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[Online ahead of print]

Shank3 Mutation in a Mouse Model of Autism Leads to Changes in the S-nitroso-proteome and Affects Key Proteins Involved in Vesicle Release and Synaptic Function

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Shank3 Mutation in a Mouse Model of Autism Leads to Changes in the S-nitroso-proteome and Affects Key Proteins Involved in Vesicle Release and Synaptic Function

Haitham Amal et al. Mol Psychiatry.

Abstract

Mutation in the SHANK3 human gene leads to different neuropsychiatric diseases including Autism Spectrum Disorder (ASD), intellectual disabilities and Phelan-McDermid syndrome. Shank3 disruption in mice leads to dysfunction of synaptic transmission, behavior, and development. Protein S-nitrosylation, the nitric oxide (NO)-mediated posttranslational modification (PTM) of cysteine thiols (SNO), modulates the activity of proteins that regulate key signaling pathways. We tested the hypothesis that Shank3 mutation would generate downstream effects on PTM of critical proteins that lead to modification of synaptic functions. SNO-proteins in two ASD-related brain regions, cortex and striatum of young and adult InsG3680(+/+) mice (a human mutation-based Shank3 mouse model), were identified by an innovative mass spectrometric method, SNOTRAP. We found changes of the SNO-proteome in the mutant compared to WT in both ages. Pathway analysis showed enrichment of processes affected in ASD. SNO-Calcineurin in mutant led to a significant increase of phosphorylated Synapsin1 and CREB, which affect synaptic vesicle mobilization and gene transcription, respectively. A significant increase of 3-nitrotyrosine was found in the cortical regions of the adult mutant, signaling both oxidative and nitrosative stress. Neuronal NO Synthase (nNOS) was examined for levels and localization in neurons and no significant difference was found in WT vs. mutant. S-nitrosoglutathione concentrations were higher in mutant mice compared to WT. This is the first study on NO-related molecular changes and SNO-signaling in the brain of an ASD mouse model that allows the characterization and identification of key proteins, cellular pathways, and neurobiological mechanisms that might be affected in ASD.

Conflict of interest statement

Conflict of interest The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Schematic describes the steps done in this study. SNOTRAP sample preparation was conducted to identify SNO-proteins in the cortex and striatum of 6 week- and 4 month-old mice in both WT and InsG3680(+/+) (KO) groups. Each SNO-proteins set shown in the table was analyzed by GO, KEGG pathway and STRING protein–protein interactions. WB and IHC were conducted to validate SNO-signaling mechanisms. IHC was performed to identify differences in 3-Nitrotyrosine in 6 different regions of the cortex and striatum. Using an LC–MS quantitative method, we analyzed GSNO concentrations in all WT and KO samples. nNOS expression levels were measured by WB. nNOS co-localization with NeuN was examined
Fig. 2
Fig. 2
a BP and KEGG analysis of the SNO-proteins that were found exclusively in 6w-cor-KO. b Functional and physical protein interaction analysis of the SNO-proteins that were found exclusively in 6wcor-KO. c BP and KEGG analysis of the SNO-proteins that were found exclusively in 4m-cor-KO. d Functional and physical protein interaction analysis of the SNO-proteins that found exclusively in 4m-cor-KO. e BP and KEGG analysis of the shared SNO-proteins between 6w-cor-KO and 4m-cor-KO. f Functional and physical protein interaction analysis of the shared SNO-proteins between 6w-cor-KO and 4m-cor-KO. * Bars represent the −log10 of the Benjamini corrected false discovery rate (FDR) and the squares on bars represent the fold enrichment
Fig. 3
Fig. 3
a Representative WB from eluted SNO-proteins prepared from 4m-WT and 4m-KO showing SNO-CN (Ppp3ca) in KO and not present in WT. CN shows similar levels in both groups. b Representative WB from eluted SNO-proteins prepared from 4m-WT and 4m-KO showing SNO-Stx1a in KO and not present in WT. Stx1a shows similar levels in both groups. c The relative average WB intensity of P-synapsin1 (Ser 62, Ser 67) comparing 6w-cor-WT to 6w-cor-KO and 4m-cor-WT to 4m-cor-KO. The data shows significant increase of P-synapsin1 in 4m-cor-KO compared to 4m-cor-WT. The data is normalized to synapsin1 and GAPDH and presented as mean ± SEM. One tailed t-test was conducted. *P < 0.05. WT mice (n = 4) and KO mice (n = 4). d Representative WB bands of P-synapin1, synapsin1, and GAPDH from cortex tissue prepared from 6w-WT and 6w-KO mice groups. e Representative WB bands of P-synapin1, synapsin1, and GAPDH from cortex tissue prepared from 4m-WT and 4m-KO. f The relative average WB intensity of P-CREB (Ser133) comparing 6w-cor-WT to 6w-cor-KO and 4m-cor-WT to 4m-cor-KO. The data shows significant increase of P-CREB in 4m-cor-KO compared to 4m-cor-WT. The data is normalized to CREB and GAPDH and presented as mean ± SEM. One tailed t-test was conducted. *P < 0.05. WT mice (n = 4) and KO mice (n = 4). g Representative WB bands P-synapin1, synapsin1, and GAPDH from cortex tissue prepared from 6w-cor and 6w-KO. h Representative WB from cortex tissue prepared from 4m-WT and 4m-KO
Fig. 4
Fig. 4
Representative staining of 3-nitrotyrosine (green) and DAPI (blue) for (a) Cortex and for (b) Striatum in 4m-WT and 4m-KO groups. c The average fluorescent intensity of 3-nitrotyrosine comparing 4m-WT to 4m-KO of the cortical regions (prefrontal cortex, motor cortex and somatosensory cortex) and striatal regions (central, medial, and lateral striatum). Significant increase was found in 4m-cor-KO compared to 4m-cor-WT. An increase was found in 4m-str-KO compared to 4m-str-WT. Presented as mean and ±SEM. One tailed t-test was conducted. *P < 0.05, WT mice (n = 9) and KO mice (n = 9). d Representative WB (for nNOS) from cortex and striatum tissues prepared from 6w-WT, 6w-KO, 4m-WT, and 4m-KO. See Supplementary Figure 9 for quantification. e Representative staining of the co-localization of nNOS (green), neurons (NeuN) (red), and DAPI (blue) in the cortex and f striatum of 6w-WT, 6w-KO, 4m-WT, and 4m-KO. g GSNO concentration of the 8 different tested groups. The multiple reaction monitoring (MRM) mode of a Triple Quadrupole mass spectrometer (QqQ MS) was used to establish a sensitive and selective quantification method. Y axis represents the concentration of GSNO in pmol/mg protein. Significant increase in the cor-KO compared to the cor-WT groups. Increase in the str-KO compared to the str-WT groups. Presented as mean and ±SEM. One tailed t-test was conducted. *P < 0.05, WT mice (n = 3) and KO mice (n = 3)
Fig. 5
Fig. 5
Schematic model summarizing the findings of the SNO-proteomics analysis and its consequences in 4m-cor-KO mice. In the pre-synapse, right side: S-nitrosylation of mGluR7 inhibits the receptor activity, once mGluR7 in a rest (or inhibited), β and γ subunits will not move to the calcium channel and inhibit the calcium channel activity. SNO-mGluR7 may lead to increase of calcium influx in the pre-synapse. In the pre-synapse, middle: S-nitrosylation of calcineurin (CN) leads to the inhibition of the phosphatase activity. This leads to an increase of its phosphorylated substrate P-synapsin1 (Syn1) (Ser62, Ser67). Phosphorylation of synapsin1 disconnects it from the vesicle and this leads to vesicle mobilization from the reserved pool to the readily released pool. In the pre-synapse, left side: SNO-Syntaxin1a prevents munc18 (also known as n-sec1) from binding to the closed conformation of syntaxin1a once SNOed. This allows syntaxin1a to unfold and bind to both VAMP on the vesicle and SNAP25 at the release site, which in turn enables the vesicle to dock to the membrane. In the post-synapse: The prolonged phosphorylation of CREB on Ser133 is a process mediated by Ca2+ dependent and -independent protein kinases. CREB phosphorylation is terminated by CN possibly through direct CREB dephosphorylation. SNO-CN inhibits CN activity and leads to increase of P-CREB (Ser 133) that leads to an increase of recruiting transcriptional co-activators

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