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: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet: ( A, B ) Hippocampal neurons were subjected to ( A ) chemical LTP (cLTP) or ( B ) chemical LTD (cLTD). DKK3 levels were analyzed in the supernatant (extracell) and cell lysates by western blot. Phospho-GluA1 Ser845 was used as a readout of LTP and LTD induction, and tubulin as a loading control for the cell lysate. Graphs show the levels of DKK3 relative to control and the ratio of extracellular/lysate DKK3 levels (Mann-Whitney Test, n=3 independent cultures for cLTP and Student’s T-test, n=6 independent cultures for cLTD). ( C ) Representative immunoblots show DKK3 levels in the cell lysate and extracellular (extracell) fraction of acute WT hippocampal slices treated with vehicle (Ctrl) or NMDA (cLTD) and/or Brefeldin A (BFA) for 60 min. Actin was used as a loading control in homogenates. Graphs show densiometric quantifications of DKK3 relative to the control condition and the ratio of extracellular/lysate DKK3 levels (Kruskal-Wallis followed by Dunn’s multiple comparisons; n=5 animals). ( D ) Immunoblot showing DKK3 is less abundant in the extracellular fraction when compared to the cell lysate fraction. Representatives immunoblot of J20 brain slices treated with control or APV for 3 hr. Time exposure for obtaining DKK3 chemiluminescent images is indicated. Figure 2—figure supplement 3—source data 1. Uncropped western blot gels. Raw and annotated WB images. The representative western blot images for are indicated within a blue square.

Article Snippet: Primary antibodies and dilutions used for western blot were: Mouse Anti- Aβ (6E10 clone, 1:1000, Biolegend, Cat# 803001, RRID: AB_2564653 ), goat anti- Mouse DKK-3 (1:1000, R and D Systems, Cat# AF948, RRID: AB_355734 ), goat anti- Human DKK-3 (1:1000, R&D Systems, Cat# AF1118, RRID: AB_354610 ), rabbit anti-GAPDH (1:5000, Abcam, Cat# ab181602, RRID: AB_2630358 ), rabbit anti-GluA1 (1:1000, Cell Signaling Technology, Cat# 13185, RRID: AB_2732897 ), rabbit anti-phospho GluA1 Serine 845 (1:1000, Cell Signaling Technology, Cat# 8084, RRID: AB_10860773 ), rabbit anti- SAPK/JNK (1:1,000, Cell Signaling Technology, Cat# 9252, RRID: AB_2250373 ), mouse anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:500, Cell Signaling Technology, Cat# 9255, RRID: AB_2307321 ), mouse anti-Tubulin (1:5000, Sigma-Aldrich, Cat# T9026, RRID: AB_477593 ), mouse Anti-Vinculin (1:2000, Sigma Aldrich, Cat# v4505, RRID: AB_477617 ), HRP mouse anti-beta Actin (1:10,000, Abcam, Cat# ab8224, RRID: AB_449644 ).

Techniques: Western Blot, Control, MANN-WHITNEY

Journal: eLife

Article Title: Downregulation of Dickkopf-3, a Wnt antagonist elevated in Alzheimer’s disease, restores synapse integrity and memory in a disease mouse model

doi: 10.7554/eLife.89453

Figure Lengend Snippet:

Article Snippet: Primary antibodies and dilutions used for western blot were: Mouse Anti- Aβ (6E10 clone, 1:1000, Biolegend, Cat# 803001, RRID: AB_2564653 ), goat anti- Mouse DKK-3 (1:1000, R and D Systems, Cat# AF948, RRID: AB_355734 ), goat anti- Human DKK-3 (1:1000, R&D Systems, Cat# AF1118, RRID: AB_354610 ), rabbit anti-GAPDH (1:5000, Abcam, Cat# ab181602, RRID: AB_2630358 ), rabbit anti-GluA1 (1:1000, Cell Signaling Technology, Cat# 13185, RRID: AB_2732897 ), rabbit anti-phospho GluA1 Serine 845 (1:1000, Cell Signaling Technology, Cat# 8084, RRID: AB_10860773 ), rabbit anti- SAPK/JNK (1:1,000, Cell Signaling Technology, Cat# 9252, RRID: AB_2250373 ), mouse anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:500, Cell Signaling Technology, Cat# 9255, RRID: AB_2307321 ), mouse anti-Tubulin (1:5000, Sigma-Aldrich, Cat# T9026, RRID: AB_477593 ), mouse Anti-Vinculin (1:2000, Sigma Aldrich, Cat# v4505, RRID: AB_477617 ), HRP mouse anti-beta Actin (1:10,000, Abcam, Cat# ab8224, RRID: AB_449644 ).

Techniques: Isolation, Knock-Out, Control, Sequencing, shRNA, Recombinant, TUNEL Assay, cDNA Synthesis, Fluorsave, Software, Microscopy, Immunofluorescence, Patch Clamp

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