Aberrant calcium signaling and neuronal activity in the L271H CACNA1D (Cav1.3) iPSC model of neurodevelopmental disease

Voltage-gated calcium channels (VGCCs) are essential for neuronal excitability and synapse transmission as well as gene transcription regulation controlling cellular differentiation and survival. Recently, genetic variants in the CACNA1D gene, which encodes the α₁-subunit of voltage-gated Ca_v1.3 L-type Ca²⁺-channels, were linked to neurodevelopmental disorders, but their pathophysiological role on neuronal activity and development in a human background remains unknown. Here, we report the first functional characterization of a patient-derived iPSC-based disease model of the CACNA1D L271H variant. We observed that Ca_v1.3 is the dominantly expressed L-type calcium channel isoform in neural progenitor cells (NPCs). NPCs expressing the L271H variant exhibit increased spontaneous calcium transients compared to the WT controls. Differentiated L271H-variant midbrain neurons show a more depolarized resting membrane potential and reduced excitability. Cortical organoids generated from the L271H-iPSCs contain fewer and smaller ventricular-like structures indicating impaired cellular organization. We identify spatial distortion of radial glial cell distribution and accelerated neuronal differentiation in patient-derived organoids as judged by premature intermediate progenitor cell and neuron emergence. Unbiased transcriptomic analysis revealed numerous dysregulated genes that according to gene ontology analysis were associated with "transcriptional regulation", "CNS development", and "neurogenesis" including PTN, POU3F2, CNTN4 and AUTS2. These findings imply that disease-causing Ca_v1.3 variants alter ion homeostasis, result in aberrant neuronal function and distort human neurodevelopment, contributing to the complex disease phenotype observed in patients with high-risk CACNA1D variants.