Riassunto analitico
Episodic ataxia is an autosomal dominant ion channel disorder characterised by episodes of imbalance and incoordination. The disease is classified as episodic ataxia type 2 (EA2) when it is caused by a mutation in the CACNA1A gene, encoding the α1A subunit of the P/Q-type voltage-gated calcium channel Cav2.1. Although CACNA1A is a heavily spliced gene, the impact of many of its splicing variants on synaptic transmission, which crucially depends on VGCCs, is unclear. Modulating splicing of CACNA1A may therefore represent a promising new strategy to understand VGCCs’ variants roles in brain function, and lastly, to develop improved EA2 therapies. This study was focused on two mutually exclusive expressed splice isoforms of exon 37 in CACNA1A (37A/37B) encoding for two different versions of the calcium channel EF-hand motif. More specifically, non-homologous end joining-based CRISPR strategy was used to generate a model of isogenic human induced pluripotent stem cell (hiPSCs) lines carrying either monoallelic or biallelic frameshift variants for each respective isoforms of exon 37. Clones selected for these variants, were cultured until they were differentiated to a hiPSC-derived cortical excitatory neurons (iNeurons) network. Further, 3 different hiPS control lines were differentiated to a homogeneous monolayer of iNeurons by transfecting them with lentiviral vectors to analyse the relative expression levels of the two isoforms over time. The characterization of the expression levels has started with the design of qPCR primers specific for each isoform in human cDNA and by testing their efficiency with the qPCR efficiency test. The aim was to investigate the role of each isoform during the generation of an iPs derived iNeurons network, and further, how each isoform contributes to the development of the neuronal network starting from an early stage, the network physiological activity, and eventually how this can be related to the episodic ataxia type 2 phenotype.
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