Dietary Zinc Supplementation Prevents Autism Related Behaviors and Striatal Synaptic Dysfunction in Shank3 Exon 13-16 Mutant Mice

Abstract

The SHANK family of synaptic proteins (SHANK1-3) are master regulators of the organizational structure of excitatory synapses in the brain. Mutations in SHANK1-3 are prevalent in patients with autism spectrum disorders (ASD), and loss of one copy of SHANK3 causes Phelan-McDermid Syndrome, a syndrome in which Autism occurs in >80% of cases. The synaptic stability of SHANK3 is highly regulated by zinc, driving the formation of postsynaptic protein complexes and increases in excitatory synaptic strength. As ASD-associated SHANK3 mutations retain responsiveness to zinc, here we investigated how increasing levels of dietary zinc could alter behavioral and synaptic deficits that occur with ASD. We performed behavioral testing together with cortico-striatal slice electrophysiology on a Shank3 ^-/- mouse model of ASD (Shank3 ^ex13-1616-/-), which displays ASD-related behaviors and structural and functional deficits at striatal synapses. We observed that 6 weeks of dietary zinc supplementation in Shank3 ^ex13-16-/- mice prevented ASD-related repetitive and anxiety behaviors and deficits in social novelty recognition. Dietary zinc supplementation also increased the recruitment of zinc sensitive SHANK2 to synapses, reduced synaptic transmission specifically through N-methyl-D-aspartate (NMDA)-type glutamate receptors, reversed the slowed decay tau of NMDA receptor (NMDAR)-mediated currents and occluded long term potentiation (LTP) at cortico-striatal synapses. These data suggest that alterations in NMDAR function underlie the lack of NMDAR-dependent cortico-striatal LTP and contribute to the reversal of ASD-related behaviors such as compulsive grooming. Our data reveal that dietary zinc alters neurological function from synapses to behavior, and identifies dietary zinc as a potential therapeutic agent in ASD.

Keywords: NMDA receptor; SHANK3; autism; synapse; zinc.

Figure 1

Shank3^{ex13–16−/−} autism spectrum disorder (ASD) mice repetitive grooming behaviors and reduced exploration behaviors are prevented with supplementation of dietary zinc. (A) Repetitive grooming in $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ mice was prevented in mice fed 150 ppm dietary zinc ($Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$). (B) Reduced exploratory behaviors in $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ mice, measured as a reduction in mean activity levels were no longer significantly different from Wild-type (WT) after dietary zinc supplementation. (C) Total distance moved by each mouse during the grooming test. Distance moved by Shank3^{ex13–16−/−} mice was increased with 150 ppm dietary zinc ($Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$). All data represent mean ± SEM. Statistics: one-way ANOVA with Dunnett’s post hoc test for (A) Kruskal-Wallis one-way ANOVA with Dunn’s post hoc test was applied for (B,C) as not all groups were normally distributed and variances were not equal (see “Materials and Methods” section). WT_{30 ppm} n = 10, $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 7, WT_{150 ppm} n = 8, $Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 6 animals, *p < 0.05, **p < 0.01, ****p < 0.0001, NS, not significant.

Figure 2

ASD anxiety behaviors in Shank3^{ex13–16−/−} mice are prevented with supplementation of dietary zinc. (A) Increased anxiety-type behaviors in $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ mice, reflected as a reduced percentage time spent in the light chamber, was prevented in mutant mice fed 150 ppm dietary zinc ($Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$). (B) Heat map example of WT and Shank3^{ex13–16−/−} mice on 30 or 150 ppm dietary zinc. Note the significantly decreased time the $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ mice spend in the light, and how this increases in $Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$ mice. (C) $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ mice take significantly longer to first exit the dark chamber (latency), reflecting heightened anxiety, and this was prevented in mutant mice fed 150 ppm dietary zinc ($Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$). (D) The number of transitions between the light and dark chambers are significantly reduced in $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ mice, but no longer significantly different from WT controls in Shank3^{ex13–16−/−} mice fed 150 ppm dietary zinc. All data represent mean ± SEM. Statistics: one-way ANOVA with Dunnett’s post hoc test. WT_{30 ppm} n = 10, $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 7, WT_{150 ppm} n = 8, $Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 6 animals, *p < 0.05, **p < 0.01, NS, not significant.

Figure 3

ASD social interaction deficits in Shank3^{ex13–16−/−} mice are prevented by increased dietary zinc. (A) All mice display normal social interaction during phase two of the three-chamber test. (B) $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ mice lack social novelty recognition during phase three, displaying no preference for the stranger or familiar mouse. Increased dietary zinc recovered the social novelty recognition behavior in Shank3^{ex13–16−/−} mice. (C) Example heat maps of phase three of the social interaction test. WT mice on the normal and high zinc diet prefer the novel mouse as depicted by increased presence close to stranger 2. Shank3^{ex13–16−/−} mice on the normal zinc diet show no preference for the novel mouse (stranger 2), but do show increased interaction with the novel mouse when fed the high zinc diet. All data represent mean ± SEM, two-tailed paired t-test. WT_{30 ppm} n = 9, $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 6, WT_{150 ppm} n = 8, $Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 7 mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS, not significant.

Figure 4

Effect of dietary zinc on cortico-striatal AMPAR mediated EPSCs in WT and Shank3^{ex13–16−/−} mice. (A) Schematic of electrode placement in dorsolateral striatum for all electrophysiology experiments. (B) Shank3^{ex13–16−/−} induces a decrease in AMPAR EPSC amplitude, and this decrease is not altered when animals are fed a high zinc diet (WT_{30 ppm} n = 11 cells from nine animals, $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 8 cells from seven animals, WT_{150 ppm} n = 14 cells from nine animals, $Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 13 cells from eight animals). (C) Example AMPAR mediated EPSCs from each animal genotype and zinc diet. (D) Frequency histograms of AMPAR EPSC amplitudes from WT and Shank3^{ex13–16−/−} mice fed normal (left) or high zinc diet (right), illustrating the shift towards smaller AMPAR EPSC amplitudes in Shank3^{ex13–16−/−} mice on either zinc diet. All data represent mean ± SEM. *p < 0.05, student’s t-test.

Figure 5

Increased dietary zinc specifically decreases cortico-striatal N-methyl-D-aspartate receptor (NMDA) receptor (NMDAR) mediated EPSCs in $Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$ mice. (A) Shank3^{ex13–16−/−} deletion ($Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$) had no effect on mean NMDAR mediated EPSC amplitude, but Shank3^{ex13–16−/−} mice fed 150 ppm dietary zinc ($Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$) exhibited a decreased mean NMDAR EPSC amplitude (Kruskal-Wallis one-way ANOVA with Dunn’s post hoc test; WT_{30 ppm} n = 19 cells from 10 animals, $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 22 cells from nine animals, WT_{150 ppm} n = 18 cells from nine animals, $Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 18 cells from eight animals). (B) NMDAR decay kinetics, measured as the weighted time constant tau (τ_w), were significantly slowed in $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ mice. Increased dietary zinc in Shank3^{ex13–16−/−} mice ($Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$) reversed the slowed kinetics (Kruskal-Wallis one-way ANOVA with Dunn’s post hoc test). (C) Example NMDAR EPSCs from both genotypes and dietary zinc levels. (i) NMDAR EPSCs from WT (black) and Shank3^{ex13–16−/−} (gray) mice on 30 ppm diet. (ii) NMDAR EPSCs from WT (black) and Shank3^{ex13–16−/−} (gray) mice on 150 ppm diet. (iii) Scaled NMDAR EPSCs from WT (black) and Shank3^{ex13–16−/−} (gray) mice on 30 ppm diet. (iv) Scaled NMDAR EPSCs from WT (black) and Shank3^{ex13–16−/−} (gray) mice on 150 ppm diet. All data represent mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001, NS, not significant.

Figure 6

Dietary zinc supplementation prevents long term potentiation (LTP) in the cortico-striatal pathway in Shank3^{ex13–16−/−} mice. (A–C) WT_{30 ppm}, WT_{150 ppm}, and $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ mice all express LTP in response to tetanic stimulation (paired t-test, WT_{30 ppm} n = 11 cells from six animals, $Shank{3}_{30\text{ ppm}}^{\text{ex1}3-16-/-}$ n = 16 cells from six animals, WT_{150 ppm} n = 7 cells from four animals). (D) No significant change in AMPAR EPSC amplitude was observed 30 min after LTP induction in Shank3^{ex13–16−/−} mice with dietary zinc supplementation ($Shank{3}_{150\text{ ppm}}^{\text{ex1}3-16-/-}$: paired t-test, n =11 cells from five animals). (E) Bar graph of average percent change in AMPAR EPSC amplitude measured at 30 min post LTP-induction. All data represent mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, NS, not significant.

Figure 7

Dietary zinc supplementation increases cortico-striatal and thalamo-striatal synaptic SHANK2 expression in Shank3^{ex13–16−/−} mice. (A–D) Example overlaid images from WT and Shank3^{ex13–16−/−} mice fed normal (30 ppm) or high dietary zinc (150 ppm), immunostained for SHANK2 (green), VGluT1 (red) or VGluT2 (blue). Red triangles denote example synaptic SHANK2 puncta colocalized with VGluT1, blue triangles denote example synaptic SHANK2 puncta colocalized with VGluT2, open triangles denote example non-synaptic SHANK2 puncta. Scale bar 5 μm. (E) SHANK2 puncta intensity at cortico-striatal synapses, i.e., puncta co-localized with VGluT1, was significantly increased in $Shank{3}_{150\text{ ppm}}^{\text{ex168}3-16-/-}$ mice. (F) SHANK2 puncta intensity at thalamocostriatal synapses, i.e., puncta co-localized with VGluT2, was significantly increased in WT and Shank3^{ex13–16−/−} mice fed 150 ppm dietary zinc. (G) Total synaptic SHANK2, puncta co-localized with VGluT1 plus VGluT2, was significantly increased in both WT and Shank3^{ex13–16−/−}mice fed 150 ppm dietary zinc. (H) Non-synaptic SHANK2, i.e., puncta not co-localized with either VGluT1 or VGluT2, was not significantly different between WT and Shank3^{ex13–16−/−} mice fed either diet. Data represent mean ± SEM from six Shank3^{ex13–16−/−} mice and six WT mice. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01. NS, not significant.