These findings establish DLG4 haploinsufficiency as a shared consequence of pathogenic DLG4 variants, while revealing additional variant-associated effects on neuronal structure and activity, rescued by AAV9-mediated neuronal restoration.
Abstract
DLG4-related Synaptopathy, or SHINE syndrome, is a neurodevelopmental disorder caused by de novo heterozygous variants in DLG4 gene, encoding the postsynaptic scaffold PSD-95. Although clinical and genetic evidence support haploinsufficiency, the consequences of pathogenic DLG4 variants in human neurons remain poorly defined. Here, we model three mutations spanning distinct protein domains: a frameshift, nonsense and a missense mutation using iPSC-derived excitatory neurons. Molecular analysis of mature neurons reveals shared PSD-95 deficiency irrespective of transcript levels, together with reduced mature spine density. High-density microelectrode array recordings further reveal convergent and mutationspecific electrophysiological signatures at both single-neuron and network levels, as mutant cultures display genotype-dependent shifts in extracellular waveform states associated with altered firing dynamics. Importantly, restoration of PSD-95 levels using an adeno-associated viral vector (AAV9) harboring human DLG4 cDNA and driven by the human neuronal Synapsin I promoter (AAV9-hSynI-DLG4) rescues PSD95 abundance and associated cellular and electrophysiological deficits. Together, these findings establish DLG4 haploinsufficiency as a shared consequence of pathogenic DLG4 variants, while revealing additional variant-associated effects on neuronal structure and activity, rescued by AAV9-mediated neuronal restoration.
Mutations in chromosome alignment maintaining phosphoprotein 1 (CHAMP1) have been linked to neurodevelopmental disorders characterized by intellectual disability, developmental delay, and autism spectrum disorder. However, the cellular and electrophysiological mechanisms by which CHAMP1 mutations disrupt human neuronal development remain poorly understood. In the present study, we used patient-derived induced pluripotent stem cells (iPSCs) carrying two pathogenic CHAMP1 mutations and generated neural progenitor cells (NPCs) and excitatory neurons to investigate the effects of each mutation on neuronal maturation and function, DNA repair, and gene expression. Proliferative capacity declines with CHAMP1 dosage, while DNA repair dysfunction is allele-specific. Whole-cell patch-clamp electrophysiology revealed that CHAMP1 mutant neurons exhibit significant alterations in intrinsic membrane properties during early developmental stages, including depolarized resting membrane potential, reduced action potential firing, and impaired waveform kinetics. These functional deficits were accompanied by reduced sodium and potassium current densities, suggesting impaired ion channel accumulation during neuronal maturation. Furthermore, recordings of spontaneous excitatory postsynaptic currents indicated altered synaptic activity and reduced proportions of synaptically active neurons. Morphological analyses showed that CHAMP1-deficient neurons exhibit impaired neurite outgrowth and branching, supporting a defect in neuronal maturation. Single-nucleus transcriptomic profiling further revealed delayed developmental trajectories and mutation-specific dysregulation of synaptic gene programs enriched for autism, ADHD, and epilepsy risk genes. Together, these findings demonstrate that CHAMP1 mutations disrupt multiple aspects of neuronal development, including homologous recombination (HR) dysfunction in NPCs, membrane excitability, ion channel function, and synaptic connectivity. Our results provide insights into the neurobiological consequences of CHAMP1 mutations and establish patient-derived neurons as a platform to investigate cellular pathophysiology and potential therapeutic strategies for CHAMP1-associated neurodevelopmental disorders.
Dailey Nettles, Christina Stanton, Z. Hunter et al.· bioRxiv· 0 citations
Using patient-derived iPSCs and multi-omics profiling, it is demonstrated that early-truncating variants cause loss-of-function via nonsense-mediated decay (NMD), while late-truncating variants that escape NMD cause gain-of-function effects.
A. Nava, Y. Pérez-Rodríguez, T. Hsieh et al.· medRxiv· 0 citations
An integrative study combining Mendelian genetics, clinical and association studies, and animal and molecular modeling supports variants in ELAVL2 as a cause of a neurodevelopmental disorder, with haploinsufficiency as the disease mechanism, and identifies crucial roles of ELAVL2 in neuronal function, cognition, and behavior.
Marina Boon, Meghan R. Mulligan, Jolijn J A Verseput et al.· American Journal of Human Ge...· 0 citations
The findings implicate DCLK1 in a previously unrecognized progressive neurodevelopmental disorder and demonstrate the power of integrative cross-species functional genomics in resolving ultra-rare disease variants.
Stephen C. Pak, David Butler, Wei-Xi Yuan et al.· Research Square· 0 citations
SYNGAP1 developmental and epileptic encephalopathy (DEE) is a severe neurodevelopmental disorder characterised by intellectual disability, developmental delay, and refractory epilepsy caused by heterozygous variants in SYNGAP1, which encodes Synaptic Ras GTPase-activating protein 1. While SYNGAP1 is best known for its role at the postsynaptic density, increasing evidence indicates that haploinsufficiency also disrupts early neurodevelopment. Here, we used patient-derived induced pluripotent stem cell (iPSC) models to investigate early neurodevelopmental and neuronal phenotypes associated with SYNGAP1 haploinsufficiency. iPSCs derived from a female patient carrying the frameshift variant p.Leu150Valfs*6 were differentiated into two complementary models: micropatterned neural rosettes representing early neuroepithelial organisation and NGN2-induced excitatory neurons representing postmitotic functional development. Patient-derived neural rosettes displayed enlarged, dysmorphic lumens, indicating disrupted neuroepithelial organisation at the earliest stages of brain development. Transcriptomic profiling revealed widespread dysregulation of genes involved in neurodevelopment, cell adhesion and ion channel regulation, including coordinated downregulation of protocadherin family members. Whole-cell patch-clamp electrophysiology demonstrated reduced input resistance, larger action potential amplitudes, and increased inward and outward current densities, consistent with accelerated intrinsic neuronal maturation rather than generalized hyperexcitability. Together, these complementary findings demonstrate that SYNGAP1 haploinsufficiency disrupts early human brain development and accelerates intrinsic neuronal maturation, with pathogenic mechanisms emerging before synaptogenesis and extending beyond SYNGAP1’s established synaptic role.
Montanna Waters, Lucas Teasdale, Sean Byars et al.· bioRxiv· 0 citations
Arid1b is a high confidence risk gene for autism spectrum disorder that encodes a subunit of a chromatin remodeling complex initially expressed in neuronal progenitors. Haploinsufficiency causes a broad range of social, behavioral, and intellectual disability phenotypes, including Coffin-Siris syndrome. Recent work suggests pathology is due to deficits in proliferation, survival, and synaptic development of cortical neurons. Here, we used transgenic mice to investigate how Arid1b dysfunction in cortical excitatory neurons impacts their intrinsic membrane properties, synaptic connectivity and physiology of local cortical circuits using paired whole-cell recordings, social behavior, and seizure susceptibility. We found that loss of both copies of Arid1b altered the proportions of different excitatory neuron cell-types in the superficial cortical layers; however, their intrinsic membrane properties were mostly unchanged. In mice with conditional Arid1b haploinsufficiency in excitatory neurons, we found an increase in the rate of synaptic connectivity between excitatory neurons and reduced strength of excitatory synapses to parvalbumin (PV)-expressing inhibitory interneurons. In the deep cortical layers, we found hyperpolarization of action potential threshold. Collectively, these data suggest an increase in the ratio of excitation to inhibition. However, we also found enhanced inhibition from PV interneurons to excitatory neurons that may rebalance this ratio. Indeed, Arid1b haploinsufficiency in excitatory neurons was insufficient to cause social deficits and seizure phenotypes observed in a preclinical germline haploinsufficient mouse model. Our data suggest that while excitatory neurons likely contribute to autistic phenotypes, pathology in these cells is not the primary cause.
Alec H. Marshall, Meretta A. Hanson, Danielle J. Boyle et al.· Experimental Neurology· 0 citations