Abstract 3744: Brain's High Constitutive Background Autophagy Level Is Disrupted by Stroke Injury and Post-stroke Pharmacological Induction of Autophagy Reduces Lesion Size
Background: Autophagy is a highly regulated and dynamic process that allows cells to recycle aging organelles, bulk proteins and lipids for nutritional reuse. Autophagy occurs in all cell types and body tissues and is induced by oxidative stress or lack of nutrients to promote survival. Excessive autophagy, however, can lead to cell death, including apoptotic (PCD type I) and autophagic cell death (PCD Type II). Recent literature reports that autophagy is disregulated during stroke and other brain injuries. We hypothesized that the normally high innate level of autophagy in the brain undergoes aberrant regulation during stroke injury and this aberrant regulation correlates to changes in stroke lesion size.
Methods: We induced ischemia in C57BL/6J mice using a permanent MCA ligation model, with sacrifice 48 hours after stroke. For time course analysis mice were sacrificed at 3, 6, 12, 24, 48, or 72 hours after stroke. Mice were treated i.p. with chloroquine (30, 60, or 90 mg/K/d for 2 days), an autophagy inhibitor, rapamycin (1.25 or 2.5 mg/K/d for 2 days), an autophagy activator via inhibition of mTOR, or treated with vehicle (saline or 10% DMSO in 100 ul). The autophagy markers LC3, p62 and Beclin-1 were analyzed by Western blot and immunohistochemistry and infarct size was measured by TTC staining.
Results: Brain showed consistently higher baseline levels of LC3II than other tissues. Chloroquine and rapamycin both reduced lesion size, the former between 28 & 34% and the later, in a dose dependent fashion, between 60 and 65%. Beclin-1 is decreased in the infarct side of the brain and treatment with rapamycin enhanced this depletion while treatment with chloroquine increased Beclin-1 levels. LC3II is decreased in the infarct side and does not increase with chloroquine treatment on the infarct side, suggesting that stroke injury may inhibit the high innate baseline level of autophagy in the brain. Rapamycin led to increased LC3II and p62, consistent with increased induction of autophagy. Time course analysis of stroked brains showed that LC3II and Beclin-1 are initially decreased at 3 to 6 hours post injury, then increased above non-injured between 12 and 24 hours before it falls below baseline at 48 to 72 hours.
Conclusions: 1) Stroke appears to inhibit the high innate baseline levels of autophagy in the brain after the injury. 2) Since chloroquine and rapamycin both decreased lesion size, this suggests cross-talk between autophagy and other programmed cell death pathways and/or direct effects of the drugs on these and other pathways. 3) The rapid decrease in autophagy markers post injury indicates that modulation of autophagy may offer a new therapeutic avenue for stroke.
- © 2012 by American Heart Association, Inc.