COMBINING BIOINFORMATICS WITH WHOLE-CELL PATCH-CLAMP RECORDINGS TO INVESTIGATE THE ROLE OF αVβ3 INTEGRIN IN MOUSE MODELS OF ASD AND EPILEPSY

Autism spectrum disorder (ASD) and epilepsy have a high degree of comorbidity. This co-occurrence is likely the result of underlying factors predisposing to both conditions. Integrins are heterodimeric receptors for extracellular matrix proteins and counter-receptors on adjacent cells. In the mouse brain, β3 integrin subunit pairs only with αV integrin subunit; loss of αV and β3 integrin subunits have been implicated in the onset of epilepsy and autism, respectively. In this project, I investigate the effects of impairing αVβ3 integrin in the mouse cortex with the goal of identifying changes that may underlie both ASD and epilepsy, focusing on deficits in αVβ3 integrin receptor signalling at synapses. I used AAV-mediated retrograde labelling in WT and constitutive Itgb3 KO mice to investigate the role of β3 integrin in dendritic spine number and morphology in corticopontine (CP) and commissural (COM) layer V pyramidal neurons of the medial prefrontal cortex (mPFC), two populations of pyramidal neurons that differ in terms of excitatory hodology. I found that COM neurons harboured more mushroom and fewer stubby spines compared to CP neurons in both WT and Itgb3 KO brains. Moreover, loss of β3 integrin induced a specific shortening of thin spines in CP neurons, hinting to a role of β3 integrin in immature spines. In two different mouse models (a conditional KO for αV integrin and the constitutive KO for β3 integrin), I used comparative quantitative mass spectrometry of cortical synapses and bioinformatic protein-protein interaction (PPI) network analyses to identify genes involved in ASD and epilepsy. I found 19 proteins in common between Itgb3 and ItgaV PPI networks that were significantly changing in mass spectrometry analyses and were associated with ASD or epilepsy or both. Among these proteins, I focused of AMPA receptor (AMPAR) subunit GluA2, and group I metabotropic glutamate receptors mGluR1 and mGluR5 since they are involved in excitatory synaptic transmission. In both mouse models, I recorded AMPAR excitatory synaptic currents in mPFC pyramidal neurons in the whole-cell patch clamp configuration upon pharmacological modulation of mGluR1/5 signalling. I isolated mGluR5 contribution using the selective antagonist MPEP in Itgb3 and ItgaV mouse models: MPEP treatment reduces AMPAR currents in both ItgaV KO and Itgb3 KO neurons. Loss of αVβ3 integrin affect selectivity mGluR5 regulation of AMPAR excitatory synaptic currents. Since AMPAR phosphorylation affects receptor trafficking, I investigated whether mGluR5 inhibition affects AMPAR phosphorylation in cortices from WT and Itgb3 KO mice. MPEP treatment increases AMPAR subunit GluA1 S831 phosphorylation in WT cortices while decreasing S831 phosphorylation in Itgb3 KO cortices. GluA1 S831 phosphorylation promotes GluA1 targeting to post synaptic density (PSD). I concluded that αVβ3 integrin regulates AMPAR synaptic currents via mGluR5 signalling. Moreover, I speculated that, upon loss of αVβ3 integrin, mGluR5 signalling leads to increased availability of GluA1 at PSD, possibly promoting excessive excitatory transmission, which can contribute to ASD and epilepsy.