Fig 2: Deficiency of TRIM32 decreases dendritic arborization and spine density of PCs. (A) Representative micrographs of stained Golgi of single PCs from TRIM32-/– and WT mice. (B,C) Quantitative analysis of the somata area (B) and dendritic tree area (C) of PCs. (D) Sholl analysis of the numbers of the dendrites at different distances from the cell body. (E) High magnified images of distal dendritic branching of PCs. (F) Quantitative analysis of the distal dendritic spine density. Scale bar = 20 µm (A), 2 µm (E). Data are presented as mean ± SEM. n = 5 mice/genotype. *p < 0.05; **p < 0.01; # p < 0.008. For the statistical analysis in D, t-test with Bonferroni correction was performed, p < 0.05/6 was considered statistically significant.

Fig 3: TRIM32 deficiency impairs motor coordination and motor learning. (A) Scores assigned to representative postures in the hindlimb clasping test. (B) Hind-limb clasping of TRIM32-/– and WT mice (aged, 10 months) was assessed. (C,D) Beam walk test. As compared with WT mice, the performance of TRIM32-/– mice was poor in regard to the duration to cross the beam (C), freeze time (D), and the number of limb slips (E) while crossing the beam. (F) For the vertical pole test, the time to turn around and climb down was quantitated. As compared with control mice, the performance of TRIM32-/– mice was significantly poorer. (G) Accelerating -rotarod performance was tested. The time TRIM32-/– and WT mice remained on the rotarod before falling off was assessed. (H) The body of weight of TRIM32-/– mice and the WT littermate controls were measured at ages of 6 and 10 months. (I) For the hangwire test, the latency to fall off by the TRIM32-/– and WT mice were assessed at the ages of 6 and 10 months. TRIM32-/– mice displayed impaired motor coordination and muscle deficits. (J) Muscular strength of TRIM32-/– and WT mice at the ages of 6 and 10 months was evaluated using the traction test. TRIM32-/– mice had weaker forelimb strength. Data are presented as mean ± SEM. n = 13 (TRIM32-/– mice), = 15 (WT mice). * p < 0.05, *** p < 0.001, # p < 0.008; ## p < 0.001. For the statistical analysis in G, t-test with Bonferroni correction was performed, p < 0.05/6 was considered statistically significant.

Fig 4: Deficiency of TRIM32 changes levels of INPP5A and IP3. (A) Western blot analysis of levels of INPP5A and calbindin in TRIM32-/– mice and their littermates. (B, C) Relative levels of INPP5A (B) and calbindin (C) were quantified. (D) ELISA assay analysis of level of IP3. Data are presented as mean ± SEM. n = 5 mice/genotype. ** p < 0.01, ***p < 0.001.

Fig 5: The numbers of PFs-PCs and CFs-PCs were reduced in the TRIM32-/– cerebellum. (A) PFs-PCs and PCs of the cerebellum were stained with antibodies against vGlut1 and calbindin. (B) Mean fluorescence intensity of vGlut1 in TRIM32-/– mice and WT mice. (C) CFs-PCs and PCs of the cerebellum were immunostained with antibodies against vGlut2 and calbindin. (D) Quantification of vGlut2-positive puncta in TRIM32-/– mice and WT mice. Scale bar = 10 µm (A), 50 µm (C). Data are presented as mean ± SEM. n = 5 mice/genotype. *** p < 0.001.