|Abstract||SHANK proteins (SHANK1-3) are known as "master"-scaffolding proteins, especially at the postsynaptic density (PSD) of excitatory synapses. The disruption of SHANK-proteins is highly associated with autism spectrum disorder (ASD). ASD represents a neuropsychiatric disease entity that is characterized by a combination of symptoms, including persistent deficits in social communication and interaction, as well as restricted, repetitive patterns of behavior, interests, or activities [DSM-V]. Originating from its major role in mediating social behavior and as a modulator of synaptic transmission it has been hypothesized that oxytocin (OXT) could have beneficial effects on the social deficits seen in ASD. In some animal models of ASD such restoration of social behavior was confirmed, although there is mixed evidence in human studies. More specifically, a recent study showed that OXT restored synaptic plasticity and alleviated behavioral deficits in a SHANK3-deficient rat model for Phelan-McDermid syndrome (PMS) [Harony-Nicolas et al., 2017]. The experiments conducted in this thesis additionally demonstrate that plain OXT-treatment regulates SHANK protein levels in synapses of rat hippocampal neurons.
More specifically, OXT-treatment resulted in increased protein levels of all members of the SHANK protein family as reflected by an increased fluorescence intensity of dendritic SHANK-puncta in hippocampal neurons. In addition, a general positive trend concerning the number of synaptic SHANK-clusters was observed after OXT-treatment, which correlates with the increased synaptic protein levels and points towards an increased amount of dendritic SHANK-protein. Since the size of dendritic SHANK1- and SHANK3-clusters were reduced after treatment, OXT might influence the degree of synaptic protein-clustering. The fact that all SHANKs were affected in a similar way suggests a cumulative effect of OXT on general synaptic SHANK-levels by influencing multiple members of this protein family. Confirming the results seen in the immunofluorescent analysis, SHANK1- and SHANK3-levels were also significantly increased in the Western Blot analysis of crude membrane fraction of primary hippocampal neurons, which also contain the PSD. Notably, the analysis of the respective whole-cell homogenate showed a general reversal of these positive trends and even revealed significantly reduced general protein-levels of SHANK3e and SHANK2. Thus, the increased amount of dendritic SHANK-protein seems to rely on a spatial shift of protein pools towards the synaptic compartment, rather than on increased translation. In response to the saturation of dendritic/synaptic protein-levels, general translation could be reduced as a result of a negative feedback loop. Further immunofluorescent analysis did not hint at a spatial shift of protein taking place within the dendritic compartment. Apart from its effects on proteins of excitatory synapses, OXT-treatment resulted in decreased protein levels of Gephyrin at GABAergic postsynapses, as reflected by a decreased fluorescence intensity of dendritic Gephyrin-puncta.
These results represent evidence for a direct influence of the OXT-system on the ASD-associated SHANK protein family. OXT-induced effects were pronounced in the analysis of SHANKs. Other excitatory synaptic proteins like VGLUT1 or PSD95 were less affected or not influenced at all. This apparent selectivity needs to be relativized by the fact that only a limited number of proteins was examined in this study (see also 4.5). Gephyrin, which mainly localizes to the postsynapse of inhibitory synapses, was oppositely influenced. This might be an additional mechanism by which OXT affects the excitation/inhibition-balance that has been hypothesized as being crucial in ASD-pathogenesis. As OXT-treatment did not influence presynaptic proteins like VGLUT1 and VGAT to the same degree, postsynaptic scaffolding proteins might be a principal target of OXT-signaling in both excitatory and inhibitory synapses. By affecting proteins like the SHANKs that interact with many other synaptic proteins, OXT could secondarily influence the composition of the PSD at a larger scale or facilitate interaction of certain molecules like AMPAR or NMDAR.
The above-mentioned findings justify the assumption that OXT influences protein-levels at excitatory and inhibitory synapses. Accordingly, OXT might have differential effects on social deficits found in patients with ASD, depending on the underlying pathophysiological dysfunction. Since synaptic SHANK3 was increased by OXT-treatment, it might be a suitable candidate for further investigation in patients with PMS (SHANK3-haploinsufficiency syndrome).||dc.description.abstract