Journal: bioRxiv

Article Title: Pathogenic tau inhibits synaptic plasticity by blocking eIF4B-mediated local protein synthesis

doi: 10.1101/2025.09.11.675671

Figure Lengend Snippet: ( a ) Workflow of protocol to collect mRNAs extracted from polysomes in human neurons after cLTP induction. RNA-seq was performed to quantify the abundance of each transcript in polysomes extracted from tauWT and tauV337M iso neurons (n = 4 cultures/group). ( b ) Representative polysome profiles from tauWT and tauV337M iso neurons with the polysome peaks shaded in grey. ( c ) Quantification of the area under the curve (AUC) of the polysome peaks normalized to tauWT neurons (n = 4 cultures/group; *, p < 0.05, Student’s t -test). ( d ) ClueGO cellular component pathway enrichment of the 1,093 transcripts extracted from polysomes that were downregulated in tauV337M iso neurons with < −0.5 log 2 FC compared to tauWT neurons. The number of downregulated transcripts detected within each GO term is indicated in parentheses. Node colors denote functionally grouped networks. ( e ) RRHO plot of the correlation between mRNAs translated in neuropil compared to somata and the differential LTP-associated translatome in tauV337M iso compared to tauWT neurons. Analyses show a significant correlation in transcripts that were translated more within neuropil and downregulated in the tauV337M iso LTP-associated translatome. ( f ) SynGO analyses of polysome-bound mRNAs that were downregulated in tauV337M iso neurons and classified in the Postsynapse term. Analyses were performed on ( d ) transcripts with < −0.5 log 2 FC and ( e ) downregulated polysome-bound transcripts that were significantly correlated with more translation in neuropil . ( g ) Select downregulated polysome-bound transcripts in tauV337M iso neuron that were classified in the Postsynapse SynGO term ( f ). Color indicates log 2 FC of transcript abundance in polysomes of tauV337M iso compared to tauWT neurons. Transcripts that are translated more within neuropil than in the soma of neurons are labeled by a star. ( h ) Representative RT-PCR results from three different fractions containing polysomes (#16-18) that were extracted from one culture of tauWT or tauV337M iso neurons. ( i ) RT-PCR analyses of Shank3, PKMζ, and Actin mRNA levels in the polysome fractions (#16-18) that were each normalized to the mRNA levels in tauWT neurons (n = 4 cultures/group; * p < 0.05, **p<0.01, Student’s t-test). ( j ) Confocal images of Shank3 (red) and Synapsin (green) immunolabeling of tauWT and tauV337M iso neurons with or without cLTP induction. Shank3 was increased at synapses after cLTP in tauWT neurons (arrows). Scale bar, 2 µm. ( k ) Quantification of synaptic Shank3 immunolabeling intensity co-localized with Synapsin (n = 15 images/group; * p < 0.05, two-way ANOVA, Bonferroni post-hoc analyses). Values were normalized to the mean Shank3 intensity at synapses in unstimulated tauWT neurons. Values are given as means ± SEM.

Article Snippet: The following primary antibodies were used: 7-methylguanosine (m7G)-Cap (RN016M, MBL), AT180 (MN1040, Thermo Fisher), eIF4A (sc-377315, Santa Cruz), eIF4B (3592T, Cell Signaling), eIF4E (9742S, Cell Signaling), eIF4E (sc-376062, Santa Cruz), eIF4G (2498S, Cell Signaling), FLAG (F1804, Sigma-Aldrich), GAPDH (MAB374, Sigma-Aldrich), GFP-conjugated 488 (A21311, Invitrogen), GluA1 (ABN241, Millipore), HA (H6908, Sigma-Aldrich), rabbit MAP2 (4542S, Cell Signaling), chicken MAP2 (nb 300 213, Novus Biologicals), puromycin (EQ0001, Kerafast), Synapsin (5297S, Cell Signaling), RPS6 (sc-74459, Santa Cruz), Shank3 (sc-377088, Santa Cruz), SV2 (University of Iowa DSHB), Tau5 (AHB0042, Thermo Fisher), HT7 (MN1000, Thermo Fisher), vGluT1 (MAB5502, Millipore), GluN1 (114 011, Synaptic Systems).

Techniques: RNA Sequencing, Labeling, Reverse Transcription Polymerase Chain Reaction, Immunolabeling

Journal: EMBO Molecular Medicine

Article Title: Wbp2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing

doi: 10.15252/emmm.201505523

Figure Lengend Snippet: Diagram showing the Wbp2 molecular pathway, including its downstream targets and their functional relationship. The blue arrows, light blue lines and green lines link data from the literature ( in vivo and in vitro ); the orange squares and red arrows indicate up‐ or down‐regulation shown in our experimental observations, as reported in this study. Quantitative real‐time PCR showing reduced mRNA levels for Esr1 , Esr2 and Pgr and up‐regulation of Shank3 and Psd‐95 in cochleae of 4‐week‐old Wbp2‐deficient mice compared to littermate controls ( n = 3 for each genotype). Hprt is used as a relative control. *P = 0.03 for Psd‐95; **P = 0.007 for Shank3 ; *P = 0.03 for Esr2 ; **P = 0.0016 for Esr1 ; *P = 0.037 for Pgr . Synapses from one mutant and one control IHC at 4 weeks of age after Psd‐95 and Ct BP 2 labelling, showing stronger Psd‐95 expression in the mutants compared to controls, representing double labelling experiments performed on 3 mutants and 3 controls. Scale bar, 10 μm. Quantification of Psd95 fluorescence in IHC synapses, representing expression in the apical (9‐ kH z best frequency region of the cochlea) and basal (24‐ kH z best frequency region of the cochlea) regions at 4 weeks of age. Data from 2 wt and 2 homs were analysed (16 synapses per cochlear region per mouse). AU , arbitrary units. Wt: 24 kH z 22.41 ± 10.70, 9 kH z 11.045 ± 2.128; mutants: 24 kH z 66.73 ± 13.70, P = 0.069; 9 kH z: 26.02 ± 5.79 P = 0.075. Data information: Data are shown as mean ± SD and were statistically analysed by two‐tailed Student's t ‐test. Source data are available online for this figure.

Article Snippet: Real‐time PCR was performed in an CFX Connect Real‐Time System (Bio‐Rad), in triplicate for each sample using the following TaqMan probes from Applied Biosystems: Mm01246338_m1 ( Wbp2 ); Mm00498775_m1 ( Shank3 ); Mm00492193_m1 ( Psd‐95 ); Mm00433149_m1 ( Esr1 ); Mm00599821_m1 ( Esr2 ); Mm00435628_m1 ( Pgr ).

Techniques: Functional Assay, In Vivo, In Vitro, Real-time Polymerase Chain Reaction, Control, Mutagenesis, Expressing, Fluorescence, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction

doi: 10.1523/JNEUROSCI.0893-19.2020

Figure Lengend Snippet: Effects of Shank3 overexpression on LTCC signaling to the nucleus. A, Shank3 was immunoprecipitated from soluble fractions of HEK293T cells expressing HA-CaV1.3-CTD with mAp-Shank3R-WT or mAp-Shank3R-ΔPDZ. Immunoblots (representative of three biological replicates) demonstrate that HA-CaV1.3-CTD coimmunoprecipitates with mAp-Shank3R-WT, but not with mAp-Shank3R-ΔPDZ. B, Schematic of experimental protocols. Primary hippocampal neurons were transfected (see below) and then incubated to stimulate LTCC signaling to the nucleus (see Materials and Methods). Neurons were either fixed and stained using DAPI and pSer133-CREB antibodies after a 90 s depolarization (top: for C), or incubated for an additional 3 h in conditioned media before fixation and staining with DAPI and c-Fos antibodies (bottom: for D). C, Overexpression of mAp-Shank3-WT, but not mAp-Shank3-AAA or mAp-Shank3-ΔPDZ, increases the levels of pCREB staining relative to nontransfected neurons under basal and depolarized conditions (two-way ANOVA with 2 factors [Mutant, Stimulation]: Mutant, F(3,179) = 17.86, p < 0.0001; Stimulation, F(1,179) = 1108, p < 0.0001; Interaction, F(3,179) = 4.442, p < 0.01). Dunnett's multiple-comparisons test: **p < 0.01, ****p < 0.0001. D, The expression of c-Fos is not affected by overexpression of mAp-Shank3-WT, mAp-Shank3-AAA, or mAp-Shank3-ΔPDZ (two-way ANOVA with 2 factors [Mutation, Stimulation]: Mutation, F(3,166) = 2.152; Stimulation, F(1,166) = 830.1, p < 0.0001; Interaction, F(3,166) = 0.3284, Dunnett's multiple-comparisons test). Error bars indicate mean ± SEM. Superimposed data points indicate values from single cells accumulated from three to five independent neuronal cultures/transfections. Images below the bar graphs are of representative nuclei for each condition. Scale bars, 5 μm.

Article Snippet: D , Synaptic (P2) fractions from WT or CaMKIIα T286A mouse forebrains were immunoprecipitated using control IgG or Shank3 (Bethyl) antibodies, and immunoblotted using Shank3 (Cell Signaling Technology) and CaMKIIα antibodies.

Techniques: Over Expression, Immunoprecipitation, Expressing, Western Blot, Transfection, Incubation, Staining, Mutagenesis

Journal: The Journal of Neuroscience

Article Title: Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction

doi: 10.1523/JNEUROSCI.0893-19.2020

Figure Lengend Snippet: Rescue of pCREB signaling and c-Fos expression after Shank3 shRNA knockdown. The Shank3 shRNA construct was transfected alone or with shRNA-resistant mAp-Shank3R-WT (green bar), mAp-Shank3R-AAA (blue bar), or mAp-Shank3R-ΔPDZ (purple bar). Neurons were depolarized for 90 s, and the levels of pCREB (A) and c-Fos (B) were determined (see Materials and Methods) in transfected (colored bars) and nearby nontransfected (black bars) neurons. A, The expression of mAp-Shank3R-WT, but not mAp-Shank3R-AAA or mAp-Shank3R-ΔPDZ, rescued signaling to pCREB relative to shRNA alone. In addition, neurons expressing Shank3-WT had significantly higher pCREB signal relative to nearby, nontransfected neurons (two-way ANOVA with 2 factors [Mutant, Transfection]: Mutant, F(3,156) = 10.14, p < 0.0001; Transfection, F(1,156) = 22.70, p < 0.0001; Interaction, F(3,156) = 12.29, p < 0.0001, comparison between mutants: Dunnett's multiple-comparisons test, ****p < 0.0001; comparison between transfected/nontransfected cells: Sidak's multiple-comparisons test, *p < 0.05). B, The expression of mAp-Shank3R-WT, but not mAp-Shank3R-AAA or mAp-Shank3R-ΔPDZ, rescued signaling to increase c-Fos expression relative to shRNA alone (two-way ANOVA with 2 factors ([Mutant, Transfection]: Mutant, F(3,150) = 13.80, p < 0.0001; Transfection, F(1,150) = 26.52, p < 0.0001; Interaction, F(3,150) = 8.149, p < 0.0001, comparison between mutants: Dunnett's multiple-comparisons test, ****p < 0.0001; comparison between transfected/nontransfected cells: Sidak's multiple-comparisons test). Error bars indicate mean ± SEM. Each data point represents a single cell, accumulated from four or five independent neuronal cultures/transfections. Images below the bar graphs are of representative nuclei for each condition. Scale bars, 5 μm.

Article Snippet: D , Synaptic (P2) fractions from WT or CaMKIIα T286A mouse forebrains were immunoprecipitated using control IgG or Shank3 (Bethyl) antibodies, and immunoblotted using Shank3 (Cell Signaling Technology) and CaMKIIα antibodies.

Techniques: Expressing, shRNA, Knockdown, Construct, Transfection, Mutagenesis, Comparison

Journal: The Journal of Neuroscience

Article Title: Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction

doi: 10.1523/JNEUROSCI.0893-19.2020

Figure Lengend Snippet: Reciprocal coimmunoprecipitation of Shank3 and CaMKIIα from mouse forebrain extracts. A, Whole forebrain lysates (Input), cytosolic (S1), membrane-associated (S2), and synaptic (P2) subcellular fractions from WT, CaMKIIα-KO, and CaMKIIαT286A mice were immunoblotted for localization of Shank3 (Cell Signaling Technology antibody), PSD-95, and CaMKIIα. The Shank3 antibody detected two bands (open and solid arrowheads) that are primarily localized in P2 fractions, which were also enriched for CaMKIIα. B, The upper and lower Shank3 bands in whole forebrain lysates from WT and CaMKIIα-KO mice (left) or WT and CaMKIIαT286A mice (right) were quantified. Signals were corrected for protein loading based on Ponceau-stained membranes and normalized to the levels of the upper Shank3 band in WT samples. C, Synaptic (P2) fractions from WT or CaMKIIα-KO mouse forebrains were immunoprecipitated using control IgG or CaMKIIα-specific antibodies, and immunoblotted using Shank3 (Cell Signaling Technology antibody) and pan-CaMKII antibodies. Arrows indicate CaMKIIα and CaMKIIβ bands. CaMKIIα and coimmunoprecipitated Shank3 were not detected in samples isolated from CaMKIIα-KO mice. D, Synaptic (P2) fractions from WT or CaMKIIαT286A mouse forebrains were immunoprecipitated using control IgG or Shank3 (Bethyl) antibodies, and immunoblotted using Shank3 (Cell Signaling Technology) and CaMKIIα antibodies. The levels of coprecipitated CaMKIIα from CaMKIIαT286A mice were significantly reduced (93 ± 4% reduction in lane 6 compared with lane 5, n = 3. p < 0.001, one-sample Student's t test with equal variance compared with theoretical value of 100). All immunoblots are representative of ≥3 biological replicates.

Article Snippet: D , Synaptic (P2) fractions from WT or CaMKIIα T286A mouse forebrains were immunoprecipitated using control IgG or Shank3 (Bethyl) antibodies, and immunoblotted using Shank3 (Cell Signaling Technology) and CaMKIIα antibodies.

Techniques: Membrane, Staining, Immunoprecipitation, Control, Isolation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction

doi: 10.1523/JNEUROSCI.0893-19.2020

Figure Lengend Snippet: T286-phosphorylated CaMKIIα specifically binds to Shank3 (829–1130). A, Domain structure of full-length Shank3 and the six GST-Shank3 fusion proteins used in these studies that span the entire Shank3 protein. Gray boxes represent canonical Shank3 domains. Residue numbers are listed in parentheses. ANK, Ankyrin repeats domain; SH3, Src homology 3 domain; PDZ, PSD95/Dlg1/zo-1 domain; PRR, proline-rich region containing binding sites for Homer and Cortactin; SAM, sterile α motif involved in multimerization of Shank3. B, Glutathione agarose cosedimentation assay shows that preactivated (Thr286-autophosphorylated) CaMKIIα specifically binds to GST-Shank3 #4 (829–1130) and positive control GST-GluN2B (1260–1309). *Full-length GST fusion proteins on the GST immunoblot. C, Glutathione agarose cosedimentation assay shows that, in the absence of Ca2+/CaM binding or Thr286 autophosphorylation, CaMKIIα (Basal) does not bind to GST-Shank3 #4; in vitro binding of CaMKIIα is partially supported by Ca2+/CaM binding (Ca/CaM), and maximally enhanced by pT286 autophosphorylation. Bar graph represents levels of each form of CaMKII bound to GST-Shank3 #4 (or the GST negative control) (mean ± SEM) relative to levels of pT286-CaMKIIα binding to GST-GluN2B. Immunoblots are representative of three or four biological replicates.

Article Snippet: D , Synaptic (P2) fractions from WT or CaMKIIα T286A mouse forebrains were immunoprecipitated using control IgG or Shank3 (Bethyl) antibodies, and immunoblotted using Shank3 (Cell Signaling Technology) and CaMKIIα antibodies.

Techniques: Residue, Binding Assay, Sterility, Positive Control, Western Blot, In Vitro, Negative Control

Journal: The Journal of Neuroscience

Article Title: Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction

doi: 10.1523/JNEUROSCI.0893-19.2020

Figure Lengend Snippet: Characterization of the CaMKII binding motif in Shank3. A, Top, Diagram of 3 truncations used to map the CaMKII interaction site within GST-Shank3 #4 (829–1130). Bottom, Sequence alignment of human Shank3 residues 941–959 with the corresponding Shank3 residues in other species and the CaMKII binding domain in the N-terminal domain of the Rattus norvegicus CaV1.3 α1 subunit (Wang et al., 2017) and the C-terminal tail of the Rattus norvegicus mGlu5 (Marks et al., 2018). Black represents conserved residues. Gray represents dissimilar residues. Red box represents the conserved tribasic residue motif. B, Glutathione agarose cosedimentation assay comparing binding of activated CaMKIIα to GST-Shank3 #4 (829–1130) and 3 nonoverlapping fragments (4a, 4b, and 4c). *Full-length GST fusion proteins. GST-Shank3 #4 (829–1130) and #4b (931–1014) bind similar amounts of pT286-autophosphorylated CaMKIIα, but there is no detectable binding to the other Shank3 fragments. C, Mutation of amino acids 949RRK951 to AAA in GST-Shank3 #4 (829–1130) blocks CaMKIIα binding in glutathione agarose cosedimentation assay (98 ± 4% reduced compared with WT, n = 3, p < 0.001, one-sample Student's t test with equal variance compared with theoretical value of 100). All immunoblots are representative of three biological replicates.

Article Snippet: D , Synaptic (P2) fractions from WT or CaMKIIα T286A mouse forebrains were immunoprecipitated using control IgG or Shank3 (Bethyl) antibodies, and immunoblotted using Shank3 (Cell Signaling Technology) and CaMKIIα antibodies.

Techniques: Binding Assay, Sequencing, Residue, Mutagenesis, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction

doi: 10.1523/JNEUROSCI.0893-19.2020

Figure Lengend Snippet: A Shank3 949RRK951 to AAA mutation disrupts association with CaMKIIα but does not with the CaV1.3 CTD. A, Soluble fractions of HEK293T cells expressing CaMKIIα with GFP or GFP-Shank3 (WT or 949RRK951 to AAA mutant) were immunoprecipitated using a GFP antibody. Coprecipitation of CaMKIIα with GFP-Shank3-AAA is significantly reduced by 95 ± 10% compared with GFP-Shank3-WT. ***p < 0.001 (one-sample Student's t test with equal variance compared with a theoretical value of 100). B, Soluble fractions of HEK293T cells expressing GFP or GFP-Shank3 (WT or AAA) with the HA-tagged CTD of the CaV1.3 α1 subunit (HA-CaV1.3-CTD) were immunoprecipitated as in A. The AAA mutation has no significant effect on the coprecipitation of HA-CaV1.3-CTD (p = 0.84). All immunoblots are representative of four biological replicates. Error bars indicate the mean ± SEM.

Article Snippet: D , Synaptic (P2) fractions from WT or CaMKIIα T286A mouse forebrains were immunoprecipitated using control IgG or Shank3 (Bethyl) antibodies, and immunoblotted using Shank3 (Cell Signaling Technology) and CaMKIIα antibodies.

Techniques: Mutagenesis, Expressing, Immunoprecipitation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction

doi: 10.1523/JNEUROSCI.0893-19.2020

Figure Lengend Snippet: Shank3 949RRK951 to AAA mutation disrupts colocalization of activated CaMKIIα. A, Immunoblots of undifferentiated, differentiated, and transfected/differentiated STHdh+/+ cells, with a WT mouse forebrain lysate as positive control. Shank3 and CaMKIIα are not expressed in nontransfected STHdh+/+ cells, and transfected mApple-tagged CaMKIIα (mAp-CaMKIIα) is T286-phosphorylated. B, Representative images of differentiated STHdh+/+ cells expressing GFP-Shank3-WT with mAp-CaMKIIα-WT (left), mAp-CaMKIIα-T286D (middle), or mAp-CaMKIIα-T286A (right). Inset, Regions (dashed line box) of the processes containing punctate GFP signals (arrowheads) that overlap with mApple signal from CaMKIIα-WT and -T286D, but not -T286A. C, Intensity correlation analysis quantifying the colocalization of GFP and mAp signals in transfected and differentiated STHdh+/+ cells in B. Each data point represents an ICQ value from a single cell, with 7–12 cells analyzed from each of three independent cultures/transfections. ICQ values for GFP-Shank3-WT and either mAp-CaMKIIα-WT (mean ± SEM: 0.29 ± 0.02) or mAp-CaMKIIα-T286D (0.36 ± 0.02) were significantly more colocalized compared with mAp-CaMKIIα-T286A (0.17 ± 0.02) (one-way ANOVA, F(2,71) = 21.35, p < 0.0001). Tukey's post hoc test: ***p < 0.001, ****p < 0.0001. D, Representative images of differentiated STHdh+/+ expressing GFP-Shank3-WT or GFP-Shank3-AAA with soluble mAp or mAp-CaMKIIα-WT. Inset, Expanded regions of the processes, as in B. E, Intensity correlation analysis quantifying the colocalization of GFP and mAp signals in transfected and differentiated STHdh+/+ cells in C. GFP-Shank3-WT and mAp-CaMKIIα-WT (0.31 ± 0.02) are significantly more colocalized than GFP-Shank3-WT and mAp (0.07 ± 0.01) or GFP-Shank3-AAA and mAp-CaMKIIα (0.09 ± 0.02) (one-way ANOVA, F(2,70) = 39.55, p < 0.0001). Tukey's post hoc test: ****p < 0.0001. Scale bars: B, D, 2.5 μm.

Article Snippet: D , Synaptic (P2) fractions from WT or CaMKIIα T286A mouse forebrains were immunoprecipitated using control IgG or Shank3 (Bethyl) antibodies, and immunoblotted using Shank3 (Cell Signaling Technology) and CaMKIIα antibodies.

Techniques: Mutagenesis, Western Blot, Transfection, Positive Control, Expressing

Journal: The Journal of Neuroscience

Article Title: Neuronal L-Type Calcium Channel Signaling to the Nucleus Requires a Novel CaMKIIα-Shank3 Interaction

doi: 10.1523/JNEUROSCI.0893-19.2020

Figure Lengend Snippet: Shank3 knockdown disrupts pCREB signaling and c-Fos expression. A, Validation of Shank3 shRNA and mApple-Shank3 shRNA-resistant (mAp-Shank3R) expression vectors. Expression of shRNA, mAp-Shank3, and mAp-Shank3R in HEK293T cells. Lysates of cells expressing (as indicated above) a control shRNA or Shank3 shRNA, along with mAp-Shank3 constructs with the WT shRNA target sequences (mAp-Shank3-WT and mAp-Shank3-AAA) or contain “silent” mutations that confer shRNA resistance (mAp-Shank3R constructs) were immunoblotted for Shank3 (NeuroMab antibody). B, DIV13 primary hippocampal neurons transfected at DIV10 with Shank3 shRNA (GFP+) and stained for Shank3 (magenta; CST antibody) and CaMKIIα (white; a marker of excitatory neurons). Neurons expressing the Shank3 shRNA contain substantially reduced levels of Shank3 (reduced by 91 ± 2% relative to nearby nontransfected excitatory neurons). ****p < 0.0001 (one-sample unpaired Student's t test with equal variance compared with a theoretical value of 100). Error bars indicate mean ± SEM. Each data point represents a single cell accumulated from three independent neuronal cultures/transfections. Scale bars, 20 μm. C, Shank3 knockdown has little effect on global Ca2+ influx. Left, fura-2-loaded hippocampal neurons transfected with control shRNA (nRNA) or Shank3 shRNA were equilibrated with Tyrode's solution containing 5 mm KCl for 3–5 min and switched (black arrow) to Tyrode's solution containing 40 mm KCl for 90 s. The graph plots mean ± SEM ΔF/F0 values for the last 30 s of the equilibration period and during depolarization from four independent experiments (12–90 cells per replicate). The data were analyzed using a two-way repeated-measures ANOVA: Factor 1 (time), F(1.342,8.049) = 32.63, p = 0.0003; Factor 2 (control/shRNA), F(1,6) = 1.703, p = 0.2398; Interaction, F(35,210) = 0.8281, p = 0.7423. Right, Comparison of average areas under the curve from each independent experiment revealed no statistically significant difference (n = 4, p = 0.13; paired Student's t test with equal variance). D, The robust increase in pCREB levels following a brief depolarization (as in Fig. 6A) is significantly reduced in cells expressing the Shank3 shRNA (red bar), but not control shRNA (gray bar) (5K vs 40K: unpaired Student's t test with equal variance, ****p < 0.0001; 40K stimulations: one-way ANOVA, F(2,98) = 38.17, p < 0.0001; Tukey's post hoc test, ****p < 0.0001). E, Similarly, the robust increase in c-Fos expression 3 h after the brief depolarization is significantly reduced in cells expressing Shank3 shRNA (red bar), but not control shRNA (gray bar) (5K vs 40K: unpaired Student's t test with equal variance, ****p < 0.0001; 40K stimulations: one-way ANOVA, F(2,90) = 9.990, p < 0.001, Tukey's post hoc test, ***p < 0.001). Error bars indicate mean ± SEM. Each data point represents a single cell accumulated from three independent neuronal cultures/transfections. Images below the bar graphs are of representative nuclei for each condition. Scale bars, 5 μm.

Article Snippet: D , Synaptic (P2) fractions from WT or CaMKIIα T286A mouse forebrains were immunoprecipitated using control IgG or Shank3 (Bethyl) antibodies, and immunoblotted using Shank3 (Cell Signaling Technology) and CaMKIIα antibodies.

Techniques: Knockdown, Expressing, Biomarker Discovery, shRNA, Control, Construct, Transfection, Staining, Marker, Comparison

Journal: Journal of Molecular Neuroscience

Article Title: The Role of Thioredoxin System in Shank3 Mouse Model of Autism

doi: 10.1007/s12031-024-02270-y

Figure Lengend Snippet: The effect of the Shank3 mutation on the levels of Trx and Prdx system proteins in the mouse cortex and primary cortical neurons, and Trx activity in the SH-SY5Y SHANK3 KO cells. A Representative western blots (WB) for Trx1, Trx2, TrxR1, Prdx1, and Prdx2 in the cortex tissues of WT and KO mice. B–F Statistical analysis of the relative abundance of Trx1 ( n = 5), Trx2 ( n = 5), TrxR1 ( n = 7), Prdx1 ( n = 5), and Prdx2 ( n = 5), respectively, in the cortex of WT and KO mice. G A scatter plot showing average RFU for Trx activity in SH-SY5Y and SH-SY5Y SHANK3 KO cells. H Statistical analysis of the Trx activity in SH-SY5Y and SH-SY5Y SHANK3 KO cells ( n = 3). I Representative confocal images of the fluorescence of Trx, MAP2 (a marker of neuronal differentiation), and DAPI (a marker of nuclei) in the primary cortical neuronal cell cultures derived from WT and Shank3 KO mice. The image was captured at × 100 magnification. The scale bar = 50 µm. * P < 0.05, ** P < 0.01

Article Snippet: Paraformaldehyde solution 4% in PBS (sc-281692), Anti-Trx1 (sc-13526), anti-Trx (sc-271281), and anti-Shank3 (sc-377088) antibodies were acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Mutagenesis, Activity Assay, Western Blot, Fluorescence, Marker, Derivative Assay

Journal: Journal of Molecular Neuroscience

Article Title: The Role of Thioredoxin System in Shank3 Mouse Model of Autism

doi: 10.1007/s12031-024-02270-y

Figure Lengend Snippet: The cytosolic and nuclear levels of Prdx1 and Trx1, and their translocation to the nucleus in the cortex of Shank3 KO and WT mice. A Representative WB for Trx1 in the cytosolic (Cyto) and nuclear (Nuc) fractions of the cortex of WT and KO mice. GAPDH and Lamin B were used as a reference for the cytosolic and nuclear protein loading, respectively. B Statistical analysis of the relative abundance of Trx1 in the cytosolic and nuclear fractions in the cortex of WT and KO mice ( n = 4). C Statistical analysis of the nuclear/cytosolic fractions ratio of the Trx1 relative abundance in the cortex of WT and KO mice ( n = 4). D Statistical analysis of the relative abundance of Prdx1 in the cytosolic and nuclear fractions of the cortex of WT and KO mice ( n = 3). E Statistical analysis of the nuclear/cytosolic ratio of the Prdx1 relative abundance in the cortex of WT and KO mice ( n = 3). GAPDH and Lamin B were used as a reference for the cytosolic and nuclear protein loading, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, non-significant

Article Snippet: Paraformaldehyde solution 4% in PBS (sc-281692), Anti-Trx1 (sc-13526), anti-Trx (sc-271281), and anti-Shank3 (sc-377088) antibodies were acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Translocation Assay

Journal: Journal of Molecular Neuroscience

Article Title: The Role of Thioredoxin System in Shank3 Mouse Model of Autism

doi: 10.1007/s12031-024-02270-y

Figure Lengend Snippet: Effects of Trx1 inhibition with PX-12 on the levels of Trx1, Nrf2, 3-Ntyr, and the molecular markers of synaptic phenotype in the cortex of WT and Shank3 KO mice. A Representative WB for Trx1 and Nrf2 from the cortex of WT, KO, WT + PX-12, and KO + PX-12 mice. β-actin was used as a reference for protein loading. B Statistical analysis of the relative abundance of Trx1 in the cortex of WT ( n = 6), KO ( n = 6), WT + PX-12 ( n = 7), and KO + PX-12 ( n = 7) mice. C Statistical analysis of the relative abundance of Nrf2 in the cortex of WT ( n = 4), KO ( n = 3), WT + PX-12 ( n = 4), and KO + PX-12 ( n = 4) mice. D Representative WB for the molecular markers of nitrosative stress (3-nitrotyrosine (3-Ntyr)) ( n = 6), synaptic phenotype (PSD95) ( n = 4), synaptophysin (SYP) ( n = 3), Homer ( n = 3)), GABAergic system (GAD67) ( n = 6) and (VGAT) ( n = 6)), and glutamatergic system (GluN1) ( n = 3) in the cortex of WT and WT + PX-12 mice. β-actin was used as a reference for protein loading. E, F Statistical analysis of the relative abundance of 3-Ntyr, PSD95, SYP, Homer, GAD67, VGAT, and GluN1, respectively, in the cortex of WT, and WT + PX-12 mice. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, non-significant

Article Snippet: Paraformaldehyde solution 4% in PBS (sc-281692), Anti-Trx1 (sc-13526), anti-Trx (sc-271281), and anti-Shank3 (sc-377088) antibodies were acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Inhibition

Journal: Journal of Molecular Neuroscience

Article Title: The Role of Thioredoxin System in Shank3 Mouse Model of Autism

doi: 10.1007/s12031-024-02270-y

Figure Lengend Snippet: PX-12 treatment induces autistic behavior abnormalities in WT mice similar to those in Shank3 KO mice. A , C , E Schematic representation of the novel object recognition test, three-chamber sociability test, and three-chamber social memory test, respectively. B Statistical analysis of the time spent by the test mouse for interaction with novel and familiar objects in WT ( n = 8), KO ( n = 9), WT + PX-12 ( n = 7), and KO + PX-12 ( n = 8) groups of mice. D Statistical analysis of close interaction time spent by the test mouse with a caged stranger mouse and an empty cage in WT ( n = 15), KO ( n = 15), WT + PX-12 ( n = 8), and KO + PX-12 ( n = 8) groups of mice. F Statistical analysis of the time spent by the test mouse for interaction with a caged stranger and familiar mouse in WT ( n = 19), KO ( n = 19), WT + PX-12 ( n = 8), and KO + PX-12 ( n = 8) groups compared to familiar mice. * P < 0.05, *** P < 0.001; ns, non-significant

Article Snippet: Paraformaldehyde solution 4% in PBS (sc-281692), Anti-Trx1 (sc-13526), anti-Trx (sc-271281), and anti-Shank3 (sc-377088) antibodies were acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques:

Journal: Journal of Molecular Neuroscience

Article Title: The Role of Thioredoxin System in Shank3 Mouse Model of Autism

doi: 10.1007/s12031-024-02270-y

Figure Lengend Snippet:

Article Snippet: Paraformaldehyde solution 4% in PBS (sc-281692), Anti-Trx1 (sc-13526), anti-Trx (sc-271281), and anti-Shank3 (sc-377088) antibodies were acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Sequencing

Journal: Nature Neuroscience

Article Title: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding

doi: 10.1038/s41593-024-01615-5

Figure Lengend Snippet: a , Immunofluorescence (IF) images of primary cortical neurons immunostained for glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor 2 (OLIG2) simultaneously with PSD95 to show that the cultures are devoid of glial cells or oligodendrocytes, respectively. DAPI for nuclei; PSD95 for excitatory neurons. Magnification, ×40. Scale bars, 50 μm. b , TurboID-PSD95 was cloned without (top row) and with (bottom row) its 5′ and 3′ UTRs and lentivirally expressed in primary cortical neurons. White dashed boxes are zoomed in areas in black&white images. DAPI for nuclei; MAP2 for dendrites; Flag for each TurboID. % dendritically localized TurboID-PSD95 is quantified by co-localization with MAP2 signal in ImageJ. 3 different areas of images per replicate ( n = 3). Magnification, ×20. Scale bars, 50 μm. Significance was derived from biological replicates, showing the center line at mean. c , IF images of TurboID-PSD95-transduced neurons immunostained for DAPI (blue, for nuclei), PSD95 (red, for endogenous PSD95) and TurboID-PSD95 (cyan, detected by Flag). Magnification, ×60. Scale bar, 50 μm. d , IF images show the expression of a presynaptic marker, Synaptophysin (cyan), and TurboID-PSD95 (red, detected by Flag antibody) in primary cortical neurons transduced with TurboID-PSD95. DAPI (blue) marker for nuclei. Three zoomed in regions are marked by the white boxes. Magnification, ×60. Scale bar, 10 μm. e , IF images show TurboID expression and biotinylation in primary cortical neurons transduced with TurboID-PSD95 or Pan-TurboID after 30 minutes of biotin incubation. DAPI (blue, nuclei); MAP2 (green, dendrites); Flag (red, TurboID); and Streptavidin (cyan, biotinylated proteins). Magnification, ×20. Scale bars, 50 μm. f , Western blots stained for Flag and β-Actin from Pan-TurboID and TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin shown to indicate the relative expression levels of TurboID proteins. Quantifications of TurboID protein levels normalized to β-Actin are shown on the right ( n = 3); relative levels are not significant by two-tailed, paired Student’s t -test. g , Western blots stained for streptavidin signal in inputs (‘in’) and streptavidin pulldowns (‘pd’) from Pan-TurboID or TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. h , Streptavidin pulldowns shown for dendritic (SHANK3, GKAP, NLGN1 and HOMER1) and negative control (GAPDH) proteins from TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. Flag signal indicates self-biotinylation of each construct. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by TurboID-PSD95 in each condition is calculated by dividing the signal in the pulldown lane by that of the input lane, after each is adjusted to total, and quantifications are shown as bar graphs ( n = 3). P values: Flag = 0.58, SHANK3 = 0.0061, GKAP = 0.018, NLGN1 = 0.00052, HOMER1 = 0.021, GAPDH = 0.42. i , Streptavidin pulldowns shown for dendritic (BAIAP2 and DLGAP3) and nuclear (TBR1, H4 and H2AX) proteins from Pan-TurboID and TurboID-PSD95-transduced neurons in the presence (+) of exogenous biotin. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by each TurboID is calculated as in (h) ( n = 3). P values: BAIAP2 = 0.0052, DLGAP3 = 0.0035, TBR1 = 0.0063, H4 = 0.018, H2AX = 0.0037. j , Phosphorylation of EEF2, eIF2α, ERK1/2 and IRE1 and total levels of ATF4 and CHOP are shown in resting (rest), activated (DHPG, Dep) and stressed (Sodium arsenite (NaAsO 2 )) cells by using phospho-specific and total antibodies. The amount of phosphorylated or total protein is shown in the bar graphs, calculated by dividing the phosphorylated signal to total and β-Actin for the phosphorylated proteins and by dividing the total to β-Actin for ATF4 and CHOP ( n = 3). Significance was calculated with respect to rest. P values: P-EEF2 (DHPG = 0.0088, Dep = 0.0023, NaAsO 2 = 0.039), P-eIF2α (DHPG = 0.018, Dep = 0.0034, NaAsO 2 = 0.028), P-ERK1/2 (DHPG = 0.015, Dep = 0.0067, NaAsO 2 = 0.00084), P-IRE1 (DHPG = 0.06, Dep = 0.37, NaAsO 2 = 0.0027), ATF4 (DHPG = 0.038, Dep = 0.42, NaAsO 2 = 0.016), CHOP (DHPG = 0.044, Dep = 0.18, NaAsO 2 = 0.024). k , Quantitative PCR (qPCR) results shown for immediate early genes, Arc , Fos and Jun . The fold changes for each gene are calculated by first normalizing to the house-keeping gene β-Actin in each condition, then dividing the value of each condition by that of the resting state ( n = 3). l , Dendritic spine size in resting and KCl-depolarized neurons are measured using the Keyence microscope. Red squares are examples of spines that are counted ( n = 3, 12 spines from each biological replicate are counted as technical replicates). Significance was derived from the biological replicates using the two-tailed, unpaired Student’s t -test. Box plots show the min and max, with the center line at median. Magnification, ×100. Scale bars, 5 μm. m , Fluo-4-AM staining in resting, KCl-depolarized and DHPG-depolarized cells. Fluo4-AM was loaded in resting cells and measurements were taken at indicated time points after Fluo4-AM removal. In depolarized cells, the dye was loaded during silencing. After silencing, fluorescence was measured during stimulus at 10, 30 and 60-minute time points for the KCl treatment and at 10-minute for the DHPG-induced activation. Fluorescence was also measured 60 minutes after the stimulus removal (60′post KCl and 60′post DHPG). Circles represent data from 2 biological and 3 technical replicates. Below: Examples of Fluo4-AM fluorescence are shown in resting, 10-minute KCl-treated and 10-minute DHPG-treated neurons. Fluo4-AM loading (45 minutes) was performed during the last 45 minutes of the silencing step prior to stimulus addition for the KCl and DHPG treatment and simultaneously for the resting neurons. Imaging was performed 10 minutes after the stimulus was added. Scale bars, 50 μm. (b,f,h-k,m) Data are mean ± s.d. Significance was calculated using the two-tailed, paired Student’s t -test. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples.

Article Snippet: Puromycin (1:3,000, mouse, Kerafast, EQ0001, RRID: AB_2620162), Flag (1:3,000, mouse, Sigma-Aldrich, F1804, RRID: AB_262044), β-Actin antibody (1:2,500, mouse, Sigma-Aldrich, A1978, RRID: AB_476692), RPL10A (1:1,000, rabbit, Abcam, ab174318), MAP2 (1:2,500, guinea pig, Synaptic Systems, 188004, RRID: AB_2138181), GFAP (1:500, rabbit, Abcam, ab7260, RRID: AB_305808), OLIG2 (1:500, rabbit, Proteintech, 13999-1-AP, RRID: AB_2157541), PSD95 (1:500, mouse, Millipore, MABN68, RRID: AB_10807979), Synaptophysin (1:300, mouse, Abcam, ab8049, RRID: AB_2198854), SHANK3 (1:500, mouse, Novus, NBP1-47610, RRID: AB_10010567), GKAP (1:500, rabbit, Novus, NBP1-76911, RRID: AB_11017331), NLGN1 (1:200, mouse, Novus, NBP2-42192), HOMER1 (1:1,000, rabbit, Proteintech, 12433-1-AP, RRID: AB_2295573), GAPDH (1:5,000, mouse, Thermo Fisher Scientific, AM4300, RRID: AB_2536381), BAIAP2 (1:500, rabbit, Proteintech, 11087-2-AP, RRID: AB_2063075), DLGAP3 (1:500, rabbit, Proteintech, 55056-1-AP, RRID: AB_10858793), TBR1 (1:500, rabbit, Proteintech, 20932-1-AP, RRID: AB_10695502), H4 (1:1,000, mouse, Abcam, ab31830, RRID: AB_1209246), H2A.X (1:1,000, rabbit, Proteintech, 10856-1-AP, RRID: AB_2114985), EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2332, RRID:AB_10693546), P-EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2331, RRID: AB_10015204), eIF2α (1:1,000, rabbit, Cell Signaling Technology, 9722, RRID: AB_2230924), P-eIF2α (1:1,000, rabbit, Cell Signaling Technology, 3398, RRID: AB_2096481), p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 4695, RRID: AB_390779), P-p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 9101, RRID: AB_331646), P-IRE1 (1:500, rabbit, Novus, NB100-2323SS, RRID: AB_10145203), IRE1 (1:500, rabbit, Novus, NB100-2324SS, RRID: AB_10000972), CHOP (1:1,000, mouse, Cell Signaling, 2895T, RRID: AB_2089254), ATF4 (1:1,000, rabbit, Cell Signaling Technology, 11815S, RRID: AB_2616025), MPHOSPH (1:300, rabbit, Biorbyt, orb100446), KCNJ9 (1:300, rabbit, LSBio, LS-C352416), KCNJ9 (1:300, mouse, Antibodies Incorporated, 75-445, RRID: AB_2686912), eIF4G2 (1:1,000, rabbit, Cell Signaling Technology, RRID: AB_10622189 and rabbit, Cell Signaling Technology, RRID: AB_2261993), NSUN3 (1:250, rabbit, LSBio, LS-C163024), MTF1 (1:300, rabbit, Novus, NBP1-86380, RRID: AB_11011361), ZFP64 (1:300, rabbit, Proteintech, 17187-1-AP, RRID: AB_2218826) and KATNBL1 (1:250, rabbit, Proteintech, 24795-1-AP, RRID: AB_2879730).

Techniques: Immunofluorescence, Clone Assay, Derivative Assay, Expressing, Marker, Transduction, Incubation, Western Blot, Staining, Two Tailed Test, Negative Control, Construct, Isolation, Real-time Polymerase Chain Reaction, Microscopy, Fluorescence, Activation Assay, Imaging