Journal: Frontiers in Cellular Neuroscience

Article Title: Ceftriaxone attenuates Poly I:C–induced neuroinflammation in vitro by modulating glutamate transport, synaptic integrity, and immunometabolic reprogramming

doi: 10.3389/fncel.2025.1684398

Figure Lengend Snippet: Ceftriaxone rescues the reduced colocalization of PSD-95 and CaMKIIα induced by Poly I:C. (A) Representative image used for colocalization analysis. The red box denotes the zoomed-in ROI highlighting a primary dendrite of a single neuron. Scale bars: 20 μm (overview) and 2 μm (zoom). (B,C) Quantification of normalized expression levels at 48 h post-treatment: PSD-95 ( B , red) and CaMKIIα ( C , green). (D) Pearson’s R coefficients for colocalization of CaMKIIα and PSD-95 within primary dendrites. (E) Representative immunocytochemical staining of AMPA-receptor subunits GluA1 and GluA2. Scale bar: 100 μm. (F,G) Normalized expression levels of GluA1 (F) and GluA2 (G) at 48 h post-treatment. Data are presented as mean ± SEM. ** p < 0.01, **** p < 0.0001; ns, not significant.

Article Snippet: The primary antibodies used were CaMKIIα (goat, 1:750, Abcam, ab111890), COX4 (rabbit, 1:500, Synaptic Systems, AB_2620041), Connexin 43 (rabbit, 1:500, Sigma-Aldrich, C6219), EAAT1/GLAST-1/SLC1A3 (rabbit, 1:500, Novus Biologicals, NB100-1869), EAAT2/GLT-1 (rabbit, 1:500, Novus Biologicals, NBP1-20136), GFAP (mouse, 1:1000, Sigma-Aldrich, G3893), GluR1 (GluA1) (guinea pig, 1:400, Alomone Labs, AGP-009), GluR2 (GluA2) (rabbit, 1:400, Alomone Labs, AGC-005), IBA-1 (mouse, 1:1000, Synaptic Systems, 234011), PSD-95 (mouse, 1:750, Novus Biologicals, NB300-556), and MAP2 (chicken, 1:1000, Synaptic Systems, 188006).

Techniques: Expressing, Staining

Journal: Journal of Neurochemistry

Article Title: Extracellular Vesicles Released From Cortical Neurons Influence Spontaneous Activity of Recipient Neurons

doi: 10.1111/jnc.70231

Figure Lengend Snippet: EVs isolated from primary cultures contain AMPA receptor subunits. (A) Glutamate receptor subunits are present in EVs isolated from primary cultures, in combination with the EV‐marker CD63. (B) Immunoelectron microscopy of EVs isolated from cultured neurons support the presence of the glutamate receptor subunit GluA2. (C) EVs isolated from mouse brains ( n = 4 mice) and validated qualitatively by the presence of the EV markers Flotillin‐1 and CD81, contain both AMPA receptor subunits and Nr2b subunit of NMDA receptors. SIgnificance use of * indicates GluA2‐negative EV.

Article Snippet: Grids with EVs were incubated with the primary antibody GluA2 (AGC‐005, Alomone Labs) for 2 h. Protein A coupled to 10 nm colloidal gold particles was used to target the primary antibody (G. Posthuma, University Medical Center Utrecht) (1:50).

Techniques: Isolation, Marker, Immuno-Electron Microscopy, Cell Culture

Journal: bioRxiv

Article Title: The Alzheimer’s disease risk gene SORL1 is a regulator of excitatory neuronal function

doi: 10.1101/2025.07.28.667194

Figure Lengend Snippet: A: Representative images of isogenic wild-type and SORL1 KO neurons, immunostained for AMPA receptor subunits GluR1, GluR2, somatodendritic neuronal marker MAP2, and DAPI. B: Quantification of GluR1 puncta sum fluorescence intensity and volume from wild-type and SORL1 KO neurons. GluR1 puncta in SORL1 KO neurons were smaller and had lower sum fluorescence intensity. C: GluR2 puncta sum fluorescence intensity and volumes from wild-type and SORL1 KO neurons. Immunostained GluR2 puncta were brighter and larger in volume in SORL1 KO neurons compared to wild-type neurons. N = 10,000-12,000 GluR1 and GluR2 puncta were analyzed from 32-34 images per genotype. Two clones per genotype were analyzed. ICC puncta data points plotted with mean values indicated by horizontal dashed line. Outliers were identified using the ROUT (Q = 1%) method and removed for statistical testing and plotting. Data were tested for normality and were determined to be not normally distributed (D’Agostino and Pearson test). Statistical testing for all quantifications was the Kolmogorov-

Article Snippet: HOMER1 (Synaptic Systems cat. #160003 1:250), SYNAPSIN1/2 (Synaptic Systems cat. #106004 1:500), GLUR1 (Millipore cat. # MAB2263 1:500), GLUR2 (ProteinTech cat. #11994-1-AP 1:500), MAP2 (Abcam cat. #ab92434 1:1000).

Techniques: Marker, Fluorescence, Clone Assay

Journal: bioRxiv

Article Title: The Alzheimer’s disease risk gene SORL1 is a regulator of excitatory neuronal function

doi: 10.1101/2025.07.28.667194

Figure Lengend Snippet: Quantification of GLUR1 and GLUR2 immunofluorescent signal captured from matured DPP 30 wild-type and SORL1 KO neuronal somatic and dendritic compartments. A-B: Soma localized GLUR1 and GLUR2 puncta sum intensity and volumes. C-D: Dendrite localized GLUR1 and GLUR2 puncta sum intensity and volumes in wild-type and SORL1 KO neurons. With the exception of GLUR1 puncta volume in dendrites, differences of puncta sum intensity and volume match whole-cell analysis , indicating that changes in GLUR1 and GLUR2 are cell-wide and not localized imbalances of somatic and dendritic levels of the AMPA subunits. N = 10,000-12,000 GLUR1 and GLUR2 puncta were analyzed from somatic and dendritic neuronal structures. 32-34 images per genotype. Two clones per genotype were analyzed. Puncta data points plotted with mean values indicated by horizontal dashed line. Outliers were identified using the ROUT (Q = 1%) method and removed for statistical testing and plotting. Data were tested for normality and were determined to be not normally distributed (D’Agostino and Pearson test). Statistical testing for all quantifications was the Kolmogorov-Smirnov test, with significance marked as follows: P < 0.0001 (****). Not significant comparisons are unmarked

Article Snippet: HOMER1 (Synaptic Systems cat. #160003 1:250), SYNAPSIN1/2 (Synaptic Systems cat. #106004 1:500), GLUR1 (Millipore cat. # MAB2263 1:500), GLUR2 (ProteinTech cat. #11994-1-AP 1:500), MAP2 (Abcam cat. #ab92434 1:1000).

Techniques: Cell Analysis, Clone Assay

Journal: bioRxiv

Article Title: Brain-derived extracellular vesicle microRNAs in Lewy body and Alzheimer’s disease

doi: 10.1101/2025.06.06.656900

Figure Lengend Snippet: (A) Outline of study time-course and sample processing for EVs. Antemortem plasma samples with postmortem pathology confirmation of neurological diagnoses were processed to isolate tissue-specific EVs via our microfluidic platform, alongside plasma protein biomarkers via commercial digital ELISA, for patients with AD, LBD, AD/LBD, AD/ALB and controls. (B) Left: schematic of operation of TENPO platform for tissue-specific EV isolation using antibody-labeled MNPs; right: SEM images of EVs isolated via GluR2 (top) and GLAST (bottom) pulldowns immobilized on TENPO. Abbreviations: LBD, Lewy body disease; AD, Alzheimer’s disease; AD/LBD, mixed Alzheimer’s and Lewy body disease; AD/ALB, Alzheimer’s disease with amygdala Lewy bodies; EV, extracellular vesicle; TENPO, track-etch magnetic nanopore; ELISA, enzyme-linked immunosorbent assay; MNP, magnetic nanoparticle; SEM, scanning electron microscopy.

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 minutes 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., The sample was then incubated with 50 μL of 50 nm magnetic nanoparticles (MNPs) at manufacturer stock concentration (Anti-Biotin MicroBeads UltraPure, Miltenyi Biotec) for another 20 minutes.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Isolation, Labeling, Electron Microscopy

Journal: bioRxiv

Article Title: Brain-derived extracellular vesicle microRNAs in Lewy body and Alzheimer’s disease

doi: 10.1101/2025.06.06.656900

Figure Lengend Snippet: (A) Heatmap of z-score of log 2 (expression) for biomarkers with FDR-corrected P value < .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 between GluR2+ EVs and GLAST+ EVs. (D) Top 30 biomarkers in all compartments ranked by descending AUC. Error bars represent standard error from bootstrapping 10x. Abbreviations: LBD, Lewy body disease; AD, Alzheimer’s disease; EV, extracellular vesicle; FDR, false discovery rate; AUC, area under the curve.

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 minutes 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., The sample was then incubated with 50 μL of 50 nm magnetic nanoparticles (MNPs) at manufacturer stock concentration (Anti-Biotin MicroBeads UltraPure, Miltenyi Biotec) for another 20 minutes.

Techniques: Expressing, Quantitative Proteomics, Clinical Proteomics

Journal: bioRxiv

Article Title: Brain-derived extracellular vesicle microRNAs in Lewy body and Alzheimer’s disease

doi: 10.1101/2025.06.06.656900

Figure Lengend Snippet: GO and KEGG pathway analyses were performed via DIANA miRPath v4.0 using the TarBase v8.0 database. FDR P values were calculated using a one-sided Fisher’s exact test. 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. Abbreviations: LBD, Lewy body disease; AD, Alzheimer’s disease; EV, extracellular vesicle; FDR, false discovery rate; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, biological process; CC, cellular component; MF, molecular function.

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 minutes 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., The sample was then incubated with 50 μL of 50 nm magnetic nanoparticles (MNPs) at manufacturer stock concentration (Anti-Biotin MicroBeads UltraPure, Miltenyi Biotec) for another 20 minutes.

Techniques: Labeling

Journal: bioRxiv

Article Title: Brain-derived extracellular vesicle microRNAs in Lewy body and Alzheimer’s disease

doi: 10.1101/2025.06.06.656900

Figure Lengend Snippet:

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 minutes 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., The sample was then incubated with 50 μL of 50 nm magnetic nanoparticles (MNPs) at manufacturer stock concentration (Anti-Biotin MicroBeads UltraPure, Miltenyi Biotec) for another 20 minutes.

Techniques: Marker

Journal: bioRxiv

Article Title: Brain-derived extracellular vesicle microRNAs in Lewy body and Alzheimer’s disease

doi: 10.1101/2025.06.06.656900

Figure Lengend Snippet: (A) (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 via 10-fold cross-validation, with error bars 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 10-fold cross validation for the 15-marker panel or from bootstrapping 10x for individual markers. Abbreviations: LBD, Lewy body disease; AD, Alzheimer’s disease; EV, extracellular vesicle; LASSO, least absolute shrinkage and selection operator; AUC, area under the curve.

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 minutes 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., The sample was then incubated with 50 μL of 50 nm magnetic nanoparticles (MNPs) at manufacturer stock concentration (Anti-Biotin MicroBeads UltraPure, Miltenyi Biotec) for another 20 minutes.

Techniques: Expressing, Biomarker Discovery, Control, Marker, Selection