okadaic-acid and 3-methyladenine

Autophagosomes are accumulated in Alzheimer's disease (AD), but the regulatory pathway of autophagy in AD remains largely unknown. By using electron microscopy, Western blotting, and immunocytochemistry, here we show that autophagosomes are accumulated in rat neurons by okadaic acid (OA), a protein phosphatase-2A inhibitor known to enhance tau phosphorylation, beta-amyloid (Abeta) deposition, and neuronal death, which are the pathological hallmarks of AD. Autophagy can be generally induced via several distinct pathways, such as inhibition of mTOR or activation of beclin-1. Interestingly, OA increased both mTOR and beclin-1 pathways simultaneously, which suggests that autophagy in OA-treated neurons is induced mainly via the beclin-1 pathway, and less so via mTOR inhibition. Finally, inhibition of autophagy by 3MA reduced cytotoxicity in OA-treated neurons. Our novel findings provide new insights into the pathology of and therapeutic intervention for AD.

Topics: Adenine; Alzheimer Disease; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Blotting, Western; Enzyme Inhibitors; Immunohistochemistry; Lysosomes; Microscopy, Electron, Transmission; Neurons; Neuroprotective Agents; Okadaic Acid; Phagosomes; Protein Kinases; Rats; TOR Serine-Threonine Kinases

The aim of the present study was to elucidate the mechanism responsible for the high mannose glycoprotein instability in undifferentiated HT-29 cells (a human colon cancer cell line) reported previously. The results presented here are consistent with lysosomal degradation of these molecular species. In addition inhibitors of the autophagic-lysosomal degradative pathway (3-methyladenine, okadaic acid and asparagine) dramatically block the degradation of proteins and N-linked glycoproteins in undifferentiated HT-29 cells. The main conclusions of this work are: 1- the autophagic-lysosomal pathway is responsible for the high mannose glycoprotein degradation in undifferentiated HT-29 cells; 2- this degradative pathway exists in differentiated cells but is greatly reduced (3.5-4 fold); 3- the HT-29 cell line is a new model to investigate the molecular regulation of autophagy.

Topics: Adenine; Adenocarcinoma; Asparagine; Autophagy; Carbon Radioisotopes; Carcinogens; Cell Differentiation; Cell Line; Chloroquine; Colonic Neoplasms; Ethers, Cyclic; Glycoproteins; Humans; Kinetics; Leucine; Lysosomes; Mannose; Okadaic Acid; Polysaccharides; Tritium; Tumor Cells, Cultured