CACNA1Ahaploinsufficiency leads to reduced synaptic function and increased intrinsic excitability

Abstract

Haploinsufficiency of theCACNA1Agene, encoding the pore-forming α1 subunit of P/Q-type voltage-gated calcium channels, is associated with a clinically variable phenotype ranging from cerebellar ataxia, to neurodevelopmental syndromes with epilepsy and intellectual disability.To understand the pathological mechanisms ofCACNA1Aloss-of-function variants, we characterized a human neuronal model forCACNA1Ahaploinsufficiency, by differentiating isogenic induced pluripotent stem cell lines into glutamatergic neurons, and investigated the effect ofCACNA1Ahaploinsufficiency on mature neuronal networks through a combination of electrophysiology, gene expression analysis, andin silicomodeling.We observed an altered network synchronization inCACNA1A+/−networks alongside synaptic deficits, notably marked by an augmented contribution of GluA2-lacking AMPA receptors. Intriguingly, these synaptic perturbations coexisted with increased non-synaptically driven activity, as characterized by inhibition of NMDA and AMPA receptors on micro-electrode arrays. Single-cell electrophysiology and gene expression analysis corroborated this increased intrinsic excitability through reduced potassium channel function and expression. Moreover, we observed partial mitigation of theCACNA1A+/−network phenotype by 4-aminopyridine, a therapeutic intervention for episodic ataxia type 2.In summary, our study pioneers the characterization of a human induced pluripotent stem cell-derived neuronal model forCACNA1Ahaploinsufficiency, and has unveiled novel mechanistic insights. Beyond showcasing synaptic deficits, this neuronal model exhibited increased intrinsic excitability mediated by diminished potassium channel function, underscoring its potential as a therapeutic discovery platform with predictive validity.

Automated inference of disease mechanisms in patient-hiPSC-derived neuronal networks

“…Second, we analyzed networks differentiated from isogenic iPSC lines with a monoallelic frameshift variant in exon 8 of CACNA1A , generated via CRISPR/Cas9 [21]. CACNA1A encodes part of the voltage-gated calcium channel Ca V 1.1, crucial for proper neurotransmitter release [22].…”

“…Utilizing hiPSC-derived neuronal networks on MEA, whether genome-edited or patient-derived, provides a rapid means to gather neuronal electrophysiological data. These data often exhibit distinct characteristics that differ between healthy and diseased networks, reflecting underlying pathological molecular mechanisms [2, 3, 5, 6, 7, 21]. While some mechanisms are explicitly linked to specific activity patterns [17, 11], characterizing them definitively through quantitative analysis of the data remains challenging.…”

“…While some mechanisms are explicitly linked to specific activity patterns [17, 11], characterizing them definitively through quantitative analysis of the data remains challenging. Additional experiments to nonetheless uncover these mechanisms, such as patch-clamp measurements or RNA-sequencing, are costly and time-consuming and may not cover all possible molecular pathways, potentially overlooking crucial mechanisms [10, 21]. Here, we propose the use of simulation-based inference (SBI) and our previously developed bio-physical computational model to automatically identify disease mechanisms in cultured neuronal networks on MEAs.…”

“…Therefore, a shift towards more influence of GluA2-lacking AMPA receptors in vitro , could be predicted by SBI as a reduction in GluA2-containing receptors, leading to a more NMDA-dominated synaptic transmission. In CACNA1A +/− neurons, evidence was also found for reduced voltage-gated potassium channel function, indicated by the slightly lower AP decay time, reduced AHP amplitude, reduced rheobase, and altered potassium channel-related RNA expression [21]. SBI predicted a significant shift towards lower values of the potassium-channel conductance.…”

“…While some mechanisms are explicitly linked to specific activity patterns [17,11], characterizing them definitively through quantitative analysis of the data remains challenging. Additional experiments to nonetheless uncover these mechanisms, such as patch-clamp measurements or RNA-sequencing, are costly and time-consuming and may not cover all possible molecular pathways, potentially overlooking crucial mechanisms [10,21].…”