Journal: Translational Psychiatry

Article Title: S100B dysregulation during brain development affects synaptic SHANK protein networks via alteration of zinc homeostasis

doi: 10.1038/s41398-021-01694-z

Figure Lengend Snippet: a Treatment of hippocampal cultures with 30 μM S100B or S100Bmut for 24 h at DIV10. The mean intracellular signal intensity of Zinpyr1 fluorescence is shown. Treatment of hippocampal cultures with S100B significantly reduces the free intracellular Zn 2+ concentration assessed by the analysis of 35 cells per condition. No significant decrease in intracellular Zinpyr1 fluorescence can be observed in cultures treated with S100Bmut (one-way ANOVA, F 2,77 = 8.86; p < 0.0001; Post-hoc analysis: control vs. S100B, p = 0.0037; control vs. S100Bmut, p = 0.2469; S100B vs. S100Bmut, p < 0.0001). b Exemplary images showing signal intensities of Zinpyr1 in a color-coded manner. c – g Increased levels of S100B lead to a decrease in zinc-dependent SHANK proteins at the synapse. c Treatment of hippocampal cultures with 30 μM S100B for 24 h. The signal intensity of SHANK2 is significantly reduced 24 h after exposure to S100B. Similarly, the signal intensity of SHANK3 puncta shows a trend toward a reduction. No significant reduction of SHANK2 and SHANK3 was observed 24 h after applying S100Bmut. For all analyses, 8–10 cells per condition were used (one-way ANOVA: SHANK2: F 2,27 = 502.009, p = 0.001; Post-hoc analysis: Control vs. S100B p = 0.0013, S100B vs. S100Bmut p = 0.0029; Shank3: F 2,27 = 278.257, p = 0.005; Post-hoc analysis: Control vs. S100B p = 0.497, S100B vs. S100Bmut p = 0.0037) ( n = 10 cells per condition). d Exemplary images showing anti-SHANK2 and SHANK3 staining of hippocampal neurons 24 h after treatment with S100B and S100Bmut. e The mean number of immunoreactive signals per dendrite length was evaluated. No significant alterations were detected. f Using Western blot experiments (from three different preparations), a significant decrease of SHANK2 protein levels in the crude membrane fraction can be seen ( t -test, p = 0.0265). g Cells were again treated with S100B for 24 h at DIV8. A significant reduction of SHANK2 and SHANK3 proteins is visible. This reduction was abolished in case cells were treated with 30 μM S100B that was saturated with 60 μM ZnCl 2 for 1 h on ice before application (one-way ANOVA: SHANK2: F 3,36 = 12.2454, p < 0.001; Post-hoc analysis: Control vs. S100B p = 0.001, S100B vs. saturated S100B p = 0.001; SHANK3: F 3,36 = 15.9105, p = 0.005; Post-hoc analysis: Control vs. S100B p = 0.001, S100B vs. saturated S100Bmut p = 0.001, p = 0.0037) ( n = 10 cells per condition).

Article Snippet: In brief, pLenti S100b vector (OriGene) was used for the mutagenesis of S100B zinc-binding sites and carried a C-terminal Myc-DKK tag.

Techniques: Fluorescence, Concentration Assay, Control, Staining, Western Blot, Membrane

Journal: Translational Psychiatry

Article Title: S100B dysregulation during brain development affects synaptic SHANK protein networks via alteration of zinc homeostasis

doi: 10.1038/s41398-021-01694-z

Figure Lengend Snippet: a – c Measurement of trace element levels in whole blood and brain tissue of mice using ICP-MS. a The average whole blood concentration of Zn is significantly different between saline- and S100B-injected pregnant mice ( t -test, p = 0.0335; n = 3 per group). b The average whole blood concentration of Zn is not significantly different between pups from Ctrls ( n = 7), and S100B-injected mice ( n = 4). c The average concentration of Zn and Cu in whole-brain tissue is not significantly different between pups from Ctrls ( n = 10) and S100B-injected mice ( n = 7). Fe and Se levels are significantly increased in pups from S100B-injected mice ( t -test, p Fe = 0.0356; p Se = 0.0084). d The average free Zn concentration measured by Zinpyr1 fluorescence assays shows significantly lower free Zn levels in the brain of pups from mothers with S100B injection compared to Ctrls ( n = 3 per group) ( t -test, p = 0.0032).

Article Snippet: In brief, pLenti S100b vector (OriGene) was used for the mutagenesis of S100B zinc-binding sites and carried a C-terminal Myc-DKK tag.

Techniques: Concentration Assay, Saline, Injection, Fluorescence

Journal: Translational Psychiatry

Article Title: S100B dysregulation during brain development affects synaptic SHANK protein networks via alteration of zinc homeostasis

doi: 10.1038/s41398-021-01694-z

Figure Lengend Snippet: a Increased blood S100B levels were detected in pups using S100B specific Luminex MAGPIX assays and normalized to the sample’s total protein concentration measured by BCA assays ( t -test, p = 0.0343). b Western blot analysis of whole-brain lysate of pups from Ctrls and S100B mothers reveals significantly higher levels of S100B in the brain of pups exposed to S100B ( t -test, p = 0.0432; n = 3 per group). c, d Western blot analysis of whole-brain homogenate of pups from Ctrls and S100B-injected mothers reveals no significant changes in SHANK2 ( c ) and SHANK3 ( d ) in the brain of pups exposed to S100B. e, f Using crude membrane (P2) fractions, we detected significantly lower synaptic SHANK2 levels ( e ) in S100B pups compared to Ctrls ( t -test, p SHANK2 = 0.046; n = 3 per group). Both isoforms (SHANK2A and SHANK2) were similarly affected (right panel ( t -test, p SHANK2A = 0.0487; p SHANK2 = 0.0362; n = 3 per group)). f The levels of total SHANK3 were significantly reduced ( t -test, p SHANK3 = 0.0018; n = 4 Ctrls, n = 3 S100B) in S100B pups compared to Ctrls. All higher MW isoforms (SHANK3a, SHANK3c/d and SHANK3e) were similarly affected (right panel ( t -test, p SHANK3a = 0.0487; p SHANK3c.d = 0.0362; p SHANK3c.d = 0.0362)).

Article Snippet: In brief, pLenti S100b vector (OriGene) was used for the mutagenesis of S100B zinc-binding sites and carried a C-terminal Myc-DKK tag.

Techniques: Luminex, Protein Concentration, Western Blot, Injection, Membrane

Journal: Translational Psychiatry

Article Title: S100B dysregulation during brain development affects synaptic SHANK protein networks via alteration of zinc homeostasis

doi: 10.1038/s41398-021-01694-z

Figure Lengend Snippet: The motor performance and anxiety-like behavior were assessed in the open field task (Ctrls, n = 15; S100B, n = 36): a S100B mice show increased total distance ( p = 0.045), ( b ) increased latency to the first immobility episode ( p = 0.012) and ( c ) higher number of line crossings ( p < 0.001) compared to Ctrls. d No significant difference was detected for the time spent in the center zone between the two groups ( p = 0.126). e Occupancy plots of the two groups of mice. The center zone is indicated by the dashed square. Statistical analysis according to Mann–Whitney U test.

Article Snippet: In brief, pLenti S100b vector (OriGene) was used for the mutagenesis of S100B zinc-binding sites and carried a C-terminal Myc-DKK tag.

Techniques: MANN-WHITNEY

Journal: Translational Psychiatry

Article Title: S100B dysregulation during brain development affects synaptic SHANK protein networks via alteration of zinc homeostasis

doi: 10.1038/s41398-021-01694-z

Figure Lengend Snippet: In the resident-intruder task, although ( a ) the latency to the first attack is not different between the two groups ( p = 0.202), the percentage of mice that does not display an aggressive behavior during the interaction with the intruder is statistically higher in S100B group ( e , Fisher’s test, p < 0.001). A significant increase in stereotypic behaviors such as ( b ) rearing ( p = 0.021) and ( c ) self-grooming ( p = 0.008) events were detected in S100B mice during both intruder (not shown) and marble burying tests. In addition, ( d ) the percentage of mice with jumping behavior is statistically higher in the S100B group ( f , Fisher’s test, p < 0.001). Statistical analysis according to Mann–Whitney U test (when not indicated) or Fisher’s test.

Article Snippet: In brief, pLenti S100b vector (OriGene) was used for the mutagenesis of S100B zinc-binding sites and carried a C-terminal Myc-DKK tag.

Techniques: MANN-WHITNEY

Journal: Translational Psychiatry

Article Title: S100B dysregulation during brain development affects synaptic SHANK protein networks via alteration of zinc homeostasis

doi: 10.1038/s41398-021-01694-z

Figure Lengend Snippet: a No significant difference was observed in the entries into the newly opened arm between S100B and Ctrl mice ( p = 0.148) while ( b ) a trend for a significant decrease in time spent in the newly opened arm was observed in S100B mice ( p = 0.092). c Analysis of the learning curve showed a significant effect of S100B exposure (repeated-measures ANOVA, Time F (5, 43) = 80.862, p < 0.001, Treatment F (1, 43) = 3.715, p = 0.061, Time × Treatment F (5, 43) = 3,340, p = 0.006). d Heat map of the time passed by the Ctrls and S100B mice in the maze during the probe test. e S100B mice spent more time in the adjacent left ( p = 0.042) and opposite ( p = 0.065) sectors. f S100B mice spent more time in the maze center ( p = 0.033). g The analysis of search strategy showed an increase in non-spatially targeted strategies in S100B mice (Chi-square test, p = 0.057). h No significant difference between S100B and Ctrls was shown for the time to reach the platform in the reversal test, but ( i ) a significantly higher percentage of S100B mice continued to target the old platform location before finding the platform in the new location on the second day of the test (log-rank test, p = 0.037; note that the quadrants indicated are referred to the new location of the platform). j The percentage of S100B mice building only a partial nest structure is significantly higher than that of Ctrl mice ( p = 0.009, Fisher’s test; Ctrls, n = 15; S100B, n = 36). Statistical analysis according to Mann–Whitney U test (when not indicated), Fisher’s test, Chi-square test. Log-rank test or repeated-measures ANOVA.

Article Snippet: In brief, pLenti S100b vector (OriGene) was used for the mutagenesis of S100B zinc-binding sites and carried a C-terminal Myc-DKK tag.

Techniques: MANN-WHITNEY