Roles Of An Autophagy/lysosomal Pathway In Excitotoxicity Mediated By Kainic Acid Receptor

Aim: To study the role of an autophagy/lysosomal pathway in excitotoxicity mediated by kainic acid (KA) receptor.Methods: Rat excitotoxic model was produced with stereotaxic administration of KA into unilateral striatum. KA induced internucleosomal DNA fragmentation was detected with agarose gel electrophoresis. Autophagosomes were observed with electron microscopy. Protein levels of cathepsin B and cathepsin L were determined with Western blot analysis. Cellular location of cathepsin B was detected with double immunofluoresnce. Effects of 3-MA or Z-FA-FMK on KA-induced cathepsin B activation were detected with enzyme activity assay. 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 KA-induced increases in the level of lysosomal-associated membrane protein 1 (Lamp-1) and decreases in the level of Bcl-2 were detected with immunohistochemistry. Effects of 3-MA or Z-FA-FMK on KA-induced Bcl-2 downregulation, caspase-3 activation, and releasing of cyto-c from mitochondria to cytoplasm were detected with Western blot analysis.Results: Internucleosomal DNA fragmentation occurred in the KA-injected (1.25-5.0 nmol) striatum in a dose-dependent fashion. Electron microscopy revealed that the formation of autophagosomes in the ipsilateral striatum started 1 hour and reached maximum 8 hours after injection of KA. Western blot analysis revealed the increase in cathepsin B level in a time-dependent fashion, while there was no change in the level of cathepsin L. A number of cathepsin B-positive neurons were seen 24 hr after KA administration, especially in those excitotoxic damaged striatal neurons. Pre-treatment with 3-MA and Z-FA-FMK inhibited KA-induced increases in cathepsin B activity (P<0.01). Pretreatment with 3-MA and Z-FA-FMK attenuated KA-induced internucleosomal DNA fragmentation and significantly reduced the loss of striatal neurons (P<0.01). Immunohistochemistry demonstrated that 3-MA and Z-FA-FMK blocked KA-induced increases in protein level of Lamp-1 and prevented decreases in protein level ofBcl-2. 3-MA and Z-FA-FMK inhibited KA-induced Bcl-2 downregulation, caspase-3 activation, and releasing of cyto-c from mitochondria to cytoplasm.Conclusions: The increase of autophagosomes and the activation of lysosomes may be involved in the excitotoxicity mediated by KA receptor. Autophagy inhibitors and lysosomal inhibitors have neuroprotective actions in against KA-induced apoptotic death of rat stritatal neurons by inhibiting autophagy/lysosome-mediated apoptotic signaling pathways.