Epilepsy is one of the most common neurological disorders. Although many factors contribute to epileptogenesis, seizure generation is mostly linked to hyperexcitability due to alterations in ...excitatory/inhibitory (E/I) balance. The common hypothesis is that reduced inhibition, increased excitation, or both contribute to the etiology of epilepsy. Increasing evidence shows that this view is oversimplistic, and that increased inhibition through depolarizing γ‐aminobutyric acid (GABA) similarly contributes to epileptogenisis. In early development, GABA signaling is depolarizing, inducing outward Cl− currents due to high intracellular Cl− concentrations. During maturation, the mechanisms of GABA action shift from depolarizing to hyperpolarizing, a critical event during brain development. Altered timing of this shift is associated with both neurodevelopmental disorders and epilepsy. Here, we consider the different ways that depolarizing GABA contributes to altered E/I balance and epileptogenesis, and discuss that alterations in depolarizing GABA could be a common denominator underlying seizure generation in neurodevelopmental disorders and epilepsies.
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.
Mammalian neocortex is a highly layered structure. Each layer is populated by distinct subtypes of principal cells that are born at different times during development. While the differences between ...principal cells across layers have been extensively studied, it is not known how the developmental profiles of neurons in different layers compare. Here, we provide a detailed morphological and functional characterisation of pyramidal neurons in mouse mPFC during the first postnatal month, corresponding to known critical periods for synapse and neuron formation in mouse sensory neocortex. Our data demonstrate similar maturation profiles of dendritic morphology and intrinsic properties of pyramidal neurons in both deep and superficial layers. In contrast, the balance of synaptic excitation and inhibition differs in a layer-specific pattern from one to four postnatal weeks of age. Our characterisation of the early development and maturation of pyramidal neurons in mouse mPFC not only demonstrates a comparable time course of postnatal maturation to that in other neocortical circuits, but also implies that consideration of layer- and time-specific changes in pyramidal neurons may be relevant for studies in mouse models of neuropsychiatric and neurodevelopmental disorders.
We implicated the X-chromosome THOC2 gene, which encodes the largest subunit of the highly-conserved TREX (Transcription-Export) complex, in a clinically complex neurodevelopmental disorder with ...intellectual disability as the core phenotype. To study the molecular pathology of this essential eukaryotic gene, we generated a mouse model based on a hypomorphic Thoc2 exon 37-38 deletion variant of a patient with ID, speech delay, hypotonia, and microcephaly. The Thoc2 exon 37-38 deletion male (Thoc2
) mice recapitulate the core phenotypes of THOC2 syndrome including smaller size and weight, and significant deficits in spatial learning, working memory and sensorimotor functions. The Thoc2
mouse brain development is significantly impacted by compromised THOC2/TREX function resulting in R-loop accumulation, DNA damage and consequent cell death. Overall, we suggest that perturbed R-loop homeostasis, in stem cells and/or differentiated cells in mice and the patient, and DNA damage-associated functional alterations are at the root of THOC2 syndrome.
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.
•MEAs are a robust tool to model neuronal network functioning•Neuronal networks from different healthy donors show comparable network activity•MEAs are able to distinguish disease-specific neuronal network phenotypes•We provide recommendations to standardize neuronal network recordings on MEA
In this article, Mossink and colleagues demonstrate that micro-electrode arrays (MEAs) are a highly robust tool to uncover genotype/phenotype interactions in hiPSC-derived excitatory neuronal networks, and provide an important resource for the design, execution, and analysis of hiPSC-derived neuronal networks studies on MEA.
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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•High m.3243A > G heteroplasmy leads to mitochondrial dysfunction in human neurons•High heteroplasmy reduces synapses, mitochondria, and dendritic complexity•High heteroplasmy leads to reduced single-cell neuronal activity•High heteroplasmy leads to lower neuronal network activity and synchronicity
Using human-inducible-pluripotent-stem-cell-derived neurons with high levels of m.3243A > G heteroplasmy, Klein Gunnewiek et al. show neuron-specific mitochondrial dysfunction as well as structural and functional impairments ranging from reduced dendritic complexity and fewer synapses and mitochondria to reduced neuronal activity and impaired network synchronicity.
Dravet syndrome is a severe epileptic encephalopathy, characterized by (febrile) seizures, behavioural problems and developmental delay. Eighty per cent of patients with Dravet syndrome have a ...mutation in SCN1A, encoding Nav1.1. Milder clinical phenotypes, such as GEFS+ (generalized epilepsy with febrile seizures plus), can also arise from SCN1A mutations. Predicting the clinical phenotypic outcome based on the type of mutation remains challenging, even when the same mutation is inherited within one family. This clinical and genetic heterogeneity adds to the difficulties of predicting disease progression and tailoring the prescription of anti-seizure medication. Understanding the neuropathology of different SCN1A mutations may help to predict the expected clinical phenotypes and inform the selection of best-fit treatments. Initially, the loss of Na+-current in inhibitory neurons was recognized specifically to result in disinhibition and consequently seizure generation. However, the extent to which excitatory neurons contribute to the pathophysiology is currently debated and might depend on the patient clinical phenotype or the specific SCN1A mutation. To examine the genotype-phenotype correlations of SCN1A mutations in relation to excitatory neurons, we investigated a panel of patient-derived excitatory neuronal networks differentiated on multi-electrode arrays. We included patients with different clinical phenotypes, harbouring various SCN1A mutations, along with a family in which the same mutation led to febrile seizures, GEFS+ or Dravet syndrome. We hitherto describe a previously unidentified functional excitatory neuronal network phenotype in the context of epilepsy, which corresponds to seizurogenic network prediction patterns elicited by proconvulsive compounds. We found that excitatory neuronal networks were affected differently, depending on the type of SCN1A mutation, but did not segregate according to clinical severity. Specifically, loss-of-function mutations could be distinguished from missense mutations, and mutations in the pore domain could be distinguished from mutations in the voltage sensing domain. Furthermore, all patients showed aggravated neuronal network responses at febrile temperatures compared with controls. Finally, retrospective drug screening revealed that anti-seizure medication affected GEFS+ patient- but not Dravet patient-derived neuronal networks in a patient-specific and clinically relevant manner. In conclusion, our results indicate a mutation-specific excitatory neuronal network phenotype, which recapitulates the foremost clinically relevant features, providing future opportunities for precision therapies.
Abstract
Purpose
To characterize a novel neurodevelopmental syndrome due to loss-of-function (LoF) variants in Ankyrin 2 (ANK2), and to explore the effects on neuronal network dynamics and ...homeostatic plasticity in human-induced pluripotent stem cell-derived neurons.
Methods
We collected clinical and molecular data of 12 individuals with heterozygous de novo LoF variants in ANK2. We generated a heterozygous LoF allele of ANK2 using CRISPR/Cas9 in human-induced pluripotent stem cells (hiPSCs). HiPSCs were differentiated into excitatory neurons, and we measured their spontaneous electrophysiological responses using micro-electrode arrays (MEAs). We also characterized their somatodendritic morphology and axon initial segment (AIS) structure and plasticity.
Results
We found a broad neurodevelopmental disorder (NDD), comprising intellectual disability, autism spectrum disorders and early onset epilepsy. Using MEAs, we found that hiPSC-derived neurons with heterozygous LoF of ANK2 show a hyperactive and desynchronized neuronal network. ANK2-deficient neurons also showed increased somatodendritic structures and altered AIS structure of which its plasticity is impaired upon activity-dependent modulation.
Conclusions
Phenotypic characterization of patients with de novo ANK2 LoF variants defines a novel NDD with early onset epilepsy. Our functional in vitro data of ANK2-deficient human neurons show a specific neuronal phenotype in which reduced ANKB expression leads to hyperactive and desynchronized neuronal network activity, increased somatodendritic complexity and AIS structure and impaired activity-dependent plasticity of the AIS.