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.

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

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-ß1 and Integrin-ß3, 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.

Brunner syndrome associated MAOA mutations result in NMDAR hyperfunction and increased network activity in human dopaminergic neurons.

Monoamine neurotransmitter abundance affects motor control, emotion, and cognitive function and is regulated by monoamine oxidases. Among these, Monoamine oxidase A (MAOA) catalyzes the degradation of dopamine, norepinephrine, and serotonin into their inactive metabolites. Loss-of-function mutations in the X-linked MAOA gene have been associated with Brunner syndrome, which is characterized by various forms of impulsivity, maladaptive externalizing behavior, and mild intellectual disability. Impaired MAOA activity in individuals with Brunner syndrome results in bioamine aberration, but it is currently unknown how this affects neuronal function, specifically in dopaminergic (DA) neurons. Here we generated human induced pluripotent stem cell (hiPSC)-derived DA neurons from three individuals with Brunner syndrome carrying different mutations and characterized neuronal properties at the single cell and neuronal network level in vitro. DA neurons of Brunner syndrome patients showed reduced synaptic density but exhibited hyperactive network activity. Intrinsic functional properties and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission were not affected in DA neurons of individuals with Brunner syndrome. Instead, we show that the neuronal network hyperactivity is mediated by upregulation of the GRIN2A and GRIN2B subunits of the N-methyl-d-aspartate receptor (NMDAR), resulting in increased NMDAR-mediated currents. By correcting a MAOA missense mutation with CRISPR/Cas9 genome editing we normalized GRIN2A and GRIN2B expression, NMDAR function and neuronal population activity to control levels. Our data suggest that MAOA mutations in Brunner syndrome increase the activity of dopaminergic neurons through upregulation of NMDAR function, which may contribute to the etiology of Brunner syndrome associated phenotypes.

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.

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.

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.

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.

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

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.Abbreviations: APO: apocynin; ATG: autophagy related; BAF: bafilomycin A1; 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.

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

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.

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

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.

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

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.

Glucocorticoid-endocannabinoid uncoupling mediates fear suppression deficits after early - Life stress.

Early-life stress (ELS) creates life-long vulnerability to stress-related anxiety disorders through altering stress and fear systems in the brain. The endocannabinoid system has emerged as an important regulator of the stress response through a crosstalk with the glucocorticoid system, yet whether it plays a role in the persistent effects of ELS remains unanswered. By combining, behavioral, pharmacological and biochemical approaches in adult male rats, we examined the impact of ELS on the regulation of endocannabinoid function by stress and glucocorticoids. We employed a postnatal limited-nesting/bedding induced ELS between postnatal days 2-9 in rats. Exposure to postnatal ELS compromised the ability of both acute stress and glucocorticoid administration to mobilize the endocannabinoid ligand 2-arachidonoyl glycerol (2-AG) in the hippocampus of adult male rats. These findings suggest that ELS compromises the coupling of the glucocorticoid and endocannabinoid systems in the hippocampus. Since 2-AG signaling is essential in mediating glucocorticoid-induced suppression of fear recall, we further examined the impact of ELS on the ability of glucocorticoids to suppress fear memory recall. While ELS did not affect normative fear recall, it impaired the ability of glucocorticoids to dampen fear recall. Notably, bypassing glucocorticoids and directly amplifying hippocampal 2-AG signaling with a monoacyl glycerol lipase inhibitor produced a suppression of fear memory recall in animals exposed to ELS. These findings suggest that ELS results in an uncoupling of glucocorticoid-endocannabinoid signaling in the hippocampus, which, in turn, relates to alterations in stress regulation of memory recall. These data provide compelling evidence that ELS-induced deficits in the glucocorticoid-endocannabinoid coupling following stress could predispose susceptibility to stress-related psychopathology.