MODULATION OF CA<SUP>2+</SUP> CHANNELS’ SPLICE ISOFORMS AS A STRATEGY TO RESTORE SYNAPTIC TRANSMISSION IN A CRISPR/CAS9-GENERATED MOUSE MODEL OF CA<SUB>V</SUB>2.1-RELATED DISEASES

Ca_V2.1 channels are voltage-gated Ca²⁺ channels (VGCCs) which are crucial for fast synaptic transmission at central synapses. The CACNA1A gene, encoding the primary α₁ subunit of Ca_V2.1, undergoes extensive alternative splicing, potentially generating thousands of channel isoforms, including Ca_V2.1[EFa] and Ca_V2.1[EFb], which are expressed throughout the adult brain. Ca_V2.1[EFa] supports synchronous release and short-term synaptic depression, whereas Ca_V2.1[EFb] boosts asynchronous release and short-term facilitation. Loss-of-function mutations in CACNA1A are linked to neurological disorders such as episodic ataxia type 2 (EA2) and absence epilepsy. Given the complexity of CACNA1A gene and the CACNA1A-related disorders, focusing on the channel dysfunction alone presents challenges in understanding disease mechanisms and developing effective therapeutic strategies. Through CRISPR/Cas9 technology we developed a mouse model of episodic ataxia and absence epilepsy by selectively knocking down Ca_V2.1 expression in cortical pyramidal neurons. Electrophysiological recordings combined with optogenetics confirmed a marked reduction of AMPAR- and GABAR-mediated postsynaptic currents, leading to impaired synaptic transmission and disrupted excitation-inhibition (E/I) ratio. To counteract these deficits, we exploited the CRISPR/Cas9 technology to modulate the splicing of Ca_V2.2, which cooperates with Ca_V2.1 to regulate neurotransmitter release. By selectively upregulating the expression of its highly efficient isoform Ca_V2.2[EFa], synaptic transmission was restored and the E/I ratio rebalanced in the Ca_V2.1 knockdown model. These findings highlight the therapeutic potential of targeting alternative splicing in VGCCs to compensate for CACNA1A loss-of-function mutations, offering new perspectives for developing novel treatments for CACNA1A-related diseases.