NRXN1α+/- is associated with increased excitability in ASD iPSC-derived neurons

Abstract Background NRXN1 deletions are identified as one of major rare risk factors for autism spectrum disorder (ASD) and other neurodevelopmental disorders. ASD has 30% co-morbidity with epilepsy, and the latter is associated with excessive neuronal firing. NRXN1 encodes hundreds of presynaptic neuro-adhesion proteins categorized as NRXN1α/β/γ. Previous studies on cultured cells show that the short NRXN1β primarily exerts excitation effect, whereas the long NRXN1α which is more commonly deleted in patients involves in both excitation and inhibition. However, patient-derived models are essential for understanding functional consequences of NRXN1α deletions in human neurons. We recently derived induced pluripotent stem cells (iPSCs) from five controls and three ASD patients carrying NRXN1α+/- and showed increased calcium transients in patient neurons. Methods In this study we investigated the electrophysiological properties of iPSC-derived cortical neurons in control and ASD patients carrying NRXN1α+/- using patch clamping. Whole genome RNA sequencing was carried out to further understand the potential underlying molecular mechanism. Results NRXN1α+/- cortical neurons were shown to display larger sodium currents, higher AP amplitude and accelerated depolarization time. RNASeq analyses revealed transcriptomic changes with significant upregulation glutamatergic synapse and ion channels/transporter activity including voltage-gated potassium channels (GRIN1, GRIN3B, SLC17A6, CACNG3, CACNA1A, SHANK1), which are likely to couple with the increased excitability in NRXN1α+/- cortical neurons. Conclusions Together with recent evidence of increased calcium transients, our results showed that human NRXN1α+/- isoform deletions altered neuronal excitability and non-synaptic function, and NRXN1α+/- patient iPSCs may be used as an ASD model for therapeutic development with calcium transients and excitability as readouts.

Download Full-text

P.135 Autism-associated mutations in SHANK2 increase synaptic connectivity and dendrite complexity in human neurons

Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques ◽

10.1017/cjn.2018.237 ◽

2018 ◽

Vol 45 (s2) ◽

pp. S52-S52

Author(s):

K Zaslavsky ◽

W Zhang ◽

E Deneault ◽

M Zhao ◽

DC Rodrigues ◽

...

Keyword(s):

Cortical Neurons ◽

Autism Spectrum ◽

Dermal Fibroblasts ◽

Neuronal Morphology ◽

Synaptic Connectivity ◽

Loss Of Function ◽

Knock Out ◽

Electrophysiological Properties ◽

Dendrite Length ◽

Human Neurons

Background: Heterozygous loss-of-function mutations in the synaptic scaffolding gene SHANK2 are strongly associated with autism spectrum disorder (ASD). However, their impact on the function of human neurons is unknown. Derivation of induced pluripotent stem cells (iPSC) from affected individuals permits generation of live neurons to answer this question. Methods: We generated iPSCs by reprogramming dermal fibroblasts of neurotypic and ASD-affected donors. To isolate the effect of SHANK2, we used CRISPR/Cas9 to knock out SHANK2 in control iPSCs and correct a heterozygous nonsense mutation in ASD-affected donor iPSCs. We then derived cortical neurons from SOX1+ neural precursor cells differentiated from these iPSCs. Using a novel assay that overcomes line-to-line variability, we compared neuronal morphology, total synapse number, and electrophysiological properties between SHANK2 mutants and controls. Results: Relative to controls, SHANK2 mutant neurons have increased dendrite complexity, dendrite length, total synapse number (1.5-2-fold), and spontaneous excitatory postsynaptic current (sEPSC) frequency (3-7.6-fold). Conclusions: ASD-associated heterozygous loss-of-function mutations in SHANK2 increase synaptic connectivity among human neurons by increasing synapse number and sEPSC frequency. This is partially supported by increased dendrite length and complexity, providing evidence that SHANK2 functions as a suppressor of dendrite branching during neurodevelopment.

Download Full-text

Increased Ca2+ signaling in NRXN1α+/− neurons derived from ASD induced pluripotent stem cells

Molecular Autism ◽

10.1186/s13229-019-0303-3 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Sahar Avazzadeh ◽

Katya McDonagh ◽

Jamie Reilly ◽

Yanqin Wang ◽

Stephanie D. Boomkamp ◽

...

Keyword(s):

Clinical Symptoms ◽

Neurodevelopmental Disorder ◽

Autism Spectrum ◽

Healthy Donors ◽

Human Neurons ◽

Co Morbidity ◽

Functional Consequences ◽

Skin Biopsies ◽

Screening Assays ◽

Induced Pluripotent

Abstract Background Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a high co-morbidity of epilepsy and associated with hundreds of rare risk factors. NRXN1 deletion is among the commonest rare genetic factors shared by ASD, schizophrenia, intellectual disability, epilepsy, and developmental delay. However, how NRXN1 deletions lead to different clinical symptoms is unknown. Patient-derived cells are essential to investigate the functional consequences of NRXN1 lesions to human neurons in different diseases. Methods Skin biopsies were donated by five healthy donors and three ASD patients carrying NRXN1α+/− deletions. Seven control and six NRXN1α+/− iPSC lines were derived and differentiated into day 100 cortical excitatory neurons using dual SMAD inhibition. Calcium (Ca2+) imaging was performed using Fluo4-AM, and the properties of Ca2+ transients were compared between two groups of neurons. Transcriptome analysis was carried out to undercover molecular pathways associated with NRXN1α+/− neurons. Results NRXN1α+/− neurons were found to display altered calcium dynamics, with significantly increased frequency, duration, and amplitude of Ca2+ transients. Whole genome RNA sequencing also revealed altered ion transport and transporter activity, with upregulated voltage-gated calcium channels as one of the most significant pathways in NRXN1α+/− neurons identified by STRING and GSEA analyses. Conclusions This is the first report to show that human NRXN1α+/− neurons derived from ASD patients’ iPSCs present novel phenotypes of upregulated VGCCs and increased Ca2+ transients, which may facilitate the development of drug screening assays for the treatment of ASD.

Download Full-text

Chronic lithium treatment alters the excitatory/ inhibitory balance of synaptic networks and reduces mGluR5–PKC signalling in mouse cortical neurons

Journal of Psychiatry and Neuroscience ◽

10.1503/jpn.200185 ◽

2021 ◽

Vol 46 (3) ◽

pp. E402-E414

Author(s):

Anouar Khayachi ◽

Ariel Ase ◽

Calwing Liao ◽

Anusha Kamesh ◽

Naila Kuhlmann ◽

...

Keyword(s):

Bipolar Disorder ◽

Cortical Neurons ◽

Neuronal Excitability ◽

Lithium Treatment ◽

Calcium Flux ◽

Therapeutic Benefits ◽

Human Neurons ◽

Imaging Results ◽

Holistic Understanding ◽

Depressive Episodes

Background: Bipolar disorder is characterized by cyclical alternation between mania and depression, often comorbid with psychosis and suicide. Compared with other medications, the mood stabilizer lithium is the most effective treatment for the prevention of manic and depressive episodes. However, the pathophysiology of bipolar disorder and lithium’s mode of action are yet to be fully understood. Evidence suggests a change in the balance of excitatory and inhibitory activity, favouring excitation in bipolar disorder. In the present study, we sought to establish a holistic understanding of the neuronal consequences of lithium exposure in mouse cortical neurons, and to identify underlying mechanisms of action. Methods: We used a range of technical approaches to determine the effects of acute and chronic lithium treatment on mature mouse cortical neurons. We combined RNA screening and biochemical and electrophysiological approaches with confocal immunofluorescence and live-cell calcium imaging. Results: We found that only chronic lithium treatment significantly reduced intracellular calcium flux, specifically by activating metabotropic glutamatergic receptor 5. This was associated with altered phosphorylation of protein kinase C and glycogen synthase kinase 3, reduced neuronal excitability and several alterations to synapse function. Consequently, lithium treatment shifts the excitatory–inhibitory balance toward inhibition. Limitations: The mechanisms we identified should be validated in future by similar experiments in whole animals and human neurons. Conclusion: Together, the results revealed how lithium dampens neuronal excitability and the activity of the glutamatergic network, both of which are predicted to be overactive in the manic phase of bipolar disorder. Our working model of lithium action enables the development of targeted strategies to restore the balance of overactive networks, mimicking the therapeutic benefits of lithium but with reduced toxicity.

Download Full-text

Global enhancement of cortical excitability following coactivation of large neuronal populations

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914869117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 20254-20264 ◽

Cited By ~ 1

Author(s):

Deng Zhang ◽

Xingjian Yan ◽

Liang She ◽

Yunqing Wen ◽

Mu-ming Poo

Keyword(s):

Cortical Neurons ◽

Neuronal Excitability ◽

Cortical Excitability ◽

Large Population ◽

Neuronal Firing ◽

Enhancement Effect ◽

Neuronal Populations ◽

Mouse Cortex ◽

The Brain

Correlated activation of cortical neurons often occurs in the brain and repetitive correlated neuronal firing could cause long-term modifications of synaptic efficacy and intrinsic excitability. We found that repetitive optogenetic activation of neuronal populations in the mouse cortex caused enhancement of optogenetically evoked firing of local coactivated neurons as well as distant cortical neurons in both ipsilateral and contralateral hemispheres. This global enhancement of evoked responses required coactivation of a sufficiently large population of neurons either within one cortical area or distributed in several areas. Enhancement of neuronal firing was saturable after repeated episodes of coactivation, diminished by inhibition ofN-methyl-d-aspartic acid receptors, and accompanied by elevated excitatory postsynaptic potentials, all consistent with activity-induced synaptic potentiation. Chemogenetic inhibition of neuronal activity of the thalamus decreased the enhancement effect, suggesting thalamic involvement. Thus, correlated excitation of large neuronal populations leads to global enhancement of neuronal excitability.

Download Full-text

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

10.1101/585596 ◽

2019 ◽

Cited By ~ 3

Author(s):

Monica Frega ◽

Katrin Linda ◽

Jason M. Keller ◽

Güvem Gümüş-Akay ◽

Britt Mossink ◽

...

Keyword(s):

Neural Network ◽

Neuronal Network ◽

Cortical Neurons ◽

Critical Role ◽

Ips Cells ◽

Autism Spectrum ◽

Human Model ◽

Human Neurons ◽

Kleefstra Syndrome ◽

The Impact

AbstractEpigenetic regulation of gene transcription plays a critical role in neural network development and in the etiology of Intellectual Disability (ID) and Autism Spectrum Disorder (ASD). However, little is known about the mechanisms by which epigenetic dysregulation leads to neural network defects. Kleefstra syndrome (KS), caused by mutation in the histone methyltransferase EHMT1, is a neurodevelopmental disorder with the clinical features of both ID and ASD. 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. In addition, we created an isogenic set by genetically editing healthy iPS cells. Characterization of the neurons at the single-cell and neuronal network level revealed consistent discriminative properties that distinguished EHMT1-mutant from wildtype neurons. Mutant neuronal networks exhibited network bursting with a reduced rate, longer duration, and increased temporal irregularity compared to control networks. We show that these changes were mediated by the upregulation of the NMDA receptor (NMDAR) subunit 1 and correlate with reduced deposition of the repressive H3K9me2 mark, the catalytic product of EHMT1, at the GRIN1 promoter. Furthermore, we show that EHMT1 deficiency in mice leads to similar neuronal network impairments and increased NMDAR function. Finally, we could rescue the KS patient-derived neuronal network phenotypes by pharmacological inhibition of NMDARs. Together, our results demonstrate a direct link between EHMT1 deficiency in human neurons and NMDAR hyperfunction, providing the basis for a more targeted therapeutic approach to treating KS.

Download Full-text

Deficiency of autism risk factor ASH1L in prefrontal cortex induces epigenetic aberrations and seizures

Nature Communications ◽

10.1038/s41467-021-26972-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Luye Qin ◽

Jamal B. Williams ◽

Tao Tan ◽

Tiaotiao Liu ◽

Qing Cao ◽

...

Keyword(s):

Risk Factor ◽

Prefrontal Cortex ◽

Neuronal Excitability ◽

Critical Role ◽

Treatment Strategies ◽

Autism Spectrum ◽

Synaptic Function ◽

Brain Diseases ◽

Risk Genes

AbstractASH1L, a histone methyltransferase, is identified as a top-ranking risk factor for autism spectrum disorder (ASD), however, little is known about the biological mechanisms underlying the link of ASH1L haploinsufficiency to ASD. Here we show that ASH1L expression and H3K4me3 level are significantly decreased in the prefrontal cortex (PFC) of postmortem tissues from ASD patients. Knockdown of Ash1L in PFC of juvenile mice induces the downregulation of risk genes associated with ASD, intellectual disability (ID) and epilepsy. These downregulated genes are enriched in excitatory and inhibitory synaptic function and have decreased H3K4me3 occupancy at their promoters. Furthermore, Ash1L deficiency in PFC causes the diminished GABAergic inhibition, enhanced glutamatergic transmission, and elevated PFC pyramidal neuronal excitability, which is associated with severe seizures and early mortality. Chemogenetic inhibition of PFC pyramidal neuronal activity, combined with the administration of GABA enhancer diazepam, rescues PFC synaptic imbalance and seizures, but not autistic social deficits or anxiety-like behaviors. These results have revealed the critical role of ASH1L in regulating synaptic gene expression and seizures, which provides insights into treatment strategies for ASH1L-associated brain diseases.

Download Full-text

DNA Methyltransferase 1 (DNMT1) Shapes Neuronal Activity of Human iPSC-Derived Glutamatergic Cortical Neurons

International Journal of Molecular Sciences ◽

10.3390/ijms22042034 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2034

Author(s):

Sarah Bachmann ◽

Jenice Linde ◽

Michael Bell ◽

Marc Spehr ◽

Hans Zempel ◽

...

Keyword(s):

Dna Methylation ◽

Neuronal Activity ◽

Cortical Neurons ◽

Dna Methyltransferase ◽

Synaptic Function ◽

Synaptic Activity ◽

Epigenetic Mechanism ◽

Human Neurons ◽

Excitatory Neurons ◽

Methyltransferase 1

Epigenetic mechanisms are emerging key players for the regulation of brain function, synaptic activity, and the formation of neuronal engrams in health and disease. As one important epigenetic mechanism of transcriptional control, DNA methylation was reported to distinctively modulate synaptic activity in excitatory and inhibitory cortical neurons in mice. Since DNA methylation signatures are responsive to neuronal activity, DNA methylation seems to contribute to the neuron’s capacity to adapt to and integrate changing activity patterns, being crucial for the plasticity and functionality of neuronal circuits. Since most studies addressing the role of DNA methylation in the regulation of synaptic function were conducted in mice or murine neurons, we here asked whether this functional implication applies to human neurons as well. To this end, we performed calcium imaging in human induced pluripotent stem cell (iPSC)-derived excitatory cortical neurons forming synaptic contacts and neuronal networks in vitro. Treatment with DNMT1 siRNA that diminishs the expression of the DNA (cytosine-5)-methyltransferase 1 (DNMT1) was conducted to investigate the functional relevance of DNMT1 as one of the main enzymes executing DNA methylations in the context of neuronal activity modulation. We observed a lowered proportion of actively firing neurons upon DNMT1-knockdown in these iPSC-derived excitatory neurons, pointing to a correlation of DNMT1-activity and synaptic transmission. Thus, our experiments suggest that DNMT1 decreases synaptic activity of human glutamatergic neurons and underline the relevance of epigenetic regulation of synaptic function also in human excitatory neurons.

Download Full-text

Increased BDNF Protein Expression after Ischemic Or PKC Epsilon Preconditioning Promotes Electrophysiologic Changes That Lead to Neuroprotection

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2014.185 ◽

2014 ◽

Vol 35 (1) ◽

pp. 121-130 ◽

Cited By ~ 34

Author(s):

Jake T Neumann ◽

John W Thompson ◽

Ami P Raval ◽

Charles H Cohan ◽

Kevin B Koronowski ◽

...

Keyword(s):

Protein Expression ◽

Cortical Neurons ◽

Neuronal Excitability ◽

Hippocampal Slices ◽

Glucose Deprivation ◽

Neuronal Firing ◽

Trk Receptors ◽

Bdnf Mrna

Ischemic preconditioning (IPC) via protein kinase C epsilon (PKCε) activation induces neuroprotection against lethal ischemia. Brain-derived neurotrophic factor (BDNF) is a pro-survival signaling molecule that modulates synaptic plasticity and neurogenesis. Interestingly, BDNF mRNA expression increases after IPC. In this study, we investigated whether IPC or pharmacological preconditioning (PKCε activation) promoted BDNF-induced neuroprotection, if neuroprotection by IPC or PKCε activation altered neuronal excitability, and whether these changes were BDNF-mediated. We used both in vitro (hippocampal organotypic cultures and cortical neuronal-glial cocultures) and in vivo (acute hippocampal slices 48 hours after preconditioning) models of IPC or PKCε activation. BDNF protein expression increased 24 to 48 hours after preconditioning, where inhibition of the BDNF Trk receptors abolished neuroprotection against oxygen and glucose deprivation (OGD) in vitro. In addition, there was a significant decrease in neuronal firing frequency and increase in threshold potential 48 hours after preconditioning in vivo, where this threshold modulation was dependent on BDNF activation of Trk receptors in excitatory cortical neurons. In addition, 48 hours after PKCε activation in vivo, the onset of anoxic depolarization during OGD was significantly delayed in hippocampal slices. Overall, these results suggest that after IPC or PKCε activation, there are BDNF-dependent electrophysiologic modifications that lead to neuroprotection.

Download Full-text

The Autism Risk Factor CHD8 Is a Chromatin Activator in Human Neurons and Functionally Dependent on the ERK-MAPK Pathway Effector ELK1

10.21203/rs.3.rs-799225/v1 ◽

2021 ◽

Author(s):

Bahareh Haddad Derafshi ◽

Tamas Danko ◽

Soham Chanda ◽

Pedro Batista ◽

Ulrike Litzenburger ◽

...

Keyword(s):

Cortical Neurons ◽

De Novo ◽

Mapk Pathway ◽

Transcriptional Profiling ◽

Distinct Group ◽

Chromatin Accessibility ◽

Autism Spectrum ◽

Loss Of Function ◽

De Novo Mutations ◽

Human Neurons

Abstract The chromodomain helicase DNA-binding protein CHD8 is among the most frequently found de-novo mutations in autism spectrum disorder (ASD)1-4. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is believed to be a chromatin regulator, but mechanisms for its genomic targeting is also unclear. To elucidate the role of CHD8 in human neurons, we generated conditional loss-of-function alleles in pluripotent stem cells. Chromatin accessibility and transcriptional profiling showed that CHD8 is a potent chromatin opener and transcriptional activator of its direct neuronal targets, including a distinct group of ASD genes. We found the chromatin targeting of CHD8 to be highly context dependent. In human neurons, CHD8 was preferentially bound at promoter sequences which were significantly enriched in ETS motifs. Indeed, the chromatin state of ETS motif-containing promoters was preferentially affected upon loss of CHD8. Among the many ETS transcription factors, we found ELK1 to be the best correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our ES cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply the functional cooperativity between ELK1, a key downstream factor of the MAPK/ERK pathway, and CHD8 on chromatin involvement in human neurons. THEREFORE, the MAPK/ERK/ELK1 axis may also play a role in the pathogenesis caused by CHD8 mutations5 .

Download Full-text

The autism-associated gene Scn2a plays an essential role in synaptic stability and learning

10.1101/366781 ◽

2018 ◽

Cited By ~ 1

Author(s):

Perry WE Spratt ◽

Roy Ben-Shalom ◽

Caroline M Keeshen ◽

Kenneth J Burke ◽

Rebecca L Clarkson ◽

...

Keyword(s):

Action Potential ◽

Sodium Channel ◽

Cortical Neurons ◽

Pyramidal Neurons ◽

De Novo ◽

Gene Mutations ◽

Autism Spectrum ◽

Synaptic Function ◽

Cellular Mechanisms ◽

Behavioral Impairments

SummaryAutism spectrum disorder (ASD) is strongly associated with de novo gene mutations. One of the most commonly affected genes is SCN2A. ASD-associated SCN2A mutations impair the encoded protein NaV1.2, a sodium channel important for action potential initiation and propagation in developing excitatory cortical neurons. The link between an axonal sodium channel and ASD, a disorder typically attributed to synaptic or transcriptional dysfunction, is unclear. Here, we show NaV1.2 is unexpectedly critical for dendritic excitability and synaptic function in mature pyramidal neurons, in addition to regulating early developmental axonal excitability. NaV1.2 loss reduced action potential backpropagation into dendrites, impairing synaptic plasticity and synaptic stability, even when NaV1.2 expression was disrupted late in development. Furthermore, we identified behavioral impairments in learning and sociability, paralleling observations in children with SCN2A loss. These results reveal a novel dendritic function for NaV1.2, providing insight into cellular mechanisms likely underlying circuit and behavioral dysfunction in ASD.

Download Full-text