Effects of Low-Dose Gestational TCDD Exposure on Behavior and on Hippocampal Neuron Morphology and Gene Expression in Mice.

BACKGROUND: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and toxic environmental pollutant. Gestational exposure to TCDD has been linked to cognitive and motor deficits, and increased incidence of autism spectrum disorder (ASD) traits in children. Most animal studies of these neurodevelopmental effects involve acute TCDD exposure, which does not model typical exposure in humans. OBJECTIVES: The aim of the study was to establish a dietary low-dose gestational TCDD exposure protocol and performed an initial characterization of the effects on offspring behavior, neurodevelopmental phenotypes, and gene expression. METHODS: Throughout gestation, pregnant C57BL/6J mice were fed a diet containing a low dose of TCDD (9 ng TCDD/kg body weight per day) or a control diet. The offspring were tested in a battery of behavioral tests, and structural brain alterations were investigated by magnetic resonance imaging. The dendritic morphology of pyramidal neurons in the hippocampal Cornu Ammonis (CA)1 area was analyzed. RNA sequencing was performed on hippocampi of postnatal day 14 TCDD-exposed and control offspring. RESULTS: TCDD-exposed females displayed subtle deficits in motor coordination and reversal learning. Volumetric difference between diet groups were observed in regions of the hippocampal formation, mammillary bodies, and cerebellum, alongside higher dendritic arborization of pyramidal neurons in the hippocampal CA1 region of TCDD-exposed females. RNA-seq analysis identified 405 differentially expressed genes in the hippocampus, enriched for genes with functions in regulation of microtubules, axon guidance, extracellular matrix, and genes regulated by SMAD3. DISCUSSION: Exposure to 9 ng TCDD/kg body weight per day throughout gestation was sufficient to cause specific behavioral and structural brain phenotypes in offspring. Our data suggest that alterations in SMAD3-regulated microtubule polymerization in the developing postnatal hippocampus may lead to an abnormal morphology of neuronal dendrites that persists into adulthood. These findings show that environmental low-dose gestational exposure to TCDD can have significant, long-term impacts on brain development and function. https://doi.org/10.1289/EHP7352

MicroRNA-21 dysregulates the expression of MEF2C in neurons in monkey and human SIV/HIV neurological disease

MicroRNAs (miRNAs) have important roles in regulating a plethora of physiological and pathophysiogical processes including neurodegeneration. In both human immunodeficiency virus (HIV)-associated dementia in humans and its monkey model simian immunodeficiency virus encephalitis (SIVE), we find miR-21, a miRNA largely known for its link to oncogenesis, to be significantly upregulated in the brain. In situ hybridization of the diseased brain sections revealed induction of miR-21 in neurons. miR-21 can be induced in neurons by prolonged N-methyl--aspartic acid receptor stimulation, an excitotoxic process active in HIV and other neurodegenerative diseases. Introduction of miR-21 into human neurons leads to pathological functional defects. Furthermore, we show that miR-21 specifically targets the mRNA of myocyte enhancer factor 2C (MEF2C), a transcription factor crucial for neuronal function, and reduces its expression. MEF2C is dramatically downregulated in neurons of HIV-associated dementia patients, as well as monkeys with SIVE. Together, this study elucidates a novel role for miR-21 in the brain, not only as a potential signature of neurological disease, but also as a crucial effector of HIV-induced neuronal dysfunction and neurodegeneration

The intellectual disability risk gene Kdm5b regulates long term memory consolidation in the hippocampus

This is the author accepted manuscript. the final version is available from the Society for Neuroscience via the DOI in this recordData availability: RNAseq data (fastq files) were deposited at the Gene Expression Omnibus (GEO) archive under the accession number GSE240887 and made freely available upon publicationThe histone lysine demethylase KDM5B is implicated in recessive intellectual disability disorders and heterozygous, protein truncating variants in KDM5B are associated with reduced cognitive function in the population. The KDM5 family of lysine demethylases has developmental and homeostatic functions in the brain, some of which appear to be independent of lysine demethylase activity. To determine the functions of KDM5B in hippocampus-dependent learning and memory, we first studied male and female mice homozygous for a Kdm5b ΔARID allele that lacks demethylase activity. Kdm5b ΔARID/ΔARID mice exhibited hyperactivity and long-term memory deficits in hippocampus-dependent learning tasks. The expression of immediate early, activity-dependent genes was downregulated in these mice and hyperactivated upon learning stimulus compared to wildtype mice. A number of other learning-associated genes was also significantly dysregulated in the Kdm5b ΔARID/ΔARID hippocampus. Next, we knocked down Kdm5b specifically in the adult, wildtype mouse hippocampus with shRNA. Kdm5b knockdown resulted in spontaneous seizures, hyperactivity and hippocampus-dependent long-term memory and long-term potentiation deficits. These findings identify KDM5B as a critical regulator of gene expression and synaptic plasticity in the adult hippocampus and suggest that at least some of the cognitive phenotypes associated with KDM5B gene variants are caused by direct effects on memory consolidation mechanisms. Significance statement The histone lysine demethylase KDM5B has been implicated in cognitive performance and intellectual disability conditions in the human population. In the present manuscript we show that mice expressing a demethylase-deficient KDM5B and mice with a specific knockdown of KDM5B in the adult hippocampus exhibit hippocampus-dependent learning and memory phenotypes. Molecular analyses suggest a key role for KDM5B in regulating the dynamic expression of activity-regulated genes during memory consolidation. Deficits in LTP are present in mice with KDM5B knockdown. Together, these findings provide the first evidence for a direct function for KDM5B in memory consolidation in the hippocampus.Medical Research Council (MRC)National Institute of AgingWellcome Trus

The intellectual disability risk gene Kdm5b regulates long term memory consolidation in the hippocampus

The histone lysine demethylase KDM5B is implicated in recessive intellectual disability disorders and heterozygous, protein truncating variants in KDM5B are associated with reduced cognitive function in the population. The KDM5 family of lysine demethylases has developmental and homeostatic functions in the brain, some of which appear to be independent of lysine demethylase activity. To determine the functions of KDM5B in hippocampus-dependent learning and memory, we first studied male and female mice homozygous for a Kdm5bΔARID allele that lacks demethylase activity. Kdm5bΔARID/ΔARID mice exhibited hyperactivity and long-term memory deficits in hippocampus-dependent learning tasks. The expression of immediate early, activity-dependent genes was downregulated in these mice and hyperactivated upon learning stimulus compared to wildtype mice. A number of other learning-associated genes was also significantly dysregulated in the Kdm5bΔARID/ΔARID hippocampus. Next, we knocked down Kdm5b specifically in the adult, wildtype mouse hippocampus with shRNA. Kdm5b knockdown resulted in spontaneous seizures, hyperactivity and hippocampus-dependent long-term memory and long-term potentiation deficits. These findings identify KDM5B as a critical regulator of gene expression and synaptic plasticity in the adult hippocampus and suggest that at least some of the cognitive phenotypes associated with KDM5B gene variants are caused by direct effects on memory consolidation mechanisms. Significance statement The histone lysine demethylase KDM5B has been implicated in cognitive performance and intellectual disability conditions in the human population. In the present manuscript we show that mice expressing a demethylase-deficient KDM5B and mice with a specific knockdown of KDM5B in the adult hippocampus exhibit hippocampus-dependent learning and memory phenotypes. Molecular analyses suggest a key role for KDM5B in regulating the dynamic expression of activity-regulated genes during memory consolidation. Deficits in LTP are present in mice with KDM5B knockdown. Together, these findings provide the first evidence for a direct function for KDM5B in memory consolidation in the hippocampus

Tenascin-C Enhances Pancreatic Cancer Cell Growth and Motility and Affects Cell Adhesion through Activation of the Integrin Pathway

Background: Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells. Methods: Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition. Results: Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt. Conclusion: TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and migh

Top-Down Feedback in an HMAX-Like Cortical Model of Object Perception Based on Hierarchical Bayesian Networks and Belief Propagation

PubMed ID: 2313976

Improving a Natural CaMKII Inhibitor by Random and Rational Design

CaM-KIIN has evolved to inhibit stimulated and autonomous activity of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) efficiently, selectively, and potently (IC50 ∼100 nM). The CN class of peptides, derived from the inhibitory region of CaM-KIIN, provides powerful new tools to study CaMKII functions. The goal of this study was to identify the residues required for CaMKII inhibition, and to assess if artificial mutations could further improve the potency achieved during evolution.First, the minimal region with full inhibitory potency was identified (CN19) by determining the effect of truncated peptides on CaMKII activity in biochemical assays. Then, individual residues of CN19 were mutated. Most individual Ala substitutions decreased potency of CaMKII inhibition, however, P3A, K13A, and R14A increased potency. Importantly, this initial Ala scan suggested a specific interaction of the region around R11 with the CaMKII substrate binding site, which was exploited for further rational mutagenesis to generate an optimized pseudo-substrate sequence. Indeed, the potency of the optimized peptide CN19o was >250fold improved (IC50 <0.4 nM), and CN19o has characteristics of a tight-binding inhibitor. The selectivity for CaMKII versus CaMKI was similarly improved (to almost 100,000fold for CN19o). A phospho-mimetic S12D mutation decreased potency, indicating potential for regulation by cellular signaling. Consistent with importance of this residue in inhibition, most other S12 mutations also significantly decreased potency, however, mutation to V or Q did not.These results provide improved research tools for studying CaMKII function, and indicate that evolution fine-tuned CaM-KIIN not for maximal potency of CaMKII inhibition, but for lower potency that may be optimal for dynamic regulation of signal transduction

A Significant but Rather Mild Contribution of T286 Autophosphorylation to Ca2+/CaM-Stimulated CaMKII Activity

Autophosphorylation of the Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) at T286 generates partially Ca(2+)/CaM-independent "autonomous" activity, which is thought to be required for long-term potentiation (LTP), a form of synaptic plasticity thought to underlie learning and memory. A requirement for T286 autophosphorylation also for efficient Ca(2+)/CaM-stimulated CaMKII activity has been described, but remains controversial.In order to determine the contribution of T286 autophosphorylation to Ca(2+)/CaM-stimulated CaMKII activity, the activity of CaMKII wild type and its phosphorylation-incompetent T286A mutant was compared. As the absolute activity can vary between individual kinase preparations, the activity was measured in six different extracts for each kinase (expressed in HEK-293 cells). Consistent with measurements on purified kinase (from a baculovirus/Sf9 cell expression system), CaMKII T286A showed a mildly but significantly reduced rate of Ca(2+)/CaM-stimulated phosphorylation for two different peptide substrates (to ~75-84% of wild type). Additional slower CaMKII autophosphorylation at T305/306 inhibits stimulation by Ca(2+)/CaM, but occurs only minimally for CaMKII wild type during CaM-stimulated activity assays. Thus, we tested if the T286A mutant may show more extensive inhibitory autophosphorylation, which could explain its reduced stimulated activity. By contrast, inhibitory autophosphorylation was instead found to be even further reduced for the T286A mutant under our assay conditions. On a side note, the phospho-T305 antibody showed some basal background immuno-reactivity also with non-phosphorylated CaMKII, as indicated by T305/306A mutants.These results indicate that Ca(2+)/CaM-stimulated CaMKII activity is mildly (~1.2-1.3fold) further increased by additional T286 autophosphorylation, but that this autophosphorylation is not required for the major part of the stimulated activity. This indicates that the phenotype of CaMKII T286A mutant mice is indeed due to the lack of autonomous activity, as the T286A mutant showed no dramatic reduction in stimulated activity

Differential roles of the Drosophila EMT-inducing transcription factors Snail and Serpent in driving primary tumour growth.

Several transcription factors have been identified that activate an epithelial-to-mesenchymal transition (EMT), which endows cells with the capacity to break through basement membranes and migrate away from their site of origin. A key program in development, in recent years it has been shown to be a crucial driver of tumour invasion and metastasis. However, several of these EMT-inducing transcription factors are often expressed long before the initiation of the invasion-metastasis cascade as well as in non-invasive tumours. Increasing evidence suggests that they may promote primary tumour growth, but their precise role in this process remains to be elucidated. To investigate this issue we have focused our studies on two Drosophila transcription factors, the classic EMT inducer Snail and the Drosophila orthologue of hGATAs4/6, Serpent, which drives an alternative mechanism of EMT; both Snail and GATA are specifically expressed in a number of human cancers, particularly at the invasive front and in metastasis. Thus, we recreated conditions of Snail and of Serpent high expression in the fly imaginal wing disc and analysed their effect. While either Snail or Serpent induced a profound loss of epithelial polarity and tissue organisation, Serpent but not Snail also induced an increase in the size of wing discs. Furthermore, the Serpent-induced tumour-like tissues were able to grow extensively when transplanted into the abdomen of adult hosts. We found the differences between Snail and Serpent to correlate with the genetic program they elicit; while activation of either results in an increase in the expression of Yorki target genes, Serpent additionally activates the Ras signalling pathway. These results provide insight into how transcription factors that induce EMT can also promote primary tumour growth, and how in some cases such as GATA factors a ‘multi hit’ effect may be achieved through the aberrant activation of just a single gene