Neural and metabolic dysregulation in PMM2-deficient human in vitro neural models.

Phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is a rare inborn error of metabolism caused by deficiency of the PMM2 enzyme, which leads to impaired protein glycosylation. While the disorder presents with primarily neurological symptoms, there is limited knowledge about the specific brain-related changes caused by PMM2 deficiency. Here, we demonstrate aberrant neural activity in 2D neuronal networks from PMM2-CDG individuals. Utilizing multi-omics datasets from 3D human cortical organoids (hCOs) derived from PMM2-CDG individuals, we identify widespread decreases in protein glycosylation, highlighting impaired glycosylation as a key pathological feature of PMM2-CDG, as well as impaired mitochondrial structure and abnormal glucose metabolism in PMM2-deficient hCOs, indicating disturbances in energy metabolism. Correlation between PMM2 enzymatic activity in hCOs and symptom severity suggests that the level of PMM2 enzyme function directly influences neurological manifestations. These findings enhance our understanding of specific brain-related perturbations associated with PMM2-CDG, offering insights into the underlying mechanisms and potential directions for therapeutic interventions.

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.

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.

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.

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

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.

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.

MicroRNA-338 Attenuates Cortical Neuronal Outgrowth by Modulating the Expression of Axon Guidance Genes.

MicroRNAs (miRs) are small non-coding RNAs that confer robustness to gene networks through post-transcriptional gene regulation. Previously, we identified miR-338 as a modulator of axonal outgrowth in sympathetic neurons. In the current study, we examined the role of miR-338 in the development of cortical neurons and uncovered its downstream mRNA targets. Long-term inhibition of miR-338 during neuronal differentiation resulted in reduced dendritic complexity and altered dendritic spine morphology. Furthermore, monitoring axon outgrowth in cortical cells revealed that miR-338 overexpression decreased, whereas inhibition of miR-338 increased axonal length. To identify gene targets mediating the observed phenotype, we inhibited miR-338 in cortical neurons and performed whole-transcriptome analysis. Pathway analysis revealed that miR-338 modulates a subset of transcripts involved in the axonal guidance machinery by means of direct and indirect gene targeting. Collectively, our results implicate miR-338 as a novel regulator of cortical neuronal maturation by fine-tuning the expression of gene networks governing cortical outgrowth.