A role for protein arginine methyltransferase 7 in repetitive and mild traumatic brain injury

Abstract

Mild traumatic brain injury affects the largest proportion of individuals in the United States and world-wide. Pre-clinical studies of repetitive and mild traumatic brain injury (rmTBI) have been limited in their ability to recapitulate human pathology (i.e. diffuse rotational injury). We used the closed-head impact model of engineered rotation acceleration (CHIMERA) to simulate rotational injury observed in patients and to study the pathological outcomes post-rmTBI using C57BL/6J mice. Enhanced cytokine production was observed in both the cortex and hippocampus to suggest neuroinflammation. Furthermore, microglia were assessed via enhanced iba1 protein levels and morphological changes using immunofluorescence. In addition, LC/MS analyses revealed excess glutamate production, as well as diffuse axonal injury via Bielschowsky's silver stain kit. Moreover, the heterogeneous nature of rmTBI has made it challenging to identify drug therapies that address rmTBI, therefore we sought to identify novel targets in the concurrent rmTBI pathology. The pathophysiological findings correlated with a time-dependent decrease in protein arginine methyltransferase 7 (PRMT7) protein expression and activity post-rmTBI along with dysregulation of PRMT upstream mediators s-adenosylmethionine and methionine adenosyltransferase 2 (MAT2) in vivo. In addition, inhibition of the upstream mediator MAT2A using the HT22 hippocampal neuronal cell line suggest a mechanistic role for PRMT7 via MAT2A in vitro. Collectively, we have identified PRMT7 as a novel target in rmTBI pathology in vivo and a mechanistic link between PRMT7 and upstream mediator MAT2A in vitro.

Keywords: Methionine adenosyltransferase 2; PF-9366; Protein arginine methyltransferase; S-adenosylmethionine; Traumatic brain injury.

Fig. 1.

Loss of righting reflex (LRR) indicates loss of consciousness post-injury to validate repetitive and mild traumatic brain injury (rmTBI) in C57BL/6 male mice. (A) Cartoon of experimental design illustrating 3 repetitive hits separated by 24 h intervals at 0.7 J followed by assessment at 1, 3, and 7 days post-rmTBI. (B) We used a closed head impact model (CHIMERA) for the proposed TBI studies. The CHIMERA is a novel surgeiy-free model that allows for free rotation of the mouse brain to simulate real-world rotational and diffuse injury. (C) Loss of righting reflex was significantly increased in rmTBI (black line) mice as compared to age-matched (pink line) SHAM mice. Results were expressed as mean ± SEM. *p ≤ 0.05 as compared to age-matched rmTBI mice, evaluated by two-way ANOVA with Tukey’s post-hoc test.

Fig. 2.

Repetitive and mild traumatic brain injury (rmTBI) enhanced iba1 protein expression in the hippocampus and optic tract. (A) Iba1 was enhanced in the hippocampus 3-day & 7 day post-rmTBI as compared to age-matched 8–12-week SHAM mice (black bar). (B) Iba1 was enhanced in the cortex but was not statistically significant. (C–N) Iba1 was assessed via immunofluorescence to demonstrate enhanced microglia within the optic tract at 1, 3, and 7 days post-rmTBI relative to SHAM mice. Results were expressed as mean ± SEM. *p ≤ 0.05, as compared to age-matched rmTBI mice, evaluated by one-way ANOVA with Tukey’s post-hoc test, (n = 3). Scale bar = 100 μm.

Fig. 3.

Repetitive and mild traumatic brain injuiy (rmTBI) induced cytokine production in the cortex and hippocampus. Cytokine array was performed in both the hippocampus and cortex of SHAM and rmTBI mice. Cortex: (A) ICAM-1 was significantly increased at 7 days post-rmTBI (blue violin) relative to sham (black violin). (B) IL-6 was significantly enhanced at 1 and 3 days post-rmTBI (blue violins) relative to SHAM (black violin). (C) CD30L was significantly elevated 1 day post-rmTBI (blue violin) relative to SHAM (black violin). (D) IL-5 was significantly elevated 1-day post-rmTBI relative to SHAM (black violin) and significantly decreased at 3 days relative to 1 day (blue violin) post-rmTBI. (E) IL-10 was significantly enhanced at all time points 1, 3, and 7 days post-rmTBI relative to SHAM (black violin). Hippocampus: (F) IL-1α was significantly enhanced at 1-day post-rmTBI (blue violin) relative to sham (black violin). (G) CD30L was significantly elevated at 1-day post-rmTBI (blue violin) relative to SHAM (black violin), followed by a significant decrease at 7 day relative to 1-day post-rmTBI. (H) IL-12p70 was significantly increased at 1-day post-rmTBI (blue bar) relative to SHAM (black violin). Violin polts represent the distribution of individual mice, and the middle dotted line represents the median; with the other two dotted lines representing quartiles. Results were expressed as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 evaluated by one-way ANOVA with Tukey’s post-hoc test with SEM descriptive statistics, (n = 3–6).

Fig. 4.

PRMT7 protein was altered in the cerebral cortex after repetitive and mild traumatic brain injury (rmTBI) (A) PRMT7 protein expression was significantly decreased in the cortex at 3 and 7 days (blue bar) relative to SHAM (black bar) and remain unchanged in the (B) hippocampus as compared to 8–12 weeks age-matched (black bar) SHAM mice. (C) PRMT7 mRNA levels remain unchanged at 3 and 7 days post-rmTBI in the cortex (blue bars) and D) unchanged at 3 and 7 days (blue bars) in the hippocampus post-rmTBI as compared to sham (black bar). Results were expressed as mean ± SEM. *p ≤ 0.05, evaluated by one-way ANOVA with Tukey’s post-hoc test, (n = 3–8).

Fig. 5.

Monomethylarginine (MMA) production was decreased in the hippocampus after repetitive and mild traumatic brain injury (rmTBI). MMA, the catalytic end-product of PRMT7 was measured using capillary electrophoresis in the cortex and hippocampus. (A) MMA was unchanged in the cortex and significantly decreased in the (B) hippocampus (1, 3 and 7 day) as compared to SHAM mice (black bar). Results were expressed as mean ± SEM. *p ≤ 0.05, evaluated by one-way ANOVA with Tukey’s post-hoc test, (n = 3–6).

Fig. 6.

S-adenosylmethionine was increased 3 days post-rmTBI in the hippocampus after repetitive and mild traumatic brain injury (rmTBI). ELISA was performed to measure S-adenosylmethionine (SAM) levels in the cortex and hippocampus of mice tissue lysates. (A) There were no significant differences in SAM concentration in the cortex, however, the levels followed the same trend seen in the hippocampus. (B) There was a significant elevation of SAM levels within the hippocampus 3 days (blue bar) post-rmTBI relative to SHAM (black bar), followed by a significant decrease at 7 days (blue bar) relative to 3 days post-rmTBI. Results were expressed as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, evaluated by one-way ANOVA with Tukey’s post-hoc test, (n = 4–5).

Fig. 7.

MAT2A was decreased and MAT2B was increased in the hippocampus post-rmTBI. MAT2A and MATB were assessed via capillary electrophoresis. (A) MAT2A (catalytic subunit) had a trending decrease at 7 days (blue bar) relative to SHAM (black bar). (B) MAT2B (regulatory subunit) was significantly enhanced 7 days (blue bar) post-rmTBI relative to SHAM (black bar). (C–D) MAT2A and MAT2B were unchanged in the cerebral cortex post-rmTBI. Results were expressed as mean ± SEM. *p ≤ 0.05, evaluated by one-way ANOVA with Tukey’s post-hoc test, (n = 3–6).

Fig. 8.

MAT2A inhibitor reduced PRMT7 protein expression and MMA production. PF-9366 allosteric inhibitor was used (25 μM) to inhibit MAT2A in HT22 cells for 24 hrs. Inhibition of MAT2A significantly decreased (A) PRMT7 protein expression (B) decreased mono-methylarginine (MMa) production (C) increased MAT2A protein expression and significantly decreased (D) MAT2B protein expression. Results were expressed as mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 evaluated by student’s t-test. (n = 3).

Fig. 9.

Schematic diagram of methionine-adenosyltransferase 2 A/B in vivo. (A) MAT2A is the catalytic subunit and MAT2B is the regulatory subunit and can associate to form a heterotetramer to synthesize s-adenosylmethionine (SAM) via methionine and ATP. Our in vivo studies of rmTBI reveal a dysregulation in the methionine metabolism that is potentially contributing to downstream disruption of PRMT7 protein expression and activity. Our results suggest PRMT7 protein expression and activity are downregulated post-rmTBI and could be attributed to the upstream dysregulation in methionine metabolism. (B) Methionine-adenosyltransferase 2 A/B in vitro. Our in vitro studies in HT22 cells using the MAT2A inhibitor (PF-9366) indicate that MAT2A inhibition affects not only PRMT7 protein expression, but activity as indicated by the significant decrease in mono-methylarginine (MMA, PRMT7 end-product). Purple arrows = upregulation, red arrows = down-regulation. These results suggest a possible mechanism of PRMT7 post-rmTBI.