The present study supports a “gain of function” mechanism for synaptic and behavioral dysfunction in a mouse model designed to mimic human mutations of Shank3 associated with ASD(Durand et al., 2007
). In combination with observations from Homer KO mice, we propose a model in which perturbations that reduce the stability of the Homer-Shank3 protein complex result in increased ubiquitination of Shank3 and reduced expression at the synapse. Changes of NR1 expression and ubiquitination are coupled to changes in Shank3. Determinants of the stability of the Homer-Shank protein complex have been reported in biochemical and crystallographic studies(Hayashi et al., 2009
), and are consistent with our findings. Our model is also consistent with a report that Shank proteins undergo ubiquitination in physiological adaptations to neuronal activity(Ehlers, 2003
). Data regarding effects of reduced expression of Homer cross-linking proteins include reduced Shank3 and NR1 expression and increased ubiquitination in Homer triple KO, Homer 1 KO and Homer 2/3 KO brain. Moreover, Homer1a expression, which inhibits the formation of Homer-Shank3 complex(Hayashi et al., 2009
) mimics Homer KO to reduce Shank3 expression, while selective genetic deletion of Homer1a results in increased Shank3 and reduced ubiquitination in brain. In extending this model to include the effect of Shank3 mutation, we note that Shank3ΔC protein is stably expressed in the Shank3(+/ΔC) mice, and physically associates with full-length Shank3 from brain. Since Shank3ΔC lacks the Homer-binding domain and associates with full-length Shank3, its incorporation with WT Shank3 effectively reduces Homer cross-linking of the Homer-Shank3 protein complex. Acute over-expression of Shank3ΔC in neurons reduces WT Shank3, providing evidence that down-regulation of WT Shank3 is due to Shank3ΔC expression. We do not detect ubiquitination of Shank3ΔC even from Shank3(ΔC/ΔC) brain, which mitigates the notion that Shank3ΔC is primarily ubiquitinated and “drags” the rest of the proteins along. This model predicts that any mutation of Shank3 or Homer that effectively reduces the stability of the Homer-Shank complex will lead to increased ubiquitination and proteasomal degradation of Shank3 and NR1. This could include deletions or point mutations of Shank3 that limit their physical assembly with other Shank3 molecules or Homer. Consistent with this model, Homer1 KO mice exhibit behavioral phenotypes (Szumlinski et al., 2005
) similar to Shank3(+/ΔC) mice.
Our model begs the question of why Shank3 expression and ubiquitination should be sensitive to physical association with Homer. We note that manipulations of Homer1a can influence Shank3 expression and ubiquitination, and this reinforces the notion that Shank3 is a nodal point for regulation of synaptic function in response to natural activity. Homer1a expression is tightly linked to behavioral experience(Brakeman et al., 1997
) and can also be induced by a variety of pharmacological and pathological stimuli. Thus, dynamic expression of Homer1a could contribute to the disassembly of Homer-Shank3 complexes, especially if the complex is weakened by mutations of Shank or Homer. By this mechanism, Homer1a expression could contribute to the pathogenesis of ASD.
Our studies also suggest a link between Shank3 and group I mGluR function. The molecular basis of enhanced mGluR-LTD in Shank3(+/ΔC) does not appear to be linked to altered expression of mGluRs. mGluR-LTD is increased in Fmr1 KO mice and this has been linked to neurological deficits in this mouse and in patients with fragile X disease (Bear et al., 2004
). Thus, enhanced mGluR-LTD may contribute to behavioral deficits in the Shank3(+/ΔC) mice.
Shank3(+/ΔC) mice exhibit behavioral phenotypes that parallel symptoms of ASD. The most striking differences between Shank3(+/ΔC) and WT mice were observed in a social recognition test that allowed for engagement in reciprocal interactions. Shank3(+/ΔC) mice reacted to novel conspecifics by a sharp increase in aggression rather than an increase in social investigation. Aggressive behaviors of Shank3(+/ΔC) mice were not generalized across different tasks indicating that the exaggerated aggression is likely a reaction to changes in social routine rather than a result of their generalized aggressiveness. Inferences regarding phenotypic relevance between mouse and human behaviors, such as aggression in individuals with ASD (Kanne and Mazurek, 2010
) are difficult, however, the findings of exaggerated aggressiveness to novel conspecifics as well as deficits in ultrasound vocalization and alterations in social approach behaviors indicate that the effects of Shank3(+/ΔC) mutation are detected on multiple domains of social interactions tested in a species-specific manner.
The decrease in NR1 observed in Shank3(+/ΔC) mice invites comparisons with mice hypomorphic for NR1 (Mohn et al., 1999
). The preserved structure of synapses and spines that are observed in Shank3(+/Δ C) mice is consistent with reports from the conditional NR1 KO where NR1 excision within CA1 did not effect spine density (Rampon et al., 2000
). The most striking difference between mice hypomorphic for NR1 and Shank3(+/ΔC) mice is that behavioral phenotypes of Shank3(+/ΔC) mice occur in the context of normal cognitive function as assayed in multiple tests of learning and memory. NMDAR-dependent LTP and LTD are reduced in Shank3(+/ΔC) hippocampus, but are not absent, and plasticity is apparently sufficient for normal cognitive function. The preserved cognition in these mice mitigates the concern that aberrant social behaviors might arise non-specifically in a mouse with major cognitive dysfunction, and suggests that Shank3ΔC models will be useful to explore the cellular and developmental determinants of social deficits associated with ASD, and relate these to a molecular model that includes enhanced ubiquitination of NR1. Our studies, however, should not be taken as direct evidence of a role of Shank3 in schizophrenia since human genetic evidence remains incomplete. Genetic studies report linkage of schizophrenia at 22q11-13 locus, which includes Shank3
(Condra et al., 2007
; DeLisi et al., 2002
), but the single reported association of Shank3 mutations with schizophrenia (Gauthier et al., 2010
) is notable for inclusion of patients with early onset symptoms that may overlap with ASD (Rapoport et al., 2009
). Nevertheless, our study supports the notion that patients with ASD or schizophrenia may share the molecular pathology of reduced NMDAR function.