Neuroprotection in cerebral ischemia by neutralization of 3-aminopropanal

Abstract

Cerebral ischemia stimulates increased activity of polyamine oxidase, a ubiquitous enzyme that catabolizes polyamines to produce 3-aminopropanal. 3-Aminopropanal is a reactive aldehyde that mediates progressive neuronal necrosis and glial apoptosis. Here we report that increased levels of 3-aminopropanal-modified protein levels in humans after aneurysmal subarachnoid hemorrhage correlate with the degree of cerebral injury as measured by admission Hunt/Hess grade. In vitro screening of clinically approved drugs reveals that N-2-mercaptopropionyl glycine (N-2-MPG), an agent clinically approved for prevention of renal stones in patients with cysteinuria, significantly inhibits the cytotoxicity of 3-aminopropanal. N-2-MPG reacts with 3-aminopropanal to yield a nontoxic thioacetal adduct, as confirmed by electrospray ionization mass spectroscopy. Administration of N-2-MPG in clinically relevant doses to rats significantly reduces cerebral 3-aminopropanal-modified protein immunoreactivity and infarct volume in a standardized model of middle cerebral artery occlusion, even when the agent is administered after the onset of ischemia. These results implicate 3-aminopropanal as a therapeutic target for cerebral ischemia.

Figures

Figure 1

3-Aminopropanal (3-AP)-protein adducts are elevated in CSF of patients with severe acute neurological injury and poor grade SAH patients, as compared with good-grade patients. Levels of 3-aminopropanal-protein adducts were estimated immunochemically by using dot blots as described in Materials and Methods.P < 0.0001 good prognosis vs. poor prognosis; P < 0.05 Hunt/Hess grade I/II vs. Hunt/Hess grade IV/V.

Figure 3

N-2-MPG reduces 3-aminopropanal production during cerebral ischemia. Experimental MCAO, and N-2-MPG treatment where indicated, were performed as described inMaterials and Methods. At 24 h, the brains were removed, and a section of the brain corresponding to the area of MCAO was sectioned and stained by immunofluorescence using antibodies against 3-aminopropanal-modified proteins as described inMaterials and Methods. Pictures taken are representative of observations from 3–4 animals/group. All pictures were taken at ×10 magnification. (A) Detection of intense immunofluorescence for 3-aminopropanal-modified proteins in the anatomic region of cerebral ischemia in the ischemic hemisphere. The arrow shows intracellular 3-aminopropanal-modified protein fluorescence of scattered pattern in the ischemic penumbra. (B) 3-Aminopropanal-protein immunofluorescence was not detected in the opposite, nonischemic brain hemisphere. (C)N-2-MPG treatment reduces the overall area and intensity of 3-aminopropanal-modified protein immunofluorescence. (D) 3-Aminopropanal-protein immunofluorescence was not detected in the opposite, nonischemic brain hemisphere ofN-2-MPG treated animals. Schematic diagrams designate in green the approximate area and location of the fluorescent pictures shown, and the hatched areas represent the approximate area of intense immunofluorescence for 3-aminopropanal-modified proteins.