Mechanisms of ischemic neuroprotection by acetyl-L-carnitine

Abstract

Acetyl-L-carnitine is a naturally occurring substance that, when administered at supraphysiologic concentrations, is neuroprotective in several animal models of global and focal cerebral ischemia. Three primary mechanisms of action are supported by neurochemical outcome measures performed with these models and with in vitro models of acute neuronal cell death. The metabolic hypothesis is based on the oxidative metabolism of the acetyl component of acetyl-L-carnitine and is a simple explanation for the reduction in postischemic brain lactate levels and elevation of ATP seen with drug administration. The antioxidant mechanism is supported by reduction of oxidative stress markers, for example, protein oxidation, in both brain tissue and cerebrospinal fluid. The relatively uncharacterized mechanism of inhibiting excitotoxicity could be extremely important in both acute brain injury and chronic neurodegenerative disorders. New experiments performed with primary cultures of rat cortical neurons indicate that the presence of acetyl-L-carnitine significantly inhibits both acute and delayed cell death following exposure to NMDA, an excitotoxic glutamate antagonist. Finally, several other mechanisms of action are possible, including a neurotrophic effect of acetyl-L-carnitine and inhibition of mitochondrial permeability transition. While the multiple potential mechanisms of neuroprotection by acetyl-L-carnitine limit an accurate designation of the most important mode of action, they are compatible with the concept that several brain injury pathways must be inhibited to optimize therapeutic efficacy.

FIGURE 1

Possible metabolism of acetyl-L-carnitine (ALCAR) after cerebral ischemia. ALCAR may serve as an exogenous, alternative source of acetyl-CoA, thereby promoting aerobic energy metabolism via the electron transport chain (ETC), reducing tissue acidosis, and improving neurologic outcome after cerebral ischemia due to cardiac arrest or stroke. The pyruvate dehydrogenase complex (PDH) is a target of reactive oxygen species (ROS) and is inhibited following cerebral ischemia. Such inhibition may be responsible for chronically elevated brain lactate levels following ischemic episodes as this enzyme constitutes the bridge between aerobic and anaerobic cerebral energy metabolism.

FIGURE 2

Possible shift in cellular redox state caused by ALCAR metabolism. ALCAR can be metabolized to acetyl-CoA by the carnitine acetyltransferase (CAT) reaction. Metabolism of acetyl-CoA by the tricarboxylic acid (TCA) cycle provides NADH that is then used for oxidative phosphorylation (OXPHOS) and for reducing intramitochondrial NADP to form NADPH via the transhydrogenase (TH) reaction. Elevation of intramitochondrial citrate results in transport out to the cytosol, where it is converted to isocitrate. The cytosolic isocitrate dehydrogenase (ICDH) reaction oxidizes isocitrate to form α-ketoglutarate (αKG) and NADPH. NADPH provides the reducing power for converting oxidized glutathione (GSSG) to reduced glutathione (GSH) via the glutathione reductase (GR) reaction. Reduced glutathione provides the reducing power for detoxifying H₂O₂ and organic peroxides via the glutathione peroxidase (GP) reaction. Intramitochondrial NADPH is also used to detoxify peroxides by an intramitochondrial glutathione reductase/peroxidase system (not shown).

FIGURE 3

Protection by ALCAR against NMDA-induced acute neuronal death. Cultured cortical neurons (DIV 10–14) were exposed to 100 μM NMDA for 30 min and immediately examined for cell death using the calcein-AM/PI ratio (live/dead). Exposure to NMDA resulted in significant neuronal cell death as compared to controls: 64.3± 12.9% live cells in the NMDA group vs. 91.8± 2.4% live cells in the control group (*P = 0.007 vs. control). In the presence of 1 mM ALCAR, cell death after exposure to NMDA was not significant: 86.2± 3.4% live cells (P = 0.5 vs. control). Differences between the mean were determined by ANOVA, and a Holm-Sidak test was applied for pairwise multiple comparisons. Results are shown as the mean ± SEM for n = 3 experiments.

FIGURE 4

Protection by ALCAR against NMDA-induced delayed neuronal death. Cultured cortical neurons (DIV 10–14) were exposed to 100 μM NMDA for 30 min and examined for cell death at 24 h postinjury using the calcein-AM/PI ratio (live/dead). Exposure to NMDA for 30 min followed by a 24-h recovery period resulted in significant neuronal cell death as compared to controls: 33.7± 9.6% live cells in the NMDA group vs. 91.8± 12.9% live cells in the control group (*P = 0.005 vs. control). In neurons treated with ALCAR during NMDA exposure and during the recovery period, cell death was not significantly different than controls (74.4± 9.9% live cells; P = 0.09 vs. control) and significantly less than with NMDA in the absence of ALCAR (*P = 0.006 vs. NMDA). Differences between the mean were determined by ANOVA, and a Holm-Sidak test was applied for pairwise multiple comparisons. Results are shown as the mean ± SEM for n = 3 experiments.