Publications by authors named "Nael Nadif Kasri"

72 Publications

NGN2 induces diverse neuron types from human pluripotency.

Stem Cell Reports 2021 Sep 5;16(9):2118-2127. Epub 2021 Aug 5.

Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland; Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany; Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. Electronic address:

Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.
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http://dx.doi.org/10.1016/j.stemcr.2021.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452516PMC
September 2021

The continued need for animals to advance brain research.

Neuron 2021 08;109(15):2374-2379

Radboud University Medical Center, Nijmegen, the Netherlands.

Policymakers aim to move toward animal-free alternatives for scientific research and have introduced very strict regulations for animal research. We argue that, for neuroscience research, until viable and translational alternatives become available and the value of these alternatives has been proven, the use of animals should not be compromised.
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http://dx.doi.org/10.1016/j.neuron.2021.07.015DOI Listing
August 2021

Sonlicromanol improves neuronal network dysfunction and transcriptome changes linked to m.3243A>G heteroplasmy in iPSC-derived neurons.

Stem Cell Reports 2021 Sep 29;16(9):2197-2212. Epub 2021 Jul 29.

Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, Nijmegen, 6500 HB, the Netherlands. Electronic address:

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is often caused by an adenine to guanine variant at m.3243 (m.3243A>G) of the MT-TL1 gene. To understand how this pathogenic variant affects the nervous system, we differentiated human induced pluripotent stem cells (iPSCs) into excitatory neurons with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function from MELAS patients with the m.3243A>G pathogenic variant. We combined micro-electrode array (MEA) measurements with RNA sequencing (MEA-seq) and found reduced expression of genes involved in mitochondrial respiration and presynaptic function, as well as non-cell autonomous processes in co-cultured astrocytes. Finally, we show that the clinical phase II drug sonlicromanol can improve neuronal network activity when treatment is initiated early in development. This was intricately linked with changes in the neuronal transcriptome. Overall, we provide insight in transcriptomic changes in iPSC-derived neurons with high m.3243A>G heteroplasmy, and show the pathology is partially reversible by sonlicromanol.
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http://dx.doi.org/10.1016/j.stemcr.2021.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452519PMC
September 2021

Human neuronal networks on micro-electrode arrays are a highly robust tool to study disease-specific genotype-phenotype correlations in vitro.

Stem Cell Reports 2021 Sep 29;16(9):2182-2196. Epub 2021 Jul 29.

Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behavior, 6500 HB Nijmegen, the Netherlands; Department of Clinical Genomics, Mayo Clinic, Rochester, MN 55905, USA. Electronic address:

Micro-electrode arrays (MEAs) are increasingly used to characterize neuronal network activity of human induced pluripotent stem cell (hiPSC)-derived neurons. Despite their gain in popularity, MEA recordings from hiPSC-derived neuronal networks are not always used to their full potential in respect to experimental design, execution, and data analysis. Therefore, we benchmarked the robustness of MEA-derived neuronal activity patterns from ten healthy individual control lines, and uncover comparable network phenotypes. To achieve standardization, we provide recommendations on experimental design and analysis. With such standardization, MEAs can be used as a reliable platform to distinguish (disease-specific) network phenotypes. In conclusion, we show that MEAs are a powerful and robust tool to uncover functional neuronal network phenotypes from hiPSC-derived neuronal networks, and provide an important resource to advance the hiPSC field toward the use of MEAs for disease phenotyping and drug discovery.
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http://dx.doi.org/10.1016/j.stemcr.2021.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452490PMC
September 2021

Imbalanced autophagy causes synaptic deficits in a human model for neurodevelopmental disorders.

Autophagy 2021 Jul 21:1-20. Epub 2021 Jul 21.

Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behavior, Nijmegen, The Netherlands.

Macroautophagy (hereafter referred to as autophagy) is a finely tuned process of programmed degradation and recycling of proteins and cellular components, which is crucial in neuronal function and synaptic integrity. Mounting evidence implicates chromatin remodeling in fine-tuning autophagy pathways. However, this epigenetic regulation is poorly understood in neurons. Here, we investigate the role in autophagy of KANSL1, a member of the nonspecific lethal complex, which acetylates histone H4 on lysine 16 (H4K16ac) to facilitate transcriptional activation. Loss-of-function of KANSL1 is strongly associated with the neurodevelopmental disorder Koolen-de Vries Syndrome (KdVS). Starting from KANSL1-deficient human induced-pluripotent stem cells, both from KdVS patients and genome-edited lines, we identified SOD1 (superoxide dismutase 1), an antioxidant enzyme, to be significantly decreased, leading to a subsequent increase in oxidative stress and autophagosome accumulation. In KANSL1-deficient neurons, autophagosome accumulation at excitatory synapses resulted in reduced synaptic density, reduced GRIA/AMPA receptor-mediated transmission and impaired neuronal network activity. Furthermore, we found that increased oxidative stress-mediated autophagosome accumulation leads to increased MTOR activation and decreased lysosome function, further preventing the clearing of autophagosomes. Finally, by pharmacologically reducing oxidative stress, we could rescue the aberrant autophagosome formation as well as synaptic and neuronal network activity in KANSL1-deficient neurons. Our findings thus point toward an important relation between oxidative stress-induced autophagy and synapse function, and demonstrate the importance of H4K16ac-mediated changes in chromatin structure to balance reactive oxygen species- and MTOR-dependent autophagy.: APO: apocynin; ATG: autophagy related; BAF: bafilomycin A; BSO: buthionine sulfoximine; CV: coefficient of variation; DIV: days in vitro; H4K16ac: histone 4 lysine 16 acetylation; iPSC: induced-pluripotent stem cell; KANSL1: KAT8 regulatory NSL complex subunit 1; KdVS: Koolen-de Vries Syndrome; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEA: micro-electrode array; MTOR: mechanistic target of rapamycin kinase; NSL complex: nonspecific lethal complex; 8-oxo-dG: 8-hydroxydesoxyguanosine; RAP: rapamycin; ROS: reactive oxygen species; sEPSCs: spontaneous excitatory postsynaptic currents; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; SYN: synapsin; WRT: wortmannin.
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http://dx.doi.org/10.1080/15548627.2021.1936777DOI Listing
July 2021

Cadherin-13 is a critical regulator of GABAergic modulation in human stem-cell-derived neuronal networks.

Mol Psychiatry 2021 May 10. Epub 2021 May 10.

Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behavior, Nijmegen, The Netherlands.

Activity in the healthy brain relies on a concerted interplay of excitation (E) and inhibition (I) via balanced synaptic communication between glutamatergic and GABAergic neurons. A growing number of studies imply that disruption of this E/I balance is a commonality in many brain disorders; however, obtaining mechanistic insight into these disruptions, with translational value for the patient, has typically been hampered by methodological limitations. Cadherin-13 (CDH13) has been associated with autism and attention-deficit/hyperactivity disorder. CDH13 localizes at inhibitory presynapses, specifically of parvalbumin (PV) and somatostatin (SST) expressing GABAergic neurons. However, the mechanism by which CDH13 regulates the function of inhibitory synapses in human neurons remains unknown. Starting from human-induced pluripotent stem cells, we established a robust method to generate a homogenous population of SST and MEF2C (PV-precursor marker protein) expressing GABAergic neurons (iGABA) in vitro, and co-cultured these with glutamatergic neurons at defined E/I ratios on micro-electrode arrays. We identified functional network parameters that are most reliably affected by GABAergic modulation as such, and through alterations of E/I balance by reduced expression of CDH13 in iGABAs. We found that CDH13 deficiency in iGABAs decreased E/I balance by means of increased inhibition. Moreover, CDH13 interacts with Integrin-β1 and Integrin-β3, which play opposite roles in the regulation of inhibitory synaptic strength via this interaction. Taken together, this model allows for standardized investigation of the E/I balance in a human neuronal background and can be deployed to dissect the cell-type-specific contribution of disease genes to the E/I balance.
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http://dx.doi.org/10.1038/s41380-021-01117-xDOI Listing
May 2021

Biallelic variants in TMEM222 cause a new autosomal recessive neurodevelopmental disorder.

Genet Med 2021 07 6;23(7):1246-1254. Epub 2021 Apr 6.

Department of Human Genetics, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, The Netherlands.

Purpose: To elucidate the novel molecular cause in families with a new autosomal recessive neurodevelopmental disorder.

Methods: A combination of exome sequencing and gene matching tools was used to identify pathogenic variants in 17 individuals. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and subcellular localization studies were used to characterize gene expression profile and localization.

Results: Biallelic variants in the TMEM222 gene were identified in 17 individuals from nine unrelated families, presenting with intellectual disability and variable other features, such as aggressive behavior, shy character, body tremors, decreased muscle mass in the lower extremities, and mild hypotonia. We found relatively high TMEM222 expression levels in the human brain, especially in the parietal and occipital cortex. Additionally, subcellular localization analysis in human neurons derived from induced pluripotent stem cells (iPSCs) revealed that TMEM222 localizes to early endosomes in the synapses of mature iPSC-derived neurons.

Conclusion: Our findings support a role for TMEM222 in brain development and function and adds variants in the gene TMEM222 as a novel underlying cause of an autosomal recessive neurodevelopmental disorder.
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http://dx.doi.org/10.1038/s41436-021-01133-wDOI Listing
July 2021

Generation of induced pluripotent stem cell (iPSC) lines carrying a heterozygous (UKWMPi002-A-1) and null mutant knockout (UKWMPi002-A-2) of Cadherin 13 associated with neurodevelopmental disorders using CRISPR/Cas9.

Stem Cell Res 2021 03 11;51:102169. Epub 2021 Jan 11.

Division of Molecular Psychiatry, Center of Mental Health, University Hospital Würzburg, Germany; Laboratory of Psychiatric Neurobiology, Institute of Molecular Medicine, I.M Sechenov First Moscow State Medical University, Moscow, Russia; Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience (MHeNs), Maastricht University, Maastricht, The Netherlands. Electronic address:

Fibroblasts isolated from a skin biopsy of a healthy 46-year-old female were infected with Sendai virus containing the Yamanaka factors to produce transgene-free human induced pluripotent stem cells (iPSCs). CRISPR/Cas9 was used to generate isogenic cell lines with a gene dose-dependent deficiency of CDH13, a risk gene associated with neurodevelopmental and psychiatric disorders. Thereby, a heterozygous CDH13 knockout (CDH13) and a CDH13 null mutant (CDH13) iPSC line was obtained. All three lines showed expression of pluripotency-associated markers, the ability to differentiate into cells of the three germ layers in vitro, and a normal female karyotype.
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http://dx.doi.org/10.1016/j.scr.2021.102169DOI Listing
March 2021

Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes.

Elife 2021 Jan 20;10. Epub 2021 Jan 20.

Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.

We generated induced excitatory neurons (iNeurons, iNs) from chimpanzee, bonobo, and human stem cells by expressing the transcription factor neurogenin-2 (NGN2). Single-cell RNA sequencing showed that genes involved in dendrite and synapse development are expressed earlier during iNs maturation in the chimpanzee and bonobo than the human cells. In accordance, during the first 2 weeks of differentiation, chimpanzee and bonobo iNs showed repetitive action potentials and more spontaneous excitatory activity than human iNs, and extended neurites of higher total length. However, the axons of human iNs were slightly longer at 5 weeks of differentiation. The timing of the establishment of neuronal polarity did not differ between the species. Chimpanzee, bonobo, and human neurites eventually reached the same level of structural complexity. Thus, human iNs develop slower than chimpanzee and bonobo iNs, and this difference in timing likely depends on functions downstream of NGN2.
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http://dx.doi.org/10.7554/eLife.59323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870144PMC
January 2021

The emerging role of chromatin remodelers in neurodevelopmental disorders: a developmental perspective.

Cell Mol Life Sci 2021 Mar 2;78(6):2517-2563. Epub 2020 Dec 2.

Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition and Behaviour, Geert Grooteplein 10, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands.

Neurodevelopmental disorders (NDDs), including intellectual disability (ID) and autism spectrum disorders (ASD), are a large group of disorders in which early insults during brain development result in a wide and heterogeneous spectrum of clinical diagnoses. Mutations in genes coding for chromatin remodelers are overrepresented in NDD cohorts, pointing towards epigenetics as a convergent pathogenic pathway between these disorders. In this review we detail the role of NDD-associated chromatin remodelers during the developmental continuum of progenitor expansion, differentiation, cell-type specification, migration and maturation. We discuss how defects in chromatin remodelling during these early developmental time points compound over time and result in impaired brain circuit establishment. In particular, we focus on their role in the three largest cell populations: glutamatergic neurons, GABAergic neurons, and glia cells. An in-depth understanding of the spatiotemporal role of chromatin remodelers during neurodevelopment can contribute to the identification of molecular targets for treatment strategies.
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http://dx.doi.org/10.1007/s00018-020-03714-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004494PMC
March 2021

EHMT1 regulates Parvalbumin-positive interneuron development and GABAergic input in sensory cortical areas.

Brain Struct Funct 2020 Dec 25;225(9):2701-2716. Epub 2020 Sep 25.

Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, 6500 HB, Nijmegen, The Netherlands.

Mutations in the Euchromatic Histone Methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a rare form of intellectual disability (ID) with strong autistic traits and sensory processing deficits. Proper development of inhibitory interneurons is crucial for sensory function. Here we report a timeline of Parvalbumin-positive (PV) interneuron development in the three most important sensory cortical areas in the Ehmt1 mouse. We find a hitherto unreported delay of PV neuron maturation early in sensory development, with layer- and region-specific variability later in development. The delayed PV maturation is also reflected in a delayed maturation of GABAergic transmission in Ehmt1 auditory cortex, where we find a reduced GABA release probability specifically in putative PV synapses. Together with earlier reports of excitatory impairments in Ehmt1 neurons, we propose a shift in excitatory-inhibitory balance towards overexcitability in Ehmt1 sensory cortices as a consequence of early deficits in inhibitory maturation.
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http://dx.doi.org/10.1007/s00429-020-02149-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674571PMC
December 2020

The Object Space Task reveals increased expression of cumulative memory in a mouse model of Kleefstra syndrome.

Neurobiol Learn Mem 2020 09 9;173:107265. Epub 2020 Jun 9.

Donders Institute for Brain Cognition and Behaviour, Radboud University, Nijmegen, Netherlands; Department of Human Genetics and Department of Cognitive Neuroscience, Radboudumc, Donders Institute for Brain, Cognition and Behaviour, 6500 HB Nijmegen, Netherlands. Electronic address:

Kleefstra syndrome is a disorder caused by a mutation in the EHMT1 gene characterized in humans by general developmental delay, mild to severe intellectual disability and autism. Here, we characterized cumulative memory in the Ehmt1 mouse model using the Object Space Task. We combined conventional behavioral analysis with automated analysis by deep-learning networks, a session-based computational learning model, and a trial-based classifier. Ehmt1 mice showed more anxiety-like features and generally explored objects less, but the difference decreased over time. Interestingly, when analyzing memory-specific exploration, Ehmt1 show increased expression of cumulative memory, but a deficit in a more simple, control memory condition. Using our automatic classifier to differentiate between genotypes, we found that cumulative memory features are better suited for classification than general exploration differences. Thus, detailed behavioral classification with the Object Space Task produced a more detailed behavioral phenotype of the Ehmt1 mouse model.
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http://dx.doi.org/10.1016/j.nlm.2020.107265DOI Listing
September 2020

m.3243A > G-Induced Mitochondrial Dysfunction Impairs Human Neuronal Development and Reduces Neuronal Network Activity and Synchronicity.

Cell Rep 2020 04;31(3):107538

Department of Anatomy, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, 6500 HB Nijmegen, the Netherlands; Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA; Department of Clinical Genomics, Mayo Clinic, Rochester, MN 55905, USA; Department of Biochemistry and Molecular Biology, Mayo Clinic, 55905 Rochester, MN, USA. Electronic address:

Epilepsy, intellectual and cortical sensory deficits, and psychiatric manifestations are the most frequent manifestations of mitochondrial diseases. How mitochondrial dysfunction affects neural structure and function remains elusive, mostly because of a lack of proper in vitro neuronal model systems with mitochondrial dysfunction. Leveraging induced pluripotent stem cell technology, we differentiated excitatory cortical neurons (iNeurons) with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function on an isogenic nuclear DNA background from patients with the common pathogenic m.3243A > G variant of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). iNeurons with high heteroplasmy exhibited mitochondrial dysfunction, delayed neural maturation, reduced dendritic complexity, and fewer excitatory synapses. Micro-electrode array recordings of neuronal networks displayed reduced network activity and decreased synchronous network bursting. Impaired neuronal energy metabolism and compromised structural and functional integrity of neurons and neural networks could be the primary drivers of increased susceptibility to neuropsychiatric manifestations of mitochondrial disease.
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http://dx.doi.org/10.1016/j.celrep.2020.107538DOI Listing
April 2020

Distinct Pathogenic Genes Causing Intellectual Disability and Autism Exhibit a Common Neuronal Network Hyperactivity Phenotype.

Cell Rep 2020 01;30(1):173-186.e6

Department of Cognitive Neuroscience, Radboudumc, Donders Institute for Brain, Cognition and Behaviour, 6525 HR Nijmegen, the Netherlands; Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, 6500 HB Nijmegen, the Netherlands. Electronic address:

Pathogenic mutations in either one of the epigenetic modifiers EHMT1, MBD5, MLL3, or SMARCB1 have been identified to be causative for Kleefstra syndrome spectrum (KSS), a neurodevelopmental disorder with clinical features of both intellectual disability (ID) and autism spectrum disorder (ASD). To understand how these variants lead to the phenotypic convergence in KSS, we employ a loss-of-function approach to assess neuronal network development at the molecular, single-cell, and network activity level. KSS-gene-deficient neuronal networks all develop into hyperactive networks with altered network organization and excitatory-inhibitory balance. Interestingly, even though transcriptional data reveal distinct regulatory mechanisms, KSS target genes share similar functions in regulating neuronal excitability and synaptic function, several of which are associated with ID and ASD. Our results show that KSS genes mainly converge at the level of neuronal network communication, providing insights into the pathophysiology of KSS and phenotypically congruent disorders.
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http://dx.doi.org/10.1016/j.celrep.2019.12.002DOI Listing
January 2020

Modeling Psychiatric Diseases with Induced Pluripotent Stem Cells.

Adv Exp Med Biol 2019 ;1192:297-312

Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, 6500 HB, Nijmegen, The Netherlands.

Neuropsychiatric disorders are a heterogeneous group of disorders that are challenging to model and treat, due to their underlying complex genetic architecture and clinical variability. Presently, increasingly more studies are making use of induced pluripotent stem cell (iPSC)-derived neurons, reprogrammed from patient somatic cells, to model neuropsychiatric disorders. iPSC-derived neurons offer the possibility to recapitulate relevant disease biology in the context of the individual patient genetic background. In addition to disease modeling, iPSC-derived neurons offer unprecedented opportunities in drug screening. In this chapter, the current status of iPSC disease modeling for neuropsychiatric disorders is presented. Both 2D and 3D disease modeling approaches are discussed as well as the generation of different neuronal cell types that are relevant for studying neuropsychiatric disorders. Moreover, the advantages and limitations are highlighted in addition to the future perspectives of using iPSC-derived neurons in the uncovering of robust cellular phenotypes that consecutively have the potential to lead to clinical developments.
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http://dx.doi.org/10.1007/978-981-32-9721-0_15DOI Listing
November 2019

Neuronal network dysfunction in a model for Kleefstra syndrome mediated by enhanced NMDAR signaling.

Nat Commun 2019 10 30;10(1):4928. Epub 2019 Oct 30.

Department of Human Genetics, Radboudumc, Donders Institute for Brain, Cognition, and Behaviour, 6500, HB Nijmegen, Netherlands.

Kleefstra syndrome (KS) is a neurodevelopmental disorder caused by mutations in the histone methyltransferase EHMT1. To study the impact of decreased EHMT1 function in human cells, we generated excitatory cortical neurons from induced pluripotent stem (iPS) cells derived from KS patients. Neuronal networks of patient-derived cells exhibit network bursting with a reduced rate, longer duration, and increased temporal irregularity compared to control networks. We show that these changes are mediated by upregulation of NMDA receptor (NMDAR) subunit 1 correlating with reduced deposition of the repressive H3K9me2 mark, the catalytic product of EHMT1, at the GRIN1 promoter. In mice EHMT1 deficiency leads to similar neuronal network impairments with increased NMDAR function. Finally, we rescue the KS patient-derived neuronal network phenotypes by pharmacological inhibition of NMDARs. Summarized, we demonstrate a direct link between EHMT1 deficiency and NMDAR hyperfunction in human neurons, providing a potential basis for more targeted therapeutic approaches for KS.
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http://dx.doi.org/10.1038/s41467-019-12947-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821803PMC
October 2019

ONECUT transcription factors induce neuronal characteristics and remodel chromatin accessibility.

Nucleic Acids Res 2019 06;47(11):5587-5602

Department of Human Genetics, Donders Institute for Brain, Cognition and Behavior, Radboud University Medical Center, Nijmegen, The Netherlands.

Remodeling of chromatin accessibility is necessary for successful reprogramming of fibroblasts to neurons. However, it is still not fully known which transcription factors can induce a neuronal chromatin accessibility profile when overexpressed in fibroblasts. To identify such transcription factors, we used ATAC-sequencing to generate differential chromatin accessibility profiles between human fibroblasts and iNeurons, an in vitro neuronal model system obtained by overexpression of Neurog2 in induced pluripotent stem cells (iPSCs). We found that the ONECUT transcription factor sequence motif was strongly associated with differential chromatin accessibility between iNeurons and fibroblasts. All three ONECUT transcription factors associated with this motif (ONECUT1, ONECUT2 and ONECUT3) induced a neuron-like morphology and expression of neuronal genes within two days of overexpression in fibroblasts. We observed widespread remodeling of chromatin accessibility; in particular, we found that chromatin regions that contain the ONECUT motif were in- or lowly accessible in fibroblasts and became accessible after the overexpression of ONECUT1, ONECUT2 or ONECUT3. There was substantial overlap with iNeurons, still, many regions that gained accessibility following ONECUT overexpression were not accessible in iNeurons. Our study highlights both the potential and challenges of ONECUT-based direct neuronal reprogramming.
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http://dx.doi.org/10.1093/nar/gkz273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582315PMC
June 2019

Loss of function of SVBP leads to autosomal recessive intellectual disability, microcephaly, ataxia, and hypotonia.

Genet Med 2019 08 4;21(8):1790-1796. Epub 2019 Jan 4.

Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

Purpose: Identifying and characterizing novel causes of autosomal recessive intellectual disability based on systematic clinical and genetic evaluation, followed by functional experiments.

Methods: Clinical examinations, genome-wide positional mapping, and sequencing were followed by quantitative polymerase chain reaction and western blot of the protein SVBP and its interaction partners. We then knocked down the gene in rat primary hippocampal neurons and evaluated the consequences on synapses.

Results: We identified a founder, homozygous stop-gain variant in SVBP (c.82C>T; p.[Gln28*]) in four affected individuals from two independent families with intellectual disability, microcephaly, ataxia, and muscular hypotonia. SVBP encodes a small chaperone protein that transports and stabilizes two angiogenesis regulators, VASH1 and VASH2. The altered protein is unstable and nonfunctional since transfected HeLa cells with mutant SVBP did not reveal evidence for immunoreactive SVBP protein fragments and cotransfection with VASH1 showed a severe reduction of VASH1 in medium and cell lysate. Knocking down Svbp in rat primary hippocampal neurons led to a significant decrease in the number of excitatory synapses.

Conclusion: SVBP is not only involved in angiogenesis, but also has vital functions in the central nervous system. Biallelic loss-of-function variants in SVBP lead to intellectual disability.
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http://dx.doi.org/10.1038/s41436-018-0415-8DOI Listing
August 2019

Foxp2 loss of function increases striatal direct pathway inhibition via increased GABA release.

Brain Struct Funct 2018 Dec 5;223(9):4211-4226. Epub 2018 Sep 5.

Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboudumc, 6525 HR, Nijmegen, The Netherlands.

Heterozygous mutations of the Forkhead-box protein 2 (FOXP2) gene in humans cause childhood apraxia of speech. Loss of Foxp2 in mice is known to affect striatal development and impair motor skills. However, it is unknown if striatal excitatory/inhibitory balance is affected during development and if the imbalance persists into adulthood. We investigated the effect of reduced Foxp2 expression, via a loss-of-function mutation, on striatal medium spiny neurons (MSNs). Our data show that heterozygous loss of Foxp2 decreases excitatory (AMPA receptor-mediated) and increases inhibitory (GABA receptor-mediated) currents in D1 dopamine receptor positive MSNs of juvenile and adult mice. Furthermore, reduced Foxp2 expression increases GAD67 expression, leading to both increased presynaptic content and release of GABA. Finally, pharmacological blockade of inhibitory activity in vivo partially rescues motor skill learning deficits in heterozygous Foxp2 mice. Our results suggest a novel role for Foxp2 in the regulation of striatal direct pathway activity through managing inhibitory drive.
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http://dx.doi.org/10.1007/s00429-018-1746-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6267273PMC
December 2018

Basolateral amygdala noradrenergic activity is required for enhancement of object recognition memory by histone deacetylase inhibition in the anterior insular cortex.

Neuropharmacology 2018 10 18;141:32-41. Epub 2018 Aug 18.

Dept. Cognitive Neuroscience, Radboud University Medical Center, Nijmegen, the Netherlands; Donders Institute for Brain, Cognition and Behaviour, Radboud University, Nijmegen, the Netherlands. Electronic address:

Extensive evidence indicates that noradrenergic activation of the basolateral amygdala (BLA) is essential for mediating emotional arousal effects on memory consolidation in different target regions. However, the mechanism by which BLA activation regulates such information storage processes remains largely elusive. Here we demonstrate, in male Sprague-Dawley rats, that noradrenergic activation of the BLA is critically involved in enabling facilitation of memory consolidation induced by histone acetylation, a form of chromatin modification, within the insular cortex (IC) on object recognition memory. The histone deacetylase (HDAC) inhibitor sodium butyrate (NaB) administered either systemically or directly into the anterior, but not posterior, IC immediately after object recognition training enhanced long-term memory for the identity of the object. Systemic NaB administration also enhanced memory for the location of the object. This NaB-induced enhancement of both object recognition and object location memory was selectively associated with an increased ability to assess the familiarity of the training stimulus, without affecting interaction with a novel stimulus. The β-adrenoceptor antagonist propranolol infused into the BLA concurrently abolished the NaB-induced enhancement of familiarity detection underlying both object recognition and object location memory. These findings indicate that noradrenergic activity within the BLA induced by emotional arousal interacts with chromatin modification mechanisms in its target regions to affect post-learning consolidation processes underlying long-term recognition memory and discrimination of a familiar stimulus.
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http://dx.doi.org/10.1016/j.neuropharm.2018.08.018DOI Listing
October 2018

Connecting the dots in mental illness: The synapse as the intersection of brain function and disease.

Prog Neuropsychopharmacol Biol Psychiatry 2018 06;84(Pt B):305

Florey Institute of Neuroscience and Mental Health, Florey Department of Neuroscience, University of Melbourne, Parkville Victoria Australia.

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http://dx.doi.org/10.1016/j.pnpbp.2018.03.025DOI Listing
June 2018

Increased H3K9 methylation and impaired expression of Protocadherins are associated with the cognitive dysfunctions of the Kleefstra syndrome.

Nucleic Acids Res 2018 06;46(10):4950-4965

Radboud University, Department of Molecular Biology, Faculty of Science, 6500 HB Nijmegen, the Netherlands.

Kleefstra syndrome, a disease with intellectual disability, autism spectrum disorders and other developmental defects is caused in humans by haploinsufficiency of EHMT1. Although EHMT1 and its paralog EHMT2 were shown to be histone methyltransferases responsible for deposition of the di-methylated H3K9 (H3K9me2), the exact nature of epigenetic dysfunctions in Kleefstra syndrome remains unknown. Here, we found that the epigenome of Ehmt1+/- adult mouse brain displays a marked increase of H3K9me2/3 which correlates with impaired expression of protocadherins, master regulators of neuronal diversity. Increased H3K9me3 was present already at birth, indicating that aberrant methylation patterns are established during embryogenesis. Interestingly, we found that Ehmt2+/- mice do not present neither the marked increase of H3K9me2/3 nor the cognitive deficits found in Ehmt1+/- mice, indicating an evolutionary diversification of functions. Our finding of increased H3K9me3 in Ehmt1+/- mice is the first one supporting the notion that EHMT1 can quench the deposition of tri-methylation by other Histone methyltransferases, ultimately leading to impaired neurocognitive functioning. Our insights into the epigenetic pathophysiology of Kleefstra syndrome may offer guidance for future developments of therapeutic strategies for this disease.
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http://dx.doi.org/10.1093/nar/gky196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6007260PMC
June 2018

Inhibitory control of the excitatory/inhibitory balance in psychiatric disorders.

F1000Res 2018 8;7:23. Epub 2018 Jan 8.

Department of Human Genetics & Department of Cognitive Neuroscience, Radboudumc, Geert Grooteplein 10, Box 9101, 6500 HB Nijmegen, Netherlands.

Neuronal networks consist of different types of neurons that all play their own role in order to maintain proper network function. The two main types of neurons segregate in excitatory and inhibitory neurons, which together regulate the flow of information through the network. It has been proposed that changes in the relative strength in these two opposing forces underlie the symptoms observed in psychiatric disorders, including autism and schizophrenia. Here, we review the role of alterations to the function of the inhibitory system as a cause of psychiatric disorders. First, we explore both patient and post-mortem evidence of inhibitory deficiency. We then discuss the function of different interneuron subtypes in the network and focus on the central role of a specific class of inhibitory neurons, parvalbumin-positive interneurons. Finally, we discuss genes known to be affected in different disorders and the effects that mutations in these genes have on the inhibitory system in cortex and hippocampus. We conclude that alterations to the inhibitory system are consistently identified in animal models of psychiatric disorders and, more specifically, that mutations affecting the function of parvalbumin-positive interneurons seem to play a central role in the symptoms observed in these disorders.
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http://dx.doi.org/10.12688/f1000research.12155.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760969PMC
January 2018

Integrated transcriptional analysis unveils the dynamics of cellular differentiation in the developing mouse hippocampus.

Sci Rep 2017 12 22;7(1):18073. Epub 2017 Dec 22.

Radboud University, Department of Molecular Biology, Faculty of Science, 6500 HB, Nijmegen, The Netherlands.

The ability to assign expression patterns to the individual cell types that constitute a tissue is a major challenge. This especially applies to brain, given its plethora of different, functionally interconnected cell types. Here, we derived cell type-specific transcriptome signatures from existing single cell RNA data and integrated these signatures with a newly generated dataset of expression (bulk RNA-Seq) of the postnatal developing mouse hippocampus. This integrated analysis allowed us to provide a comprehensive and unbiased prediction of the differentiation drivers for 11 different hippocampal cell types and describe how the different cell types interact to support crucial developmental stages. Our results provide a reliable resource of predicted differentiation drivers and insights into the multifaceted aspects of the cells in hippocampus during development.
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http://dx.doi.org/10.1038/s41598-017-18287-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741714PMC
December 2017

The promise of induced pluripotent stem cells for neurodevelopmental disorders.

Prog Neuropsychopharmacol Biol Psychiatry 2018 06 8;84(Pt B):382-391. Epub 2017 Nov 8.

Department of Human Genetics, Department of Cognitive Neuroscience, Radboudumc, 6500 HB, Nijmegen, The Netherlands; Donders Institute for Brain, Cognition, and Behaviour, Centre for Neuroscience, 6525 AJ, Nijmegen, The Netherlands. Electronic address:

A major challenge in clinical genetics and medicine is represented by genetically and phenotypically highly diverse neurodevelopmental disorders, like for example intellectual disability and autism. Intellectual disability is characterized by substantial limitations in cognitive function and adaptive behaviour. At the cellular level, this is reflected by deficits in synaptic structure and plasticity and therefore has been coined as a synaptic disorder or "synaptopathy". In this review, we summarize the findings from recent studies in which iPSCs have been used to model specific neurodevelopmental syndromes, including Fragile X syndrome, Rett syndrome, Williams-Beuren syndrome and Phelan-McDermid syndrome. We discuss what we have learned from these studies and what key issues need to be addressed to move the field forward.
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http://dx.doi.org/10.1016/j.pnpbp.2017.11.009DOI Listing
June 2018

Epigenetic Etiology of Intellectual Disability.

J Neurosci 2017 11;37(45):10773-10782

Instituto de Neurociencias, Molecular Neurobiology Unit, s/n 03550, San Juan de Alicante, Alicante, Spain

Intellectual disability (ID) is a prevailing neurodevelopmental condition associated with impaired cognitive and adaptive behaviors. Many chromatin-modifying enzymes and other epigenetic regulators have been genetically associated with ID disorders (IDDs). Here we review how alterations in the function of histone modifiers, chromatin remodelers, and methyl-DNA binding proteins contribute to neurodevelopmental defects and altered brain plasticity. We also discuss how progress in human genetics has led to the generation of mouse models that unveil the molecular etiology of ID, and outline the direction in which this field is moving to identify therapeutic strategies for IDDs. Importantly, because the chromatin regulators linked to IDDs often target common downstream genes and cellular processes, the impact of research in individual syndromes goes well beyond each syndrome and can also contribute to the understanding and therapy of other IDDs. Furthermore, the investigation of these disorders helps us to understand the role of chromatin regulators in brain development, plasticity, and gene expression, thereby answering fundamental questions in neurobiology.
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http://dx.doi.org/10.1523/JNEUROSCI.1840-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678009PMC
November 2017

Mouse models of 17q21.31 microdeletion and microduplication syndromes highlight the importance of Kansl1 for cognition.

PLoS Genet 2017 Jul 13;13(7):e1006886. Epub 2017 Jul 13.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Illkirch, France.

Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches.
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http://dx.doi.org/10.1371/journal.pgen.1006886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5531616PMC
July 2017

Basal ryanodine receptor activity suppresses autophagic flux.

Biochem Pharmacol 2017 05 18;132:133-142. Epub 2017 Mar 18.

KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, B-3000 Leuven, Belgium. Electronic address:

The inositol 1,4,5-trisphosphate receptors (IPRs) and intracellular Ca signaling are critically involved in regulating different steps of autophagy, a lysosomal degradation pathway. The ryanodine receptors (RyR), intracellular Ca-release channels mainly expressed in excitable cell types including muscle and neurons, have however not yet been extensively studied in relation to autophagy. Yet, aberrant expression and excessive activity of RyRs in these tissues has been implicated in the onset of several diseases including Alzheimer's disease, where impaired autophagy regulation contributes to the pathology. In this study, we determined whether pharmacological RyR inhibition could modulate autophagic flux in ectopic RyR-expressing models, like HEK293 cells and in cell types that endogenously express RyRs, like C2C12 myoblasts and primary hippocampal neurons. Importantly, RyR3 overexpression in HEK293 cells impaired the autophagic flux. Conversely, in all cell models tested, pharmacological inhibition of endogenous or ectopically expressed RyRs, using dantrolene or ryanodine, augmented autophagic flux by increasing lysosomal turn-over (number of autophagosomes and autolysosomes measured as mCherry-LC3 punctae/cell increased from 70.37±7.81 in control HEK RyR3 cells to 111.18±7.72 and 98.14±7.31 after dantrolene and ryanodine treatments, respectively). Moreover, in differentiated C2C12 cells, transmission electron microscopy demonstrated that dantrolene treatment decreased the number of early autophagic vacuoles from 5.9±2.97 to 1.8±1.03 per cellular cross section. The modulation of the autophagic flux could be linked to the functional inhibition of RyR channels as both RyR inhibitors efficiently diminished the number of cells showing spontaneous RyR3 activity in the HEK293 cell model (from 41.14%±2.12 in control cells to 18.70%±2.25 and 9.74%±2.67 after dantrolene and ryanodine treatments, respectively). In conclusion, basal RyR-mediated Ca-release events suppress autophagic flux at the level of the lysosomes.
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http://dx.doi.org/10.1016/j.bcp.2017.03.011DOI Listing
May 2017

Reduced Inhibition within Layer IV of Sert Knockout Rat Barrel Cortex is Associated with Faster Sensory Integration.

Cereb Cortex 2017 02;27(2):933-949

Department of Cognitive Neuroscience, Radboudumc, Donders Institute for Brain, Cognition and Behaviour, Nijmegen, The Netherlands.

Neural activity is essential for the maturation of sensory systems. In the rodent primary somatosensory cortex (S1), high extracellular serotonin (5-HT) levels during development impair neural transmission between the thalamus and cortical input layer IV (LIV). Rodent models of impaired 5-HT transporter (SERT) function show disruption in their topological organization of S1 and in the expression of activity-regulated genes essential for inhibitory cortical network formation. It remains unclear how such alterations affect the sensory information processing within cortical LIV. Using serotonin transporter knockout (Sert-/-) rats, we demonstrate that high extracellular serotonin levels are associated with impaired feedforward inhibition (FFI), fewer perisomatic inhibitory synapses, a depolarized GABA reversal potential and reduced expression of KCC2 transporters in juvenile animals. At the neural population level, reduced FFI increases the excitatory drive originating from LIV, facilitating evoked representations in the supragranular layers II/III. The behavioral consequence of these changes in network excitability is faster integration of the sensory information during whisker-based tactile navigation, as Sert-/- rats require fewer whisker contacts with tactile targets and perform object localization with faster reaction times. These results highlight the association of serotonergic homeostasis with formation and excitability of sensory cortical networks, and consequently with sensory perception.
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http://dx.doi.org/10.1093/cercor/bhx016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390402PMC
February 2017

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays.

J Vis Exp 2017 01 8(119). Epub 2017 Jan 8.

Department of Cognitive Neurosciences, Radboudumc; Donders Institute for Brain, Cognition and Behaviour, Radboud University; Department of Human Genetics, Radboudumc;

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-2. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (>95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.
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http://dx.doi.org/10.3791/54900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5407693PMC
January 2017
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