Journal: Npj Biosensing

Article Title: Microfluidic nanomagnetically isolated neuron- and astrocyte-derived extracellular vesicles to differentiate Lewy body and Alzheimer’s disease

doi: 10.1038/s44328-026-00086-x

Figure Lengend Snippet: A Outline of study time-course and sample processing for EV isolation. Antemortem plasma samples with postmortem pathological confirmation of neurological diagnoses were processed to isolate cell-specific EVs using our mTENPO microfluidic platform, alongside plasma protein biomarkers using commercial digital ELISA, for patients with LBD ( n = 30), AD ( n = 31), AD/LBD ( n = 30), AD/ALB ( n = 19), and controls ( n = 27). B The mTENPO platform, illustrating the external magnet, inlet reservoir, outlet ports, and tubing connections to syringe pumps. Syringes are connected to the waste outlet for blocking, washing, and sample addition steps, and then replaced and switched to the lysate outlet before captured EVs are lysed on-chip. The inset shows a photo of the mTENPO chip with a quarter for scale. C Schematic of operation of the mTENPO platform for cell-specific EV isolation using antibody-labeled magnetic nanoparticles (MNPs) for GluR2+ (top) and GLAST+ (bottom) EV pulldowns. D Scanning electron microscopy (SEM) images of GluR2+ EVs immobilized on the edges of pores of the mTENPO device’s surface. E SEM images of GLAST+ EVs immobilized on the edges of pores of the mTENPO device’s surface. F Representative cropped western blot images showing protein expression of GluR2, GLAST, and EV-associated marker TSG101 using mTENPO-isolated GluR2+ or GLAST + EV lysates from n = 2 human plasma samples. Full-length western blot images are shown in Supplementary Fig. .

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 min at a concentration of 1 μg/mL with either biotinylated GluR2 capture antibody (GluR1 + GluR2 polyclonal antibody, Bioss bs-10042R-Biotin) for neuron-derived EVs per our previous work or biotinylated GLAST capture antibody [GLAST (ACSA-1) antibody, anti-human/mouse/rat Biotin, Miltenyi Biotec, 130-118-984] for astrocyte-derived EVs, where the use of GLAST as a protein target for astrocyte EV isolation has been previously reported , – , .

Techniques: Isolation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Blocking Assay, Labeling, Electron Microscopy, Western Blot, Expressing, Marker

Journal: Npj Biosensing

Article Title: Microfluidic nanomagnetically isolated neuron- and astrocyte-derived extracellular vesicles to differentiate Lewy body and Alzheimer’s disease

doi: 10.1038/s44328-026-00086-x

Figure Lengend Snippet: A Heatmap of z-score of log 2 (expression) for biomarkers with Benjamini-Hochberg FDR-corrected P value < 0.1. Subjects (columns) are hierarchically clustered within cohort and biomarkers within each compartment (rows) are sorted by descending fold-change. B Volcano plot demonstrating differential expression of GluR2+ EV miRNAs, GLAST + EV miRNAs, and plasma proteins. C Venn diagram showing overlap in FDR P value significant miRNAs ( P value < 0.1) between GluR2+ EVs and GLAST+ EVs. D Top 30 biomarkers in all compartments ranked by descending area under the curve (AUC). Error bars represent standard error from bootstrapping 10x.

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 min at a concentration of 1 μg/mL with either biotinylated GluR2 capture antibody (GluR1 + GluR2 polyclonal antibody, Bioss bs-10042R-Biotin) for neuron-derived EVs per our previous work or biotinylated GLAST capture antibody [GLAST (ACSA-1) antibody, anti-human/mouse/rat Biotin, Miltenyi Biotec, 130-118-984] for astrocyte-derived EVs, where the use of GLAST as a protein target for astrocyte EV isolation has been previously reported , – , .

Techniques: Expressing, Quantitative Proteomics, Clinical Proteomics

Journal: Npj Biosensing

Article Title: Microfluidic nanomagnetically isolated neuron- and astrocyte-derived extracellular vesicles to differentiate Lewy body and Alzheimer’s disease

doi: 10.1038/s44328-026-00086-x

Figure Lengend Snippet: GO and KEGG pathway analyses were performed on differentially expressed miRNAs using DIANA miRPath v4.0 using the TarBase v8.0 database. FDR P values for identified GO terms and KEGG pathways were calculated using a one-sided Fisher’s exact test and considered significant at P value < 0.05. The top 10 (ranked by number of target genes) terms within each of the three GO categories (BP, CC, MF) and top 10 (ranked by number of target genes) KEGG pathways were identified for each pulldown. A Top 10 terms within each GO category for GluR2+ EV miRNAs. B Top 10 KEGG pathways for GluR2+ EV miRNAs. C Top 10 terms within each GO category for GLAST + EV miRNAs. D Top 10 KEGG pathways for GLAST + EV miRNAs. In all panels, each bar is labeled to the right with the number of differentially expressed miRNAs associated with the given GO term or KEGG pathway.

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 min at a concentration of 1 μg/mL with either biotinylated GluR2 capture antibody (GluR1 + GluR2 polyclonal antibody, Bioss bs-10042R-Biotin) for neuron-derived EVs per our previous work or biotinylated GLAST capture antibody [GLAST (ACSA-1) antibody, anti-human/mouse/rat Biotin, Miltenyi Biotec, 130-118-984] for astrocyte-derived EVs, where the use of GLAST as a protein target for astrocyte EV isolation has been previously reported , – , .

Techniques: Labeling

Journal: Npj Biosensing

Article Title: Microfluidic nanomagnetically isolated neuron- and astrocyte-derived extracellular vesicles to differentiate Lewy body and Alzheimer’s disease

doi: 10.1038/s44328-026-00086-x

Figure Lengend Snippet: A Heatmap of z-score of log 2 (expression) for LASSO-selected biomarkers. Subjects (columns) are hierarchically clustered within cohort and biomarkers within each compartment (rows) are sorted by descending AUC. B Kendall correlation staircase plots identifying the extent to which biomarker information was correlated between the LASSO-selected GluR2+ EV, GLAST + EV, and protein biomarkers. Biomarkers are sorted within compartments by AUC. The inset shows the correlation distribution of Kendall’s τ, where the dotted line represents the median count. C LASSO panel accuracy versus panel size for classifying LBD versus AD, shown in blue; accuracy is assessed through tenfold cross-validation, with error bars representing standard error from 5 repeats of panel training on the LBD vs AD patient groups. Average accuracy and standard error for control experiments performed by scrambling patient labels 10x are shown in orange. D LASSO panel AUC versus panel size for classifying LBD versus AD, shown in blue with error bars as described in ( C ). Average AUC and standard error for the same control experiments described in ( C ) are shown in orange. E AUCs for the 15-marker LASSO panel and individual LASSO biomarkers, sorted by descending AUC. Error bars represent 95% confidence intervals, calculated from 5x repeats of tenfold cross-validation for the 15-marker panel or from bootstrapping 10x for individual markers.

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 min at a concentration of 1 μg/mL with either biotinylated GluR2 capture antibody (GluR1 + GluR2 polyclonal antibody, Bioss bs-10042R-Biotin) for neuron-derived EVs per our previous work or biotinylated GLAST capture antibody [GLAST (ACSA-1) antibody, anti-human/mouse/rat Biotin, Miltenyi Biotec, 130-118-984] for astrocyte-derived EVs, where the use of GLAST as a protein target for astrocyte EV isolation has been previously reported , – , .

Techniques: Expressing, Biomarker Discovery, Control, Marker

Journal: Molecular Psychiatry

Article Title: The schizophrenia risk gene C4 induces pathological synaptic loss by impairing AMPAR trafficking

doi: 10.1038/s41380-024-02701-7

Figure Lengend Snippet: A Model depicting effects of C4 overexpression on GluR1 recycling in dendritic spines. B Representative images (60X) showing a GFP-positive dendritic spine (white), GluR1 (green) and Rab11a (magenta) of P21–23 apical tufts in GFP-controls (blue frame). C Representative images (60X) showing a dendritic spine identified with GFP signal (white), GluR1 (green) and Rab11a (magenta) in C4-OE (red frame). B , C Yellow arrowhead, GluR1 or Rab11a clusters in spines. White-filled arrowhead, GluR1/Rab11a colocalization. White empty arrowhead, non-colocalized GluR1/Rab11a. Spine silhouette, white dotted line. Orthogonal views are shown (XY, YZ and XZ). Scale bar = 2 μm. D C4-OE caused a 47% decrease in the amount of GluR1 colocalized with Rab11a compared to GFP-control. E There was no change in the amount of Rab11a colocalized with GluR1 in C4-OE relative to GFP-control. F C4-OE increased the minimum distance between GluR1 and Rab11a clusters by 35% relative to GFP-control. Green circle: GluR1, Magenta circle: Rab11a. G C4-OE led to a 25% decrease in the overlapping volume between GluR1 and Rab11a relative to GFP-control. Green circle: GluR1, Magenta circle: Rab11a. H Schematic showing the effects of C4-OE on GluR1 degradation in dendritic spines. I Representative images (60X) showing a GFP-positive spine (white), GluR1 (green) and LAMP1 (magenta) in GFP-control (blue frame). J Representative images (60X) showing a GFP-positive spine (white), GluR1 (green) and LAMP1 (magenta) in C4-OE (red frame). I , J Yellow arrowhead, GluR1 or Rab11a clusters in spines. White empty arrowhead, non-colocalized GluR1/LAMP1. White-filled arrowhead, GluR1/LAMP1 colocalization. Spine silhouette, white dotted line. Orthogonal views are shown (XY, YZ and XZ). Scale bar = 2 μm. K C4-OE led to a 145% increase in the amount of GluR1 colocalized with LAMP1. L C4-OE caused a 103% increase in the amount of LAMP1 colocalized with GluR1. M C4-OE induced a 31% decrease in the minimum distance between GluR1 and LAMP1 clusters. Green circle: GluR1, Magenta circle: LAMP1. N Compared to GFP-control, C4-OE did not alter the overlapping volume between GluR1 and LAMP1. Green circle: GluR1, Magenta circle: LAMP1. D , E , K , L N = 8 dendrites, 3 animals for Con and C4-OE. F , G , M , N N = 7 dendrites, 3 animals, Con; and N = 8 dendrites, 3 animals, C4-OE. D – G , K – N t- test. * p < 0.05, ** p < 0.01, *** p < 0.001. All graphs, Mean ± SEM.

Article Snippet: For GluR1 expression in HEK293T cells, we used Gria1 (NM_008165 ) under the CMV promoter (pCMV6-Gria1, OriGene Technologies plasmid #MC203472).

Techniques: Over Expression, Control

Journal: Molecular Psychiatry

Article Title: The schizophrenia risk gene C4 induces pathological synaptic loss by impairing AMPAR trafficking

doi: 10.1038/s41380-024-02701-7

Figure Lengend Snippet: A Left: Schematic of dissected GFP-positive tissue from IUE animals, used for isolating cytosolic and synaptosome fractions. Right: Western blot (WB) showing levels of GluR1 with C4 overexpression. GluR1 levels were detected in synaptosome and cytosolic fractions in control and C4-OE conditions. Since C4 was expressed at relatively low levels, it could only be detected when several brains were pooled. Therefore, each lane corresponds to pooled brain lysate from a single litter (6 mice). Protein molecular weights, kilodaltons (kDa, left column). “+” indicates the presence of C4 (top row). Numbers in the bottom row indicate fold change in GluR1 levels relative to control synaptosome and cytosolic fraction. B Left: Schematic of degradation assay in HEK293T cells. Right: WB quantified protein levels of GluR1 in GS and GSC cells with CHX and CHX + MG132 treatment. Protein molecular weights, kilodaltons (kDa, left column). “+” indicates the presence of C4, CHX or MG132. C Left: CHX treatment led to decreased GluR1 levels in GS cells, which was rescued upon application of MG132. Right: GluR1 levels in GSC cells were unaffected by CHX or CHX + MG132 treatments. Light gray circles: DMSO treated cells, Dark gray triangles: CHX treated cells, Dark gray squares: CHX + MG132 treated cells. N = 3 sample replicates per condition. One-way ANOVA. * p < 0.05, ** p < 0.01. All graphs, Mean ± SEM.

Article Snippet: For GluR1 expression in HEK293T cells, we used Gria1 (NM_008165 ) under the CMV promoter (pCMV6-Gria1, OriGene Technologies plasmid #MC203472).

Techniques: Western Blot, Over Expression, Control, Degradation Assay

Journal: Journal of Neuroscience

Article Title: Regulation of Nucleus Accumbens Activity by the Hypothalamic Neuropeptide Melanin-Concentrating Hormone

doi: 10.1523/jneurosci.5858-09.2010

Figure Lengend Snippet: Figure 2. MCH acts via Gi/o to reduce GluR1 pSer 845 in the AcbSh. Graphs represent the ratio of phosphorylated signal to total protein signal for all treatments. A, Basal or SKF 81297-mediated GluR1 pSer 845 reduction by MCH is blocked by in vivo PTX treatment (n 12–15 slices per group). B, MCH-mediated reduction is not affected by inhibition of PLC–PKC pathway with U73122 (n 5 slices per group). *p 0.05, **p 0.01. Error bars indicate SEM.

Article Snippet: For GluR1 protein detection, membranes were first blotted with the phospho-antibodies at 1:500 (rabbit polyclonal, GluR1 Ser 845 and Ser 831 antibodies from PhosphoSolutions).

Techniques: In Vivo, Inhibition

Journal: Journal of Neuroscience

Article Title: Regulation of Nucleus Accumbens Activity by the Hypothalamic Neuropeptide Melanin-Concentrating Hormone

doi: 10.1523/jneurosci.5858-09.2010

Figure Lengend Snippet: Figure4. SurfacelevelsofGluR1andmEPSCamplitudearereducedbyMCHinMSNsoftheAcbSh.A,Datarepresenttheratioof surface(cross-linked)tointernal(un-cross-linked)GluR1signal,andisnormalizedtountreatedcontrol.Treatmentconditionsare indicated below the graph along with representative surface and internal GluR1 signal (n 7–10 slices per group). B, C, Repre- sentativetracesbefore(B)andafter(C)bathapplicationofMCH(3M).D,E,Cumulativeprobabilityhistogramforamplitude(D) and interevent interval (E). n 8 neurons for mEPSC recordings; *p 0.05., **p 0.01. Error bars indicate SEM.

Article Snippet: For GluR1 protein detection, membranes were first blotted with the phospho-antibodies at 1:500 (rabbit polyclonal, GluR1 Ser 845 and Ser 831 antibodies from PhosphoSolutions).

Techniques:

Journal: Science Advances

Article Title: CaMKII holoenzyme mechanisms that govern the LTP versus LTD decision

doi: 10.1126/sciadv.abe2300

Figure Lengend Snippet: Quantifications show means ± SEM. * P < 0.05 and ** P < 0.01. Scale bars, 10 μm. ( A ) Schematic of CaMKII movement in response excitatory LTD stimuli. The LTD-induced pT305/306 blocks CaMKII movement to glutamatergic excitatory synapses and instead enables movement to GABAergic inhibitory synapses. ( B ) cLTD stimulation increased surface GABA A R accumulation in WT, but not T305/6AV, CA1 mini-slices (unpaired two-tailed t test, P = 0.0033 for WT and P = 0.6760 for AV, n = 5 and 6 samples (two slices/sample), as detected by immunoblot after surface biotinylation. ( C ) cLTD stimulation decreased surface GluA1 accumulation in WT, but not T305/6AV, CA1 mini-slices (unpaired two-tailed t test, P = 0.0062 for WT and P = 0.6252 for AV, n = 5 and 6 samples (two slices/sample). ( D ) cLTP decreased surface GABA A R cluster size in nonpermeabilized neurons from WT mouse hippocampal cultures (fixed 5 min after washout), while cLTD increased the cluster size (fixed 20 min after washout) (one-way ANOVA, Tukey’s post hoc test versus control, P = 0.0273 for cLTP and P = 0.0046 for cLTD; n = 12, 12, and 13 neurons), as detected by immunocytochemistry. ( E ) By contrast, in nonpermeabilized neurons from T305/5AV hippocampal cultures, cLTD did not increase surface GABA A R clusters. However, cLTP still decreased surface GABA A R clusters ( P = 0.0147 for cLTP and P = 0.7121 for cLTD; n = 15, 13, and 14 neurons).

Article Snippet: The following antibodies were used: GABA A R α1 (1:1000; Synaptic Systems, 224 211), GluA1 (Millipore, AB1504), GluA1 pS845 (1:1000; PhosphoSolutions, p1160-845), GluA1 pS831 (1:1000, PhosphoSolutions, p1160-831), CaMKII pT286 (1:1000, PhosphoSolutions, p1005-286), CaMKII pT305 (1:800; Assay BioTech, A0005; but see fig. S2 and the note below), CaMKII pT306 (1:800; PhosphoSolutions, p1005-306), DAPK1 (1:1000; Sigma-Aldrich, D1319), DAPK1 pS308 (1:1000, Sigma-Aldrich, D4941), GluA1 pS567 (1:1000, provided by K. Roche) , and CaMKIIα (1:5000; CBα2, made in-house), 2° anti-rabbit (1:600; GE Healthcare, NA934V), anti-mouse secondary (1:10,000, GE Healthcare, NA931V), and Alexa Fluor 647 anti-mouse (1:5000; Molecular Probes, A21236).

Techniques: Two Tailed Test, Western Blot, Control, Immunocytochemistry

Journal: Science Advances

Article Title: CaMKII holoenzyme mechanisms that govern the LTP versus LTD decision

doi: 10.1126/sciadv.abe2300

Figure Lengend Snippet: Quantifications show means ± SEM. ** P < 0.01. ( A ) Phosphorylation of the LTD-related GluA1 S567 site by pT286-CaMKII (10 nM kinase subunits) with Ca 2+ /CaM present [stimulated (Stim)] or absent [autonomous (Auton)]. No differences in pS567 were detected between autonomous versus stimulated targeting by either WT or T305/306AV (two-way ANOVA, Bonferroni post hoc test, P = 0.5249 for WT and P = 0.0730 for T305/306AV, n = 5 reactions), although the mutant showed a trend toward increased pS567 with autonomous activity. ( B ) Phosphorylation of the LTP-related GluA1 S831 site by pT286-CaMKII (10 nM kinase subunits) with Ca 2+ /CaM present (stimulated) or absent (autonomous). Enhanced pS831 under stimulated versus autonomous conditions was seen with CaMKII WT (two-way ANOVA, Bonferroni post hoc test, P = 0.0039, n = 5) but not with the T305/306AV mutant ( P = 0.7519, n = 5 reactions).

Article Snippet: The following antibodies were used: GABA A R α1 (1:1000; Synaptic Systems, 224 211), GluA1 (Millipore, AB1504), GluA1 pS845 (1:1000; PhosphoSolutions, p1160-845), GluA1 pS831 (1:1000, PhosphoSolutions, p1160-831), CaMKII pT286 (1:1000, PhosphoSolutions, p1005-286), CaMKII pT305 (1:800; Assay BioTech, A0005; but see fig. S2 and the note below), CaMKII pT306 (1:800; PhosphoSolutions, p1005-306), DAPK1 (1:1000; Sigma-Aldrich, D1319), DAPK1 pS308 (1:1000, Sigma-Aldrich, D4941), GluA1 pS567 (1:1000, provided by K. Roche) , and CaMKIIα (1:5000; CBα2, made in-house), 2° anti-rabbit (1:600; GE Healthcare, NA934V), anti-mouse secondary (1:10,000, GE Healthcare, NA931V), and Alexa Fluor 647 anti-mouse (1:5000; Molecular Probes, A21236).

Techniques: Phospho-proteomics, Mutagenesis, Activity Assay

Journal: Aging cell

Article Title: Phenylbutyric acid reduces amyloid plaques and rescues cognitive behavior in AD transgenic mice.

doi: 10.1111/j.1474-9726.2011.00680.x

Figure Lengend Snippet: Fig. 5 Glutamate receptors up-regulated in the phenylbutyric acid (PBA)-treated animals. In the four treatment cohorts, lysates from the cortex and hippocampus were probed for several synaptic proteins to determine potential changes in expression levels. (A) Neither PSD95 nor synaptotagmin levels changed with PBA treatment (bottom two blots). However, the levels metabotropic glutamate receptor 1 (mGluR1) and two AMPA receptors (GluR1 and GluR2 ⁄ 3) increased in the PBA-treated animals (top three blots). There was no observed variation in levels because of genotype; consequently, the levels of each glutamate receptor were analyzed relative to treatment cohort. (B) The expression level of all three glutamate receptors was quantitated by pixel density showing increased levels of each receptor in the PBA-treated animals. The mGluR1 levels increased by 359.6% and were significantly different than water-treated animals (P < 0.0001, Student’s t-test). The GluR1 levels increased significantly by 65% in the PBA-treated animals (P < 0.0012, Student’s t-test). The GluR2 ⁄ 3 levels also increased significantly by 99.2% with PBA treatment (P < 0.0002, Student’s t-test).

Article Snippet: The three glutamate receptor antibodies such as mGluR1 (Chemicon), GluR1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and GluR2 ⁄ 3 (Millipore ⁄ Upstate, Waltham, MA, USA) were all used at 1:1000 dilutions.

Techniques: Expressing

Journal: Oncotarget

Article Title: Fluoxetine, an antidepressant, suppresses glioblastoma by evoking AMPAR-mediated calcium-dependent apoptosis

doi:

Figure Lengend Snippet: (A) Cells were pretreated with NBQX, NS-102 or MK-801 and then exposed to fluoxetine. Cell viability was quantified by an MTT assay. Only NBQX protected glioblastoma cells from fluoxetine-induced cell death. Data were collected from three independent experiments, and are expressed as the mean ± SD. Results were statistically analyzed by Student's t -test; ** , ## p <0.01, compared to the control group. (B-C) Time course measurements of [Ca 2+ ] i , using fluorescence spectrophotometry, made from Fura-2-loaded cells cultured in the absence or presence of 50 μM NBQX. Arrows indicate time of the addition fluoxetine (30 μM). Treatment with NBQX abrogated the [Ca 2+ ] i elevation induced by fluoxetine treatment. (D-E) mRNA expression levels of GluR1, an AMPAR subunit, were analyzed in cultured cells and in brain tissue samples from patients by RT-PCR (D), or a real-time PCR (E) The numbers below the bands indicate the relative intensities normalized to loading control. Results were statistically analyzed by Student's t -test; *** p <0.001 compared to the primary astrocyte or normal brain tissue #16, as a represented normal control. (F) In astrocyte, most of GluR1 proteins were expressed in the cytosol. In glioblastoma cell, most of GluR1 protein was expressed on the cell membrane. Blue: DAPI, Green: GluR1. (G) The cell surface (non-permeabilized) and cytosolic (permeabilized) GluR1 expression level on glioblastoma cell lines and astrocyte were analyzed by flow cytometry.

Article Snippet: After blocked by 1% BSA, cells were incubated with anti-GluR1 antibody overnight followed by incubated with FITC-conjugated anti-rabbit antibody (Santa Cruz Biotechnology).

Techniques: MTT Assay, Control, Fluorescence, Spectrophotometry, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Membrane, Flow Cytometry

Journal: Oncotarget

Article Title: Fluoxetine, an antidepressant, suppresses glioblastoma by evoking AMPAR-mediated calcium-dependent apoptosis

doi:

Figure Lengend Snippet: (A) Representative images of IHC analysis of normal brain and different grades of gliomas stained with anti-GluR1 antibody. (B) Comparison of GluR1 expression on different grades of human glioma and normal brain tissues by immunohistochemistry (IHC) staining. The human tissue microarray were obtained from US Biomax and contained normal brain (n=18) and grade II (n=73), grade III (n=20), grade IV (n=51) gliomas. * p <0.05, *** p <0.001 when compared with the normal brain group.

Article Snippet: After blocked by 1% BSA, cells were incubated with anti-GluR1 antibody overnight followed by incubated with FITC-conjugated anti-rabbit antibody (Santa Cruz Biotechnology).

Techniques: Staining, Comparison, Expressing, Immunohistochemistry, Microarray

Journal: Oncotarget

Article Title: Fluoxetine, an antidepressant, suppresses glioblastoma by evoking AMPAR-mediated calcium-dependent apoptosis

doi:

Figure Lengend Snippet: (A-B) Cells were treated with fluoxetine for 24 h, and DiOC6 staining was used to examine the damage done to mitochondrial membranes. The percentage of mitochondrial membranes damaged cells, which are within M1, is shown on each panel of the figure and summarized in histograms. Data were collected from three independent experiments, and are expressed as the mean ± SD. Results were statistically analyzed by Student's t -test. *, # p <0.001 compared to the control group. (C) A Western blot analysis of cytochrome c in both cytosolic and mitochondrial fractions, pro- and cleaved caspase-3 and -9, and PARP cleavage of glioblastoma cells treated with fluoxetine in the absence or presence of Ca 2+ in the medium. (D-E) A Western blot analysis of PARP cleavage and a MTT assay to exam pan-caspase inhibitor, zVAD, effects on fluoxetine-induced apoptosis (F) A Western blot analysis of proteins as described in (C) was made from cells after transfection with the control siRNA or with GluR1 siRNA, followed by fluoxetine (30 μM) treatment.

Article Snippet: After blocked by 1% BSA, cells were incubated with anti-GluR1 antibody overnight followed by incubated with FITC-conjugated anti-rabbit antibody (Santa Cruz Biotechnology).

Techniques: Staining, Control, Western Blot, MTT Assay, Transfection

Journal: Oncotarget

Article Title: Fluoxetine, an antidepressant, suppresses glioblastoma by evoking AMPAR-mediated calcium-dependent apoptosis

doi:

Figure Lengend Snippet: (A and B) Computer modeling of protein-ligand interaction among GluR1 ligand-binding domain (LBD) and ligands. Localization of the protein-ligand binding site and estimation of free energy required for the binding were determined using AutoDock. The predicted structure and estimated free energy of GluR1 LBD docked with either fluoxetine or AMPA were modeled. (C) Surface plasmon resonance (SPR) sensorgram shows the association and dissociation between fluoxetine and GluR1. Indicated concentrations of fluoxetine were injected into the sensor chip. Binding is expressed as the differential response unit (RU) between the bindings of fluoxetine to the GluR1-immobilized sensor chip or to a blank sensor chip. Results show that the apparent association rate constant (K a ) was 4.8 × 10 4 M −1 s −1 and the dissociation rate constant (K d ) was 1.27× 10 −3 s −1 , giving an equilibrium dissociation constant (K D ) of 2.66 × 10 −8 M. (D) The equilibrium-state response unit was plotted versus the concentration of fluoxetine.

Article Snippet: After blocked by 1% BSA, cells were incubated with anti-GluR1 antibody overnight followed by incubated with FITC-conjugated anti-rabbit antibody (Santa Cruz Biotechnology).

Techniques: Ligand Binding Assay, Binding Assay, SPR Assay, Injection, Concentration Assay

Journal: Human Molecular Genetics

Article Title: Loss of fragile X protein FMRP impairs homeostatic synaptic downscaling through tumor suppressor p53 and ubiquitin E3 ligase Nedd4-2

doi: 10.1093/hmg/ddy189

Figure Lengend Snippet: FMRP is required for homeostatic synaptic downscaling. Patch-clamp recording from WT (A) or Fmr1 KO (B) mouse hippocampal neurons treated with vehicle (DMSO) or PTX for 48 h. Representative mEPSC traces and quantification of mEPSC amplitude and frequency are shown (n = 16–18 for WT and Fmr1 KO neurons). Immunocytochemistry showing total (t) and surface (s) GluA1 and GluA2 from WT (C) or Fmr1 KO (D) hippocampal neurons treated with DMSO or PTX for 48 h. Magnified images of dendrites are shown to the right of the composite image. Quantification on the right (n = 20 cells for WT and Fmr1 KO neurons). Data were analyzed by Student’s t-test and represented as mean ± SEM with *P < 0.05, **P < 0.01, ***P < 0.001, and ns: non-significant.

Article Snippet: Ub activating enzyme (UBE1) (Boston Biochem), UbcH5b/UBE2D2 (Boston Biochem) and recombinant GluA1 (Origene) was used as substrate as described previously ( 12 , 36 ).

Techniques: Patch Clamp, Immunocytochemistry

Journal: Human Molecular Genetics

Article Title: Loss of fragile X protein FMRP impairs homeostatic synaptic downscaling through tumor suppressor p53 and ubiquitin E3 ligase Nedd4-2

doi: 10.1093/hmg/ddy189

Figure Lengend Snippet: Nedd4-2-mediated GluA1 ubiquitination during homeostatic synaptic downscaling is impaired in Fmr1 KO neurons. (A) Western blots of Ub after IP using anti-GluA1 (A1) or GluA2 (A2) antibody from WT or Fmr1 KO cortical neuron cultures treated with vehicle (DMSO) or PTX for 48 h (n = 4). (B) Western blots of recombinant GluA1 pulled down by Nedd4-2 (B1) or Nedd4-1 (B2) immunoprecipitated from WT or Fmr1 KO cortical neuron cultures treated with DMSO or PTX for 48 h (n = 5 for B1 and n = 3 for B2). Coomasie blue staining showing the purity of recombinant GluA1 is on the right (B3). The two distinct bands in Nedd4-2 blots represent two major isoforms of Nedd4-2 as previously reported (12). (C) Western blots of recombinant Nedd4-2 (top) pulled down by GluA1 (bottom) immunoprecipitated from WT or Fmr1 KO cortical neuron cultures treated with DMSO or PTX for 48 h. The difference between WT and Fmr1 KO cultures was analyzed after normalizing PTX-treated groups to DMSO-treated groups (n = 4). Coomasie blue staining showing the purity of recombinant Nedd4-2 is on the right (C3). (D) Western blots of Ub or GluA1 after IP with anti-GluA1 antibody following in vitro ubiquitination with recombinant GluA1 and Nedd4-2 or Nedd4-1 immunoprecipitated from WT or Fmr1 KO cortical neuron cultures treated with PTX for 48 h (n = 4). For all experiments, data were analyzed by two-way ANOVA with Tukey’s test and represented as mean ± SEM with *P < 0.05, **P < 0.01 and ns: non-significant.

Article Snippet: Ub activating enzyme (UBE1) (Boston Biochem), UbcH5b/UBE2D2 (Boston Biochem) and recombinant GluA1 (Origene) was used as substrate as described previously ( 12 , 36 ).

Techniques: Ubiquitin Proteomics, Western Blot, Recombinant, Immunoprecipitation, Staining, In Vitro

Journal: Human Molecular Genetics

Article Title: Loss of fragile X protein FMRP impairs homeostatic synaptic downscaling through tumor suppressor p53 and ubiquitin E3 ligase Nedd4-2

doi: 10.1093/hmg/ddy189

Figure Lengend Snippet: Loss-of-function dephosphorylation of Nedd4–2 disrupts GluA1 ubiquitination in Fmr1 KO neurons. (A) Western blots of 14-3-3 after co-IP of Nedd4-2 using lysates from WT or Fmr1 KO cortical neuron cultures treated with vehicle (DMSO) or 14-3-3 inhibitor (R18) (0.025 mg/ml) for 1 h following vehicle (DMSO) or PTX treatment for 48 h (two-way ANOVA with Tukey’s test, n = 4). (B) Western blots of Ub or GluA1 after IP with anti-GluA1 antibody following in vitro ubiquitination with recombinant GluA1 and Nedd4-2 immunoprecipitated from WT or Fmr1 KO cortical neuron cultures treated with PTX for 48 h. An addition of recombinant 14-3-3ε or R18 was applied prior to the ubiquitination reaction as labeled in the figure (Two-way ANOVA with Tukey’s test, n = 4). Coomasie blue staining showing the purity of recombinant 14-3-3ε is on the right (B3). (C) Western blots of phospho-Nedd4-2-S342 and phospho-Nedd4-2-S448 from WT or Fmr1 KO cortical neuron cultures treated with DMSO or PTX for 48 h (one-sample t-test for each genotype and Student’s t-test for comparison between genotypes, n = 4). (D) Western blots of GluA1 after co-IP of HA-Nedd4-2 using lysates from HEK cells transfected with GluA1 and control vector, HA-WT-Nedd4-2 or HA-S342A-S448A-Nedd4-2 for 2 days. The intensity of precipitated GluA1 from all samples was normalized to HA-WT-Nedd4-2-transfected cultures (Student’s t-test, n = 4). (E) Western blots of Ub after IP using anti-GluA1 antibody and lysates from HEK cells transfected with GluA1 and control vector, HA-WT-Nedd4-2 or HA-S342A-S448A-Nedd4-2 for 2 days Student’s t-test, n = 3). Data are represented as mean ± SEM with *P < 0.05, **P < 0.01, ***P < 0.001 and ns: non-significant.

Article Snippet: Ub activating enzyme (UBE1) (Boston Biochem), UbcH5b/UBE2D2 (Boston Biochem) and recombinant GluA1 (Origene) was used as substrate as described previously ( 12 , 36 ).

Techniques: De-Phosphorylation Assay, Ubiquitin Proteomics, Western Blot, Co-Immunoprecipitation Assay, In Vitro, Recombinant, Immunoprecipitation, Labeling, Staining, Comparison, Transfection, Control, Plasmid Preparation

Journal: Human Molecular Genetics

Article Title: Loss of fragile X protein FMRP impairs homeostatic synaptic downscaling through tumor suppressor p53 and ubiquitin E3 ligase Nedd4-2

doi: 10.1093/hmg/ddy189

Figure Lengend Snippet: Phosphorylation of Nedd4-2 reduces GluA1 levels and excitatory synaptic strength in Fmr1 KO neurons. (A) Western blots of Ub after IP using anti-GluA1 antibody from Fmr1 KO cortical neuron cultures lentivirally transfected with control GFP, HA-WT-Nedd4-2 (WT-Nedd4-2), or HA-S342D-S448D-Nedd4-2 (DD-Nedd4-2) and treated with vehicle (DMSO) or PTX for 48 h (n = 5). (B, C) Immunocytochemistry showing total (t) and surface (s) GluA1 and GluA2 in representative dendrites of Fmr1 KO hippocampal neurons transiently transfected with control GFP, WT-Nedd4-2 or DD-Nedd4-2 and treated with DMSO or PTX for 48 h. Quantification of total or surface GluA1 and GluA2 normalized to the average intensity of that in GFP-transfected, DMSO-treated neurons are on the bottom (n = 18–26 cells for Fmr1 KO neurons). (D) Patch-clamp recording from Fmr1 KO hippocampal neurons transiently transfected with control GFP, WT-Nedd4-2 or DD-Nedd4-2 and treated with DMSO or PTX for 48 h. Representative mEPSC traces and quantification of mEPSC amplitude are shown (n = 18–20 for Fmr1 KO neurons). Data were analyzed by two-way ANOVA with Tukey’s test and represented as mean ± SEM with *P < 0.05, **P < 0.01, ***P < 0.001 and ns: non-significant.

Article Snippet: Ub activating enzyme (UBE1) (Boston Biochem), UbcH5b/UBE2D2 (Boston Biochem) and recombinant GluA1 (Origene) was used as substrate as described previously ( 12 , 36 ).

Techniques: Phospho-proteomics, Western Blot, Transfection, Control, Immunocytochemistry, Patch Clamp

Journal: Human Molecular Genetics

Article Title: Loss of fragile X protein FMRP impairs homeostatic synaptic downscaling through tumor suppressor p53 and ubiquitin E3 ligase Nedd4-2

doi: 10.1093/hmg/ddy189

Figure Lengend Snippet: Inhibition of p53 prevents Nedd4-2 dephosphorylation and restores GluA1 ubiquitination in Fmr1 KO neurons. (A) Western blots (left) and quantification (right) of phospho-Akt-S473, total Akt, phospho-Nedd4-2-S342, phospho-Nedd4-2-S448, total Nedd4-2, phospho-Mdm2-S163, total Mdm2, p53 and internal control Gapdh from WT or Fmr1 KO cortical neuron cultures treated with vehicle (DMSO), PTX, pifithrin-α, or PTX + pifithrin-α for 48 h (n = 3–5 independent cultures). (B) Western blots of Ub after IP using anti-GluA1 antibody from WT or Fmr1 KO cortical neuron cultures treated with DMSO, PTX, pifithrin-α or PTX + pifithrin-α for 48 h (n = 4). Data were analyzed by two-way ANOVA with Tukey’s test and represented as mean ± SEM with *P < 0.05, **P < 0.01 and ns: non-significant.

Article Snippet: Ub activating enzyme (UBE1) (Boston Biochem), UbcH5b/UBE2D2 (Boston Biochem) and recombinant GluA1 (Origene) was used as substrate as described previously ( 12 , 36 ).

Techniques: Inhibition, De-Phosphorylation Assay, Ubiquitin Proteomics, Western Blot, Control

Journal: Human Molecular Genetics

Article Title: Loss of fragile X protein FMRP impairs homeostatic synaptic downscaling through tumor suppressor p53 and ubiquitin E3 ligase Nedd4-2

doi: 10.1093/hmg/ddy189

Figure Lengend Snippet: Inhibition of p53 restores homeostatic synaptic downscaling in Fmr1 KO neurons. (A) Immunocytochemistry showing total (t) and surface (s) GluA1 in representative dendrites of Fmr1 KO hippocampal neurons treated with vehicle (DMSO), PTX, pifithrin-α, or PTX + pifithrin-α for 48 h. Quantification of total and surface GluA1 normalized to the average intensity of that in DMSO-treated neurons are on the bottom (n = 40–41 cells). (B) Patch-clamp recording from Fmr1 KO hippocampal neurons treated with DMSO, PTX, pifithrin-α or PTX + pifithrin-α for 48 h. Representative mEPSC traces and quantification of mEPSC amplitude are shown (n = 19–23 cells). (C) Immunocytochemistry showing total (t) and surface (s) GluA2 in representative dendrites of Fmr1 KO hippocampal neurons treated with DMSO, PTX, pifithrin-α, or PTX + pifithrin-α for 48 h. Quantification of total and surface GluA2 normalized to the average intensity of that in DMSO-treated neurons are on the bottom (n = 34–37 cells). (D) Patch-clamp recording from Fmr1 KO hippocampal neurons transfected with non-target shRNA (control shRNA) or p53 shRNA with DMSO or PTX for 48 h. Representative mEPSC traces and quantification of mEPSC amplitude are shown (n = 20–22 cells). Data were analyzed by two-way ANOVA with Tukey’s test and represented as mean ± SEM with *P < 0.05, **P < 0.01, ***P < 0.001 and ns: non-significant.

Article Snippet: Ub activating enzyme (UBE1) (Boston Biochem), UbcH5b/UBE2D2 (Boston Biochem) and recombinant GluA1 (Origene) was used as substrate as described previously ( 12 , 36 ).

Techniques: Inhibition, Immunocytochemistry, Patch Clamp, Transfection, shRNA, Control

Journal: Human Molecular Genetics

Article Title: Loss of fragile X protein FMRP impairs homeostatic synaptic downscaling through tumor suppressor p53 and ubiquitin E3 ligase Nedd4-2

doi: 10.1093/hmg/ddy189

Figure Lengend Snippet: Working model of p53 and Nedd4-2 in FMRP-dependent homeostatic synaptic downscaling. In WT neurons, chronic activity stimulation triggers the Akt-Mdm2 pathway and leads to p53 down-regulation. A feedforward mechanism subsequently stabilizes Akt and promotes Nedd4-2 phosphorylation and the interaction between Nedd4-2 and 14-3-3. In Fmr1 KO neurons, chronic activity stimulation fails to further elevate Mdm2 phosphorylation or trigger p53 down-regulation. Destabilized Akt leads to Nedd4-2 dephosphorylation and occludes GluA1 ubiquitination even in the presence of 14-3-3. Although Mdm2 is basally phosphorylated in Fmr1 KO neurons, it is predicted that a compensatory mechanism is maintaining p53 level or an inhibitory molecule is interfering with Mdm2-p53 interaction in Fmr1 KO neurons.

Article Snippet: Ub activating enzyme (UBE1) (Boston Biochem), UbcH5b/UBE2D2 (Boston Biochem) and recombinant GluA1 (Origene) was used as substrate as described previously ( 12 , 36 ).

Techniques: Activity Assay, Phospho-proteomics, De-Phosphorylation Assay, Ubiquitin Proteomics