SHANK3 Mutations Associated with Autism and Schizophrenia Lead to Shared and Distinct Changes in Dendritic Spine Dynamics in the Developing Mouse Brain

Abstract

Autism Spectrum Disorders (ASD) and schizophrenia are distinct neurodevelopmental disorders that share certain symptoms and genetic components. Both disorders show abnormalities in dendritic spines, which are the main sites of excitatory synaptic inputs. Recent studies have identified the synaptic scaffolding protein Shank3 as a leading candidate gene for both disorders. Mutations in the SHANK3 gene have been linked to both ASD and schizophrenia; however, how patient-derived mutations affect the structural plasticity of dendritic spines during brain development is unknown. Here we use live two photon in vivo imaging to examine dendritic spine structural plasticity in mice with SHANK3 mutations associated with ASD and schizophrenia. We identified shared and distinct phenotypes in dendritic spine morphogenesis and plasticity in the ASD-associated InsG3680 mutant mice and the schizophrenia-associated R1117X mutant mice. No significant changes in dendritic arborization were observed in either mutant, raising the possibility that synaptic dysregulation may be a key contributor to the behavioral defects previously reported in these mice. These findings shed light on how patient-linked mutations in SHANK3 affect dendritic spine dynamics in the developing brain, which provides insight into the synaptic basis for the distinct phenotypes observed in ASD and schizophrenia.

Keywords: SHANK3; autism; dendritic spines; in vivo imaging; schizophrenia; synaptic plasticity.

Figure 1:

SHANK3 InsG3680 and R1117X mutations alter dendritic spine morphogenesis. (A) Schematic of in vivo two-photon imaging. (B) Shank3 structural domains including InsG3680 and R1117X mutations in the proline rich region. (C) Representative images of in vivo dendritic spine morphogenesis. Scale bar = 5μm. (D–F) Quantifications of in vivo dendritic spine (D) density, (E) spine length, and (F) spine head width. In (D) N = 21 total dendrites from 12 brains wild type, 28 total dendrites from 7 brains InsG3680, and 25 total dendrites from 9 brains R1117X. In (E) N = 47 total spines from 4 wild type brains, 50 total spines from 3 InsG3680 brains, and 62 total spines from 5 R1117X brains. In (F) N = 69 total spines from 4 wild type brains, 78 total spines from 4 InsG3680 brains, and 69 total spines from 5 R1117X brains. Data represented as violin plots with thick line as median and thin lines as quartiles. Analyzed via mixed model to account for data nesting. *p<0.05.

Figure 2:

SHANK3 InsG3680 and R1117X mutations differentially affect glutamatergic synaptogenesis. (A) Representative images of presynaptic VGluT1 (magenta) and postsynaptic PSD-95 (cyan) staining. Scale bar = 20 μm. (B) Inset from dashed box in (A). Dashed circles indicate example areas of co-localized overlap between synaptic markers. Scale bar = 10 μm. (C) Quantification of presynaptic VGluT1, postsynaptic PSD-95, and colocalization puncta. N = 46 total images from 4 wild type brains, 48 total images from 4 InsG3680 brains, and 43 total images from 4 R1117X brains. Data represented as individual measurements as data points and mean ±SEM as bar graph with error bars. Analyzed via mixed model to account for data nesting. *p<0.05.

Figure 3:

SHANK3 InsG3680 and R1117X mutations alter dendritic spine dynamics. (A) Representative images of in vivo dendritic spine images including dendritic spines gained (blue arrows), lost (red arrows), or stable (yellow arrows) throughout the 60-minute imaging session. Scale bar = 5μm. (B) Representative images of single dendritic spine dynamics across imaging session. (C–F) Quantifications of in vivo dendritic spine dynamics including (C) dendritic spines gained, (D) dendritic spines lost, (E) dendritic spine turnover rate (TOR), and (F) average change in dendritic spine length. In (C–E) N = 21 total dendrites from 12 wild type brains, 28 total dendrites from 7 InsG3680 brains, and 25 total dendrites from 9 R1117X brains. In (F) N = 47 total spines from 4 wild type brains, 50 total spines from 3 InsG3680 brains, and 62 total spines from 5 R1117X brains. Data represented as individual measurements as data points and mean ±SEM as bar graph with error bars. Analyzed via mixed model to account for data nesting. *p<0.05 and **p<0.01.

Figure 4:

SHANK3 InsG3680 and R1117X mutations do not alter dendritic arborization. (A) Representative images of reconstructed pyramidal neuron in the prefrontal association area. Scale bar = 100μm. (B) Quantification of basal and apical dendrite reconstructions including number of intersections, length, number of nodes, and number of endings per 10μm radius and also number of primary basal dendrites. N = 20 total neurons from 4 wild type brains, 20 total neurons from 4 InsG3680 brains, and 20 total neurons from 4 R1117X brains. Data represented as mean ±SEM or individual data points plus mean ±SEM. Analyzed via mixed model to account for data nesting.