Stroke, defined as an abnormality in brain function resulting from disruption of cerebral circulation, is one of the leading causes of death in the U.S. Even when a stroke does not result in death, the costs it imposes on the victim may include serious physical and emotional damage, which may result in loss of productivity. These costs stem from the tremendous damage done to the victim's brain by the stroke. With a reduction in oxygen and glucose, cells display a rapid disruption of protein synthesis, depletion of intracellular energy stores, destabilization of the cell membrane, and activation of the NMDA receptor, leading to excitotoxic and oxidative cell damage in the brain. In an attempt to survive and repair the oxidative damage and return the cell to homeostasis, numerous compensatory energy-consuming processes are activated. However, over-activation of these pathways can deleterious, further depleting cellular energy, and resulting in further brain damage. Such brain damage is, generally, irreversible. Accordingly, a method of protecting brain tissue from damage during a stroke (neuroprotection) would be tremendously important.
AMP-activated protein kinase (AMPK), a member of a metabolite-sensing protein kinase family, is a known sensor of peripheral energy balance (Carting D., “The AMP-activated protein kinase cascade—a unifying system for energy control.” Trends Biochem Sci 6:314 (2): 580-585, 2004.) AMPK is a heterotrimeric protein composed of a catalytic α subunit (α1 or α2), and 2 regulatory subunits (β and γ). AMPK is phosphorylated and activated when cellular energy levels are low. AMPK in turn regulates cellular metabolism and chronically regulates gene expression to restore ATP levels. Increases in the AMP/ATP ratio, changes in cellular pH and redox status, and increases in the creatine/phosphocreatine ratio are known to activate AMPK (Hardie D G, Salt I P, Hawley S A, Davies S P, “AMP-activated protein kinase: an ultrasensitive system for monitoring cellular energy charge,” Biochem J 338:717-22, 1999; Hawley S A, Davison M, Woods A, et al., “Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase,” J Biol Chem 271:27879-87, 1996.) AMPK increases fatty acid oxidation and restricts fatty acid synthesis in an attempt to augment ATP levels in energy-depleted cells. However, in neurons that have a restricted capacity for fatty acid oxidation, this effect could be deleterious (Almeida A, Moncada S, Bolanos J P, “Nitric oxide switches on glycolysis through the AMP protein kinase and 6-phosphofructo-2-kinase pathway,” Nature Cell Biology 6:45-51, 2004).
Inhibition of fatty acid synthase (FAS), the enzyme responsible for the de novo synthesis of palmitate, with C75, a synthetic FAS inhibitor disclosed in U.S. Pat. No. 5,981,575 (incorporated herein by reference), increases ATP levels in a number of cell types, including neurons. AMPK is highly expressed in neurons in the hypothalamus, where it appears to play a role in the regulation of food intake. Hypothalamic phosphorylated AMPK (pAMPK) is increased with starvation; C75 treatment inactivates and dephosphorylates AMPK, and induces profound anorexia.
The consequences of AMPK activation in neurons that do not have access to energy supplies is unknown. Until the present invention, it has been unclear whether AMPK activation during stress was protective or damaging. There have been no prior studies examining the role of AMPK in stroke.