D1 Receptor Agonist Ameliorates Synaptic and Behavioral Deficits in a Shank3‐Deficient Mouse Model of Autism

Dear Editor Autism spectrum disorder (ASD) has been associated with mutations in SHANK genes, which encode critical scaffolding proteins at glutamatergic synapses [1]. However, the mechanisms by which SHANK3 deficiency disrupts dopamine-dependent cortico-striatal signaling remain poorly understood. In this study, we revealed that loss of Shank3 impairs D1 receptor–mediated cortico-striatal projections, giving rise to ASD-like behavioral phenotypes. Notably, pharmacological activation of the D1 receptor with SKF-82958 restored synaptic function and ameliorated repetitive and social behavioral deficits in Shank3-deficient mice. These findings highlight D1 receptor agonism as a potential therapeutic approach for the symptomatic treatment of ASD. SHANK proteins (SHANK1, SHANK2, and SHANK3) are critical components of the postsynaptic density (PSD) and form a key part of the PSD95/SAPAP/SHANK complex that supports glutamatergic synaptic signaling [1]. Especially, SHANK3 is highly expressed in the striatum, where its deficiency is associated with significant synaptic dysfunction. In Shank3 −/− mice, reduced expression of NMDA and AMPA receptor subunits in the striatum leads to decreased cortico-striatal synaptic transmission in striatal medium spiny neurons (MSNs) [1]. MSNs integrate glutamatergic input from the prefrontal cortex with dopaminergic signals from the substantia nigra. D1 receptor-mediated Gs signaling activates D1 MSNs, whereas D2 receptor-mediated Gi signaling inhibits D2 MSNs.S1 Here, we used Shank3B-deficient mice as a model for Shank3 −/− [1]. In the cortex, we observed no significant changes in PSD95 or the cortico-striatal projection marker vGLUT1. However, the striatum exhibited marked dysregulation of PSD95 and vGLUT1, along with downregulation of dopamine signaling components, particularly DRD1 and DAT (Figure 1A). Based on these findings, we hypothesized that the reduction in D1 receptor expression leads to diminished D1 receptor-mediated signaling. To restore this signaling pathway, we administered the D1 receptor agonist SKF-82958. In the marble burying test, which reflects repetitive behaviors, Shank3 −/− mice buried more marbles than Shank3 +/+, but this behavior was recovered after SKF-82958 treatment. Moreover, in the three-chamber social interaction tests, Shank3 −/− mice exhibited impaired social interactions, which were ameliorated following SKF-82958 administration (Figure 1B). These findings collectively indicate that the activation of D1 receptor signaling by SKF-82958 can reverse ASD-like behaviors in Shank3 −/− mice. To further explore the relationship between behavioral phenotypes and synaptic function, we utilized a mixed-culture system of co-cultured cortical and striatal neurons derived from Shank3 +/+ and Shank3 −/− mice. These mice also express TdTomato under the control of the Drd1a promoter in the striatum, allowing for visualization of D1 MSNs (Figure 1C, left). Synaptic transmission was assessed using a pHluorin-based assay (vGlut-pHlourin [vG-pH]), which provides a direct measure of synaptic activity. Following stimulation with 100 action potentials (APs), synaptic transmission was significantly reduced in Shank3 −/− neurons (16 ± 1%) compared to Shank3 +/+ neurons (22 ± 2%) (Figure 1C, left). Additionally, we investigated the rate of synaptic vesicle fusion during prolonged stimulation (2000 APs), revealing that Shank3 −/− neurons exhibited a significantly slower fusion rate compared to Shank3 +/+ neurons (∼1.8-fold slower) (Figure 1C, left). We next sought to determine whether SKF-82958 could restore synaptic function at cortico-striatal synapses in Shank3 −/− mice. Treatment with SKF-82958 led to an approximately 10% increase in synaptic transmission at cortico-striatal synapses (Figure 1C, right). These findings suggest that D1 receptor-mediated synaptic dysfunction at cortico-striatal synapses contributes to ASD-like phenotypes, which can be ameliorated through the activation of the D1 receptor. Biochemical analysis of the striatum region further revealed that Shank3 −/− mice exhibited reduced ERK-CREB signaling and BDNF expression, indicating D1 receptor signaling impairment in the striatum. Administration of SKF-82958 restored D1 receptor-mediated signaling and activated the BDNF-TrkB signaling pathway, leading to upregulation of Bdnf mRNA expression (Figure 1D). These results demonstrate that SKF-82958 effectively restores impaired D1 receptor signaling within the striatum of Shank3 −/− mice. However, several limitations remain. First, not all individuals with ASD or ASD animal models exhibit reduced dopamine signaling or diminished D1 receptor-mediated signaling. Both hyper- and hypo-dopaminergic states are associated with ASD-like behaviors [2], suggesting that dopamine homeostasis is critical for behavioral regulation in ASD. Our study underscores the behavioral improvements observed when reduced dopamine signaling is restored to physiological levels through D1 receptor agonism. Second, the optimal clinical application of SKF-82958 remains unclear. For potential clinical translation, a thorough evaluation of appropriate dosing regimens, administration frequency, and potential development of tolerance is required. To address the issue of tolerance, the D1 receptor allosteric modulators may represent a promising strategy [3]. Third, we observed an increase in D1 receptor-mediated signaling within the striatum in Shank3 −/− mice, as in a previous study [4]. However, we have newly found that in the cortico-striatal projection context, the striatal D1 receptor signaling was downregulated. Further studies are needed to understand how this regional mismatch contributes to behavioral abnormalities. Finally, although SHANK3 and D1 receptors are enriched in the striatum, contributions from other brain regions cannot be excluded. Previous studies have demonstrated that region-specific deletion of Shank3 in other brain regions can contribute to behavioral abnormalities [5]. The potential effects in these additional brain subregions were not addressed in the present study and remain to be explored. Moreover, whether these phenomena are consistently reproduced in other Shank3 mutant models has not yet been evaluated. Future studies involving conditional knockouts or patient-derived point mutations are needed to enhance translational relevance. In conclusion, Shank3 −/− mice exhibit significant deficits in dopaminergic signaling within the striatum, particularly D1 receptor-mediated cortico-striatal projection. Furthermore, administration of the D1 receptor agonist SKF-82958 restored social behaviors, synaptic function, and signaling deficits in Shank3 −/− mice. Our study proposes a therapeutic strategy based on D1 receptor agonism for ASD cases characterized by reduced striatal D1 receptor signaling. D.-G.J. and S.H.K. supervised the whole project. D.-G.J., S.H.K., J.-S.K., and S.H. designed the research and wrote the manuscript. J.-S.K. and H.S. performed the experiments, analyzed the data, and made the figures. M.A. and J.P. performed behavioral studies. M.A., Y.L., and J.L. maintained the animals. All authors have read and approved the final manuscript. This study was supported by Basic Science Research Program through the National Research Foundation of Korea (S.H.K.: 2018R1A6A1A03025124 and RS-2024-00399237; D.-G.J.: RS-2019-NR040057 and RS-2024-00345742). This study was also supported by digital bio research program through the Naver Corporation (D.-G.J.: 37-2023-0140). All animal experiments were approved by Institutional Animal Care and Use Committees of Sungkyunkwan University (SKKUIACUC2020-06-36-1, SKKUIACUC2021-03-09-1, and KHSASP-21-060). The authors declare no conflicts of interest. The data presented in this study are available upon request to the corresponding author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.