Pharmacological rescue of serotonin transporter inactivation by the autism‐associated integrin b3 Pro33 coding variant.
Authors: Carneiro Ana MD , Dohn Michael , Jessen Tammy , Edwards Digna Velez , Danny Joshua , Sutcliffe James 
Published: November 2023

Cell adhesion to the extracellular matrix (ECM) is mediated by integrins, heterodimeric transmembrane proteins that comprise a family of 18 α and 8 β subunits. The interaction of integrins with the cytoskeleton and ECM requires the presence of both subunits, where the a and b composition determines the specificity of their extracellular ligands. During CNS development, ECMs are abundant throughout the brain, and ECM/integrin interactions govern neuronal migration and axonal pathfinding. Integrin‐mediated cell adhesion is a biological phenomena consistently identified as preferentially affected by autism‐spectrum disorder (ASD)‐associated mutations. The most replicated integrin gene association with ASD is ITGB3. The minor allele of the ITGB3 coding polymorphism rs5918 (Leu33Pro, ITGB3 PlA1/A2) is associated with elevated blood serotonin levels and with increased ASD risk in males, but not females. Multiple studies have also reported associations of the Pro33 variant and serotonin transporter (SERT) polymorphisms in ASD patients. The Leu33Pro mutation enhances the adhesive functions of integrins containing the β3 subunit, including the platelet fibrinogen receptor (integrin αIIbβ3) and the vitronectin receptor (integrin αvβ3), the latter of which is expressed in multiple cells, including neurons. In the mouse brain, biochemical and immunohistochemical studies previously demonstrated enriched localization of avb3 to the pre‐ and postsynaptic terminals of neurons within and outside of the raphe nuclei in the murine midbrain, as well as in striatal, hippocampal, and cortical synapses. In this study, we utilized synaptosomal preparations isolated from knock‐in (KI) mice expressing an Itgb3 variant that phenocopies the human Pro33 variant to identify functional links between αvβ3 and SERT. Biochemical and microscopy studies revealed that the synaptic Pro33 integrin αvβ3 is constitutively activated, triggering enhanced focal adhesion kinase (FAK) signaling in the synapse. These changes parallel reduced associations of SERT with focal adhesions, and decreases in SERT uptake activity. Inhibition of FAK rescued SERT function in KI synapses, demonstrating that FAK signaling downstream of hyperactive integrin β3 suppresses SERT activity. To identify the signaling pathways involved in the downregulation of serotonin uptake, we utilized pharmacological tools to inhibit Src and ERK, two kinases downstream of FAK activation. These studies identify a complex regulation of SERT activity by integrin αvβ3, revealing an important role for integrins in modulating risk for neuropsychiatric disorders.

Support or Funding Information

Pro32Pro33 KI mice were funded by an Autism Speaks pilot grant to JS. This study was supported by the grants R01MH090256 (AMDC, MRD, TJ), and R01LM010685 (JD). BioVU is supported by institutional funding and by the Vanderbilt CTSA grant ULTR000445 from NCATS/NIH. The OMX microscope is housed in the Vanderbilt Cell Imaging Shared Resource supported by NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126.