Fig 2: 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 3: TRIM32 is highly expressed in cerebellar PCs and absence of TRIM32 decreases the number of PCs. (A) Cerebellar sagittal sections were stained with specific antibodies against TRIM32. (B) Cerebellar sagittal sections were co-stained with antibodies against TRIM32 and calbindin. (C) Cerebellar sagittal sections were co-stained with antibodies against TRIM32 and NeuN. (D) PCs in the cerebellar sagittal sections were stained with antibodies against calbindin both in 6 months and 10 months old mice. (E) The number of PCs was quantified. Scale bar = 50 µm. Data are presented as mean ± SEM. n = 5 mice/genotype. **p < 0.01. ML, molecular layer; PL, Purkinje cells layer; GCL, Granule cells layer; WM, White matter.

Fig 4: TRIM32 deficiency reduces the size of the mid-aged cerebellum. (A) Macroscopic observation of whole cerebella from mid-aged TRIM32-/– and WT mice. (B) Reduced size of the mid-aged TRIM32-/– cerebellum compared with WT in gross morphology. (C) HandE staining sagittal sections of the cerebellum from mid-aged TRIM32-/– and littermate control mice. (D) A smaller cerebellar area in adult TRIM32-/– compared with control mice in sagittal sections. (E,F) Histograms representing the quantification of cerebellar area (E) and ML thickness (F) of mid-aged TRIM32-/– and WT mice. Scale bar = 2 mm (A), 500 µm (C). Data were presented as mean ± SEM. n = 5 mice/genotype. ** p < 0.01.

Fig 5: 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.