The MAPK pathway is important in various biological functions. It is also important in regulating processes associated with gene transcription via different mechanisms such as by phosphorylation of transcription factors, coactivators/corepressors and histone modifier complexes. H3K4 methylation is highly associated with active transcription. Deposition of this mark is catalysed by SET-domain methyltransferases which consists of a WAR complex (WDR5, ASH2L and RBBP5), a catalytic SET-domain protein and other subunits. However, potential links between ERK MAPK signaling and H3K4 methylation in gene expression are not well understood. Thus, the aim of this study was to probe the potential links between these two pathways towards gene-regulatory networks. This study attempted to elucidate their direct functional interaction by studying whether components of the SETD1A complex could be phosphorylated upon ERK activation. Our results showed that the core components of SETD1A complex were not phosphorylated in vivo and in vitro by ERK. Importantly, we reported that at least two splicing variants of RBBP5 exist. ERK-dependent stabilization of exogenous RBBP5 was observed but the mechanism underlying this is unknown. Surprisingly, we found that WAR complex depletion increased the pre-mRNA expression of immediate-early (IE) genes which did not necessarily reflect changes in their mRNA levels. In addition, this occurred in an H3K4me3-independent manner. This regulation is likely to be posttranscriptional that involves pre-mRNA processing events. First, we noticed a decrease of transcription initiation in WAR complex-depleted cells upon ERK activation. Second, depletion of the WAR complex affects the splicing efficiency of FOS and EGR1. Third, RBBP5 occupancy was observed and was significantly reduced upon siRNA-mediated RBBP5 depletion at the coding region and the 3' end of FOS gene. Therefore, we propose that the WAR complex regulates the pre-mRNA processing of IE genes through an interaction between RBBP5 and a splicing factor that has yet to be identified.

No description available.

Um acúmulo de evidências sugere que animais vertebrados são capazes de detectar informações geomagnéticas, dentre eles, podemos citar os mais estudados, o caso clássico do pombo-correio (Columba livia). A descoberta de material biomagnético no epitélio do bico superior de pombos sugere a possibilidade de um mecanismo transdutor de campo magnético situado nessa região. Pelos resultados obtidos em outros contextos, a utilização de genes de resposta imediata (IEGs) combinada com tratamentos disruptivos e tarefas clássicas de orientação é tida como metodologia promissora para se conseguir uma descrição mais compreensiva dos canais sensoriais e dos mecanismos de processamento nervoso envolvidos no comportamento de orientação, entre os quais a putativa magnetocepção. Pela atividade de IEGs, especificamente a expressão da proteína ZENK, avaliamos diferencialmente áreas cerebrais ativadas em pombos expostos ou não a uma estimulação magnética, 1h e 3h após a estimulação. A análise quantitativa (teste T) mostrou um aumento no número de neurônios ZENK positivos na região do córtex pré-piriforme de pombos estimulados (p=0,051) em relação aos controles, não havendo diferença entre os grupos de 1h e 3h (ANOVA, uma via). Esses neurônios estão relacionados ao sistema olfativo, o que reforça a idéia de que esse sistema seja importante no comportamento de orientação mas ao mesmo tempo apresenta a primeira evidência experimental de um possível envolvimento da via olfatória na magnetocepção. Esse resultado indica que é necessário tomar o maior cuidado na interpretação de experimentos comportamentais inibindo a via olfativa, pois, além de causar anosmia, essas manipulações poderiam também lesar mecanismos magnetoceptivos. / Much evidence suggests that vertebrate animals are capable of detecting geomagnetic information, among them, we can cite the best studied classic example of homing pigeon (Columba livia). The discovery of biomagnetic material in the pigeon upper beak suggest the possibility of a magnetoceptor transduction mechanism situated in this area. Because of the results obtained in other contexts, the use of immediate early genes (IEGs), combined with disruptive treatments and classic orientation tasks is a promising tool towards a more accurate description of sensory channels and of neural processing mechanisms involved in orientation behaviour, particularly the putative magnetoception mechanism. Using IEG activity, specifically, the ZENK-immunoreactivity protein, we studied different pigeon brain areas activated after magnetic stimulation, compared to no stimulation, at two periods of latency after the end of the experimental session (1h and 3h). Quantitative analysis (T test) showed an increase in labeling in the prepiriform cortex (CPP) of stimulated pigeons in relatioin to controls (p=0,051), but no difference between 1h and 3h groups (one-way ANOVA). These neurons are related to the olfactory system, reforcing the idea that this system is important for orientation, but at the same time presents the first experimental evidence of a possible participation of olfactory pathways in magnetoception. This result indicates that it is important to take utmost care in interpreting results of behavioural experiments in which olfaction is inhibited, since such manipulations may not only cause anosmia, but also loss of magnetic sensitivity.

Alzheimer's disease (AD) is characterized by anterograde amnesia followed by a progressive decline in cognitive function. Post mortem examination of forebrain tissue from sufferers reveals the presence of extracellular amyloid-beta plaques, intracellular neurofibrillary tangles, activation of glial cells and massive neuron loss. Transgenic mice expressing mutated forms of the amyloid precursor protein (APP) and presenilin-1 (PS1) genes develop neuritic amyloid plaques, glial cell activation and memory deficits, without the formation of intracellular tangles and neurodegeneration. The mechanisms by which these transgenic mice develop mnemonic deficiencies are unclear. Gene expression of aged memory-deficient APP+PS1 mice compared with non-transgenic littermates measured by microarray and subsequent quantitative real-time PCR (qRT-PCR) analysis revealed 6 inducible immediated-early genes (IEGs) and 5 other more stably expressed plasticity-related genes (PRGs) that were significantly down-regulated in amyloid-containing hippocampus, but not down-regulated in amyloid-free cerebellum. Other genes linked to memory remained stably expressed in both regions. Analysis of forebrain AD tissue revealed that all genes measured were down-regulated presumably due to neurodegeneration, while the amyloid-free region maintained stable expression. IEG expression in APP+PS1 mice was sensitive to lower levels of amyloid. However, only in the presence of a substantially larger amyloid burden, when memory deficits reliably persist, were both PRGs and IEGs down-regulated. Importantly, we found that IEG expression was decreased in APP+PS1 mice following exposure to a novel environment, indicating that the induction of these IEGs was impaired, rather than the basal expression of resting mice. Na+/K+ ATPase, an enzyme critical for the maintenance of membrane potential, was identified as a down-regulated PRG. We found that activity of this enzyme was both impaired in the hippocampi of APP+PS1 mice and specifically inhibited by high concentrations of amyloid-beta. Na+/K+ ATPase immunostaining revealed decreased protein in the area surrounding the amyloid plaque, where dystrophic neurites were visible, indicating amyloid may disrupt ionic gradients resulting in neuritic dystrophia. These findings suggest that amyloid accumulation may result in the impairment of IEG induction and disruption of the Na+/K+ ATPase, possibly eliciting the memory loss developed in APP+PS1 mice.

Histone deacetylases (HDAC) 1 and 2 play crucial role in chromatin remodeling and gene expression regimes, as part of multiprotein corepressor complexes. Protein kinase CK2-driven phosphorylation of HDAC1 and 2 regulates their catalytic activities and is required to form the corepressor complexes. Phosphorylation-mediated differential distributions of HDAC1 and 2 complexes in regulatory and coding regions of transcribed genes catalyze the dynamic protein acetylation of histones and other proteins, thereby influence gene expression. During mitosis, highly phosphorylated HDAC1 and 2 heterodimers dissociate and displace from mitotic chromosomes. Our goal was to identify the kinase involved in mitotic phosphorylation of HDAC1 and 2. We postulated that CK2-mediated increased phosphorylation of HDAC1 and 2 leads to dissociation of the heterodimers, and, the mitotic chromosomal exclusions of HDAC1 and 2 are largely due to the displacement of HDAC-associated proteins and transcription factors, which recruit HDACs, from chromosomes during mitosis. We further explored the role of un- or monomodified HDAC1 and 2 complexes in immediate-early genes (IEGs), FOSL1 (FOS-like antigen-1) and MCL1 (Myeloid cell leukemia-1), regulation. Dynamic histone acetylation is an important regulator of these genes that are overexpressed in a number of diseases and cancers. We hypothesized that transcription dependent recruitment of HDAC1 and 2 complexes over the gene body regions plays a regulatory role in transcription and splicing regulation of these genes. We present evidence that CK2-catalyzed increased phosphorylation of HDAC1 and 2 regulates the formation of distinct corepressor complexes containing either HDAC1 or HDAC2 homodimers during mitosis, which might target cellular factors. Furthermore, the exclusion of HDAC-recruiting proteins is the major factor for their displacement from mitotic chromosomes. We further demonstrated that un- or monophosphorylated HDAC1 and 2 are associated with gene body of FOSL1 in a transcription dependent manner. However, HDAC inhibitors prevented FOSL1 activation independently of the nucleosome response pathway, which is required for IEG induction. Interestingly, our mass spectrometry results revealed that HDAC1 and 2 interact with a number of splicing proteins, in particular, with serine/arginine-rich splicing factor 1 (SRSF1). HDAC1 and 2 are co-occupied with SRSF1 over gene body regions of FOSL1 and MCL1, regardless of underlying splicing mechanisms. Using siRNA-mediated knockdown approaches and HDAC inhibitors, we demonstrated that alternative splicing of MCL1 is regulated by RNA-directed localized changes in the histone acetylation levels at the alternative exon. The change in histone acetylation levels correlates with the increased transcription elongation and results in change in MCL1 splicing by exon skipping mechanism. Taken together, our results contribute to further understanding of how the multi-faceted HDAC1 and 2 complexes can be regulated and function in various processes, including, but not limited to, transcription regulation and alternative splicing. This can be an exciting area of future research for therapeutic interventions.

No description available.

Histone deacetylases (HDAC) 1 and 2 play crucial role in chromatin remodeling and gene expression regimes, as part of multiprotein corepressor complexes. Protein kinase CK2-driven phosphorylation of HDAC1 and 2 regulates their catalytic activities and is required to form the corepressor complexes. Phosphorylation-mediated differential distributions of HDAC1 and 2 complexes in regulatory and coding regions of transcribed genes catalyze the dynamic protein acetylation of histones and other proteins, thereby influence gene expression. During mitosis, highly phosphorylated HDAC1 and 2 heterodimers dissociate and displace from mitotic chromosomes. Our goal was to identify the kinase involved in mitotic phosphorylation of HDAC1 and 2. We postulated that CK2-mediated increased phosphorylation of HDAC1 and 2 leads to dissociation of the heterodimers, and, the mitotic chromosomal exclusions of HDAC1 and 2 are largely due to the displacement of HDAC-associated proteins and transcription factors, which recruit HDACs, from chromosomes during mitosis. We further explored the role of un- or monomodified HDAC1 and 2 complexes in immediate-early genes (IEGs), FOSL1 (FOS-like antigen-1) and MCL1 (Myeloid cell leukemia-1), regulation. Dynamic histone acetylation is an important regulator of these genes that are overexpressed in a number of diseases and cancers. We hypothesized that transcription dependent recruitment of HDAC1 and 2 complexes over the gene body regions plays a regulatory role in transcription and splicing regulation of these genes. We present evidence that CK2-catalyzed increased phosphorylation of HDAC1 and 2 regulates the formation of distinct corepressor complexes containing either HDAC1 or HDAC2 homodimers during mitosis, which might target cellular factors. Furthermore, the exclusion of HDAC-recruiting proteins is the major factor for their displacement from mitotic chromosomes. We further demonstrated that un- or monophosphorylated HDAC1 and 2 are associated with gene body of FOSL1 in a transcription dependent manner. However, HDAC inhibitors prevented FOSL1 activation independently of the nucleosome response pathway, which is required for IEG induction. Interestingly, our mass spectrometry results revealed that HDAC1 and 2 interact with a number of splicing proteins, in particular, with serine/arginine-rich splicing factor 1 (SRSF1). HDAC1 and 2 are co-occupied with SRSF1 over gene body regions of FOSL1 and MCL1, regardless of underlying splicing mechanisms. Using siRNA-mediated knockdown approaches and HDAC inhibitors, we demonstrated that alternative splicing of MCL1 is regulated by RNA-directed localized changes in the histone acetylation levels at the alternative exon. The change in histone acetylation levels correlates with the increased transcription elongation and results in change in MCL1 splicing by exon skipping mechanism. Taken together, our results contribute to further understanding of how the multi-faceted HDAC1 and 2 complexes can be regulated and function in various processes, including, but not limited to, transcription regulation and alternative splicing. This can be an exciting area of future research for therapeutic interventions.

Um acúmulo de evidências sugere que animais vertebrados são capazes de detectar informações geomagnéticas, dentre eles, podemos citar os mais estudados, o caso clássico do pombo-correio (Columba livia). A descoberta de material biomagnético no epitélio do bico superior de pombos sugere a possibilidade de um mecanismo transdutor de campo magnético situado nessa região. Pelos resultados obtidos em outros contextos, a utilização de genes de resposta imediata (IEGs) combinada com tratamentos disruptivos e tarefas clássicas de orientação é tida como metodologia promissora para se conseguir uma descrição mais compreensiva dos canais sensoriais e dos mecanismos de processamento nervoso envolvidos no comportamento de orientação, entre os quais a putativa magnetocepção. Pela atividade de IEGs, especificamente a expressão da proteína ZENK, avaliamos diferencialmente áreas cerebrais ativadas em pombos expostos ou não a uma estimulação magnética, 1h e 3h após a estimulação. A análise quantitativa (teste T) mostrou um aumento no número de neurônios ZENK positivos na região do córtex pré-piriforme de pombos estimulados (p=0,051) em relação aos controles, não havendo diferença entre os grupos de 1h e 3h (ANOVA, uma via). Esses neurônios estão relacionados ao sistema olfativo, o que reforça a idéia de que esse sistema seja importante no comportamento de orientação mas ao mesmo tempo apresenta a primeira evidência experimental de um possível envolvimento da via olfatória na magnetocepção. Esse resultado indica que é necessário tomar o maior cuidado na interpretação de experimentos comportamentais inibindo a via olfativa, pois, além de causar anosmia, essas manipulações poderiam também lesar mecanismos magnetoceptivos. / Much evidence suggests that vertebrate animals are capable of detecting geomagnetic information, among them, we can cite the best studied classic example of homing pigeon (Columba livia). The discovery of biomagnetic material in the pigeon upper beak suggest the possibility of a magnetoceptor transduction mechanism situated in this area. Because of the results obtained in other contexts, the use of immediate early genes (IEGs), combined with disruptive treatments and classic orientation tasks is a promising tool towards a more accurate description of sensory channels and of neural processing mechanisms involved in orientation behaviour, particularly the putative magnetoception mechanism. Using IEG activity, specifically, the ZENK-immunoreactivity protein, we studied different pigeon brain areas activated after magnetic stimulation, compared to no stimulation, at two periods of latency after the end of the experimental session (1h and 3h). Quantitative analysis (T test) showed an increase in labeling in the prepiriform cortex (CPP) of stimulated pigeons in relatioin to controls (p=0,051), but no difference between 1h and 3h groups (one-way ANOVA). These neurons are related to the olfactory system, reforcing the idea that this system is important for orientation, but at the same time presents the first experimental evidence of a possible participation of olfactory pathways in magnetoception. This result indicates that it is important to take utmost care in interpreting results of behavioural experiments in which olfaction is inhibited, since such manipulations may not only cause anosmia, but also loss of magnetic sensitivity.

abstract: Schizophrenia, a debilitating neuropsychiatric disorder, affects 1% of the population. This multifaceted disorder is comprised of positive (hallucinations/psychosis), negative (social withdrawal/anhedonia) and cognitive symptoms. While treatments for schizophrenia have advanced over the past few years, high economic burdens are still conferred to society, totaling more than $34 billion in direct annual costs to the United States of America. Thus, a critical need exists to identify the factors that contribute towards the etiology of schizophrenia. This research aimed to determine the interactions between environmental factors and genetics in the etiology of schizophrenia. Specifically, this research shows that the immediate early gene, early growth response 3 (EGR3), which is upregulated in response to neuronal activity, resides at the center of a biological pathway to confer risk for schizophrenia. While schizophrenia-risk proteins including neuregulin 1 (NRG1) and N-methyl-D-aspartate receptors (NMDAR’s) have been identified upstream of EGR3, the downstream targets of EGR3 remain relatively unknown. This research demonstrates that early growth response 3 regulates the expression of the serotonin 2A-receptor (5HT2AR) in the frontal cortex following the physiologic stimulus, sleep deprivation. This effect is translated to the level of protein as 8 hours of sleep-deprivation results in the upregulation of 5HT2ARs, a target of antipsychotic medications. Additional downstream targets were identified following maximal upregulation of EGR3 through electroconvulsive stimulation (ECS). Both brain-derived neurotrophic factor (BDNF) and its epigenetic regulator, growth arrest DNA-damage-inducible 45 beta (GADD45B) are upregulated one-hour following ECS in the hippocampus and require the presence of EGR3. These proteins play important roles in both cellular proliferation and dendritic structural changes. Next, the effects of ECS on downstream neurobiological processes, hippocampal cellular proliferation and dendritic structural changes were examined. Following ECS, hippocampal cellular proliferationwas increased, and dendritic structural changes were observed in both wild-type and early growth response 3 knock-out (Egr3-/-) mice. Effects in the number of dendritic spines and dendritic complexity following ECS were not found to require EGR3. Collectively, these results demonstrate that neuronal activity leads to the regulation of schizophrenia risk proteins by EGR3 and point to a possible molecular mechanism contributing risk for schizophrenia. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2020

No description available.