Journal: npj Imaging

Article Title: Quantitative multi-metabolite imaging of Parkinson’s disease using AI boosted molecular MRI

doi: 10.1038/s44303-025-00130-x

Figure Lengend Snippet: The white dashed lines in all panels mark the cortex-striatum boundary. a Glutamate (Glu) and DAPI staining. The white arrows highlight Glu-positive signals in the striatum, presenting an increase after MPTP administration. b Glu receptor (mGluR3) and DAPI staining. c Glu and mGluR3 receptor staining. The yellow arrows indicate double-stained areas in the striatum, demonstrating enhanced expression in the MPTP-treated mouse. d GFAP and DAPI staining, with an increased GFAP signal in the MPTP group, reflecting higher astrocytic activation (with DAPI providing background cell body visualization). e A merged image combining the signals from ( a , b , d ). f Coomassie blue staining. The increased staining intensity in the MPTP-treated brain suggests an increase in total protein content.

Article Snippet: Antibodies against glutamate (#AB5018, Sigma Aldrich), GFAP (#Ab4674, Abcam), and mGluR3 glutamate receptor (#AGC-010-GP, Alomone labs) were used for IHC tissue staining.

Techniques: Staining, Expressing, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A – C ) Representative current-voltage (I-V) plots of glutamate-elicited currents ( A and B ) and quantification ( C ) show the differential effect of coexpression of α2δ-1, α2δ-2, or α2δ-3 on GluA3 currents in HEK29 cells ( n = 11 cells for GluA3/empty vector [pcDNA], GluA3/α2δ-1, and GluA3/α2δ-2; n = 12 cells for GluA3/α2δ-3). ( D – F ) Representative I-V plots of glutamate-elicited currents ( D ) and mean current density ( E ) and rectification index ( F ) in HEK293 cells transfected with GluA2/A3 with either pcDNA or α2δ-1 ( n = 12 cells for GluA2/GluA3; n = 13 cells for GluA2/GluA3/α2δ-1). * P < 0.05, *** P < 0.001; 1-way ANOVA followed by Dunnett’s post hoc test in C ; 2-tailed Student’s t test in E and F . Data are expressed as means ± SEM. ( G and H ) Original confocal immunofluorescence images show the distribution of GluA3 (red) and GFP-tagged α2δ-1 (green) in HEK293 cells transfected with either GluA3/pcDNA or GluA3/α2δ-1-GFP. Areas in yellow boxes in G are magnified in H . Scale bars: 50 μm ( G ), 10 μm ( H ).

Article Snippet: After a brief rinse, the cells were blocked with 4% normal goat serum (Vector Laboratories), followed by immunolabeling with a rabbit anti-GluA3 antibody (1:100; 5117, Cell Signaling Technology) overnight at 4°C.

Techniques: Plasmid Preparation, Transfection, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A and B ) Representative immunoblot images ( A ) and quantification ( B ) show the distinct effect of coexpression of HA-tagged α2δ-1, α2δ-2, or α2δ-3 on GluA3 protein levels in HEK293 cells ( n = 8 independent experiments per group). GAPDH was used as the internal control for normalizing the protein levels on the same gel. *** P < 0.001; 1-way ANOVA followed by Dunnett’s post hoc test.

Article Snippet: After a brief rinse, the cells were blocked with 4% normal goat serum (Vector Laboratories), followed by immunolabeling with a rabbit anti-GluA3 antibody (1:100; 5117, Cell Signaling Technology) overnight at 4°C.

Techniques: Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A ) Representative immunoblot images and quantification show the concentration-dependent reduction in GluA3 protein levels induced by α2δ-1 coexpression in HEK293 cells ( n = 7 independent experiments per group). ( B ) Representative immunoblot images and quantification show the effect of coexpression with GFP on GluA3 protein levels in HEK293 cells ( n = 7 independent experiments per group). ( C ) Representative immunoblot images and quantification show the protein levels of GluA2 and GluA3 in HEK293 cells expressing GluA2/GluA3 with either empty vectors or α2δ-1 ( n = 6 independent experiments per group). ( D ) Representative immunoblot images and quantification show the protein levels of GluA3 and α2δ-1 in HEK293 cells expressing GluA3 with either empty vectors or α2δ-1 ( n = 6 independent experiments per group). GAPDH was used as the internal control for normalizing the protein levels on the same gel. ** P < 0.01, *** P < 0.001; 1-way ANOVA followed by Dunnett’s post hoc test in A and B ; 2-tailed Student’s t test in C and D . Data are expressed as means ± SEM.

Article Snippet: After a brief rinse, the cells were blocked with 4% normal goat serum (Vector Laboratories), followed by immunolabeling with a rabbit anti-GluA3 antibody (1:100; 5117, Cell Signaling Technology) overnight at 4°C.

Techniques: Western Blot, Concentration Assay, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A ) Original confocal images show the distribution of GluA3 (green), IB4 (red), and NeuN (blue) in the spinal dorsal horn of sham control and SNL rats. Scale bars: 100 μm (upper panels), 50 μm (lower panels). ( B and C ) Representative immunoblot images ( B ) and quantification ( C ) show the protein levels of α2δ-1 and GluA3 in the dorsal spinal cord of sham control and SNL rats. β-Actin served as the internal control for normalizing the protein levels on the same gel ( n = 8 mice per group). ( D and E ) Representative immunoblot images ( D ) and quantification ( E ) show the protein levels of GluA3 and GluA2/GluA3 complexes in the dorsal spinal cord from sham and SNL rats treated intrathecally with vehicle or 10 μg pregabalin (PGB; n = 9 rats per group) 3 weeks after surgery. Protein extracts from rat spinal cord tissues were immunoprecipitated using a rabbit GluA2 antibody or IgG. Immunoblotting was then performed using mouse GluA2, mouse GluA3, and mouse β-actin antibodies. β-Actin served as the internal control for normalizing GluA3 protein levels in the input. The corresponding immunoprecipitated GluA2 protein bands were used for normalizing GluA2/GluA3 protein complex levels. * P < 0.05, ** P < 0.01, *** P < 0.001; 2-tailed Student’s t test in C ; 2-way ANOVA followed by Tukey’s post hoc test in E . Data are expressed as means ± SEM.

Article Snippet: After a brief rinse, the cells were blocked with 4% normal goat serum (Vector Laboratories), followed by immunolabeling with a rabbit anti-GluA3 antibody (1:100; 5117, Cell Signaling Technology) overnight at 4°C.

Techniques: Control, Western Blot, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: Representative immunoblot images ( A and C ) and quantification show the total ( B ) and synaptosome ( D ) protein levels of GluA3 and α2δ-1 in the dorsal spinal cord of naive rats injected intrathecally with control lentiviruses or lentiviruses expressing Cacna2d1 ( n = 6 rats per group). β-Actin served as the internal control for normalizing the GluA3 and α2δ-1 protein levels on the same gel. PSD-95, a synaptic protein marker, served as the internal control for normalizing the GluA3 and α2δ-1 protein levels in synaptosome fractions. *** P < 0.001; 2-tailed Student’s t test. Data are expressed as means ± SEM.

Article Snippet: After a brief rinse, the cells were blocked with 4% normal goat serum (Vector Laboratories), followed by immunolabeling with a rabbit anti-GluA3 antibody (1:100; 5117, Cell Signaling Technology) overnight at 4°C.

Techniques: Western Blot, Injection, Control, Expressing, Marker

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A and B ) Representative immunoblot images ( A ) and quantification ( B ) show the basal protein levels of GluA3 in the dorsal spinal cord of WT and Cana2d1- KO mice ( n = 6 mice per group). ( C and D ) Representative immunoblot images ( C ) and quantification ( D ) show the protein levels of GluA3 and α2δ-1 in dorsal spinal cord tissues from WT and Cana2d1- KO mice subjected to sham or SNI surgery ( n = 11 mice per group). β-Actin served as the internal control for normalizing the GluA3 and α2δ-1 protein levels on the same gel. ** P < 0.01; 2-tailed Student’s t test. Data are expressed as means ± SEM.

Article Snippet: After a brief rinse, the cells were blocked with 4% normal goat serum (Vector Laboratories), followed by immunolabeling with a rabbit anti-GluA3 antibody (1:100; 5117, Cell Signaling Technology) overnight at 4°C.

Techniques: Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A and B ) Representative immunoblot images ( A ) and quantification ( B ) show GluA3 and α2δ-1 protein levels in HEK293 cells coexpressing GluA3 with YFP-tagged WT α2δ-1 or chimeric constructs [α2δ-1CT (α2δ-2) and α2δ-1CT (α2δ-3) ] ( n = 8 independent experiments per group). ( C and D ) Representative immunoblot images ( C ) and quantification ( D ) show the effects of treatment with control peptide (1 μM), α2δ-1CT peptide (1 μM), pregabalin (PGB; 20 μM), and MG132 (10 μM) on the GluA3 protein levels in HEK293 cells coexpressing α2δ-1 and GluA3 ( n = 9 independent experiments per group). PT, peptide. GAPDH was used as an internal control for normalizing the GluA3 protein levels on the same gel. *** P < 0.001; 1-way ANOVA followed by Dunnett’s post hoc test. Data are expressed as means ± SEM.

Article Snippet: After a brief rinse, the cells were blocked with 4% normal goat serum (Vector Laboratories), followed by immunolabeling with a rabbit anti-GluA3 antibody (1:100; 5117, Cell Signaling Technology) overnight at 4°C.

Techniques: Western Blot, Construct, Control

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A ) Time-course effects of intrathecal injection of 20 μg MG132 or vehicle (Veh) on hindpaw nociceptive thresholds in sham and SNL rats 3 weeks after surgery ( n = 9 rats per group). * P < 0.05, ** P < 0.01, *** P < 0.001 versus baseline (time 0); # P < 0.05, ### P < 0.001 versus Veh-SNL group at the same time point; 2-way ANOVA followed by Tukey’s post hoc test. ( B ) Representative immunoblot images and quantification show the effect of MG132 treatment on GluA3 protein levels in dorsal spinal cord tissues from SNL and sham rats ( n = 9 rats per group). * P < 0.05, *** P < 0.001; 1-way ANOVA followed by Tukey’s post hoc test. Data are expressed as means ± SEM.

Article Snippet: After a brief rinse, the cells were blocked with 4% normal goat serum (Vector Laboratories), followed by immunolabeling with a rabbit anti-GluA3 antibody (1:100; 5117, Cell Signaling Technology) overnight at 4°C.

Techniques: Injection, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A ) Representative immunoblot images show the ubiquitin protein levels in GluA3 precipitates from HEK293 cells expressing GluA3 with either pcDNA or α2δ-1 (similar data were obtained from 4 independent experiments). ( B ) Representative immunoblot images and quantification show the ubiquitin protein levels in GluA3 precipitates from the dorsal spinal cord of sham control and SNL rats ( n = 9 rats per group). Protein extracts from HEK293 cells or spinal cord tissues were immunoprecipitated using a rabbit GluA3 antibody or IgG. Immunoblotting was then conducted using mouse ubiquitin or mouse GluA3 antibodies. The corresponding GluA3 protein bands were used as the internal control on the same gel. ( C and D ) Representative immunoblot images ( C ) and quantification ( D ) show GluA3 protein levels in HEK293 cells expressing WT GluA3 or GluA3 mutants (K710R, K861R, and K887R) with and without α2δ-1 ( n = 12 independent experiments per group). GAPDH was used as the internal control for normalizing GluA3 and α2δ-1 protein levels on the same gel. ** P < 0.01, *** P < 0.001; 2-tailed Student’s t test in B ; 1-way ANOVA followed by Tukey’s post hoc test in D . Data are expressed as means ± SEM.

Article Snippet: After a brief rinse, the cells were blocked with 4% normal goat serum (Vector Laboratories), followed by immunolabeling with a rabbit anti-GluA3 antibody (1:100; 5117, Cell Signaling Technology) overnight at 4°C.

Techniques: Western Blot, Ubiquitin Proteomics, Expressing, Control, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: Spinal α 2 δ -1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

doi: 10.1172/JCI193349

Figure Lengend Snippet: ( A ) Changes in the hindpaw withdrawal thresholds of sham control and SNL rats 2 and 3 weeks after intrathecal injection of control (Cont) lentiviral vectors or lentiviral vectors expressing Gria3 ( n = 13 rats per group). ** P < 0.01, *** P < 0.001; 2-way ANOVA followed by Tukey’s post hoc test. ( B and C ) Representative immunoblot images ( B ) and quantification ( C ) show the protein levels of GluA3 and GluA2/GluA3 complexes in the dorsal spinal cords of sham control and SNL rats treated with intrathecal control lentiviruses or Gria3 -expressing lentiviruses ( n = 8 rats per group). Protein extracts from spinal cord tissues were immunoprecipitated (IP) using a GluA2 antibody or IgG. Immunoblotting was then conducted using GluA2, GluA3, and β-actin antibodies. β-Actin protein bands were used as the internal control on the same gel. ** P < 0.01, *** P < 0.001; 2-way ANOVA followed by Tukey’s post hoc test. ( D and E ) Representative recording traces ( D ) and quantification ( E ) show the differential effect of bath application of IEM-1460 (50 μM) on the amplitude of monosynaptic AMPAR-EPSCs in spinal lamina II neurons from SNL rats treated with intrathecal injection of control lentiviruses or Gria3 -expressing lentiviruses ( n = 18 neurons from 4 rats per group). Data were normalized to the baseline value (100%) before IEM-1460 application. * P < 0.05, ** P < 0.01 versus control vector group at the same time point; 2-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SEM.

Article Snippet: After a brief rinse, the cells were blocked with 4% normal goat serum (Vector Laboratories), followed by immunolabeling with a rabbit anti-GluA3 antibody (1:100; 5117, Cell Signaling Technology) overnight at 4°C.

Techniques: Control, Injection, Expressing, Western Blot, Immunoprecipitation, Plasmid Preparation

Journal: Brain, behavior, and immunity

Article Title: Anti-GluA3 autoantibodies define a new sub-population of frontotemporal lobar degeneration patients with distinct neuropathological features.

doi: 10.1016/j.bbi.2024.03.018

Figure Lengend Snippet: Fig. 3. Binge-like eating and impulsivity after chronic anti-GluA3 hIgGs injection. (A) Schematic representation of the Food Seeking task in the operant boxes: over a period of 40′, mice can choose between a nose-poke that delivers one palatable pellet of food (choice A) and a nose-poke that does not produce any effect (choice B); mice performance in the food seeking task expressed as (B) preference index (two-tailed unpaired t-test; t(14) = 0.7939, p = 0.4405, n = 9–7/group), (C) average of time between one food-related response and the subsequent one (i.e., avg food latency) (two-tailed unpaired t-test; t(14) = 3.146, p = 0.0072, n = 9–7/ group) and (D) number of food-related responses (choice A) (two-tailed unpaired t-test; t(14) = 2.169, p = 0.0478, n = 9–7/group); in (E-F) the number of food- related responses within the session in operant boxes is reported: in (E), the number of food-related responses is reported as a function of time (2way ANOVA; interaction: F(3,42) = 7.561, p = 0.0004, n = 9–7/group; Bonferroni correction) whereas in (F) is highlighted the time required to perform a certain number of food- related responses (2way ANOVA; interaction: F(62,868) = 7.925, p < 0.0001, n = 9–7/group). In (A-F) the task was performed by mice that underwent food re striction. (G) Mice performance in the Sucrose Preference Task expressed as Sucrose preference in percentage (two-tailed unpaired t-test; 12 hrs: t(14) = 2.907, p = 0.0115, n = 8–8/group; 24 h: t(13) = 0.5992, p = 0.5594, n = 7–8/group). Mice weight (H) at the end of the chronic treatment (D29) (two-tailed unpaired t-test; t (37) = 3.125, p = 0.0034, n = 17–22/group) and (I) monitored once a week (2way ANOVA; treatment: F(1,23) = 4.54, p = 0.0440, n = 11–14/group; Bonferroni correction). Bar graphs show mean ± s.e.m. n = number of animals. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. To apply two-tailed unpaired t-test, normal distribution was checked using D’Agostino & Pearson normality test or (when n insufficient) Shapiro-Wilk normality test.

Article Snippet: With this aim, we over-expressed a GFP-tagged human GluA3 (GFP-hGluA3, from Origene) plasmid into a non-neuronal immortalized cell line (HEK293) and we showed that purified antiGluA3 hIgGs (but not CTRL hIgGs) specifically recognized the overexpressed GluA3 subunit.

Techniques: Injection, Two Tailed Test

Journal: Brain, behavior, and immunity

Article Title: Anti-GluA3 autoantibodies define a new sub-population of frontotemporal lobar degeneration patients with distinct neuropathological features.

doi: 10.1016/j.bbi.2024.03.018

Figure Lengend Snippet: Fig. 4. PAM administration totally rescued anti-GluA3-hIgGs-mediated detrimental effects in terms of p-tau accumulation and loss of dendritic spines. (A) Scheme representing the timeline of rescue strategy administration to chronic animal model: mice receive daily intraperitoneal (IP) injection of PAM S 47445 from day 15 to day 29. (B) Western blot representative images and (C) bar graph of densitometric quantification of p-Tau (Ser202, Thr205, AT8) in TIF obtained from mice PFC at the end of chronic treatment with CTRL hIgGs and anti-GluA3 hIgGs ± PAM (Kruskal-Wallis; p = 0.0001, n = 26–21-15/group; Dunn’s correction). (D) Representative images showing dendrites of adult mice PFC at the end of the chronic treatment with CTRL hIgGs or anti-GluA3 hIgGs ± PAM and bar graphs representing (E) protrusion densities (ordinary one-way ANOVA; treatment: F(2.104) = 6.034, p = 0.0033, n = 43–39-25/group; Tukey correction). Bar graphs show mean ± s.e.m. In (C), n = number of animals; in (E), n = number of neurons. *P < 0.05; **P < 0.01; ***P < 0.001. To apply one-way ANOVA, normal distribution was checked using D’Agostino & Pearson normality test.

Article Snippet: With this aim, we over-expressed a GFP-tagged human GluA3 (GFP-hGluA3, from Origene) plasmid into a non-neuronal immortalized cell line (HEK293) and we showed that purified antiGluA3 hIgGs (but not CTRL hIgGs) specifically recognized the overexpressed GluA3 subunit.

Techniques: Animal Model, Injection, Western Blot