Calcium channels and integrins in cortical synaptic transmission: relevance for new therapeutical targets in epilepsy

Epilepsy is a neurological disease, counting nearly 65 million people worldwide, characterized by an imbalance in the excitation/inhibition (E/I) ratio. Both presynaptic and postsynaptic proteins are involved in this balance; indeed, mutations in voltage-gated calcium channels (VGCCs) and cell adhesion molecules (CAMs) are implicated in epilepsy. Despite numerous approved anti-seizure drugs, nearly 30% of patients still face uncontrolled seizures, highlighting the need for new therapeutic strategies and molecular targets in the epilepsy field. Loss-of-function mutations in CACNA1A, the gene encoding the α1 subunit of P/Q-type voltage-gated Ca2+ channels (CaV2.1) have been implicated with absence epilepsy. Cav2.1 are crucial for synaptic transmission in several mature central synapses, collaborating with Cav2.2 (N-type VGCCs, CACNA1B). Both CACNA1A and CACNA1B generate two splice isoforms (EFa and EFb) with different expression patterns and coupling configuration. Cav2.1[EFa] and Cav2.2[EFa] establish a tight synaptic functional coupling that promotes efficient synaptic vesicle release. However, only Cav2.1[EFa] is highly expressed in the brain. In most brain regions, there is predominant expression of Cav2.2[EFb], which, much like Cav2.1[EFb], is distinguished by a loose synaptic coupling. Mouse models for absence epilepsy show a compensatory up-regulation of Cav2.2 in response to Cav2.1 mutations, but this is not sufficient to prevent the pathological symptoms, probably because the prevalence of the EFb isoform in Cav2.2 hinder a complete restoration of synaptic transmission. This thesis explores alternative splicing of Cav2.2 as a potential gene therapy target for epilepsy to overcome the limitations of personalized therapies, targeting individual mutations in the CACNA1A gene. CRISPR/Cas9 technology was applied in cortical pyramidal neurons (PNs) of an absence epilepsy mouse model to modify the splicing of Cav2.2, increasing the expression of the highly efficient isoform CaV2.2[EFa]. To test this approach, I recorded AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs) and GABA receptor (GABAR) inhibitory postsynaptic currents (IPSCs) in control, knockdown, and rescue mice. Cav2.1 knockdown reduced the amplitude of both EPSCs and IPSCs, increased their post-synaptic delay and altered short-term synaptic plasticity. Notably, increasing the expression of the Cav2.2 [EFa] isoform restored synaptic transmission and rebalance the E/I ratio. Integrins are heterodimeric receptors involved in mechanotransduction between the extracellular matrix and the intracellular cytoskeleton. In the brain, β3 integrin forms heterodimers only with αV integrin, influencing the synaptic localization of GluA2 AMPAR subunit and metabotropic glutamate receptor 5 (mGluR5). Moreover, loss of αVβ3 integrin in mice increases seizure susceptibility. In this thesis, I recorded AMPAR EPSCs in mPFC L5 PNs of constitutive β3 integrin knockout mice and conditional αV integrin knockout mice upon pharmacological modulation of mGluR1 and mGluR5 to study the interaction of αVβ3 integrin with group I mGluRs with the ultimate goal of identifying therapeutic targets for epilepsy. Pharmacological modulation of mGluR1/5 revealed a developmental switch in their contribution, with young synapses mainly relying on mGluR1, while adult synapses, on mGluR5. Inhibition of mGluR5 with the antagonist MPEP induced a comparable reduction of AMPAR EPSCs in Itgb3 and ItgaV KO neurons, suggesting a common dysfunctional mechanism of mGluR5 signalling in the absence of αVβ3 integrin. I hypothesized that loss of αVβ3 integrin impairs the synaptic function of mGluR5, involved in mGluR-long term depression, and promotes synaptic potentiation, mediated by extra-synaptic, constitutively active mGluR5. This likely increases excitability, contributing to seizure susceptibility of Itgb3 KO and ItgaV KO mice.