Reduced Neurite Arborization in Primary Dopaminergic Neurons in Autism-Like Shank3B-Deficient Mice

Abstract

Despite many studies on dopamine changes in autism, specific alterations in midbrain dopamine neurons projecting to the striatum and cortex remain unclear. Mouse models with diverse SH3 domain and ankyrin repeat containing protein 3 (Shank3) deficiencies are used for investigating autistic symptoms and underlying neurobiological mechanisms. SHANK3 belongs to postsynaptic proteins crucial for synapse formation during development, and disruptions in SHANK3 structure could lead to impaired neurite outgrowth and altered dendritic arborization and morphology. Therefore, we aimed to investigate whether Shank3 deficiency (Shank3B) leads to changes in the morphology of primary neuronal cell cultures from dopaminergic brain regions of neonatal mouse pups and whether it results in alterations in synaptic proteins in dopaminergic nerve pathway projection areas (striatum, frontal cortex). Significantly reduced neurite outgrowth was observed in primary dopaminergic neurons from the midbrain and striatum of Shank3-deficient compared to WT mice. A decrease in Synapsin I immunofluorescence signal in the cortical neurons isolated from Shank3-deficient mice was found, although neurite arborization changes were less severe. Importantly, the deficit in the length of the longest neurite was confirmed in primary cortical neurons isolated from Shank3-deficient mice. No changes in the gene expression of synaptic proteins were observed in the striatum and frontal cortex of Shank3-deficient mice, but an altered gene expression profile of dopaminergic receptors was found. These results show structural changes of dopaminergic neurons, which may explain autistic symptomatology in the used model and provide a basis for understanding the long-term development of autistic symptoms.

Keywords: Shank3; Autism spectrum disorder; Dopaminergic neurons; Neurite outgrowth.

Fig. 1

Dendritic arborization in primary dopaminergic neurons isolated from the ventral tegmentum of the midbrain in neonatal WT and Shank3-deficient mice. Neurons were incubated in vitro for 9 days and then stained. Overlapping microtubule-associated protein 2 (MAP2) and tyrosine hydroxylase (TH) positive fluorescent signal was considered a marker of the dopaminergic neurons (A). The arborization of the dendritic tree was assessed using Sholl analysis. The number of dendrite intersections with various concentric circles is represented in graph (B), longest neurites (C) and the number of neurons with the longest neurite (D) as mean ± SE (n = 40 neurons per group isolated from 4 (WT) or 3 (Shank3^−/−) mice). Statistical differences between groups were determined by two-way ANOVA (factor genotype F_{(1, 8658)} = 264.8; p < 0.001; factor arborization F_{(110, 8658)} = 39.69; p < 0.001) for Sholl analysis or Mann–Whitney test (*p < 0.05) for the longest neurite. WT, wild type

Fig. 2

Dendritic arborization in primary striatal neurons isolated from WT and Shank3-deficient mice. Neurons were incubated in vitro for 9 days and then stained. Microtubule-associated protein 2 (MAP2) was used to identify neurons (B). Cell nuclei were stained with DAPI (blue). The arborization of the dendritic tree was assessed using Sholl analysis. The number of dendrite intersections with various concentric circles is represented in graph (A), longest neurites (C) and the number of neurons with the longest neurite (D) as mean ± SE (n = 30 per group in Sholl analysis, n = 50 per group in longest neurite evaluation isolated from 4 (WT) or 3 (Shank3^−/−) mice). Statistical differences between groups were determined by two-way ANOVA (factor genotype F_{(1, 6438)} = 112.1; p < 0.001; factor arborization F_{(110, 6438)} = 53.83; p < 0.001) for Sholl analysis or Mann–Whitney test (**p < 0.01) for the longest neurite. WT, wild type

Fig. 3

Dendritic arborization in primary cortical neurons isolated from WT and Shank3-deficient mice. Neurons were incubated in vitro for 9 days and then stained. Microtubule-associated protein 2 (MAP2) was used to identify neurons (B). Cell nuclei were stained with DAPI (blue). The arborization of the dendritic tree was assessed using Sholl analysis. The number of dendrite intersections with various concentric circles is represented in graph (A), longest neurites (C) and the number of neurons with the longest neurite (D) as mean ± SE (n = 30 per group in Sholl analysis, n = 50 per group in longest neurite evaluation isolated from 4 (WT) or 3 (Shank3^−/−) mice). Statistical differences between groups were determined by two-way ANOVA in factors arborization F_{(110, 6438)} = 82.13; p < 0.001 and interaction F_{(110, 6438)} = 2.084; p < 0.001. Furthermore, the length of the longest neurite was significantly shorter (***p < 0.001, Mann–Whitney) in Shank3-deficient mice. WT, wild type

Fig. 4

Synapsin I immunofluorescence signal in primary cortical neurons isolated from WT and Shank3-deficient mice. Neurons were incubated in vitro for 9 days and then stained. Microtubule-associated protein 2 (MAP2) was used to identify neurons. Cell nuclei were stained with DAPI. Quantitative assessment of Synapsin I immunofluorescence signal was performed in three regions of interest (ROI) per cell (n = 65 cells isolated from 4 (WT) or 3 (Shank3^−/−) mice), with the size of 20 × 5 µm. Data are represented as a percentage of the control group signal (A). Representative neurons (scale bar = 20 µm) isolated from WT and Shank3-deficient mice (B); *p < 0.05, Student’s t-test. WT, wild type

Fig. 5

Changes of gene expressions of dopamine receptors and enzymes involved in dopamine metabolism. Frontal cortex (A, B) and striatum (C, D) were isolated from Shank3-deficient and WT 21-day old male mice. Figures represent relative changes compared to WT group calculated by the 2^−∆∆CT method. Glyceraldehyde 3-phosphate dehydrogenase was selected as the reference gene. Column scatter dot plot represents values of individual samples and bar shows means ± SE (n = 8–9). Significantly different values are marked with *p < 0.05, Student’s t-test. WT, wild type; Comt, catechol-O-methyltransferase; D1-D5, dopamine receptors (1–5); Mao-a, b, monoamine oxidase