Fig. 7

Altered expression of genes associated with Purkinje cell function and neurological disorders are manifest in Smox/Sat1-dKO mice. The changes in the expression of mRNAs and proteins of interest identified by RNA-seq were confirmed by Northern blot, Western blot, or immunofluorescence microscopic analyses. a Northern blot analysis of selected transcripts known to be associated with Purkinje cell function and movement disorders in RNA samples from the cerebellum and cerebrum of Wt, Sat1-KO, Smox-KO, and Smox/Sat1-dKO mice revealed that while their levels remained unchanged in the cerebrum their expression was reduced in the cerebellum of Smox/Sat1-dKO mice. b Western blot and immunofluorescence microscopy studies indicate that the expression of CALB1, a Purkinje cell marker, decreased in the cerebellum of Smox/Sat1-dKO versus Wt mice (white arrows point out the areas of Purkinje cell drop out). c Western blot analyses indicate that the expression of IBA1, a microglia marker, and GFAP, an indicator of astrocytosis, increased in the cerebellum of Smox/Sat1-dKO mice as early as 8 weeks of age. Immunofluorescence microscopy studies indicate that the expression of IBA1 (red) and GFAP (green) increases in the cerebellum of Smox/Sat1-dKO mice as early as 8 weeks of age. The image of Smox/Sat1-dKO cerebellum at 14+ weeks of age was taken at a reduced exposure to mitigate the loss of definition caused by intensity of the fluorescence signal. d Northern blot analysis of Tgm2 and Tgm1 mRNA in the cerebellum and cerebrum of Wt, Sat1-KO, Smox-KO, and Smox/Sat1-dKO mice (top panel) indicates that the expression of both transcripts is elevated in the cerebellum of Smox/Sat1-dKO mice only. Also, it should be noted that the mRNA levels of Tgm1 are significantly lower than Tgm2 mRNA levels. Comparison of age-matched Wt and Smox/Sat1-dKO mice reveals that increased expression of Tgm2 is apparent as early as 4 weeks of age in Smox/Sat1-dKO mice (middle panel). Immunofluorescence microscopic images of comparable cerebellar regions examining the expression of TGM2 in the cerebellum of Wt, 4 weeks, 8 weeks, and 14+-week-old Smox/Sat1-dKO mice (white arrows point to TGM2 positive cells). Northern blots contained 20 μg of RNA/lane and Western blots contained 50 μg of protein/lane. All analyses are representative of three independent sets of experiments