ABSTRACT
Immune system dysfunction has been described in autism spectrum disorder. Here we tested the hypothesis that cerebellar defects are accompanied by immune dysfunction in adult mice lacking the autism-candidate gene Engrailed 2 (En2). Gene ontology analyses revealed that biological processes related to immune function were over-represented in the cerebellar transcriptome of En2-/- mice. Pro-inflammatory molecules and chemokines were reduced in the En2-/- cerebellum compared to controls. Conversely, pro-inflammatory molecules were increased in the peripheral blood of mutant mice. Our results suggest a link between immune dysfunction and cerebellar defects detected in En2-/- mice.
Subject(s)
Autism Spectrum Disorder , Autistic Disorder , Homeodomain Proteins , Nerve Tissue Proteins , Animals , Autistic Disorder/genetics , Cerebellum/immunology , Cerebellum/physiopathology , Homeodomain Proteins/genetics , Mice , Mice, Knockout , Nerve Tissue Proteins/geneticsABSTRACT
We identify a set of common phenotypic modifiers that interact with five independent autism gene orthologs (RIMS1, CHD8, CHD2, WDFY3, ASH1L) causing a common failure of presynaptic homeostatic plasticity (PHP) in Drosophila. Heterozygous null mutations in each autism gene are demonstrated to have normal baseline neurotransmission and PHP. However, PHP is sensitized and rendered prone to failure. A subsequent electrophysiology-based genetic screen identifies the first known heterozygous mutations that commonly genetically interact with multiple ASD gene orthologs, causing PHP to fail. Two phenotypic modifiers identified in the screen, PDPK1 and PPP2R5D, are characterized. Finally, transcriptomic, ultrastructural and electrophysiological analyses define one mechanism by which PHP fails; an unexpected, maladaptive up-regulation of CREG, a conserved, neuronally expressed, stress response gene and a novel repressor of PHP. Thus, we define a novel genetic landscape by which diverse, unrelated autism risk genes may converge to commonly affect the robustness of synaptic transmission.
Subject(s)
Autistic Disorder/genetics , Neuronal Plasticity/genetics , Animals , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Genes/genetics , Genetic Predisposition to Disease/genetics , Homeostasis/genetics , Humans , Mutation/genetics , Risk Factors , Synaptic Transmission/geneticsABSTRACT
Progressive synapse loss is an inevitable and insidious part of age-related neurodegenerative disease. Typically, synapse loss precedes symptoms of cognitive and motor decline. This suggests the existence of compensatory mechanisms that can temporarily counteract the effects of ongoing neurodegeneration. Here, we demonstrate that presynaptic homeostatic plasticity (PHP) is induced at degenerating neuromuscular junctions, mediated by an evolutionarily conserved activity of presynaptic ENaC channels in both Drosophila and mouse. To assess the consequence of eliminating PHP in a mouse model of ALS-like degeneration, we generated a motoneuron-specific deletion of Scnn1a, encoding the ENaC channel alpha subunit. We show that Scnn1a is essential for PHP without adversely affecting baseline neural function or lifespan. However, Scnn1a knockout in a degeneration-causing mutant background accelerated motoneuron loss and disease progression to twice the rate observed in littermate controls with intact PHP. We propose a model of neuroprotective homeostatic plasticity, extending organismal lifespan and health span.
Subject(s)
Epithelial Sodium Channels/metabolism , Homeostasis/physiology , Neuronal Plasticity/physiology , Neuroprotection/physiology , Presynaptic Terminals/metabolism , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Disease Models, Animal , Disease Progression , Drosophila melanogaster , Mice , Mice, Knockout , Neuromuscular Junction/metabolismABSTRACT
Axon regeneration is a necessary step toward functional recovery after spinal cord injury. The AP-1 transcription factor c-Jun has long been known to play an important role in directing the transcriptional response of Dorsal Root Ganglion (DRG) neurons to peripheral axotomy that results in successful axon regeneration. Here we performed ChIPseq for Jun in mouse DRG neurons after a sciatic nerve crush or sham surgery in order to measure the changes in Jun's DNA binding in response to peripheral axotomy. We found that the majority of Jun's injury-responsive changes in DNA binding occur at putative enhancer elements, rather than proximal to transcription start sites. We also used a series of single polypeptide chain tandem transcription factors to test the effects of different Jun-containing dimers on neurite outgrowth in DRG, cortical and hippocampal neurons. These experiments demonstrated that dimers composed of Jun and Atf3 promoted neurite outgrowth in rat CNS neurons as well as mouse DRG neurons. Our work provides new insight into the mechanisms underlying Jun's role in axon regeneration.
Subject(s)
Neuronal Outgrowth , Protein Multimerization , Proto-Oncogene Proteins c-jun/metabolism , Activating Transcription Factor 3/metabolism , Animals , Brain/cytology , Brain/metabolism , Cells, Cultured , Enhancer Elements, Genetic , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Neurons/cytology , Neurons/metabolism , Protein Binding , Rats , Rats, Sprague-DawleyABSTRACT
Histone methyltransferases (HMTs) are present in heterogeneous cell populations within the adult brain including neurogenic niches. Yet the question remains whether loss of HMTs and the resulting changes in histone methylation alter cell fate in a region-specific manner. We utilized stereotaxic injection of Cre recombinant protein into the adult neurogenic niches, the subventricular zone (SVZ) adjacent to the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus. We confirmed that Cre protein was enzymatically active in vivo and recombination events were restricted to the vicinity of injection areas. In this study, we focus on using Cre mediated recombination in mice harboring floxed HMT: enhancer of zeste homolog 2 (EZH2) or suppressor of variegation homolog (Suv4-20h). Injectable Cre protein successfully knocked out either EZH2 or Suv4-20h, allowing assessment of long-term effects in a region-specific fashion. We performed meso-scale imaging and flow cytometry for phenotype analysis and unbiased quantification. We demonstrated that regional loss of EZH2 affects the differentiation paradigm of neural stem progenitor cells as well as the maintenance of stem cell population. We further demonstrated that regional loss of Suv4-20h influences the cell cycle but does not affect stem cell differentiation patterns. Therefore, Cre protein mediated knock-out a given HMT unravel their distinguishable and important roles in adult neurogenic niches. This Cre protein-based approach offers tightly-controlled knockouts in multiple cell types simultaneously for studying diverse regulatory mechanisms and is optimal for region-specific manipulation within complex, heterogeneous brain architectures.
Subject(s)
Aging/physiology , Chromatin/metabolism , Gene Knockout Techniques , Integrases/metabolism , Neurogenesis , Organ Specificity , Recombinant Proteins/metabolism , Animals , Cell Differentiation , Cell Movement , Enhancer of Zeste Homolog 2 Protein/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Homologous Recombination/genetics , Lysine/metabolism , Methylation , Mice , Neurons/metabolism , S PhaseABSTRACT
Engrailed 1 (En1) and 2 (En2) code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.
Subject(s)
Autistic Disorder/metabolism , Dendrites/genetics , Gene Expression Regulation/genetics , Homeodomain Proteins/metabolism , Nerve Tissue Proteins/metabolism , Signal Transduction , Animals , Cells, Cultured , Fluorescent Antibody Technique , Hippocampus/cytology , Hippocampus/metabolism , Homeodomain Proteins/genetics , Mechanistic Target of Rapamycin Complex 1 , Mice , Microscopy, Confocal , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Nerve Tissue Proteins/genetics , Neuroglia/cytology , Neuroglia/metabolism , Synapses/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
High-throughput, target-based screening techniques have been utilized extensively for drug discovery in the past several decades. However, the need for more predictive in vitro models of in vivo disease states has generated a shift in strategy towards phenotype-based screens. Phenotype based screens are particularly valuable in studying complex conditions such as CNS injury and degenerative disease, as many factors can contribute to a specific cellular response. In this review, we will discuss different screening frameworks and their relative utility in examining mechanisms of neurodegeneration and axon regrowth, particularly in cell-based in vitro disease models.
Subject(s)
Drug Discovery , Drug Evaluation, Preclinical , Nerve Degeneration/drug therapy , Nerve Regeneration/drug effects , Animals , Humans , PhenotypeABSTRACT
Many evidences indicate that mice lacking the homeobox transcription factor engrailed-2 (En2(-/-) mice) represent a reliable model to investigate neurodevelopmental basis and gene expression changes relevant to autism spectrum disorders. Dysfunctions in fragile X mental retardation protein (FMRP), metabotropic glutamate receptor 5 (mGluR5), and GABAergic signaling pathways have been proposed as a possible pathogenic mechanism of autism spectrum disorders. Here, we exploited En2(-/-) mice to investigate hippocampal expression of FMRP, mGluR5, and GABA(A) receptor ß3 subunit (GABRB3). Quantitative reverse-transcription PCR showed that all these mRNAs were significantly downregulated in En2(-/-) mice compared with wild-type littermates. Western blot and immunohistochemistry confirmed the downregulation of FMRP and GABRB3 proteins, while showing a significant increase of mGluR5 protein in the En2(-/-) hippocampus. Our results suggest that the dysregulation of FMRP-mGluR5 signaling pathway, accompanied with a downregulation of GABRB3 expression, may contribute to the 'autistic-like' features observed in En2 mice, providing possible molecular targets for future pharmacological studies.
Subject(s)
Autism Spectrum Disorder/metabolism , Fragile X Mental Retardation Protein/metabolism , Hippocampus/metabolism , Receptor, Metabotropic Glutamate 5/metabolism , Receptors, GABA-A/metabolism , Animals , Disease Models, Animal , Female , Fragile X Mental Retardation Protein/genetics , Male , Mice , Mice, Knockout , RNA, Messenger/metabolism , Signal TransductionABSTRACT
Hyperpolarizing and inhibitory GABA regulates critical periods for plasticity in sensory cortices. Here we examine the role of early, depolarizing GABA in the control of plasticity mechanisms. We report that brief interference with depolarizing GABA during early development prolonged critical-period plasticity in visual cortical circuits without affecting the overall development of the visual system. The effects on plasticity were accompanied by dampened inhibitory neurotransmission, downregulation of brain-derived neurotrophic factor (BDNF) expression and reduced density of extracellular matrix perineuronal nets. Early interference with depolarizing GABA decreased perinatal BDNF signaling, and a pharmacological increase of BDNF signaling during GABA interference rescued the effects on plasticity and its regulators later in life. We conclude that depolarizing GABA exerts a long-lasting, selective modulation of plasticity of cortical circuits by a strong crosstalk with BDNF.
Subject(s)
Critical Period, Psychological , Neuronal Plasticity/physiology , Visual Cortex/physiology , gamma-Aminobutyric Acid/physiology , Animals , Animals, Newborn , Bumetanide/pharmacology , Contrast Sensitivity/drug effects , Contrast Sensitivity/physiology , Diuretics/pharmacology , Evoked Potentials, Visual/drug effects , Evoked Potentials, Visual/physiology , Female , GABA Antagonists/pharmacology , Male , Maze Learning , Neuronal Plasticity/drug effects , Rats , Rats, Long-Evans , Visual Cortex/drug effects , Visual Cortex/growth & developmentABSTRACT
Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly, En2 knock-out (En2(-/-)) mice show anatomical and behavioral "ASD-like" features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits in En2(-/-) mice are not known. Deficits in signaling pathways involving neurofibromin and extracellular-regulated kinase (ERK) have been associated with impaired learning. Here we investigated the neurofibromin-ERK cascade in the hippocampus of wild-type (WT) and En2(-/-) mice before and after spatial learning testing. When compared with WT littermates, En2(-/-) mice showed impaired performance in the Morris water maze (MWM), which was accompanied by lower expression of the activity-dependent gene Arc. Quantitative RT-PCR, immunoblotting, and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naive and MWM-treated En2(-/-) mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus of En2(-/-) mice after MWM. Treatment of En2(-/-) mice with lovastatin, an indirect inhibitor of ERK phosphorylation, markedly reduced ERK phosphorylation in the dentate gyrus, but was unable to rescue learning deficits in MWM-trained mutant mice. Further investigation is needed to unravel the complex molecular mechanisms linking dysregulation of neurofibromin-dependent pathways to spatial learning deficits in the En2 mouse model of ASD.
Subject(s)
Hippocampus/metabolism , Learning Disabilities/genetics , Learning Disabilities/pathology , Nerve Tissue Proteins/deficiency , Neurofibromin 1/metabolism , Signal Transduction/genetics , Analysis of Variance , Animals , Cell Count , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Hippocampus/drug effects , Hippocampus/pathology , Homeodomain Proteins/genetics , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Learning Disabilities/drug therapy , Lovastatin/therapeutic use , Maze Learning/drug effects , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , RNA, Messenger/metabolism , Signal Transduction/drug effectsABSTRACT
The maturation of the GABAergic system is a crucial determinant of cortical development during early postnatal life, when sensory circuits undergo a process of activity-dependent refinement. An altered excitatory/inhibitory balance has been proposed as a possible pathogenic mechanism of autism spectrum disorders (ASD). The homeobox-containing transcription factor Engrailed-2 (En2) has been associated to ASD, and En2 knockout (En2 (-/-)) mice show ASD-like features accompanied by a partial loss of cortical GABAergic interneurons. Here we studied GABAergic markers and cortical function in En2 (-/-) mice, by exploiting the well-known anatomical and functional features of the mouse visual system. En2 is expressed in the visual cortex at postnatal day 30 and during adulthood. When compared to age-matched En2 (+/+) controls, En2 (-/-) mice showed an increased number of parvalbumin (PV(+)), somatostatin (SOM(+)), and neuropeptide Y (NPY(+)) positive interneurons in the visual cortex at P30, and a decreased number of SOM(+) and NPY(+) interneurons in the adult. At both ages, the differences in distinct interneuron populations observed between En2 (+/+) and En2 (-/-) mice were layer-specific. Adult En2 (-/-) mice displayed a normal eye-specific segregation in the retino-geniculate pathway, and in vivo electrophysiological recordings showed a normal development of basic functional properties (acuity, response latency, receptive field size) of the En2 (-/-) primary visual cortex. However, a significant increase of binocularity was found in P30 and adult En2 (-/-) mice, as compared to age-matched controls. Differently from what observed in En2 (+/+) mice, the En2 (-/-) primary visual cortex did not respond to a brief monocular deprivation performed between P26 and P29, during the so-called "critical period." These data suggest that altered GABAergic circuits impact baseline binocularity and plasticity in En2 (-/-) mice, while leaving other visual functional properties unaffected.
ABSTRACT
BACKGROUND: Transcriptome analysis has been used in autism spectrum disorder (ASD) to unravel common pathogenic pathways based on the assumption that distinct rare genetic variants or epigenetic modifications affect common biological pathways. To unravel recurrent ASD-related neuropathological mechanisms, we took advantage of the En2-/- mouse model and performed transcriptome profiling on cerebellar and hippocampal adult tissues. METHODS: Cerebellar and hippocampal tissue samples from three En2-/- and wild type (WT) littermate mice were assessed for differential gene expression using microarray hybridization followed by RankProd analysis. To identify functional categories overrepresented in the differentially expressed genes, we used integrated gene-network analysis, gene ontology enrichment and mouse phenotype ontology analysis. Furthermore, we performed direct enrichment analysis of ASD-associated genes from the SFARI repository in our differentially expressed genes. RESULTS: Given the limited number of animals used in the study, we used permissive criteria and identified 842 differentially expressed genes in En2-/- cerebellum and 862 in the En2-/- hippocampus. Our functional analysis revealed that the molecular signature of En2-/- cerebellum and hippocampus shares convergent pathological pathways with ASD, including abnormal synaptic transmission, altered developmental processes and increased immune response. Furthermore, when directly compared to the repository of the SFARI database, our differentially expressed genes in the hippocampus showed enrichment of ASD-associated genes significantly higher than previously reported. qPCR was performed for representative genes to confirm relative transcript levels compared to those detected in microarrays. CONCLUSIONS: Despite the limited number of animals used in the study, our bioinformatic analysis indicates the En2-/- mouse is a valuable tool for investigating molecular alterations related to ASD.
ABSTRACT
The homeobox-containing transcription factor Engrailed-2 (En2) is involved in patterning and neuronal differentiation of the midbrain/hindbrain region, where it is prominently expressed. En2 mRNA is also expressed in the adult mouse hippocampus and cerebral cortex, indicating that it might also function in these brain areas. Genome-wide association studies revealed that En2 is a candidate gene for autism spectrum disorders (ASD), and mice devoid of its expression (En2(-/-) mice) display anatomical, behavioral and clinical "autistic-like" features. Since reduced GABAergic inhibition has been proposed as a possible pathogenic mechanism of ASD, we hypothesized that the phenotype of En2(-/-) mice might include defective GABAergic innervation in the forebrain. Here we show that the Engrailed proteins are present in postnatal GABAergic neurons of the mouse hippocampus and cerebral cortex, and adult En2(-/-) mice show reduced expression of GABAergic marker mRNAs in these areas. In addition, reduction in parvalbumin (PV), somatostatin (SOM) and neuropeptide Y (NPY) expressing interneurons is detected in the hippocampus and cerebral cortex of adult En2(-/-) mice. Our results raise the possibility of a link between altered function of En2, anatomical deficits of GABAergic forebrain neurons and the pathogenesis of ASD.
Subject(s)
Autistic Disorder/genetics , Autistic Disorder/pathology , Cerebral Cortex/cytology , GABAergic Neurons/pathology , Hippocampus/cytology , Nerve Tissue Proteins/deficiency , Animals , Disease Models, Animal , Homeodomain Proteins , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuropeptide Y/metabolism , Parvalbumins/metabolism , Somatostatin/metabolismABSTRACT
Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental diseases characterized by a triad of specific behavioral traits: abnormal social interactions, communication deficits and stereotyped or repetitive behaviors. Several recent studies showed that ASDs have a strong genetic basis, contributing to the discovery of a number of ASD-associated genes. Due to the genetic complexity of these disorders, mouse strains with targeted deletion of ASD genes have become an essential tool to investigate the molecular and neurodevelopmental mechanisms underlying ASD. Here we will review the most relevant genetic mouse models developed by targeted inactivation of ASD-associated genes, and discuss their importance for the development of novel pharmacological therapies of these disorders.