Effect of intracerebellar microinjection of cdc2-siRNA on the coordination of mice with Niemann-Pick disease type C

Abstract

Objective To observe the effect of inhibiting the abnormal activation of cdc2 gene on the coordination of mice with Niemann-Pick disease type C(NPC). Methods Recombinant adeno-associated virus( rAAV) encoding cdc2-siRNA was packaged, and then was injected into the cerebellum of 2 weeks old npc-/- mice. Footprint test and vertical screen test were performed to assess the coordination of mice at the age of 8 weeks. Purkinje cells visualized by HE staining in cerebellum were counted, and the phosphorylation of microtubule-associated protein Tau recognized by PHF-1 antibody was detected by immunoblotting technology. Results (1) Footprint test showed that the stride length in cdc2-siRNA npc-/- group((4.92±0.31) cm) was markedly longer than that in empty vector npc-/- group((4.05±0.19) cm)(P<0.05). (2)Vertical screen test showed that the latency to turn head upwards or reach the upper edge of the screen in cdc2-siRNA npc-/- group((26.01±1.82) s, (50.93±1.98) s) was significantly shorter than that in the empty vector npc-/- group((31.96±3.47)s, (56.89±2.97)s), respectively(P<0.05 for all comparisons). (3)The number of Purkinje cells in cerebellum was dramatically increased in cdc2-siRNA npc-/- group(11.0±2.5) compared with the empty vector npc-/- group(5.1±2.2)(P<0.05). (4)The relative optical densities of cdc2 and phosphorylated Tau immunoreactive bands in cdc2-siRNA npc-/- group(1.42±0.22, 0.95±0.31)were significantly lower than those in the empty vector npc-/- group(2.11±0.29, 2.61±0.62), respectively(P<0.05 for all comparisons). Conclusion Inhibiting the abnormal activation of cdc2 gene can improve the coordination of npc-/- mice by ameliorating Purkinje cell’s loss and reducing the hyperphosphorylation of Tau in cerebellum. Key words: Niemann-Pick disease type C; Coordination; Cell division cycle 2; Purkinje cell