Compromised transcription-mRNA export factor THOC2 causes R-loop accumulation, DNA damage and adverse neurodevelopment.

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 (Thoc2Δ/Y) 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 Thoc2Δ/Y 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.

A new blood DNA methylation signature for Koolen-de Vries syndrome: Classification of missense KANSL1 variants and comparison to fibroblast cells.

Pathogenic variants in KANSL1 and 17q21.31 microdeletions are causative of Koolen-de Vries syndrome (KdVS), a neurodevelopmental syndrome with characteristic facial dysmorphia. Our previous work has shown that syndromic conditions caused by pathogenic variants in epigenetic regulatory genes have identifiable patterns of DNA methylation (DNAm) change: DNAm signatures or episignatures. Given the role of KANSL1 in histone acetylation, we tested whether variants underlying KdVS are associated with a DNAm signature. We profiled whole-blood DNAm for 13 individuals with KANSL1 variants, four individuals with 17q21.31 microdeletions, and 21 typically developing individuals, using Illumina's Infinium EPIC array. In this study, we identified a robust DNAm signature of 456 significant CpG sites in 8 individuals with KdVS, a pattern independently validated in an additional 7 individuals with KdVS. We also demonstrate the diagnostic utility of the signature and classify two KANSL1 VUS as well as four variants in individuals with atypical clinical presentation. Lastly, we investigated tissue-specific DNAm changes in fibroblast cells from individuals with KdVS. Collectively, our findings contribute to the understanding of the epigenetic landscape related to KdVS and aid in the diagnosis and classification of variants in this structurally complex genomic region.

Epigenetic regulation of autophagy-related genes: Implications for neurodevelopmental disorders.

Macroautophagy/autophagy is an evolutionarily highly conserved catabolic process that is important for the clearance of cytosolic contents to maintain cellular homeostasis and survival. Recent findings point toward a critical role for autophagy in brain function, not only by preserving neuronal health, but especially by controlling different aspects of neuronal development and functioning. In line with this, mutations in autophagy-related genes are linked to various key characteristics and symptoms of neurodevelopmental disorders (NDDs), including autism, micro-/macrocephaly, and epilepsy. However, the group of NDDs caused by mutations in autophagy-related genes is relatively small. A significant proportion of NDDs are associated with mutations in genes encoding epigenetic regulatory proteins that modulate gene expression, so-called chromatinopathies. Intriguingly, several of the NDD-linked chromatinopathy genes have been shown to regulate autophagy-related genes, albeit in non-neuronal contexts. From these studies it becomes evident that tight transcriptional regulation of autophagy-related genes is crucial to control autophagic activity. This opens the exciting possibility that aberrant autophagic regulation might underly nervous system impairments in NDDs with disturbed epigenetic regulation. We here summarize NDD-related chromatinopathy genes that are known to regulate transcriptional regulation of autophagy-related genes. Thereby, we want to highlight autophagy as a candidate key hub mechanism in NDD-related chromatinopathies.Abbreviations: ADNP: activity dependent neuroprotector homeobox; ASD: autism spectrum disorder; ATG: AutTophaGy related; CpG: cytosine-guanine dinucleotide; DNMT: DNA methyltransferase; EHMT: euchromatic histone lysine methyltransferase; EP300: E1A binding protein p300; EZH2: enhancer of zeste 2 polycomb repressive complex 2 subunit; H3K4me3: histone 3 lysine 4 trimethylation; H3K9me1/2/3: histone 3 lysine 9 mono-, di-, or trimethylation; H3K27me2/3: histone 3 lysine 27 di-, or trimethylation; hiPSCs: human induced pluripotent stem cells; HSP: hereditary spastic paraplegia; ID: intellectual disability; KANSL1: KAT8 regulatory NSL complex subunit 1; KAT8: lysine acetyltransferase 8; KDM1A/LSD1: lysine demethylase 1A; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; NDD: neurodevelopmental disorder; PHF8: PHD finger protein 8; PHF8-XLID: PHF8-X linked intellectual disability syndrome; PTM: post-translational modification; SESN2: sestrin 2; YY1: YY1 transcription factor; YY1AP1: YY1 associated protein 1.

A human in vitro neuronal model for studying homeostatic plasticity at the network level.

Mechanisms that underlie homeostatic plasticity have been extensively investigated at single-cell levels in animal models, but are less well understood at the network level. Here, we used microelectrode arrays to characterize neuronal networks following induction of homeostatic plasticity in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with rat astrocytes. Chronic suppression of neuronal activity through tetrodotoxin (TTX) elicited a time-dependent network re-arrangement. Increased expression of AMPA receptors and the elongation of axon initial segments were associated with increased network excitability following TTX treatment. Transcriptomic profiling of TTX-treated neurons revealed up-regulated genes related to extracellular matrix organization, while down-regulated genes related to cell communication; also astrocytic gene expression was found altered. Overall, our study shows that hiPSC-derived neuronal networks provide a reliable in vitro platform to measure and characterize homeostatic plasticity at network and single-cell levels; this platform can be extended to investigate altered homeostatic plasticity in brain disorders.

Generation of an induced pluripotent stem cell line carrying biallelic deletions (SCTCi019-B) in ALDH7A1 using CRISPR/Cas9.

Biallelic pathogenic variants in ALDH7A1 are associated with pyridoxine-dependent epilepsy (PDE). ALDH7A1 encodes for the third enzyme of the lysine catabolism pathway. In this study a human isogenic ALDH7A1 knock-out iPSC line was created using CRISPR/Cas9 technology. One clone (SCTCi019-B) with biallelic deletions in ALDH7A1 was obtained and fully characterized, showing expression of pluripotency markers, a normal karyotype and no off-targets. Human-based models derived from this iPSC line will contribute to gain insights in the molecular mechanism of disease underlying PDE.

Sialic acid biosynthesis pathway blockade disturbs neuronal network formation in human iPSC-derived excitatory neurons.

N-acetylneuraminic acid (sialic acid) is present in large quantities in the brain and plays a crucial role in brain development, learning, and memory formation. How sialic acid contributes to brain development is not fully understood. The purpose of this study was to determine the effects of reduced sialylation on network formation in human iPSC-derived neurons (iNeurons). Using targeted mass spectrometry and antibody binding, we observed an increase in free sialic acid and polysialic acid during neuronal development, which was disrupted by treatment of iNeurons with a synthetic inhibitor of sialic acid biosynthesis. Sialic acid inhibition disturbed synapse formation and network formation on microelectrode array (MEA), showing short but frequent (network) bursts and an overall lower firing rate, and higher percentage of random spikes. This study shows that sialic acid is necessary for neuronal network formation during human neuronal development and provides a physiologically relevant model to study the role of sialic acid in patient-derived iNeurons.

SCN1A-deficient excitatory neuronal networks display mutation-specific phenotypes.

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.

An in silico and in vitro human neuronal network model reveals cellular mechanisms beyond NaV1.1 underlying Dravet syndrome.

Human induced pluripotent stem cell (hiPSC)-derived neuronal networks on multi-electrode arrays (MEAs) provide a unique phenotyping tool to study neurological disorders. However, it is difficult to infer cellular mechanisms underlying these phenotypes. Computational modeling can utilize the rich dataset generated by MEAs, and advance understanding of disease mechanisms. However, existing models lack biophysical detail, or validation and calibration to relevant experimental data. We developed a biophysical in silico model that accurately simulates healthy neuronal networks on MEAs. To demonstrate the potential of our model, we studied neuronal networks derived from a Dravet syndrome (DS) patient with a missense mutation in SCN1A, encoding sodium channel NaV1.1. Our in silico model revealed that sodium channel dysfunctions were insufficient to replicate the in vitro DS phenotype, and predicted decreased slow afterhyperpolarization and synaptic strengths. We verified these changes in DS patient-derived neurons, demonstrating the utility of our in silico model to predict disease mechanisms.

Excitatory/inhibitory balance in epilepsies and neurodevelopmental disorders: Depolarizing γ-aminobutyric acid as a common mechanism.

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.

ANK2 loss-of-function variants are associated with epilepsy, and lead to impaired axon initial segment plasticity and hyperactive network activity in hiPSC-derived neuronal networks.

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.

Generation of glutamatergic/GABAergic neuronal co-cultures derived from human induced pluripotent stem cells for characterizing E/I balance in vitro.

Obtaining mechanistic insights into the disruptions of neuronal excitation and inhibition (E/I) balance in brain disorders has remained challenging. Here, we present a protocol for in vitro characterization of E/I balance. Using human induced pluripotent stem cells, we describe the generation of glutamatergic excitatory/GABAergic inhibitory neuronal co-cultures at defined ratios, followed by analyzing E/I network properties using immunocytochemistry and multi-electrode array recording. This approach allows for studying cell-type-specific contribution of disease genes to E/I balance in human neurons. For complete details on the use and execution of this protocol, please refer to Mossink et al. (2022)1 and Wang et al. (2022).2.

Generation of an induced pluripotent stem cell line carrying a biallelic deletion (SCTCi019-A) in GCDH using CRISPR/Cas9.

GCDH encodes for the enzyme catalyzing the sixth step of the lysine catabolism pathway. Biallelic pathogenic variants in GCDH have been associated with glutaric aciduria type 1 (GA1). In this study CRISPR/Cas9 technology was used to create an isogenic GCDH knock-out human iPSC line. One clone with a biallelic deletion (SCTCi019-A) in GCDH was obtained and fully characterized, revealing a normal karyotype, no off-targets detected and expression of pluripotency markers. This iPSC line can contribute to gain insights in the molecular mechanism of disease.

Neuronal network activity and connectivity are impaired in a conditional knockout mouse model with PCDH19 mosaic expression.

Mutations in PCDH19 gene, which encodes protocadherin-19 (PCDH19), cause Developmental and Epileptic Encephalopathy 9 (DEE9). Heterogeneous loss of PCDH19 expression in neurons is considered a key determinant of the disorder; however, how PCDH19 mosaic expression affects neuronal network activity and circuits is largely unclear. Here, we show that the hippocampus of Pcdh19 mosaic mice is characterized by structural and functional synaptic defects and by the presence of PCDH19-negative hyperexcitable neurons. Furthermore, global reduction of network firing rate and increased neuronal synchronization have been observed in different limbic system areas. Finally, network activity analysis in freely behaving mice revealed a decrease in excitatory/inhibitory ratio and functional hyperconnectivity within the limbic system of Pcdh19 mosaic mice. Altogether, these results indicate that altered PCDH19 expression profoundly affects circuit wiring and functioning, and provide new key to interpret DEE9 pathogenesis.

Generation of induced pluripotent stem cell line carrying frameshift variants in NPHP1 (UCSFi001-A-68) using CRISPR/Cas9.

NPHP1 (Nephrocystin 1) is a protein that localizes to the transition zone of the cilium, a small organelle that projects from the plasma membrane of most cells and allows for integration and coordination of signalling pathways during development and homeostasis. Loss of NPHP1 function due to biallelic NPHP1 gene mutations can lead to the development of ciliopathies - a heterogeneous spectra of disorders characterized by ciliary dysfunction. Here we report the generation of an NPHP1-null hiPSC line (UCSFi001-A-68) via CRISPR/Cas9-mediated non-homologous end joining in the UCSFi001-A background, for study of the role that this protein plays in different tissues.

Loss-of-function variants in the schizophrenia risk gene SETD1A alter neuronal network activity in human neurons through the cAMP/PKA pathway.

Heterozygous loss-of-function (LoF) mutations in SETD1A, which encodes a subunit of histone H3 lysine 4 methyltransferase, cause a neurodevelopmental syndrome and increase the risk for schizophrenia. Using CRISPR-Cas9, we generate excitatory/inhibitory neuronal networks from human induced pluripotent stem cells with a SETD1A heterozygous LoF mutation (SETD1A+/-). Our data show that SETD1A haploinsufficiency results in morphologically increased dendritic complexity and functionally increased bursting activity. This network phenotype is primarily driven by SETD1A haploinsufficiency in glutamatergic neurons. In accordance with the functional changes, transcriptomic profiling reveals perturbations in gene sets associated with glutamatergic synaptic function. At the molecular level, we identify specific changes in the cyclic AMP (cAMP)/Protein Kinase A pathway pointing toward a hyperactive cAMP pathway in SETD1A+/- neurons. Finally, by pharmacologically targeting the cAMP pathway, we are able to rescue the network deficits in SETD1A+/- cultures. Our results demonstrate a link between SETD1A and the cAMP-dependent pathway in human neurons.

Basolateral amygdala activation enhances object recognition memory by inhibiting anterior insular cortex activity.

Noradrenergic activation of the basolateral amygdala (BLA) by emotional arousal enhances different forms of recognition memory via functional interactions with the insular cortex (IC). Human neuroimaging studies have revealed that the anterior IC (aIC), as part of the salience network, is dynamically regulated during arousing situations. Emotional stimulation first rapidly increases aIC activity but suppresses it in a delayed fashion. Here, we investigated in male Sprague-Dawley rats whether the BLA influence on recognition memory is associated with an increase or suppression of aIC activity during the postlearning consolidation period. We first employed anterograde and retrograde viral tracing and found that the BLA sends dense monosynaptic projections to the aIC. Memory-enhancing norepinephrine administration into the BLA following an object training experience suppressed aIC activity 1 h later, as determined by a reduced expression of the phosphorylated form of the transcription factor cAMP response element-binding (pCREB) protein and neuronal activity marker c-Fos. In contrast, the number of perisomatic γ-aminobutyric acid (GABA)ergic inhibitory synapses per pCREB-positive neuron was significantly increased, suggesting a dynamic up-regulation of GABAergic tone. In support of this possibility, pharmacological inhibition of aIC activity with a GABAergic agonist during consolidation enhanced object recognition memory. Norepinephrine administration into the BLA did not affect neuronal activity within the posterior IC, which receives sparse innervation from the BLA. The evidence that noradrenergic activation of the BLA enhances the consolidation of object recognition memory via a mechanism involving a suppression of aIC activity provides insight into the broader brain network dynamics underlying emotional regulation of memory.

Generation of induced pluripotent stem cell lines carrying monoallelic (UCSFi001-A-60) or biallelic (UCSFi001-A-61; UCSFi001-A-62) frameshift variants in CACNA1A using CRISPR/Cas9.

CACNA1A encodes a P/Q-type voltage-gated calcium channel. Heterozygous loss-of-function variants in this gene have been associated with episodic ataxia type 2. In this study, we used CRISPR/Cas9 to generate isogenic human induced pluripotent stem cell lines with a gene-dosage dependent deficiency of CACNA1A. We obtained one clone with monoallelic (UCSFi001-A-60) and two clones with biallelic (UCSFi001-A-61; UCSFi001-A-62) frameshift variants in CACNA1A. All three lines showed expression of pluripotency markers and a normal karyotype.

Following Excitation/Inhibition Ratio Homeostasis from Synapse to EEG in Monogenetic Neurodevelopmental Disorders.

Pharmacological options for neurodevelopmental disorders are limited to symptom suppressing agents that do not target underlying pathophysiological mechanisms. Studies on specific genetic disorders causing neurodevelopmental disorders have elucidated pathophysiological mechanisms to develop more rational treatments. Here, we present our concerted multi-level strategy 'BRAINMODEL', focusing on excitation/inhibition ratio homeostasis across different levels of neuroscientific interrogation. The aim is to develop personalized treatment strategies by linking iPSC-based models and novel EEG measurements to patient report outcome measures in individual patients. We focus our strategy on chromatin- and SNAREopathies as examples of severe genetic neurodevelopmental disorders with an unmet need for rational interventions.