Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: Synapse volumes increase correlatively during synaptic maturation. (A–D) Representative distribution of RIM1/2 and GluA1 under stochastic optical reconstruction microscopy (STORM). Scale 2 μm in top panels and 500 nm in lower panels. (E–G) Volumes of identified synaptic RIM1/2, GluA1, and PSD-95 clusters across different developmental stages. Numbers in bars denote the synapse numbers. (H,I) Correlations between the volumes of GluA1 and RIM1/2 clusters (H) and the volumes of PSD-95 and RIM1/2 (I) within the same synapses. Linear regressions were conducted on synapses of DIV7 (gray circles and line) and DIV18 (dark blue circles and line). Data from synapses of DIV10 and 14 were plotted with dark yellow and dark red crosses. Also refer to , and for more details on correlations and statistics. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques: Microscopy

Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: The heterogeneity of synaptic protein distribution increases with development. (A–D) Representative density maps of synaptic GluA1 at different developmental stages. Scale bars, 200 nm. (E–G) Normalized autocorrelation functions of RIM1/2 (E) , GluA1 (F) , and PSD-95 (G) . g a above 1 suggests a significant non-uniform distribution. (H–J) Developmental changes of nanocluster number (left), normalized density within nanocluster (middle), and nanocluster volume of RIM1/2 (H) , GluA1 (I) , and PSD-95 (J) . Numbers in bars denote the synapse numbers. Also refer to for more statistical details. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques:

Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: Correlation between presynaptic and postsynaptic protein heterogeneity in mature synapses. (A,B) Scatter plots for heterogeneity of GluA1 (A) and PSD-95 (B) against that of RIM1/2. All data points across all developmental stages could be fitted with linear functions as shown with lines. (C,D) Linear regressions of the relationships between heterogeneity of GluA1/PSD-95 and RIM1/2 at DIV7 (gray) and DIV18 (dark blue). (E) Relationship between heterogeneity and cluster volume of RIM1/2 at DIV7 (gray) and DIV18 (dark blue). Data points with cluster volume >1 × 10 7 nm 3 were fitted with linear functions. It is noted that g a of immature synapses was significantly lower than that of matured synapses. (F) Averaged g a of synapses with cluster volume of 1–4 × 10 7 nm 3 for immature (DIV7-10) and mature synapses (DIV18). Also refer to for more details. *** p < 0.001, t-test. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques:

Journal: Frontiers in Synaptic Neuroscience

Article Title: Correlative Assembly of Subsynaptic Nanoscale Organizations During Development

doi: 10.3389/fnsyn.2022.748184

Figure Lengend Snippet: Evolvement of trans-synaptic nano-alignment during synaptic maturation. (A–D) Representative examples of synapses with RIM1/2 (red) and GluA1 (blue) co-labeled and imaged with STORM. Thick color denotes detected nanoclusters. Scale bar, 200 nm. (E) Normalized local density of GluA1 along with distances from RIM1/2 nanoclusters for synapses at different developmental stages. (F) , Averaged enrichment of GluA1 within 50 nm from peaks of RIM1/2 nanoclusters ( n = 85, 60, 30, 54, and 54 nanoclusters). The open bar represents the enrichment indices of synapses with the position of nanoclusters randomized within synaptic clusters. (G) Fraction of nanoclusters that were enriched with protein across the cleft. (H–J) Enrichment between RIM1/2 and PSD-95 for synapses at different developmental stages. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey's multiple comparisons test in (F,I) , z -test in (G,J) . Also refer to for more statistical details. All experiments were repeated ≥3 times.

Article Snippet: For co-staining of GluA1 and RIM1/2, as both antibodies were from rabbits, staining was performed separately and the first primary antibody (GluA1) was blocked and converted with a goat anti-rabbit Fab fragment (JacksonImmuno 111-007-003) for 2 h at RT, and then recognized by secondary antibodies.

Techniques: Labeling

Journal: Molecular Pain

Article Title: Promotion of bone cancer pain development by decorin is accompanied by modification of excitatory synaptic molecules in the spinal cord

doi: 10.1177/1744806919864253

Figure Lengend Snippet: Decorin promoted the phosphorylation level of GluR1 at serine 831 in BCP rats. (a) Representative triple staining images of decorin (red), presynaptic marker of vesicle protein synapsin I (rose red), and excitatory postsynaptic marker PSD95 (green) in the superficial laminae of the spinal dorsal horn of the rats. Scale bar: 4 μm. (b) Western blot analysis of GluR1and pGluR1 in the sham, BCP, BCP+shctrl, and BCP+shdecorin groups 21-day postsurgery. pGluR1: phosphorylation of GluR1 at the site of serine 831. Values represent the mean ± standard error of the mean (SEM), n = 4. * p < 0.05 compared with the sham group; # p < 0.05 compared with the BCP group, n.s. indicates not significant. (c and d) Decorin negatively regulated sema3a in carcinoma-implanted rats. The effect of decorin on sema3a was confirmed by real-time polymerase chain reaction and western blot analysis in the four groups 21-day postsurgery. Values represent the mean ± SEM, n = 4. * p < 0.05 compared with the sham group; # p < 0.05 compared with the BCP group. (e) Sema3a had no effect on decorin in the carcinoma-implanted rats. The effect of sema3a on decorin was confirmed by western blot analysis in the sham, BCP, BCP+ctrl-LV, and BCP+sema-LV rats 21-day postsurgery. BCP+ctrl-LV rats: BCP rats treated with the control lentivirus and BCP+sema-LV rats: BCP rats treated with the recombinant lentivirus overexpressing sema3a.Values represent the mean ± SEM, n = 4. * p < 0.05 compared with the sham group, # p < 0.05 compared with the BCP group, n.s. indicates not significant ( p > 0.05). BCP: bone cancer pain; PSD95: postsynaptic density protein 95; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The primary antibodies were diluted and used at the following dilutions: rabbit antidecorin (1:100, ab175404, Abcam), goat antisynapsin I (1:50, sc-8295,Santa), rabbit anti-gluR1 (1:50, ab109450, Abcam), rabbit anti-P-gluR1 (1:50, A4352, Sigma), mouse antipostsynaptic density protein 95 (PSD95; 1:50, MABN68, Millipore), rabbit anti-Bassoon (1:500, 141002, Synaptic System), guinea pig anti-MAP2 (1:800, 188004, Synaptic System), and mouse anti-VGlut2 (1:50, MAB5504, Millipore).

Techniques: Staining, Marker, Western Blot, Real-time Polymerase Chain Reaction, Recombinant

Journal: Molecular Pain

Article Title: Promotion of bone cancer pain development by decorin is accompanied by modification of excitatory synaptic molecules in the spinal cord

doi: 10.1177/1744806919864253

Figure Lengend Snippet: Decorin knockdown impaired the excitatory synaptogenesis and the insertion of pGluR1-ser831 into the excitatory postsynaptic membranes in vitro. Neurons were infected with lentivirus at DIV 3, and the immunofluorescence assay were performed at DIV 12: (a) Immunocytochemistry of dendrites from neurons in the shctrl and shdecorin groups at DIV 12 colabeled with antibodies against Bassoon (red) and PSD95 (blue) to visualize pre- and postsynaptic levels. Scale bar: 5 μm; (b) Quantification of Bassoon, PSD95 and colocalization puncta in B per 30 μm dendrite length. Values represent the mean ± SEM, n = 30 in three independent experiments. **P < 0.01 compared with the shctrl group; n.s. indicates not significant (P > 0.05); (c) Immunofluorescence images of dendrites in DIV 12 neurons from the shctrl and shdecorin groups colabeled with antibodies against GluR1 (red) and PSD95 (blue). Scale bar: 5 μm; (d) Quantification of GluR1 and colocalization puncta in B per 30 μm dendrite length. Values represent the mean ± SEM, n = 30 in three independent experiments. n.s. indicates not significant (P > 0.05); (e) Representative images of dendrites in DIV12 neurons from the shctrl and shdecorin groups colabeled with antibodies against GluR1 (red) and PSD95 (blue). Scale bar: 5 μm and (f) Quantification of GluR1 and colocalization puncta in D per 30 μm dendrite. Values represent the mean ± SEM, n = 25 in three independent experiments.**P < 0.01 compared with the shctrl group.

Article Snippet: The primary antibodies were diluted and used at the following dilutions: rabbit antidecorin (1:100, ab175404, Abcam), goat antisynapsin I (1:50, sc-8295,Santa), rabbit anti-gluR1 (1:50, ab109450, Abcam), rabbit anti-P-gluR1 (1:50, A4352, Sigma), mouse antipostsynaptic density protein 95 (PSD95; 1:50, MABN68, Millipore), rabbit anti-Bassoon (1:500, 141002, Synaptic System), guinea pig anti-MAP2 (1:800, 188004, Synaptic System), and mouse anti-VGlut2 (1:50, MAB5504, Millipore).

Techniques: In Vitro, Infection, Immunofluorescence, Immunocytochemistry

Journal: Cell Reports

Article Title: Activity-driven synaptic translocation of LGI1 controls excitatory neurotransmission

doi: 10.1016/j.celrep.2024.114186

Figure Lengend Snippet: Stable localization of LGI1 at the cleft depends on the history of synaptic activity (A) Example response of a single presynaptic arborization expressing LGI1-pH during 1,000 AP 50 Hz electrical stimulation. Dotted line indicates the baseline before stimulation. (B) LGI1-pH fraction present at the synaptic surface before and after electrical stimulation (1,000 AP 50 Hz) for individual neurons. (C) Example response of a single presynaptic arborization expressing vGlut-pH during 1,000 AP 50 Hz electrical stimulation. Dotted line indicates the baseline before stimulation. (D) vGlut-pH fraction present at the synaptic surface before and after electrical stimulation (1,000 AP 50 Hz) for individual neurons. (E) Example images of LGI1-pHluorin expression levels at the surface (middle panel) and total (right panel, revealed by NH 4 Cl pH 7.4 in control neurons versus neurons treated with TTX for 5 days. Presynaptic boutons are identified by synapsin-mRuby (left, in red). Pseudocolor scale shows low to high intensity. Scale bar, 4.8 μm. (F) Fraction of LGI1-pH present at the synaptic surface in control versus neurons treated with TTX for 5 days. (G) Fraction of ADAM23-pH present at the synaptic surface in control versus TTX-treated neurons. (H) Diagram showing experimental flow to biochemically isolate endogenous synaptic cleft proteins. (I) Example blot for total biotinylation rates in neurons expressing LRRTM-HRP treated with or without TTX for 5 days; minus signal (−) indicates a negative control in which reaction was not run. (J) Example western blot experiment showing endogenous LGI1 levels isolated from the synaptic surface in control and neurons treated with TTX for 5 days (top panel), with the same experimental conditions shown in (H). As a control, endogenous levels of GluR1 remained unchanged (lower panel). (K) Example western blot experiment showing total LGI1 levels in control and neurons treated with TTX for 5 days. V5 shows the expression of LRRTM-HRP-V5 and β-actin is a loading control. (L) Quantification of endogenous levels of LGI1 at the synaptic surface in TTX-treated neurons. (M) Quantification of endogenous total levels of LGI1 obtained from whole cell lysate of the experiments shown in (L). Table S1 shows number of experiments and replicates, means and error, and statistical tests used.

Article Snippet: Mouse anti-GRIA1 antibody , UC Davis/NIH NeuroMab Facility , Cat# 75-327; RRID: AB_2877405.

Techniques: Activity Assay, Expressing, Control, Negative Control, Western Blot, Isolation

Journal: Cell Reports

Article Title: Activity-driven synaptic translocation of LGI1 controls excitatory neurotransmission

doi: 10.1016/j.celrep.2024.114186

Figure Lengend Snippet:

Article Snippet: Mouse anti-GRIA1 antibody , UC Davis/NIH NeuroMab Facility , Cat# 75-327; RRID: AB_2877405.

Techniques: Virus, Recombinant, Transfection, Magnetic Beads, Western Blot, Mutagenesis, Plasmid Preparation, Software, Imaging, Microscopy

Journal: Brain Sciences

Article Title: Arc-Mediated Synaptic Plasticity Regulates Cognitive Function in a Migraine Mouse Model

doi: 10.3390/brainsci13020331

Figure Lengend Snippet: Determination of hippocampal protein in mice. **: p < 0.01, *: p < 0.05. ( A ): WB results of hippocampal protein. ( B ): NR2B/GAPDH ratio. ( C ): Arc/GAPDH ratio. ( D ): GluR1/GAPDH ratio. ( E ): SYP/β-Actin ratio.

Article Snippet: Then, the sections were closed and incubated overnight with the following primary antibody: mice anti-mice Arc (1:100; Santa Cruz, USA), mice anti-mice GluR1 (1:100; Santa Cruz, USA), and rabbit anti-mice SYP (1:100, Wanleibio, China).

Techniques:

Journal: Brain Sciences

Article Title: Arc-Mediated Synaptic Plasticity Regulates Cognitive Function in a Migraine Mouse Model

doi: 10.3390/brainsci13020331

Figure Lengend Snippet: Determination of protein in the prefrontal cortex and hippocampus. ***: p < 0.001, **: p < 0.01, *: p < 0.05. ( A ): WB results of prefrontal cortex protein. ( B ): NR2B/GAPDH ratio. ( C ): GluR1/GAPDH ratio. ( D ): Arc/GAPDH ratio. ( E ): WB results of hippocampus protein. ( F ): NR2B/GAPDH ratio. ( G ): GluR1/GAPDH ratio. ( H ): Arc/GAPDH ratio.

Article Snippet: Then, the sections were closed and incubated overnight with the following primary antibody: mice anti-mice Arc (1:100; Santa Cruz, USA), mice anti-mice GluR1 (1:100; Santa Cruz, USA), and rabbit anti-mice SYP (1:100, Wanleibio, China).

Techniques:

Journal: Brain Sciences

Article Title: Arc-Mediated Synaptic Plasticity Regulates Cognitive Function in a Migraine Mouse Model

doi: 10.3390/brainsci13020331

Figure Lengend Snippet: Immunofluorescence detection of GluR1 protein in the prefrontal cortex and hippocampal CA1 region. ***: p < 0.001. The blue light point in the figure is the nucleus. The red light dot is GluR1, and the bottom left corner is a zoomed-in view of it. ( A ): GluR1 protein in the prefrontal cortex of the IS+S. ( B ): GluR1 protein in the prefrontal cortex of the IS. ( C ): GluR1 protein in the prefrontal cortex of the IS+M. ( D ): GluR1 protein in the prefrontal cortex of the Control. ( E ): Comparison of GluR1’s mean optical density in each group’s prefrontal cortex. ( F ): GluR1 expression in the hippocampal CA1 region of the IS+S. ( G ): GluR1 expression in the hippocampal CA1 region of the IS. ( H ): GluR1 expression in the hippocampal CA1 region of the IS+M. ( I ): GluR1 expression in the hippocampal CA1 region of the Control. ( J ): Comparison of GluR1’s mean optical density in each group’s hippocampal CA1 region.

Article Snippet: Then, the sections were closed and incubated overnight with the following primary antibody: mice anti-mice Arc (1:100; Santa Cruz, USA), mice anti-mice GluR1 (1:100; Santa Cruz, USA), and rabbit anti-mice SYP (1:100, Wanleibio, China).

Techniques: Immunofluorescence, Control, Comparison, Expressing

Journal: Brain Sciences

Article Title: Arc-Mediated Synaptic Plasticity Regulates Cognitive Function in a Migraine Mouse Model

doi: 10.3390/brainsci13020331

Figure Lengend Snippet: Cellular protein expression. NGF: nerve growth factor group, n = 4; Control: Control group, n = 4; N + M: NGF + memantine group, n = 4. ***: p < 0.001, **: p < 0.01, *: p < 0.05. ( A ): Western blot results of cultured cells. ( B ): The ratio of NR2B, NR1, Arc, GluR1, SYP to β-Actin, respectively.

Article Snippet: Then, the sections were closed and incubated overnight with the following primary antibody: mice anti-mice Arc (1:100; Santa Cruz, USA), mice anti-mice GluR1 (1:100; Santa Cruz, USA), and rabbit anti-mice SYP (1:100, Wanleibio, China).

Techniques: Expressing, Control, Western Blot, Cell Culture

Journal: bioRxiv

Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

doi: 10.1101/2021.01.18.427223

Figure Lengend Snippet: A) Western blot of GluAi and β-actin in cortical lysates of VKI mice (i) revealed no genotype effect on GluA1 protein levels (ii). B) Co-immunoprecipitation of GluA1 with VPS35 (i) revealed no genotype effect (ii). C) Cultured cortical neurons immunostained for MAP2 (blue), VPS35 (cyan), and GluA1 (magenta)(i). There was a significant reduction in GluA1 cluster density in homozygous VKI neurons (ii, ** p <0.005), and no genotype effect on VPS35-GluAi co-cluster density of (iii), or Pearson’s coefficient (iv).

Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

Techniques: Western Blot, Immunoprecipitation, Cell Culture

Journal: bioRxiv

Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

doi: 10.1101/2021.01.18.427223

Figure Lengend Snippet: A) Western blot of cortical lysates and coIPs were probed for VPS35, D2R, GluA1, GluN1, GluA1, and GAPDH(i). There was no genotype effect on VPS35 levels or IP (ii-iii, 1-way ANOVA p >0.99; Kruskal-Wallis p =0.62, respectively). NMDA-receptor subunit GluN1 association with retromer has not previously been published; the mutation did not affect GluN1 levels nor coIP with VPS35 (iv-v, Kruskal-Wallis p =0.76; p =0.44, respectively). D2-type dopamine receptors are a novel cargo; there was no significant genotype effect on D2R levels or CoIP with VPS35 (vi-vii, Kruskal-Wallis p =0.80; p =0.44, respectively). B) CoIP of cortical lysates in A probed for LRRK2 (i). There were no significant genotype effects on LRRK2 levels or association of LRRK2 with VPS35 by coIP (ii-iii Kruskal-Wallis p =0.76; p =0.52, respectively). C) Striatal lysates quantified as in A & B (i). There were no genotype effects on VPS35 levels or pull by the antibody (ii-iii, Kruskal-Wallis p =0.97; p =0.13, respectively); GluN1 levels or coIP (iv-v, Kruskal-Wallis p =0.51; p =0.42, respectively); D2R levels or coIP (vi-vii Kruskal-Wallis p =0.70; p =0.45, respectively); GluA1 levels or coIP (viii-ix, Kruskal-Wallis p =0.83; p =0.44, respectively); or LRRK2 levels or coIP (x-xi, Kruskal-Wallis p >0.99; p =0.40, respectively). For all panels, n= number of experimental animals.

Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

Techniques: Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

doi: 10.1101/2021.01.18.427223

Figure Lengend Snippet: A) Cultured cortical neurons immunostained for PSD95 (cyan) and VGluT1 (magenta) (i). There was no genotype effect on synapse density defined as VGluT1-PSD95 co-clusters (ii), nor on PSD95 or VGluT1 density (iii-iv).VGluT1 cluster intensity was significantly reduced in homozygous cells (v, ** p <0.003). B) Representative traces from whole-cell patch voltage clamp recording of mEPSCs in cortical neurons (i). There were significant genotype effects on mean mEPSC frequency (Hz) due to increases in heterozygous cells (ii, * p <0.02). Significant genotype effects were also seen in mEPSC amplitudes due to increases in heterozygous cells (iii, * p <0.02). Mean mEPSC decay times (τ) were not affected by genotype (iv). Representative mean-variance plots of mEPSC amplitude used for peak-scaled non-stationary fluctuation analysis, allowing calculation of single channel conductance (i). There was no significant effect of genotype on single channel conductance (vi, Kruskal-Wallis p =0.78). C) GFP-filled (cyan) cultured cortical cells immunostained for MAP2 (blue; to ensure no permeabilization) and surface GluAi (magenta)(i, left panel); in silico neurite outlines with only GluA1 staining displayed (i, right panel). There was a significant genotype effect on surface GluA1 cluster intensity (synaptic GluA1) due to significant increases in heterozygous cells (ii, ** p <0.02). There was a significant genotype effect on GluA1 cluster density, due to opposing effects on heterozygous and homozygous cells (iii, * p <0.05).

Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

Techniques: Cell Culture, In Silico, Staining

Journal: bioRxiv

Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

doi: 10.1101/2021.01.18.427223

Figure Lengend Snippet: A) Western blot of whole brain lysate following acute MLi-2 treatment were probed for LRRK2, LRRK2 phospho-S935, GluA1, VPS35, VGluTl, Rab10, Rab10 phospho-T73, and β-actin. B) There were no significant effects of genotype or treatment on LRRK2 levels (2-way ANOVA genotype x treatment p =0.93; genotype p =0.90; treatment p =0.24). C) ML12 treatment significantly reduced pLRRK2 in all genotypes (2-way ANOVA treatment p <0.0001; Uncorrected Fisher’s LSD WT-WTMLi2 **** p <0.0001; Het-HetMLi2 *** p <0.0002; Ho-HoMLi2 **** p <0.0001). D-E) There was similarly no effect of genotype or treatment on Rab10 protein levels (D, 2-way ANOVA genotype x treatment p =0.5258; genotype p =0.4683; treatment p =0.9659), but significant genotype and treatment effects on pRab10 due to significant reductions in homozygous cells (E, 2-way ANOVA genotype p =0.04; treatment p <0.009; Uncorrected Fisher’s LSD WT-WTMLi2 p =0.82; Het-HetMLi2 p =0.10; Ho-HoMLi2 ** p =0.007). F-H) There were no significant effects of genotype or treatment on protein levels of VPS35 (F, 2-way ANOVA interaction p =0.23; genotype p =0.94; treatment p =0.89), VGluT1 (G, 2-way ANOVA interaction p =0.55; genotype p =0.39; treatment p =0.69), or GluA1 (H, 2-way ANOVA interaction p =0.45; genotype p =0.61; treatment p >0.99). For all experiments, n represents number of experimental animals and are as follows: WTCap n=5, WTMLi2 n=6, HetCap n=6, HetMLi2 n=6, HoCap n=5, HoMLi2 n=5.

Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

doi: 10.1101/2021.01.18.427223

Figure Lengend Snippet: A) Cultured cortical neurons immunostained for MAP2 (blue), PSD95 (cyan), and VGluT1 (magenta) following acute MLi-2 or vehicle treatment (i). There were interaction effects on PSD95 density due to increases in PSD95 density in heterozygous controls over WT (ii, *p<0.05) and a reduction in heterozygous neurons treated with MLi-2 *p<0.04. There were also interaction effects on VGluT1 density due to a reduction in cluster density in homozygous cells following MLi-2 treatment (iii, * p =0.03); however, there were no statistically significant effects on synapse (PSD95-VGluT1 co-cluster) density (iv). B) Non-permeabilized cultured cortical cells immunostained for MAP2 (blue), and surface GluA1 (magenta) following acute MLi-2 or vehicle treatment (i). There was a significant interaction effect on surface GluA1 cluster density (ii) due to an increase in WT surface GlulA1 clusters (** p <0.004); There was a significant interaction effect on surface GluA1 intensity (iii), though in this case it was due to underlying differences in control heterozygous and homozygous cells (*** p <0.0004) and a treatment effect in homozygous cells (** p <0.004). Normalization of treatment effects within genotype revealed that changes to GluA1 surface density (iv) were significantly larger in WT cells than heterozygous (** p <0.003) and homozygous mutant cells (**** p <0.0001), and that treatment had opposite effects on GluA1 intensity (v) in heterozygous cells than it did in WT (* p <0.02) and homozygous cells (* p <0.02). For Aii-v, n=40(4) for all groups and for Bii-iii, n=30(3).

Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

Techniques: Cell Culture, Mutagenesis

Journal: bioRxiv

Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

doi: 10.1101/2021.01.18.427223

Figure Lengend Snippet: A) There was no genotype effect on Rab10 cluster intensity (i); however, Rab10 cluster density was increased in both mutant genotypes, falling just shy of statistical significance (ii, p <0.06). B) GFP-filled (blue) cortical neurons immunostained for Rab10 (magenta), and VPS35 (cyan)(i). There were no genotype effects on co-cluster density (ii) or Pearson’s coefficient (ii). C) GFP-filled (blue) cortical neurons immunostained for Rab10 (magenta), and GluA1 (cyan)(i). There were no genotype effects on co-cluster density or Pearson’s coefficient (ii-iii).

Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: SRF-deficient astrocytes provide neuroprotection in mouse models of excitotoxicity and neurodegeneration

doi: 10.1101/2023.05.17.541074

Figure Lengend Snippet: (A) Coronal sections showing NeuN immunostaining in control and Srf mutant mice at 12 mpi. (B) Quantification of NeuN+ cell numbers in the neocortex and striatum, and DAPI+ cell numbers in the CA1 and CA3 regions of the hippocampus. (C) Quantitative PCR of astrocyte-secreted synaptogenic factors shows no difference in their expression in the mutant mice when compared to control mice. n=3 mice. (D) Representative images of neocortical sections immunostained for the presynaptic marker, piccolo (red) and postsynaptic marker, GluA1 (green). Co-localization of staining (yellow puncta) was counted as a synapse. Scale bar, 10 µm. (E) Quantification of the number of synapses in the neocortex in control and Srf GFAP- ER CKO mutant mice at 3 and 15 mpi. n=3 mice. Unpaired t-test. Data are represented as mean ± SEM. ns, not significant.

Article Snippet: The following primary antibodies were used: mouse anti-GFAP (1:1000; Sigma, #G-145), rabbit anti-GFAP (1:1000; Z0334, Dako), goat anti-Sox9 (1:500, R&D Systems, #AF3075), mouse anti-Vimentin (1:50; DSHB, #40E-C), rabbit anti-S100β (1:1000; Sigma, #S2644), rabbit anti-Iba1 (1:1000; Wako, #019-19741), mouse anti-NeuN (1:1000; Chemicon, #MAB-377), chicken anti-β-gal (1:1000; Aves, #BGL-1040), rabbit anti-PhosphoHistoneH3 (1:500; Sigma, #H0412), guinea pig anti-Piccolo (1:400, Synaptic systems, #142104), mouse anti-GluA1 (1:200, Synaptic systems, # 182011), rabbit anti-Tyrosine hydroxylase (1:500, Merck, #AB152), and anti-MOAB2 (1:500, Novus Bio, #NBP2-13075).

Techniques: Immunostaining, Mutagenesis, Real-time Polymerase Chain Reaction, Expressing, Marker, Staining