Mild impairment of the citric acid cycle in neurodegenerative diseases promotes mitophagy/autophagy
Tuesday, Nov 12, 2013, 10:00 AM -11:00 AM
++C.03.m. Alzheimer's disease: In vitro therapeutics
, T. DENTON
, G. E. GIBSON
Neurosci., Weill-Cornell Med. Col. at Burke Med. Res. Inst., White Plains, NY;
Eastern Washington Univ., Cheney, WA
: Decreased mitochondrial function, as reflected by reduced brain glucose utilization, is an invariant feature of Alzheimer’s disease (AD). Indeed, diminished mitochondrial function is a common feature of many age-related neurodegenerative diseases. The alpha-ketoglutarate dehydrogenase complex (KGDHC), a key enzyme complex in mitochondrial energy production, is diminished in AD and many other age-related neurodegenerative disorders. If diminished mitochondrial function is either an initiating event or a critical step in a cascade of events, then the mitochondrial deficit should be linked to the abnormal accumulation of protein aggregates as well as to oxidative and mitophagic/autophagic stress, which are hallmarks of these disorders.
The goal of this study was to determine if inhibiting just one key mitochondrial enzyme, KGDHC, would initiate these processes.
Migration of cytosolic proteasomal proteins to the mitochondria initiates mitophagy (mitochondrial breakdown). Mitochondria and cytosol from SHSY5Y cells (control and treated) were isolated by differential centrifugation. Immunoblotting with antibodies to parkin and microtubule-associated protein light chain 3 (LC3) were performed to detect migration of several proteins from cytosol to different subcellular fractions. An increased LC3II/LC3I ratio in the mitochondrial fraction is an indicator of mitophagy. Confocal imaging was performed to confirm autophagy using green "autophagy detecting dye" that binds with autophagosomes. Cell-death was measured by trypan blue exclusion method and tetramethylrhodamine (TMRM) was used for detecting mitochondrial membrane potential.
: Carboxyethyl succinyl phosphonate (CESP) is a specific inhibitor of KGDHC that has been shown to penetrate into cells. CESP (100 µM, 5 h) diminished the mitochondrial membrane potential by about 40%. Under these conditions, cytosolic parkin declined by 25% while mitochondrial parkin increased by 20%. The LC3II/LC3I ratio increased in the mitochondrial fraction suggesting CESP induced mitophagy. Confocal imaging using the “autophagy detecting dye” confirmed the presence of autophagy in mitochondrial fraction after CESP treatment. This dye showed that other areas of the cell were also involved in autophagy. CESP treatment increased cell death by about 15%.
Conclusion & Significance:
The results suggest that proteasomal activators and/or activation of KGDHC may protect against toxicity due to altered protein translocation. An understanding of the link between mitochondria and proteasomal activity may promote the development of new therapeutic strategies.
Alpha-Ketoglutarate Deydrogenase Complex (KGDHC)
NIH grant AG14930
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