level. Then it will "zoom-in" on the molecular circuitries (AMPK/mTOR, Sirtuins, CREB/Sirt1) whereby neuronal cells perceive the reduced availability of nutrients and translate this information into protective adaptive responses. As a further development of this aspect, the emerging connection between cell metabolism and chromatin remodeling will be analyzed, together with its relevance for our understanding of how food intake affects neuronal gene expression and brain health.

There is strong biochemical, pharmacological and genetic evidence for the involvement of the endocannabinoid system (ECS) in alcohol dependence. However, the majority of studies have been performed in animal models. The aim of the present study was to assess the state of the CB1 receptor, the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), and the extracellular signal-regulated kinase (ERK) and cyclic-AMP response element-binding protein (CREB) in the post-mortem prefrontal cortex of alcoholic subjects. Experiments were performed in samples from 44 subjects classified in four experimental groups: (1) non-suicidal alcoholic subjects (n=11); (2) suicidal alcoholic subjects (n=11); (3) non-alcoholic suicide victims (n=11); and (4) control subjects (n=11). We did not observe statistically significant differences in CB1 mRNA relative expression among the four experimental groups. Conversely, our results showed an increase in CB1 receptor protein expression in the prefrontal cortex of the suicidal alcoholic group (127.2±7.3%), with no changes in functionality with regard to either G protein activation or the inhibition of adenylyl cyclase. In parallel, alcoholic subjects presented lower levels of MAGL activity, regardless of the cause of death. A significant decrease in the active form of ERK and CREB levels was also observed in both alcoholic groups. Taken together, our data are consistent with a role for the ECS in the neurobiological mechanisms underlying alcoholism. Moreover, the alterations reported here should be of great interest for the therapeutic treatment of this chronic psychiatric disease.

Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) are widespread environmental contaminants linked to neuropsychological dysfunction in children. NDL PCBs increase spontaneous Ca(2+) oscillations in neurons by stabilizing ryanodine receptor (RyR) calcium release channels in the open configuration, which results in CREB-dependent dendritic outgrowth. In this study, we address the question of whether activation of CREB by NDL PCBs also triggers dendritic spine formation. Nanomolar concentrations of PCB 95, a NDL congener with potent RyR activity, significantly increased spine density and the frequency of miniature EPSCs in primary dissociated rat hippocampal cultures coincident with upregulation of miR132. Inhibition of RyR, CREB, or miR132 as well as expression of a mutant p250GAP cDNA construct that is not suppressed by miR132 blocked PCB 95 effects on spines and miniature EPSCs. PCB 95 also induced spine formation via RyR- and miR132-dependent mechanisms in hippocampal slice cultures. These data demonstrate a novel mechanism of PCB developmental neurotoxicity whereby RyR sensitization modulates spine formation and synaptogenesis via CREB-mediated miR132 upregulation, which in turn suppresses the translation of p250GAP, a negative regulator of synaptogenesis. In light of recent evidence implicating miR132 dysregulation in Rett syndrome and schizophrenia, these findings identify NDL PCBs as potential environmental risk factors for neurodevelopmental disorders.

Agomelatine, a novel antidepressant with established clinical efficacy, acts as an agonist of melatonergic MT1 and MT2 receptors and as an antagonist of 5-HT2C receptors. The present study was undertaken to investigate whether chronic treatment with agomelatine would block unpredict-able chronic mild stress (UCMS)-induced cognitive deterioration in mice in passive avoidance (PA), modified elevated plus maze (mEPM), novel object recognition (NOR), and Morris water maze (MWM) tests. Moreover, the effects of stress and agomelatine on brain-derived neurotrophic factor (BDNF) and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) messenger ribonucleic acid (mRNA) levels in the hippocam-pus was also determined using quantitative real-time polymerase chain reaction (RT-PCR). Male inbred BALB/c mice were treated with agomelatine (10 mg/kg, i.p.), melatonin (10 mg/kg), or vehicle daily for five weeks. The results of this study revealed that UCMS-exposed animals exhibited memory deterioration in the PA, mEPM, NOR, and MWM tests. The chronic administration of melatonin had a positive effect in the PA and +mEPM tests, whereas agomelatine had a partial effect. Both agomelatine and melatonin blocked stress-induced impairment in visual memory in the NOR test and reversed spatial learning and memory impairment in the stressed group in the MWM test. Quantitative RT-PCR revealed that CREB and BDNF gene expression levels were downregulated in UCMS-exposed mice, and these alterations were reversed by chronic agomelatine or melatonin treatment. Thus, agomelatine plays an important role in blocking stress-induced hippocampal memory deterioration and activates molecular mechanisms of memory storage in response to a learning experience., non disponibile

Zinc is one of the most important trace elements in our body. Patients suffering from depression show lower serum zinc levels compared to healthy controls. Zincs antagonism to the glutamatergic system seems to be responsible for mood recovery. Recent years have shown that zinc may regulate neurotransmission via the metabotropic GPR39 receptor. Activation of the GPR39-Zn(2+)-sensing receptor (GPR39) triggers diverse neuronal pathways leading to a cAMP-responsive element binding the protein (CREB) expression, which then induces synthesis of the brain-derived neurotrophic factor and, in turn, activation of the Tropomyosin receptor kinase B (TrkB) receptor. In the present study, we investigated the alteration of the GPR39 in different models of depression, such as zinc deficiency and olfactory bulbectomy and in suicide victims. Additionaly, we focused on CREB-BDNF/TrkB under zinc deficient conditions in mice. To demonstrate depressive-like behaviour, a standard and modified forced swim test (FST) was performed. To evaluate expression of GPR39, CREB, BDNF and TrkB, Western Blot analysis was used. Zinc deficient mice and rats showed decreased GPR39 expression in the hippocampus and frontal cortex. A decreased level of hippocampal and cortical GPR39 was also observed in suicide victims. In contrast, increased GPR39 in the hippocampus of olfactory bulbectomized rats was observed. Additionally, we found a decreased expression of CREB, BDNF and TrkB only in the hippocampus of zinc-deficient mice. Our present study demonstrates the associacion of the GPR39 Zn(2+)-sensing receptor in the pathomechanism of depression. Down-regulation of CREB, BDNF, TrkB and GPR39 receptor found under zinc-deficient conditions in the hippocampus, may play an important role in the pathophysiology of mood disorders, since most of patients suffering from depression show lower serum zinc.

SOX3 is one of the earliest neural markers in vertebrates, playing the role in specifying neuronal fate. In this study we have established first functional link between CREB and human SOX3 gene which both have important roles in the nervous system throughout development and in the adulthood. Here we demonstrate both in vitro and in vivo that CREB binds to CRE half-site located -195 to -191 within the human SOX3 promoter. Overexpression studies with CREB or its dominant-negative inhibitor A-CREB indicate that this transcription factor acts as a positive regulator of basal SOX3 gene expression in NT2/D1 cells. This is further confirmed by mutational analysis where mutation of CREB binding site results in reduction of SOX3 promoter activity. Our results point at CREB as a positive regulator of SOX3 gene transcription in NT2/D1 cells, while its contribution to RA induction of SOX3 promoter is not prominent.

Throughout life adult neurogenesis generates new neurons in the dentate gyrus of hippocampus that have a critical role in memory formation. Strategies able to stimulate this endogenous process have raised considerable interest because of their potential use to treat neurological disorders entailing cognitive impairment. We previously reported that mice exposed to extremely low-frequency electromagnetic fields (ELFEFs) showed increased hippocampal neurogenesis. Here we demonstrate that the ELFEF-dependent enhancement of hippocampal neurogenesis improves spatial learning and memory. To gain insights on the molecular mechanisms underlying ELFEFs’ effects we extended our studies to an in vitro model of neural stem cells (NSCs) isolated from the hippocampi of newborn mice. We found that ELFEFs enhanced proliferation and neuronal differentiation of hippocampal NSCs by regulation of epigenetic mechanisms leading to pro-neuronal gene expression. Upon ELFEF stimulation of NSCs, we observed a significant enhancement of expression of the pro-proliferative gene Hes1 and the neuronal determination genes NeuroD1 and Neurogenin1. These events were preceded by increased acetylation of H3K9 and binding of the phosphorylated transcription factor cAMP response element-binding protein (CREB) on the regulatory sequence of these genes. Such ELFEF-dependent epigenetic modifications were prevented by the Cav1-channel blocker nifedipine, and were associated with increased occupancy of CREB binding protein (CBP) to the same loci within the analyzed promoters. Our results unravel the molecular mechanisms underlying the ELFEFs’ ability to improve endogenous neurogenesis, pointing to histone acetylation–related chromatin remodeling as a critical determinant. These findings could pave the way to the development of novel therapeutic approaches in regenerative medicine

Neuropeptide Y (NPY) has been implicated in depression, emotional processing and stress response. Part of this evidence originates from human single-nucleotide polymorphism (SNP) studies. In the present study, we report that a SNP in the rat Npy promoter (C/T; rs105431668) affects in vitro transcription and DNA–protein interactions. Genotyping studies showed that the C-allele of rs105431668 is present in a genetic rat model of depression (Flinders sensitive line; FSL), while the SNP’s T-allele is present in its controls (Flinders resistant line; FRL). In vivo experiments revealed binding of a transcription factor (CREB2) and a histone acetyltransferase (Ep300) only at the SNP locus of the FRL. Accordingly, the FRL had increased hippocampal levels of Npy mRNA and H3K18 acetylation; a gene-activating histone modification maintained by Ep300. Next, based on previous studies showing antidepressant-like effects of physical activity in the FSL, we hypothesized that physical activity may affect Npy’s epigenetic status. In line with this assumption, physical activity was associated with increased levels of Npy mRNA and H3K18 acetylation. Physical activity was also associated with reduced mRNA levels of a histone deacetylase (Hdac5). Conclusively, the rat rs105431668 appears to be a functional Npy SNP that may underlie depression-like characteristics. In addition, the achieved epigenetic reprogramming of Npy provides molecular support for the putative effectiveness of physical activity as a non-pharmacological antidepressant.

While the molecular and cellular mechanisms underlying the acquisition, consolidation, reconsolidation and retrieval of memory have attracted a considerable amount of attention in neuroscience, very little is known about memory allocation, the process that determines which neurons in a network store a given memory. Computationally, it is highly advantageous to be able to correctly "file" and "cross-reference" memories in the brain, and memory allocation provides for this. Previous results from our laboratory have suggested that the transcription factor CREB (cAMP-response element binding protein) has a role in memory allocation; increasing the level of CREB in a subset of neurons increased the probability that these neurons stored a memory. Initially, I explored the functionality of these CREB-biased memory neurons by specifically activating them, utilizing a novel technique called optogenetics. I demonstrated that activation of only the CREB-biased memory neurons is sufficient to elicit recall. This confirms a widely held, yet hitherto largely unproven, belief in the neuroscience community that activating the specific neurons that store a memory will lead to the retrieval of that memory. Although mechanisms for CREB's role in memory allocation have been proposed, to date, none has been validated. Here, I have established that increasing neuronal excitability (the likelihood that a neuron responds to a given input) in a subset of neurons can allocate memories in a way analogous to increasing levels of CREB. These results are highly suggestive that increased neuronal excitability is the mechanism by which CREB allocates memory. This research will advance not only our understanding of CREB's role in memory allocation, it will also elucidate memory mechanisms that could ultimately aid in the development of treatments for disorders thought to be caused by abnormal memory allocation, including post-traumatic stress disorder (PTSD).

The gene encoding the collagen-tailed subunit (ColQ) of acetylcholinesterase (AChE) contains two distinct promoters that drive the production of two ColQ mRNAs, ColQ-1 and ColQ-1a, in slow- and fast-twitch muscles, respectively. ColQ-1a is expressed at the neuromuscular junction (NMJ) in fast-twitch muscle, and this expression depends on trophic factors supplied by motor neurons signaling via a cAMP-dependent pathway in muscle. To further elucidate the molecular basis of ColQ-1a's synaptic expression, here we investigated the expression and localization of cAMP-responsive element binding protein (CREB) at the synaptic and extra-synaptic regions of fast- and slow-twitch muscles from adult rats. The total amount of active, phosphorylated CREB (P-CREB) present in slow-twitch soleus muscle was higher than that in fast-twitch tibialis muscle, but P-CREB was predominantly expressed in the fast-twitch muscle at NMJs. In contrast, P-CREB was detected in both synaptic and extra-synaptic regions of slow-twitch muscle. These results reveal, for the first time, the differential distribution of P-CREB in fast- and slow-twitch muscles, which might support the crucial role of cAMP-dependent signaling in controlling the synapse-specific expression of ColQ-1 a in fast-twitch muscles. (C) 2012 Elsevier Ireland Ltd. All rights reserved.