The Mechanism Of Cystatin B Gene Deletion Induced Neuroprotection In PrP (106-126) Mediated Impairment

Prion disease is a kind of consumptive, fatal and neurodegenerative disease which caused by the aggregation of the wrong folding proteins. It is characterized by PrPSc accumulation, the vacuolization of the brain tissue and astrocyte proliferation. Pervious study revealed that autophagy plays an important role in against neurodegenerative diseases which acused by misfolded protein aggregates, while, enhanced autophagy can obviously reduce the apoptosis of neurons. Cystatins, a cysteine proteinase inhibitor, have been found to be important in neurodegeneration. This study focused on the mechanism of the Cystatin B deletion mediated neuroprotective effect. Using siRNA, Western Blot, TUNEL and Immunofluorescence technique studies the mechanism of Cystatin B gene deletion induced neuroprotection in Prp polypeptide-mediated impairment. Results showed that,(1) First useing siRNA cutely knockdown Cystatin B, knockdown efficiency was measured at the protein level by Western blotting, then PrP (106-126)-treated and assay by MTT. N2a cells were exposed to Prp (106-126), cell viability was decreased and with Cystatin B gene deletion cell viability was increased. This result showed that Cystatin B gene deletion reduced PrP (106-126)-induced neuron cell death.(2) Next expriment set three groups, such as negative group, PrP (106-126) treatments and after transfection teated with PrP(106-126). Then experiment detected the mitochondrial proteins and JC-1 by Western Blot and immunofluorescence respectively. The result was PrP (106-126)-induced Bax translocation to the mitochondria and cytochrome C release to the cytosol in N2a cells, PrP (106-126)-induced Bax translocation and cytochrome C release were inhibited by Cystatin B siRNA treatment. Fluorescence microscopy images showed cells with green fluorescence (JC-1 monomer form) after PrP (106-126) treatment, indicating lower MTP, while the negative control cells and Cystatin B siRNA-treated with PrP (106-126) cells displayed high green and red fluorescence (JC-1 aggregates form), indicating high MTP values. Collectively, these results are consistent with the idea that Cystatin B gene deletion blocked PrP (106-126)-induced mitochondrial dysfunction.(3) Using TUNEL assaied PrP (106-126)-induced apoptosis wihich treated with Cystatin B siRNA. TUNEL assay showed that PrP (106-126)-treated have more green fluorescent highlights than Cystatin B gene deletion with PrP (106-126) had more green fluorescent highlights means more cells apoptosis. Thus, Cystatin B gene deletion inhibited PrP (106-126)-induced neuron cells apoptosis.(4) Using siRNA knockdown ATG5 or adding autophagy inhibitor 3-MA do more studies about LC3 and Lamp protein level on N2a cells. The reults was that PrP (106-126)-treated cells showed LC3 and Lamp protein level higher than negative, however, Cystatin B gene deletion treated cells with PrP(106-126)group showed LC3 and Lamp protein level higher than PrP (106-126)-treated N2a cell. But when exposed Cystatin B gene deletion cells to PrP (106-126) and treatment with ATG5 siRNA or 3-MA, LC3 and Lamp protein level was drceased. Accordingly, Cystatin B gene deletion is associated with enhanced autophagy.(5) While, exposed Cystatin B gene deletion N2a cells to PrP (106-126), the cell viability was significantly higher (P<0.05) than PrP (106-126)-treated, then exposed Cystatin B gene deletion N2a cells to PrP (106-126) with ATG5 siRNA or 3-MA the cell viability was significantly lower than just treatment with Cystatin B siRNA and PrP (106-126). From the result we know that Cystatin B gene deletion reduced PrP (106-126)-induced neuron cell death via activated autophagy pathway.(6) Cells was treated similar with (5) and tested cytochrome C and JC-1. We found that exposed Cystatin B and ATG5 gene deletion to PrP (106-126), the result showed Bax translocation to the mitochondria and cytochrome C release to the cytosol in N2a cells, JC-1 test showing green fluorescence and indicating low MTP values. This result was similar with 3-MA group. This date indicating Cystatin B gene deletion reduced PrP (106-126)-induced neuron mitochondrial impairment via activated autophagy pathway.(7) testing neuron cell apoptosis by TUNEL, which PrP (106-126)-treated Cystatin B gene deletion N2a cells and treatement with 3-MA. Cystatin B gene deletion cells was PrP (106-126)-treated displayed decreased cell apoptosis, on the other hand, when added 3-MA led to increased cell apoptosis. From this result we got that Cystatin B gene deletion reduced PrP(106-126)-induced neuronal apoptosis via activated autophagy pathway.Consistent with these results, the mechanism of Cystatin B gene deletion induced neuroprotection in PrP polypeptide-mediated impairment is via activation of autophagy which regulate neural cells mitochondrial homeostasis. Taken together, this study preliminarily clarified the mechanism of Cystatin B deletion in preventing prion disease and this research provides a theoretical basis for screening of targeted blocking drugs for the Prion disease treatment.