Journal: Advanced Science

Article Title: Allosteric Modulation of Pathological Ataxin‐3 Aggregation: A Path to Spinocerebellar Ataxia Type‐3 Therapies

doi: 10.1002/advs.202502216

Figure Lengend Snippet: CLR01 reverts the loss of glutamatergic synapses in cortical neurons caused by pathological Atx3. Rat cortical neurons were transfected with plasmids encoding eGFP, wild‐type eGFP‐Atx3 28Q, or mutant eGFP‐Atx3 84Q. a) Neurons were immunolabeled for MAP2, PSD95, and vGLUT1. Excitatory synapses were detected as PSD95 puncta that colocalized with vGLUT1; scale bar = 10 µm. b) Integrated fluorescence intensity of PSD95 puncta colocalized with vGLUT1 ( n = 33–36 neurons per condition in 3 independent experiments). Statistical analyses were performed using Kruskal–Wallis and Dunn's post‐hoc test (Table , Supporting Information). c) Neurons incubated with 10 µ m CLR03 or CLR01 were immunolabeled for MAP2, PSD95, and VGluT1 clusters; scale bar = 10 µm. d) Integrated fluorescence intensity of PSD95 puncta colocalized with VGluT1 ( n = 35–41 neurons per condition in each of 3 independent experiments. Statistical analyses were performed using Kruskal–Wallis and Dunn's post‐hoc tests (Table , Supporting Information). In panels (c) and (d), boxes show the 25th and 75th percentiles, whiskers range from the minimum to the maximum values, and the horizontal line shows the median value. Mean is represented by “+.” e) GFP‐Atx3 accumulates in the cell body of a fraction of transfected cortical cultures treated with CLR03 or CLR01; scale bars = 300, 15 µm. f) Percentage of cells with GFP‐Atx3 aggregates ( n = 3 independent experiments, 31–35 neurons per condition). Statistical analyses were performed using two‐way ANOVA and Sidak's multiple comparisons post‐hoc tests (Table , Supporting Information). p ‐values are indicated in the graphs shown in panels (b), (d), and (f). Data are represented as mean ± SEM.

Article Snippet: To stain for excitatory synapses, anti‐PSD95 antibody (mouse, 1:500, Cell Signaling Technology) and anti‐VGLUT1 antibody (guinea pig, 1:1000, Merck Millipore) were used; dendrites were identified with anti‐MAP2 antibody (chicken, 1:5000, Abcam).

Techniques: Transfection, Mutagenesis, Immunolabeling, Fluorescence, Incubation