Roles Of An Autophagy/lysosomal Pathway In Neurotoxicity Mediated By Mitochondrial Dysfunction

Aim: To study the role of an autophagy/lysosomal pathway in neurotoxicity mediated by mitochondrial dysfunction induced with 3-Nitropropionic Acid (3-NP).Methods: Rats neurotoxicity model were produced with stereotaxic administration of 3-NP into unilateral striatum. 3-NP induced intemucleosomal DNA fragmentation was detected with agarose gel electrophorcsis. Autophagosomes were observed with electron microscopy. Protein levels of cathepsin B, cathepsin D, caspase-3, bcl-2 and the releasing of cyto-c from mitochondria were determined with Western blot analysis. The neuroprotective effects of the autophagy inhibitor 3-MA and the lysosomal cathepsin B inhibitor Z-FA-FMK were assessed with internucleosomal DNA fragmentation and Cresyl violet staining. Effects of 3-MA or Z-FA-FMK on 3-NP-induced increases in the level of cathepsin B, cathepsin D, and caspase-3 were detected with western blot. Effects of 3-MA or Z-FA-FMK on 3-NP-induced Bcl-2 downregulation and releasing of cyto-c from mitochondria to cytoplasm were detected with Western blot analysis.Results: Intemucleosomal DNA fragmentation occurred in the 3-NP-injected (125-500 nmol) striatum in a dose-dependent fashion. Electron microscopy revealed that the formation of autophagosomes in the ipsilateral striatum started 2 hour and reached maximum 4 hours after injection of 3-NP. Western blot analysis revealed the increase in cathepsin B, pro-caspase-3 level and cathepsin D activation. Bcl-2 protein levels decreased in a time-dependent fashion. Cyto-c released from mitochondria to cytoplasm 6h after 3-NP injection. Pretreatment with 3-MA and Z-FA-FMK attenuated 3-NP-induced intemucleosomal DNA fragmentation and significantly reduced the striatal lesion area (P<0.01). Treatment with 3-MA 2h after 3-NP injection also attenuated 3-NP-induced intemucleosomal DNA fragmentation. 3-MA and Z-FA-FMK inhibited 3-NP-induced Bcl-2 downregulation as well as pro-caspase-3, cathepsin B, cathepsinD upregulation. 3-MA also inhibited releasing of cyto-c from mitochondria to cytoplasm.