Polyamine catabolism is enhanced after traumatic brain injury

Abstract

Polyamines spermine and spermidine are highly regulated, ubiquitous aliphatic cations that maintain DNA structure and function as immunomodulators and as antioxidants. Polyamine homeostasis is disrupted after brain injuries, with concomitant generation of toxic metabolites that may contribute to secondary injuries. To test the hypothesis of increased brain polyamine catabolism after traumatic brain injury (TBI), we determined changes in catabolic enzymes and polyamine levels in the rat brain after lateral controlled cortical impact TBI. Spermine oxidase (SMO) catalyzes the degradation of spermine to spermidine, generating H2O2 and aminoaldehydes. Spermidine/spermine-N(1)-acetyltransferase (SSAT) catalyzes acetylation of these polyamines, and both are further oxidized in a reaction that generates putrescine, H2O2, and aminoaldehydes. In a rat cortical impact model of TBI, SSAT mRNA increased subacutely (6-24 h) after TBI in ipsilateral cortex and hippocampus. SMO mRNA levels were elevated late, from 3 to 7 days post-injury. Polyamine catabolism increased as well. Spermine levels were normal at 6 h and decreased slightly at 24 h, but were normal again by 72 h post-injury. Spermidine levels also decreased slightly (6-24 h), then increased by approximately 50% at 72 h post-injury. By contrast, normally low putrescine levels increased up to sixfold (6-72 h) after TBI. Moreover, N-acetylspermidine (but not N-acetylspermine) was detectable (24-72 h) near the site of injury, consistent with increased SSAT activity. None of these changes were seen in the contralateral hemisphere. Immunohistochemical confirmation indicated that SSAT and SMO were expressed throughout the brain. SSAT-immunoreactivity (SSAT-ir) increased in both neuronal and nonneuronal (likely glial) populations ipsilateral to injury. Interestingly, bilateral increases in cortical SSAT-ir neurons occurred at 72 h post-injury, whereas hippocampal changes occurred only ipsilaterally. Prolonged increases in brain polyamine catabolism are the likely cause of loss of homeostasis in this pathway. The potential for simple therapeutic interventions (e.g., polyamine supplementation or inhibition of polyamine oxidation) is an exciting implication of these studies.

FIG. 1.

Polyamine metabolism and back-conversion pathways.

FIG. 2.

SSAT mRNA levels are elevated in the cerebral cortex adjacent to the site of injury. (A) Northern blot showing continued elevation of SSAT mRNA (1.2 kb) out to 7 days post-injury. SSAT mRNA was detected on Northern blots of total RNA extracted from parietal cortex ipsilateral (top panel) and contralateral (bottom panel) to injury at serial time points after TBI. Sham animals exhibited a small, nonsignificant increase in SSAT over naïve rats. (B) Histogram showing the early and sustained changes in SSAT mRNA levels in ipsilateral cortex after TBI (normalized to shams). Results from two independent studies, n ≥ 3 per time point. Cortex was freshly dissected (A) or microdissected (B) from 300-μm frozen sections. Brain injury induced as described in Methods.

FIG. 3.

Polyamine levels change over time in the cerebral cortex adjacent to injury. (A) Spermidine (left) and spermine (right) decreased transiently during the first day after TBI, and spermidine levels increased at 72 h post-injury, compared to shams and previous time points. (B) Putrescine levels (left) increased rapidly and remained elevated for at least 72 h post-injury. N¹-acetylspermidine (N1-acetylSPD) levels (right) were detectable in ipsilateral cortex at 24 to 72 h post-injury. Dotted lines show naïve values. Two-way ANOVA showed significant time and treatment effects, post hoc tests used grouped sham values because no differences were observed between sham groups. Sham operation increased putrescine by only a small fraction of the injury-induced changes. ^§p < 0.01 vs. shams; *p ≤ 0.05 vs. shams; ^ap ≤ 0.05 shams vs. naïves.

FIG. 4.

SSAT immunoreactivity in parietal cortex at 72 h post-injury. (A) Cortical neurons (arrowheads) as well as smaller glial cells (arrows) infero-lateral to the site of injury express low levels of SSAT-ir at 72 h post-injury. (B) Control section with no primary antibody from a cortical field of the same brain as in (A). (C, D) Parietal cortex SSAT-ir ipsilateral (C) and contralateral (D) to injury (same section, same exposure and processing). (E, F) Merged FITC and Cy3 stains. (E) Injured parietal cortex showing merged neuN-ir (red), SSAT-ir (green) staining (double-stained cells are yellow). Several asterisks indicate pyknotic neuronal cells below. (F) Sham parietal cortex showing SSAT-ir (green) and neuN-ir (red). Note the paucity of neuN stain in (E), and the reduced number and intensity of SSAT-ir cells in (F). Scale bars = 50 μm.

FIG. 5.

SSAT immunoreactivity in the hippocampus at 72 h post-injury. Hippocampal neurons as well as other cells show SSAT immunoreactivity both ipsilateral and contralateral (not shown) to injury. (A) Merged SSAT-ir (green) and neuN-ir (red) stains: hippocampal neurons beneath the site of injury (upper right) show higher levels of SSAT-ir. Cells in both the corpus callosum and deep cortex (lower right) also stain positive for SSAT. (B–D) Hippocampal pyramidal neurons (B, neuN, arrow), as well as other cell types (C, SSAT, arrows) show SSAT-ir. (D) Merge of (B) and (C) showing neuN and SSAT colocalization (arrowheads). Scale bars = 50 μm.

FIG. 6.

Quantification of SSAT immunoreactivity: bilateral increases in SSAT. Increased SSAT-ir in the injured cortex and hippocampus at 72 h post-injury. Cortex shows bilateral increases, while hippocampus shows ipsilateral increases in SSAT-ir. Densitometric analyses using mean integrated pixel density per field, as described in Methods. *p < 0.01, Tukey test, §p < 0.05, Dunnett.