2009 sona meeting program

POSTER #82 SIRT3 OVEREXPRESSION PROTECTS DIFFERENTIATED PC12 CELLS FROM GLUCOSE DEPRIVATION OR OXYGEN-GLUCOSE DEPRIVATION. Natalya Shulyakova, PhD Student, Department of Physiology, University of Toronto; James H. Eubanks, PhD, University of Toronto, and Linda R. Mills, PhD, University of Toronto.

SIRT3 is a mammalian sirtuin targeted to mitochondria. In non-neuronal cells SIRT3 overexpression increases cellular respiration efficiency, and decreases levels of reactive oxygen species. SIRT3 is present in the brain but there is little data on SIRT3 in neurons. We hypothesized that over-expression of SIRT3 would be neuroprotective and diminish cell vulnerability to oxidative stress. Differentiated PC12 cells were transfected with pTracer-CMV2SIRT3 plasmid; transfection efficiency was 43.4 ± 2.7% after 24h. MTT reduction assays showed that cell viability was decreased for the first 4 days post-transfection but subsequently recovered.

Confocal imaging using Rhodamine123 revealed that mitochondrial membrane potential was decreased in SIRT3 overexpressing cells but mitochondrial morphology was normal. PC12 cells were challenged with glucose deprivation (GD) or oxygen-glucose deprivation (OGD). GD was induced by incubating cells for 4 hours in glucose/glutamine deficient medium (-/- medium) plus 10mM 2deoxy-D-glucose (2DG) and 10µM CCCP. To induce OGD cells were incubated with (-/-) medium plus 2DG, and placed in an anoxic environment for 5 hours. Cell death was quantified using confocal microscopy and PI as follows: % of dead transfected cells = (# of dead (PI+) transfected cells/ # of transfected cells) x 100%. SIRT3 overexpression was neuroprotective for GD; after a GD challenge followed by 15 hrs reperfusion (RP), cell death was 39.7 ± 9.3 % in SIRT-3 overexpressing cells versus 72.5 ± 9 % in controls transfected with plckGFP. SIRT3 overexpression also reduced cell death after OGD; 20 hrs post OGD/RP cell death in SIRT3 transfected cells was significantly less (10.4±5.1%) than in controls transfected with plckGFP (32.0 ±11.7%). These data are the first evidence that SIRT3 over-expression is protective in a neuronal model of oxidative stress.