Targeting of δ-catenin to postsynaptic sites through interaction with the Shank3 N-terminus

Abstract

Background Neurodevelopmental disorders such as autism spectrum disorder (ASD) may be caused by alterations in genes encoding proteins that are involved in synapse formation and function. This includes scaffold proteins such as Shank3, and synaptic adhesion proteins such as Neurexins or Neuroligins. An important question is whether the products of individual risk genes cooperate functionally (exemplified in the interaction of Neurexin with Neuroligin isoforms). This might suggest a common pathway in pathogenesis. For the SHANK3 gene, heterozygous loss of function, as well as missense mutations have been observed in ASD cases. Several missense mutations affect the N-terminal part of Shank3 which contains the highly conserved Shank/ProSAP N-terminal (SPN) and Ankyrin repeat (Ank) domains. The role of these domains and the relevance of these mutations for synaptic function of Shank3 are widely unknown. Methods We used purification from a synaptic protein fraction, as well as a variety of biochemical and cell biological approaches to identify proteins which associate with the Shank3 N-terminus at postsynaptic sites. Results We report here that δ-catenin, which is encoded by CTNND2, an autism candidate gene, directly interacts with the Ank domain of Shank3 at postsynaptic sites through its Armadillo-repeat domain. The interaction is not affected by well-known posttranslational modifications of δ-catenin, i.e. by phosphorylation or palmitoylation. However, an ASD-associated mutation in the SPN domain of Shank3, L68P, significantly increases the interaction of Shank3 with δ-catenin. By analysis of postsynaptic fractions from mice, we show that the lack of SPN-Ank containing, large isoforms of Shank3 results in the loss of postsynaptic δ-catenin. Further, expression of Shank3 variants containing the N-terminal domains in primary cultured neurons significantly increased the presence of coexpressed δ-catenin at postsynaptic sites. Limitations Work in model organisms such as mice, and in primary cultured neurons may not reproduce faithfully the situation in human brain neurons. Work in primary cultured neurons was also hampered by lack of a specific antibody for endogenous δ-catenin. Conclusions Our data show that the interaction between Shank3 N-terminus and δ-catenin is required for the postsynaptic targeting of δ-catenin. Failure of proper targeting of δ-catenin to postsynaptic sites may contribute to the pathogenesis of autism spectrum disorder.

Autism associated SHANK3 missense point mutations impact conformational fluctuations and protein turnover at synapses

“…The SPN domain has been shown to interact with small GTPases of the Ras superfamily including R-Ras, H-Ras or Rap1, which are involved in the regulation of synaptic F-actin structure and dynamics and in postsynaptic signal transduction (Cai et al, 2020;Lilja et al, 2017). The ARR domain binds the cytoskeletal protein α-fodrin, the synaptic adhesion molecule d-catenin and a component of the ubiquitin ligase complex, sharpin (Böckers et al, 2001;Lim et al, 2001;Hassani Nia et al, 2020). While the SH3 domain directly associates with the Ca 2+ channel Cav1.3, the PDZ domain is involved in a direct interaction with guanylate kinase-associated protein (GKAP) and synapse-associated protein 90/postsynaptic density-95-associated protein (SAPAP).…”

Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders

“…In the next step we determined binding to δ-catenin, which we have recently identified as interaction partner of the N-terminus of Shank3 21 . Binding to δ-catenin was not affected by the D69G mutation in the SPN domain, but was significantly reduced by six mutations in the Ank repeats (e.g.…”

“…No statistical method was used to determine sample size. Sample size was based on experience with the relevant type of experiment 18 , 21 , 23 , 27 , and with the objective to minimise the number of animals killed. No randomisation methods were used.…”

“…The Ank repeats interact with postsynaptic partners such as α-Fodrin and δ-catenin 20 , 21 , as well as Sharpin (the postsynaptic localisation of which has been unclear) 22 . In addition, the SPN domain is in close intramolecular contact to the Ank domain, to the effect that the SPN domain interferes with the access of some known interaction partners of the Ank repeats, namely α-Fodrin and Sharpin 18 , 23 .…”

Comparison of SHANK3 deficiency in animal models: phenotypes, treatment strategies, and translational implications

“…The longest isoform of SHANK3 in mice comprises six highly conserved domains: SHANK/ProSAP/N-terminal (SPN), ankyrin repeats (ANK), Src homology 3 (SH3), PSD95/DlgA/Zo-1 (PDZ), proline-rich (PRO), and sterile alpha motif (SAM). For instance, the ANK-domain binds to SHARPIN [ 98 ], SPTAN1 (also known as α -fodrin) [ 99 ], and CTNND2 [ 104 ], while the PDZ-domain interacts with DLGAP1 (which binds to DLG4) [ 59 ], GRIA1 (also known as GluA1 or GluR1 subunit of ionotropic AMPA type glutamate receptors) [ 100 ], and CTNNB1 as crucial constituent of the Wnt signaling pathway [ 105 ]. A SPN-domain at the N-terminus binds to the ANK-domain and limits its ability to interact with SHARPIN or SPTAN1 [ 97 ].…”