Cell surface glutamate receptor analysis in wildtype and shank knockout mice in ASD-associated brain regions

Abstract

The umbrella term autism spectrum disorder (ASD) comprises a large, heterogenous group of neurodevelopmental disorders which are characterized by abnormalities in social interaction communication and ASD patients also display repetitive, stereotypical behaviour. Despite years of intensive research, the precise molecular mechanisms responsible for causing ASD remain elusive. Nonetheless, a multitude of studies has demonstrated a strong genetic component in the disorder and, interestingly, several of the identified genes code for proteins which are directly or indirectly involved in controlling synaptic function and signaling at the synapses of the brain. Therefore synaptic abnormalities may be central to understanding the pathomechanism of ASD. In most ASD patients where a genetic component has been identified, several genes are affected concomitantly and – presumably – disruptions in all of those genes are necessary to evoke an ASD phenotype. However, in a few ASD cases, changes in a single gene appear to be sufficient in causing an ASD phenotype. For example, mutations in one of the SH3 and multiple ankyrin repeat domains (SHANK) gene family members – especially in the SHANK3 and to a lesser degree in the SHANK2 gene – have been associated with an increased probability of developing ASD. Of note, Shank molecules have a scaffolding function at the excitatory postsynapse in the brain and are responsible for interconnecting glutamate receptors, the cytoskeleton, intracellular signaling components, and other molecules at the postsynaptic density (PSD) and thereby regulate synaptic signal transmission in the brain. In order to gain a better understanding of the molecular underpinnings common to different genetic ASD forms, we utilized adult male mice deficient in Shank2 (Shank2-/-) or Shank3 (Shank3αβ-/-) as ASD mouse models and studied their cell surface glutamate and gamma-Aminobutyric acid (GABA) receptor subunit levels in several ASD-related brain regions (cortex, striatum, hippocampus, thalamus, and cerbellum) relative to wildtype controls. Importantly, both Shank2-/- and Shank3αβ-/- mice exhibit ASD-like behaviour and are, therefore, suited as ASD mouse models. For the cell surface analysis, we implemented a biotinylation assay on coronal brain slices containing the aformentioned brain regions, dissected the regions of interest, lyzed them and carried out a subsequent NeutrAvidin pull-down to isolate cell surface proteins/receptors which were then analyzed by western blot. Of note, so far this biochemical technique has rarely been used to gain insight about levels of synaptic receptors in ASD research. Instead, conventional biochemical approaches focusing on the analysis of synaptosome and PSD preparations were more common which is unfortunate since the biotinylation assay has the decisive advantage of addressing receptors exclusively located at the cell surface. After all, cell surface receptors are – of course – more important for research focusing on signaling events taking place at the synapse as opposed to intracellular receptors which are, inadvertently, always present in the biochemical fractions of conventional biochemical approaches like synaptosome and PSD preparations. The analysis of cell surface glutamate and GABA receptor subunits revealed that both the Shank2-/- and Shank3αβ-/- ASD mouse model have lower levels of cell surface glutamate receptors than wildtype mice in several of the analyzed brain regions – especially in the striatum – whereas almost no changes in GABA receptor levels could be observed. Instead, total glutamate or GABA receptor levels were not altered. Of note, the changes in cell surface glutamate receptor subunits observed in the ASD mouse models of this study differed and varied considerably in each analyzed brain region. Taken together, the results of this study suggest that molecular commonalities such as similar levels of cell surface glutamate receptor levels probably exist among ASD mouse models and may, therefore, explain some of the ASD phenotypes observed in these mice. However, striking molecular differences also appear to exist and must not be neglected. Translated to the human situation, the results of this study suggest that patients suffering from ASD may share certain molecular commonalities which could potentially be the target of future drug treatments. Nonetheless, inter-individual differences are probably very relevant, as well, which means that if ASD patients are to be treated efficiently, a highly individualized approach may be necessary.