scholarly journals Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development

2020 ◽  
Author(s):  
Jasmin Morandell ◽  
Lena A. Schwarz ◽  
Bernadette Basilico ◽  
Saren Tasciyan ◽  
Armel Nicolas ◽  
...  
Keyword(s):  
Neuronal Migration ◽  
Motor Coordination ◽  
De Novo ◽  
Critical Role ◽  
Autism Spectrum ◽  
Cytoskeletal Proteins ◽  
Inhibitory Neurons ◽  
Loss Of Function ◽  
Proteomic Approach

AbstractDe novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). Here, we used Cul3 mouse models to evaluate the consequences of Cul3 mutations in vivo. Our results show that Cul3 haploinsufficient mice exhibit deficits in motor coordination as well as ASD-relevant social and cognitive impairments. Cul3 mutant brain displays cortical lamination abnormalities due to defective neuronal migration and reduced numbers of excitatory and inhibitory neurons. In line with the observed abnormal columnar organization, Cul3 haploinsufficiency is associated with decreased spontaneous excitatory and inhibitory activity in the cortex. At the molecular level, employing a quantitative proteomic approach, we show that Cul3 regulates cytoskeletal and adhesion protein abundance in mouse embryos. Abnormal regulation of cytoskeletal proteins in Cul3 mutant neuronal cells results in atypical organization of the actin mesh at the cell leading edge, likely causing the observed migration deficits. In contrast to these important functions early in development, Cul3 deficiency appears less relevant at adult stages. In fact, induction of Cul3 haploinsufficiency in adult mice does not result in the behavioral defects observed in constitutive Cul3 haploinsufficient animals. Taken together, our data indicate that Cul3 has a critical role in the regulation of cytoskeletal proteins and neuronal migration and that ASD-associated defects and behavioral abnormalities are primarily due to Cul3 functions at early developmental stages.

scholarly journals Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmin Morandell ◽  
Lena A. Schwarz ◽  
Bernadette Basilico ◽  
Saren Tasciyan ◽  
Georgi Dimchev ◽  
...  
Keyword(s):  
Cell Migration ◽  
Transcriptional Activation ◽  
Neuronal Migration ◽  
Motor Coordination ◽  
De Novo ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Critical Window ◽  
Cytoskeleton Organization

AbstractDe novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). In mouse, constitutive Cul3 haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs.

scholarly journals UBE3A-mediated PTPA ubiquitination and degradation regulate PP2A activity and dendritic spine morphology

2019 ◽  
Vol 116 (25) ◽  
pp. 12500-12505 ◽  
Author(s):  
Jie Wang ◽  
Sen-Sen Lou ◽  
Tingting Wang ◽  
Rong-Jie Wu ◽  
Guangying Li ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Dendritic Spine ◽  
Isotope Labeling ◽  
Neurodevelopmental Disorder ◽  
Critical Role ◽  
Motor Impairment ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Pp2a Activity

Deficiency in the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while additional dosage of UBE3A is linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A gain or loss of function in these neurodevelopmental disorders are still not well understood, and effective treatments are lacking. Here, using stable-isotope labeling of amino acids in mammals and ubiquitination assays, we identify PTPA, an activator of protein phosphatase 2A (PP2A), as a bona fide ubiquitin ligase substrate of UBE3A. Maternal loss of Ube3a (Ube3am−/p+) increased PTPA level, promoted PP2A holoenzyme assembly, and elevated PP2A activity, while maternal 15q11–13 duplication containing Ube3a down-regulated PTPA level and lowered PP2A activity. Reducing PTPA level in vivo restored the defects in dendritic spine maturation in Ube3am−/p+ mice. Moreover, pharmacological inhibition of PP2A activity with the small molecule LB-100 alleviated both reduction in excitatory synaptic transmission and motor impairment in Ube3am−/p+ mice. Together, our results implicate a critical role of UBE3A-PTPA-PP2A signaling in the pathogenesis of UBE3A-related disorders and suggest that PP2A-based drugs could be potential therapeutic candidates for treatment of UBE3A-related disorders.

scholarly journals Haploinsufficiency of autism candidate gene NUAK1 impairs cortical development and behavior

2018 ◽  
Author(s):  
Virginie Courchet ◽  
Amanda J Roberts ◽  
Peggy Del Carmine ◽  
Tommy L. Lewis ◽  
Franck Polleux ◽  
...  
Keyword(s):  
Social Preference ◽  
De Novo ◽  
Sensorimotor Gating ◽  
Learning Task ◽  
Autism Spectrum ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Cortical Connectivity

SUMMARYRecently, numerous rare de novo mutations have been identified in children diagnosed with autism spectrum disorders (ASD). However, despite the predicted loss-of-function nature of some of these de novo mutations, the affected individuals are heterozygous carriers, which would suggest that most of these candidate genes are haploinsufficient and/or that these mutations lead to expression of dominant-negative forms of the protein. Here, we tested this hypothesis with the gene Nuak1, recently identified as a candidate ASD gene and that we previously identified for its role in the development of cortical connectivity. We report that Nuak1 is happloinsufficient in mice in regard to its function in cortical axon branching in vitro and in vivo. Nuak1+/− mice show a combination of abnormal behavioral traits ranging from defective memory consolidation in a spatial learning task, defects in social novelty (but not social preference) and abnormal sensorimotor gating and prepulse inhibition of the startle response. Overall, our results demonstrate that Nuak1 haploinsufficiency leads to defects in the development of cortical connectivity and a complex array of behavorial deficits compatible with ASD, intellectual disability and schizophrenia.

scholarly journals Reduced prefrontal synaptic connectivity and disturbed oscillatory population dynamics in the CNTNAP2 model of autism

2018 ◽  
Author(s):  
Maria T. Lazaro ◽  
Jiannis Taxidis ◽  
Tristan Shuman ◽  
Iris Bachmutsky ◽  
Taruna Ikrar ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Phase Locking ◽  
Autism Spectrum ◽  
Repetitive Behaviors ◽  
Neuronal Firing ◽  
Inhibitory Neurons ◽  
Loss Of Function ◽  
Knock Out ◽  
Synaptic Inputs

ABSTRACTLoss of function mutations in CNTNAP2 cause a syndromic form of autism spectrum disorder (ASD) in humans and produce social deficits, repetitive behaviors, and seizures in mice. Yet, the functional effects of these mutations at the cellular and circuit level remain elusive. Using laser scanning photostimulation, whole-cell recordings, and electron microscopy, we found a dramatic decrease in functional excitatory and inhibitory synaptic inputs in L2/3 medial prefrontal cortex (mPFC) of Cntnap2 knock-out (KO) mice. In accordance with decreased synaptic input, KO mice displayed reduced spine and synapse densities, despite normal intrinsic excitability and dendritic complexity. To determine how this decrease in synaptic inputs alters coordination of neuronal firing patterns in vivo, we recorded mPFC local field potentials (LFP) and unit spiking in head-fixed mice during locomotion and rest. In KO mice, LFP power was not significantly altered at all tested frequencies, but inhibitory neurons showed delayed phase-firing and reduced phase-locking to delta and theta oscillations during locomotion. Excitatory neurons showed similar changes but only to delta oscillations. These findings suggest that profound ASD-related alterations in synaptic inputs can yield perturbed temporal coordination of cortical ensembles.

scholarly journals The critical role of ASD-related gene CNTNAP3 in regulating synaptic development and social behavior in mice

2018 ◽  
Author(s):  
Da-li Tong ◽  
Rui-guo Chen ◽  
Yu-lan Lu ◽  
Wei-ke Li ◽  
Yue-fang Zhang ◽  
...  
Keyword(s):  
Critical Role ◽  
Autism Spectrum ◽  
Repetitive Behaviors ◽  
Inhibitory Synapses ◽  
Scaffolding Proteins ◽  
Loss Of Function ◽  
Chinese Han

AbstractAccumulated genetic evidences indicate that the contactin associated protein-like (CNTNAP) family is implicated in autism spectrum disorders (ASD). In this study, we identified genetic mutations in the CNTNAP3 gene from Chinese Han ASD cohorts and Simons Simplex Collections. We found that CNTNAP3 interacted with synaptic adhesion proteins Neuroligin1 and Neuroligin2, as well as scaffolding proteins PSD95 and Gephyrin. Significantly, we found that CNTNAP3 played an opposite role in controlling the development of excitatory and inhibitory synapses in vitro and in vivo, in which ASD mutants exhibited loss-of-function effects. In this study, we showed that Cntnap3-null mice exhibited deficits in social interaction, spatial learning and prominent repetitive behaviors. These evidences elucidate the pivotal role of CNTNAP3 in synapse development and social behaviors, providing the mechanistic insights for ASD.

BET Bromodomain Degradation As a Therapeutic Strategy in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1062-1062 ◽  
Author(s):  
Geoffrey M. Matthews ◽  
Sara Gandolfi ◽  
Johanna Bruggentheis ◽  
Ricardo De Matos Simoes ◽  
Dennis L Buckley ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Caspase 3 ◽  
De Novo ◽  
Critical Role ◽  
Cell Types ◽  
Drug Resistant ◽  
Loss Of Function ◽  
Treatment Naïve

Abstract Multiple myeloma (MM) remains an incurable malignancy with a clear need for novel therapeutic modalities. Moreover, acquired or de novo resistance to established or novel therapeutics remains a major challenge in this, and other, neoplasias. BET Bromodomain inhibitors (BBIs), including JQ1, have potent anti-MM activity in vitro and in in vivo, but do not provide curative outcome and do not induce apoptosis in many tumor cell types. Recently, a "next-generation" BBI, dBET, that causes degradation of BET Bromodomains (BRDs) through CRBN-mediated ubiquitination has been demonstrated to have potent activity in leukemia and myeloma. Here we sought to compare the mechanistic differences between BRD inhibition with BRD degradation in treatment-naive and drug-resistant MM. Additionally, we posited that resistance to dBET treatment could emerge through genetic perturbations and wished to uncover potential mechanisms prior to its clinical utilization. To address this, we compared effects of JQ1 with lead optimized compound dBET6, in a panel of human MM cell lines (± stromal cells), including clones resistant to JQ1, bortezomib and IMIDs, and assessed viability using CS-BLI/CTG assay. RNAseq and reverse phase protein arrays (RPPA) were employed to compare the transcriptional and translational effects of BRD degradation vs. inhibition. Using an open-ended unbiased genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-associated Cas9 approach, we examined whether we could uncover genes associated with resistance to dBET6. MM1.S cells were transduced with Cas9 and pooled lentiviral particles of the GeCKO library, consisting of 2 pooled sgRNA sub-libraries (~120,000 sgRNAs; targeting ~19,000 genes and ~1800 miRNAs). Using this CRISPR/Cas9-based approach we sought to expedite the isolation of MM cells resistant to dBET6. We treated the pool of cells thrice with dBET (250nM), allowing regrowth between treatments and maintaining a coverage of 1000 cells/sgRNA. dBET6-resistant cells were processed to quantify sgRNA enrichment or depletion, using deep sequencing. We observed dBET6 to have significantly greater potency against MM cells than JQ1, or its combination with lenalidomide, and that MM1S.CRBN-/- cells were resistant to dBET6. Resistance to neither JQ1 nor bortezomib conferred resistance to dBET6. We observed dBET6 to induce rapid and robust (<4hrs) degradation of BRD2, BRD3 and BRD4 and loss of c-MYC protein, compared with JQ1 which caused an apparent increase in BRD4 protein and significantly less c-MYC down-regulation. Interestingly, while dBET6 caused a time-dependent reduction in pro-survival Mcl-1 protein (among others) and increased cleavage of caspase-3/7, JQ1 caused Mcl-1 upregulation and did not induce cleavage of caspase-3/7. As predicted, our CRISPR/Cas9 screen identified significant enrichment of sgRNAs targeting CRBN, as well as several members of the Cullin-RING ligase (CRL) complex, known to play a critical role in E3 ubiquitin ligase activity. Preliminary experiments using individual sgRNAs appear to validate the role the CRL complex in dBET resistance. In summary, our data strongly support the development of dBET for the treatment of treatment-naive and drug-resistant MM. We demonstrate overlapping and distinct mechanisms of action between BRD inhibition vs. degradation and suggest that differential potencies of JQ1 vs. dBET is, at least in part, due to far greater loss of c-MYC and Mcl-1 expression, however further analysis is warranted. Additionally, our results demonstrate that loss of function of CRBN or the CRL complex induces dBET resistance by perturbing dBET-mediated BRD4 degradation. However, it is plausible that additional CRBN/CRL-independent mechanisms of dBET resistance exist that allow cells to survive despite complete degradation of BRDs and this will be a key question to be answered in future studies. Disclosures Bradner: Novartis Institutes for BioMedical Research: Employment.

scholarly journals CHD8 Regulates Cellular Homeostasis and Neuronal Function Genes Across Multiple Models of CHD8 Haploinsufficiency

2018 ◽  
Author(s):  
A. Ayanna Wade ◽  
Kenneth Lim ◽  
Rinaldo Catta-Preta ◽  
Alex S. Nord
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Chromatin Remodeling ◽  
De Novo ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Cell Functions ◽  
High Affinity

ABSTRACTThe packaging of DNA into chromatin determines the transcriptional potential of cells and is central to eukaryotic gene regulation. Recent sequencing of patient mutations has linked de novo loss-of-function mutations to chromatin remodeling factors with specific, causal roles in neurodevelopmental disorders. Characterizing cellular and molecular phenotypes arising from haploinsufficiency of chromatin remodeling factors could reveal convergent mechanisms of pathology. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeling factor gene and has among the highest de novo loss-of-function mutations rates in patients with autism spectrum disorder (ASD). Mutations to CHD8 are expected to drive neurodevelopmental pathology through global disruptions to gene expression and chromatin state, however, mechanisms associated with CHD8 function have yet to be fully elucidated. We analyzed published transcriptomic and epigenomic data across CHD8 in vitro and in vivo knockdown and knockout models to identify convergent mechanisms of gene regulation by CHD8. We found reproducible high-affinity interactions of CHD8 near promoters of genes necessary for basic cell functions and gene regulation, especially chromatin organization and RNA processing genes. Overlap between CHD8 interaction and differential expression suggests that reduced dosage of CHD8 directly relates to decreased expression of these genes. In addition, genes important for neuronal development and function showed consistent dysregulation, though there was a reduced rate and decreased affinity for CHD8 interactions near these genes. This meta-analysis verifies CHD8 as a critical regulator of gene expression and reveals a consistent set of high affinity CHD8 interaction targets observed across human and mouse in vivo and in vitro studies. Our findings highlight novel core functions of CHD8 and indicate direct and downstream gene regulatory impacts that are likely to be associated with neuropathology underlying CHD8-associated neurodevelopmental disorder.

scholarly journals In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes

Science ◽  
2020 ◽  
Vol 370 (6520) ◽  
pp. eaaz6063 ◽  
Author(s):  
Xin Jin ◽  
Sean K. Simmons ◽  
Amy Guo ◽  
Ashwin S. Shetty ◽  
Michelle Ko ◽  
...  
Keyword(s):  
De Novo ◽  
Disease Risk ◽  
Cell Types ◽  
Autism Spectrum ◽  
Loss Of Function ◽  
Risk Genes ◽  
Cellular Effects ◽  
Gene Modules ◽  
Conserved Gene

The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNA-sequencing of perturbed cells in the postnatal brain. We identified cell type–specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism.

scholarly journals In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with Autism risk genes

2019 ◽  
Author(s):  
Xin Jin ◽  
Sean K. Simmons ◽  
Amy X. Guo ◽  
Ashwin S. Shetty ◽  
Michelle Ko ◽  
...  
Keyword(s):  
De Novo ◽  
Disease Risk ◽  
Autism Spectrum ◽  
Specific Gene ◽  
Functional Evaluation ◽  
Loss Of Function ◽  
Risk Genes ◽  
Biologically Relevant ◽  
Relevant Context

AbstractThe thousands of disease risk genes and loci identified through human genetic studies far outstrip our current capacity to systematically study their functions. New experimental approaches are needed for functional investigations of large panels of genes in a biologically relevant context. Here, we developed a scalable genetic screen approach, in vivo Perturb-Seq, and applied this method to the functional evaluation of 35 autism spectrum disorder (ASD) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing brain in utero, and then performed single-cell RNA-Seq in the postnatal brain. We identified cell type-specific gene signatures from both neuronal and glial cell classes that are affected by genetic perturbations, and pointed at elements of both convergent and divergent cellular effects across this cohort of ASD risk genes. In vivo Perturb-Seq pioneers a systems genetics approach to investigate at scale how diverse mutations affect cell types and states in the biologically relevant context of the developing organism.

scholarly journals Conditional Pten knockout in parvalbumin- or somatostatin-positive neurons sufficiently leads to autism-related behavioral phenotypes

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Sangyep Shin ◽  
Andrea Santi ◽  
Shiyong Huang
Keyword(s):  
Autism Spectrum Disorder ◽  
Motor Coordination ◽  
Critical Role ◽  
Autism Spectrum ◽  
Gabaergic Neurons ◽  
Repetitive Behaviors ◽  
Inhibitory Neurons ◽  
Social Deficits ◽  
Behavioral Phenotypes ◽  
Behavioral Assessments

AbstractDisrupted GABAergic neurons have been extensively described in brain tissues from individuals with autism spectrum disorder (ASD) and animal models for ASD. However, the contribution of these aberrant inhibitory neurons to autism-related behavioral phenotypes is not well understood. We examined ASD-related behaviors in mice with conditional Pten knockout in parvalbumin (PV)-expressing or somatostatin (Sst)-expressing neurons, two common subtypes of GABAergic neurons. We found that mice with deletion of Pten in either PV-neurons or Sst-neurons displayed social deficits, repetitive behaviors and impaired motor coordination/learning. In addition, mice with one copy of Pten deletion in PV-neurons exhibited hyperlocomotion in novel open fields and home cages. We also examined anxiety behaviors and found that mice with Pten deletion in Sst-neurons displayed anxiety-like behaviors, while mice with Pten deletion in PV-neurons exhibited anxiolytic-like behaviors. These behavioral assessments demonstrate that Pten knockout in the subtype of inhibitory neurons sufficiently gives rise to ASD-core behaviors, providing evidence that both PV- and Sst-neurons may play a critical role in ASD symptoms.

Sign in / Sign up

Export Citation Format

Share Document