Cerebral ischemia enhances polyamine oxidation: identification of enzymatically formed 3-aminopropanal as an endogenous mediator of neuronal and glial cell death

S Ivanova, G I Botchkina, Y Al-Abed, M Meistrell 3rd, F Batliwalla, J M Dubinsky, C Iadecola, H Wang, P K Gregersen, J W Eaton, K J Tracey, S Ivanova, G I Botchkina, Y Al-Abed, M Meistrell 3rd, F Batliwalla, J M Dubinsky, C Iadecola, H Wang, P K Gregersen, J W Eaton, K J Tracey

Abstract

To elucidate endogenous mechanisms underlying cerebral damage during ischemia, brain polyamine oxidase activity was measured in rats subjected to permanent occlusion of the middle cerebral artery. Brain polyamine oxidase activity was increased significantly within 2 h after the onset of ischemia in brain homogenates (15.8 +/- 0.9 nmol/h/mg protein) as compared with homogenates prepared from the normally perfused contralateral side (7.4 +/- 0.5 nmol/h/mg protein) (P <0.05). The major catabolic products of polyamine oxidase are putrescine and 3-aminopropanal. Although 3-aminopropanal is a potent cytotoxin, essential information was previously lacking on whether 3-aminopropanal is produced during cerebral ischemia. We now report that 3-aminopropanal accumulates in the ischemic brain within 2 h after permanent forebrain ischemia in rats. Cytotoxic levels of 3-aminopropanal are achieved before the onset of significant cerebral cell damage, and increase in a time-dependent manner with spreading neuronal and glial cell death. Glial cell cultures exposed to 3-aminopropanal undergo apoptosis (LD50 = 160 microM), whereas neurons are killed by necrotic mechanisms (LD50 = 90 microM). The tetrapeptide caspase 1 inhibitor (Ac-YVAD-CMK) prevents 3-aminopropanal-mediated apoptosis in glial cells. Finally, treatment of rats with two structurally distinct inhibitors of polyamine oxidase (aminoguanidine and chloroquine) attenuates brain polyamine oxidase activity, prevents the production of 3-aminopropanal, and significantly protects against the development of ischemic brain damage in vivo. Considered together, these results indicate that polyamine oxidase-derived 3-aminopropanal is a mediator of the brain damaging sequelae of cerebral ischemia, which can be therapeutically modulated.

Figures

Figure 1
Figure 1
Polyamine oxidase activity increases during cerebral ischemia, and is inhibited by aminoguanidine and chloroquine. Polyamine oxidase activity was measured in brain homogenates prepared as described in Methods. Data shown are mean ± SE; n = 3. Normal, sham-operated control brain homogenate; Ischemia Vehicle, homogenate prepared 2 h after the onset of middle cerebral artery occlusion; Ischemia AG, addition of aminoguanidine (1 mM) at time = −5 min before spermine; Ischemia CHLQ, addition of chloroquine (1 mM) at time = −5 min before spermine. *, P <0.05 versus normal; #, P <0.05 versus ischemia vehicle.
Figure 2
Figure 2
(a) 1H-NMR spectroscopy (DMSO-d6 and CDCl3, 270 MHz) of the products of reacting 3-aminopropanal with 2,4-dinitrophenylhydrazine. NMR revealed the presence of anti and syn isomers with resonance at δ8.83 and δ11.35. (Inset) Structure of the principle condensation products. (b) Electrospray ionization mass spectrum of synthetic dansylated 3-aminopropanal-2,4-dinitrophenylhydrazone. 3-aminopropanal was derivatized with 2,4-dinitrophenylhydrazine and dansyl chloride, and the reaction products were subjected to HPLC as outlined in Materials and Methods. The inset shows the HPLC profile of the separable geometric isomers. Note that the EIMS of the HPLC-purified fractions revealed the expected molecular ion at m/z 251. (c) EIMS of derivatized ischemic brain homogenate. Animals were subjected to permanent focal cerebral ischemia and after 25 h, brain tissue was obtained for homogenization and derivatization as described in Materials and Methods. The inset shows the HPLC profile, and the mass spectrum confirms the expected molecular ion of the HPLC-purified fractions at m/z 251.
Figure 2
Figure 2
(a) 1H-NMR spectroscopy (DMSO-d6 and CDCl3, 270 MHz) of the products of reacting 3-aminopropanal with 2,4-dinitrophenylhydrazine. NMR revealed the presence of anti and syn isomers with resonance at δ8.83 and δ11.35. (Inset) Structure of the principle condensation products. (b) Electrospray ionization mass spectrum of synthetic dansylated 3-aminopropanal-2,4-dinitrophenylhydrazone. 3-aminopropanal was derivatized with 2,4-dinitrophenylhydrazine and dansyl chloride, and the reaction products were subjected to HPLC as outlined in Materials and Methods. The inset shows the HPLC profile of the separable geometric isomers. Note that the EIMS of the HPLC-purified fractions revealed the expected molecular ion at m/z 251. (c) EIMS of derivatized ischemic brain homogenate. Animals were subjected to permanent focal cerebral ischemia and after 25 h, brain tissue was obtained for homogenization and derivatization as described in Materials and Methods. The inset shows the HPLC profile, and the mass spectrum confirms the expected molecular ion of the HPLC-purified fractions at m/z 251.
Figure 2
Figure 2
(a) 1H-NMR spectroscopy (DMSO-d6 and CDCl3, 270 MHz) of the products of reacting 3-aminopropanal with 2,4-dinitrophenylhydrazine. NMR revealed the presence of anti and syn isomers with resonance at δ8.83 and δ11.35. (Inset) Structure of the principle condensation products. (b) Electrospray ionization mass spectrum of synthetic dansylated 3-aminopropanal-2,4-dinitrophenylhydrazone. 3-aminopropanal was derivatized with 2,4-dinitrophenylhydrazine and dansyl chloride, and the reaction products were subjected to HPLC as outlined in Materials and Methods. The inset shows the HPLC profile of the separable geometric isomers. Note that the EIMS of the HPLC-purified fractions revealed the expected molecular ion at m/z 251. (c) EIMS of derivatized ischemic brain homogenate. Animals were subjected to permanent focal cerebral ischemia and after 25 h, brain tissue was obtained for homogenization and derivatization as described in Materials and Methods. The inset shows the HPLC profile, and the mass spectrum confirms the expected molecular ion of the HPLC-purified fractions at m/z 251.
Figure 3
Figure 3
Brain 3-aminopropanal levels increase during cerebral ischemia. Brain 3-aminopropanal levels were measured by HPLC in rats subjected to permanent focal cerebral ischemia. 3-Aminopropanal was not detected in sham-operated controls. Note that 3-aminopropanal tissue levels increased markedly within 2 h after middle cerebral artery occlusion, and continued to increase for at least 25 h. Data shown are mean ± SE, n = 3 animals/group. *, P <0.05 versus t = 0 h by analysis of variance.
Figure 4
Figure 4
Brain damaging effects of intracortically administered polyamines, and protection with polyamine oxidase inhibitors. (a) Brain damage mediated by intracortical microinjection of spermine, spermidine, and 3-aminopropanal, but not putrescine. Data shown are volume of brain damage (mm3) as measured by integrating the area of negative TTC staining over the entire brain hemisphere in animals injected with the polyamines shown; mean ± SE, n = 6–8/group. *, P <0.05 versus vehicle. (b) Aminoguanidine and chloroquine protection against intracortical spermine toxicity. All animals received intracortical spermine (25 μg in 2 μl) by stereotactically guided microinjection. Experimental animals were treated with aminoguanidine or chloroquine simultaneously with the intracortical spermine injectate in the following doses: systemic aminoguanidine was 320 mg/kg intraperitoneal 30-min pretreatment followed by subsequent doses of 110 mg/kg intraperitoneally each 8 h after intracortical spermine; intracortical aminoguanidine was administered by a single dose (320 mg/kg) given simultaneously with intracortical spermine; chloroquine was administered by a single intraperitoneal dose (25 mg/kg) 30 min before the spermine injection. Data shown are infarct volume (mm3) assessed quantitatively 48 h after the intracortical spermine injection (mean ± SE, n = 6–8/group). *, P <0.05 versus spermine/vehicle.
Figure 4
Figure 4
Brain damaging effects of intracortically administered polyamines, and protection with polyamine oxidase inhibitors. (a) Brain damage mediated by intracortical microinjection of spermine, spermidine, and 3-aminopropanal, but not putrescine. Data shown are volume of brain damage (mm3) as measured by integrating the area of negative TTC staining over the entire brain hemisphere in animals injected with the polyamines shown; mean ± SE, n = 6–8/group. *, P <0.05 versus vehicle. (b) Aminoguanidine and chloroquine protection against intracortical spermine toxicity. All animals received intracortical spermine (25 μg in 2 μl) by stereotactically guided microinjection. Experimental animals were treated with aminoguanidine or chloroquine simultaneously with the intracortical spermine injectate in the following doses: systemic aminoguanidine was 320 mg/kg intraperitoneal 30-min pretreatment followed by subsequent doses of 110 mg/kg intraperitoneally each 8 h after intracortical spermine; intracortical aminoguanidine was administered by a single dose (320 mg/kg) given simultaneously with intracortical spermine; chloroquine was administered by a single intraperitoneal dose (25 mg/kg) 30 min before the spermine injection. Data shown are infarct volume (mm3) assessed quantitatively 48 h after the intracortical spermine injection (mean ± SE, n = 6–8/group). *, P <0.05 versus spermine/vehicle.
Figure 5
Figure 5
Histology of 3-aminopropanal–induced cell death. Animals were anesthetized and perfused intracardially with 100 ml saline followed by 200–300 ml of 4% formaldehyde in PBS. Brains were removed, postfixed for 2 hr in the same fixative, and then incubated overnight in 30% sucrose at 4°C. Ten micrometer sections were prepared and airdried. TUNEL staining was performed as outlined in Methods. (A) Low magnification microphotograph demonstrating the site of microinjecting needle (large arrow) and areas of 3-aminopropanal–induced prominent cell death (small arrows). (B) High power microphotograph taken within the marked area showing coexistence of apoptosis (TUNEL-positive cell on the right) and necrosis (eosin-stained cell on the left). (C) High magnification of area indicated in A showing the extent of cellular degeneration.
Figure 6
Figure 6
Apoptosis in glial cells exposed to 3-aminopropanal. (a) DNA gel electrophoresis of glial-like and neuronal-like cells exposed to 3-aminopropanal for 13 h as described in Materials and Methods; 7.5 μg of DNA was loaded per lane for the glial cells (lanes B and C), and 15 μg was loaded per lane for the neuronal cells (lanes D and E). Lane A, 1 kb DNA size marker; lane B, 3-aminopropanal–treated (160 μM); lane C, vehicle control; lane D, vehicle-treated; lane E, 3-aminopropanal–treated (90 μM). (b) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated glial cells (160 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, 76% of the cell population stained TUNEL positive. (c) FACS® histogram of vehicle-treated glial cells revealed no evidence of apoptosis (0.13% TUNEL-positive cells). (d) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated neuronal cells (90 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, only 5.02% of the cell population of stained TUNEL positive, though a forward/side scatter of the same cell population revealed 55.7% cell death upon electronic gating (data not shown). (e) FACS® histogram of vehicle-treated neuronal cells showed no evidence of apoptosis (0.53% TUNEL-positive cells). Electronic gating for forward/side scatter showed 9.1% cell death under these conditions. (  f  ) Annexin V FITC/propidium iodide (PI) staining of 3-aminopropanal– treated neuronal cells, as above, revealed no evidence of apoptosis (1.8% apoptotic cells). Necrotic cells comprised 68.97% of the cell population. (g) Annexin V FITC/propidium iodide staining of vehicle-treated neuronal cells revealed no evidence of apoptosis (1.02% of the cell population) or necrosis (1.58% of the cell population).
Figure 6
Figure 6
Apoptosis in glial cells exposed to 3-aminopropanal. (a) DNA gel electrophoresis of glial-like and neuronal-like cells exposed to 3-aminopropanal for 13 h as described in Materials and Methods; 7.5 μg of DNA was loaded per lane for the glial cells (lanes B and C), and 15 μg was loaded per lane for the neuronal cells (lanes D and E). Lane A, 1 kb DNA size marker; lane B, 3-aminopropanal–treated (160 μM); lane C, vehicle control; lane D, vehicle-treated; lane E, 3-aminopropanal–treated (90 μM). (b) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated glial cells (160 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, 76% of the cell population stained TUNEL positive. (c) FACS® histogram of vehicle-treated glial cells revealed no evidence of apoptosis (0.13% TUNEL-positive cells). (d) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated neuronal cells (90 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, only 5.02% of the cell population of stained TUNEL positive, though a forward/side scatter of the same cell population revealed 55.7% cell death upon electronic gating (data not shown). (e) FACS® histogram of vehicle-treated neuronal cells showed no evidence of apoptosis (0.53% TUNEL-positive cells). Electronic gating for forward/side scatter showed 9.1% cell death under these conditions. (  f  ) Annexin V FITC/propidium iodide (PI) staining of 3-aminopropanal– treated neuronal cells, as above, revealed no evidence of apoptosis (1.8% apoptotic cells). Necrotic cells comprised 68.97% of the cell population. (g) Annexin V FITC/propidium iodide staining of vehicle-treated neuronal cells revealed no evidence of apoptosis (1.02% of the cell population) or necrosis (1.58% of the cell population).
Figure 6
Figure 6
Apoptosis in glial cells exposed to 3-aminopropanal. (a) DNA gel electrophoresis of glial-like and neuronal-like cells exposed to 3-aminopropanal for 13 h as described in Materials and Methods; 7.5 μg of DNA was loaded per lane for the glial cells (lanes B and C), and 15 μg was loaded per lane for the neuronal cells (lanes D and E). Lane A, 1 kb DNA size marker; lane B, 3-aminopropanal–treated (160 μM); lane C, vehicle control; lane D, vehicle-treated; lane E, 3-aminopropanal–treated (90 μM). (b) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated glial cells (160 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, 76% of the cell population stained TUNEL positive. (c) FACS® histogram of vehicle-treated glial cells revealed no evidence of apoptosis (0.13% TUNEL-positive cells). (d) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated neuronal cells (90 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, only 5.02% of the cell population of stained TUNEL positive, though a forward/side scatter of the same cell population revealed 55.7% cell death upon electronic gating (data not shown). (e) FACS® histogram of vehicle-treated neuronal cells showed no evidence of apoptosis (0.53% TUNEL-positive cells). Electronic gating for forward/side scatter showed 9.1% cell death under these conditions. (  f  ) Annexin V FITC/propidium iodide (PI) staining of 3-aminopropanal– treated neuronal cells, as above, revealed no evidence of apoptosis (1.8% apoptotic cells). Necrotic cells comprised 68.97% of the cell population. (g) Annexin V FITC/propidium iodide staining of vehicle-treated neuronal cells revealed no evidence of apoptosis (1.02% of the cell population) or necrosis (1.58% of the cell population).
Figure 6
Figure 6
Apoptosis in glial cells exposed to 3-aminopropanal. (a) DNA gel electrophoresis of glial-like and neuronal-like cells exposed to 3-aminopropanal for 13 h as described in Materials and Methods; 7.5 μg of DNA was loaded per lane for the glial cells (lanes B and C), and 15 μg was loaded per lane for the neuronal cells (lanes D and E). Lane A, 1 kb DNA size marker; lane B, 3-aminopropanal–treated (160 μM); lane C, vehicle control; lane D, vehicle-treated; lane E, 3-aminopropanal–treated (90 μM). (b) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated glial cells (160 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, 76% of the cell population stained TUNEL positive. (c) FACS® histogram of vehicle-treated glial cells revealed no evidence of apoptosis (0.13% TUNEL-positive cells). (d) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated neuronal cells (90 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, only 5.02% of the cell population of stained TUNEL positive, though a forward/side scatter of the same cell population revealed 55.7% cell death upon electronic gating (data not shown). (e) FACS® histogram of vehicle-treated neuronal cells showed no evidence of apoptosis (0.53% TUNEL-positive cells). Electronic gating for forward/side scatter showed 9.1% cell death under these conditions. (  f  ) Annexin V FITC/propidium iodide (PI) staining of 3-aminopropanal– treated neuronal cells, as above, revealed no evidence of apoptosis (1.8% apoptotic cells). Necrotic cells comprised 68.97% of the cell population. (g) Annexin V FITC/propidium iodide staining of vehicle-treated neuronal cells revealed no evidence of apoptosis (1.02% of the cell population) or necrosis (1.58% of the cell population).
Figure 6
Figure 6
Apoptosis in glial cells exposed to 3-aminopropanal. (a) DNA gel electrophoresis of glial-like and neuronal-like cells exposed to 3-aminopropanal for 13 h as described in Materials and Methods; 7.5 μg of DNA was loaded per lane for the glial cells (lanes B and C), and 15 μg was loaded per lane for the neuronal cells (lanes D and E). Lane A, 1 kb DNA size marker; lane B, 3-aminopropanal–treated (160 μM); lane C, vehicle control; lane D, vehicle-treated; lane E, 3-aminopropanal–treated (90 μM). (b) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated glial cells (160 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, 76% of the cell population stained TUNEL positive. (c) FACS® histogram of vehicle-treated glial cells revealed no evidence of apoptosis (0.13% TUNEL-positive cells). (d) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated neuronal cells (90 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, only 5.02% of the cell population of stained TUNEL positive, though a forward/side scatter of the same cell population revealed 55.7% cell death upon electronic gating (data not shown). (e) FACS® histogram of vehicle-treated neuronal cells showed no evidence of apoptosis (0.53% TUNEL-positive cells). Electronic gating for forward/side scatter showed 9.1% cell death under these conditions. (  f  ) Annexin V FITC/propidium iodide (PI) staining of 3-aminopropanal– treated neuronal cells, as above, revealed no evidence of apoptosis (1.8% apoptotic cells). Necrotic cells comprised 68.97% of the cell population. (g) Annexin V FITC/propidium iodide staining of vehicle-treated neuronal cells revealed no evidence of apoptosis (1.02% of the cell population) or necrosis (1.58% of the cell population).
Figure 6
Figure 6
Apoptosis in glial cells exposed to 3-aminopropanal. (a) DNA gel electrophoresis of glial-like and neuronal-like cells exposed to 3-aminopropanal for 13 h as described in Materials and Methods; 7.5 μg of DNA was loaded per lane for the glial cells (lanes B and C), and 15 μg was loaded per lane for the neuronal cells (lanes D and E). Lane A, 1 kb DNA size marker; lane B, 3-aminopropanal–treated (160 μM); lane C, vehicle control; lane D, vehicle-treated; lane E, 3-aminopropanal–treated (90 μM). (b) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated glial cells (160 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, 76% of the cell population stained TUNEL positive. (c) FACS® histogram of vehicle-treated glial cells revealed no evidence of apoptosis (0.13% TUNEL-positive cells). (d) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated neuronal cells (90 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, only 5.02% of the cell population of stained TUNEL positive, though a forward/side scatter of the same cell population revealed 55.7% cell death upon electronic gating (data not shown). (e) FACS® histogram of vehicle-treated neuronal cells showed no evidence of apoptosis (0.53% TUNEL-positive cells). Electronic gating for forward/side scatter showed 9.1% cell death under these conditions. (  f  ) Annexin V FITC/propidium iodide (PI) staining of 3-aminopropanal– treated neuronal cells, as above, revealed no evidence of apoptosis (1.8% apoptotic cells). Necrotic cells comprised 68.97% of the cell population. (g) Annexin V FITC/propidium iodide staining of vehicle-treated neuronal cells revealed no evidence of apoptosis (1.02% of the cell population) or necrosis (1.58% of the cell population).
Figure 6
Figure 6
Apoptosis in glial cells exposed to 3-aminopropanal. (a) DNA gel electrophoresis of glial-like and neuronal-like cells exposed to 3-aminopropanal for 13 h as described in Materials and Methods; 7.5 μg of DNA was loaded per lane for the glial cells (lanes B and C), and 15 μg was loaded per lane for the neuronal cells (lanes D and E). Lane A, 1 kb DNA size marker; lane B, 3-aminopropanal–treated (160 μM); lane C, vehicle control; lane D, vehicle-treated; lane E, 3-aminopropanal–treated (90 μM). (b) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated glial cells (160 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, 76% of the cell population stained TUNEL positive. (c) FACS® histogram of vehicle-treated glial cells revealed no evidence of apoptosis (0.13% TUNEL-positive cells). (d) FACS® histogram showing TUNEL staining of 3-aminopropanal–treated neuronal cells (90 μM, 13 h) as outlined in Materials and Methods. As indicated by the marker, only 5.02% of the cell population of stained TUNEL positive, though a forward/side scatter of the same cell population revealed 55.7% cell death upon electronic gating (data not shown). (e) FACS® histogram of vehicle-treated neuronal cells showed no evidence of apoptosis (0.53% TUNEL-positive cells). Electronic gating for forward/side scatter showed 9.1% cell death under these conditions. (  f  ) Annexin V FITC/propidium iodide (PI) staining of 3-aminopropanal– treated neuronal cells, as above, revealed no evidence of apoptosis (1.8% apoptotic cells). Necrotic cells comprised 68.97% of the cell population. (g) Annexin V FITC/propidium iodide staining of vehicle-treated neuronal cells revealed no evidence of apoptosis (1.02% of the cell population) or necrosis (1.58% of the cell population).
Figure 7
Figure 7
Inhibition of caspase 1 but not of caspase 3 blocks 3-aminopropanal–induced glial apoptosis. Cells were pretreated with (a) the caspase 1 inhibitor (Ac-YVAD-CMK) or (b) the caspase 3 inhibitor (Ac-DEVD-CHO) at concentrations 0.4 (triangles) or 40 μM (circles) for 3 h, followed by treatment with 3-aminopropanal for an additional 5 h, and then were analyzed for cell viability by the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay. Controls consisted of DMSO-treated cells (squares) to assess for nonspecific solvent effects. Data are mean ± SE, n = 3 wells/experiment.
Figure 7
Figure 7
Inhibition of caspase 1 but not of caspase 3 blocks 3-aminopropanal–induced glial apoptosis. Cells were pretreated with (a) the caspase 1 inhibitor (Ac-YVAD-CMK) or (b) the caspase 3 inhibitor (Ac-DEVD-CHO) at concentrations 0.4 (triangles) or 40 μM (circles) for 3 h, followed by treatment with 3-aminopropanal for an additional 5 h, and then were analyzed for cell viability by the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay. Controls consisted of DMSO-treated cells (squares) to assess for nonspecific solvent effects. Data are mean ± SE, n = 3 wells/experiment.

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