Neuroprotective Effects and Treatment Potential of Incretin Mimetics in a Murine Model of Mild Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) is a commonly occurring injury in sports, victims of motor vehicle accidents, and falls. TBI has become a pressing public health concern with no specific therapeutic treatment. Mild TBI (mTBI), which accounts for approximately 90% of all TBI cases, may frequently lead to long-lasting cognitive, behavioral, and emotional impairments. The incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gastrointestinal hormones that induce glucose-dependent insulin secretion, promote β-cell proliferation, and enhance resistance to apoptosis. GLP-1 mimetics are marketed as treatments for type 2 diabetes mellitus (T2DM) and are well tolerated. Both GLP-1 and GIP mimetics have shown neuroprotective properties in animal models of Parkinson's and Alzheimer's disease. The aim of this study is to evaluate the potential neuroprotective effects of liraglutide, a GLP-1 analog, and twincretin, a dual GLP-1R/GIPR agonist, in a murine mTBI model. First, we subjected mice to mTBI using a weight-drop device and, thereafter, administered liraglutide or twincretin as a 7-day regimen of subcutaneous (s.c.) injections. We then investigated the effects of these drugs on mTBI-induced cognitive impairments, neurodegeneration, and neuroinflammation. Finally, we assessed their effects on neuroprotective proteins expression that are downstream to GLP-1R/GIPR activation; specifically, PI3K and PKA phosphorylation. Both drugs ameliorated mTBI-induced cognitive impairments evaluated by the novel object recognition (NOR) and the Y-maze paradigms in which neither anxiety nor locomotor activity were confounds, as the latter were unaffected by either mTBI or drugs. Additionally, both drugs significantly mitigated mTBI-induced neurodegeneration and neuroinflammation, as quantified by immunohistochemical staining with Fluoro-Jade/anti-NeuN and anti-Iba-1 antibodies, respectively. mTBI challenge significantly decreased PKA phosphorylation levels in ipsilateral cortex, which was mitigated by both drugs. However, PI3K phosphorylation was not affected by mTBI. These findings offer a new potential therapeutic approach to treat mTBI, and support further investigation of the neuroprotective effects and mechanism of action of incretin-based therapies for neurological disorders.

Keywords: concussive head injury; glucagon-like peptide-1; glucose-dependent insulinotropic polypeptide; incretin; liraglutide; traumatic brain injury; twincretin.

FIGURE 1

Scheme of study design time line. Animals were exposed to sham/mTBI and 30 min later were given liraglutide (247.6 μg/kg), twincretin (50 μg/kg), or saline once daily via s.c. injections for 7 days {the human dose of liraglutide and twincretin is 1.8 mg s.c. daily that, for a 88.8 kg human [the mean weight of a male American (Yuan et al., 2017)], following normalization to body surface area across species in line with U.S. Department of Health and Human Services Food and Drug Administration guidelines (Han et al., 2016), translates to a mouse dose of approximately 247.6 μg/kg}. Behavioral tests to assess behavior and cognitive abilities were carried out 7 and 30 days following mTBI in separate cohorts of animals. Immunohistochemical staining to evaluate neurodegeneration and neuroinflammation was performed 72 h following mTBI challenge. Western blot analysis to assess changes in neuroprotective protein levels following mTBI and drug treatments was performed starting from 1 h to 1 week post mTBI. Each experimental time point was performed using a different group of mice (NOR: novel object recognition paradigm; EPM: elevated plus maze paradigm).

FIGURE 2

mTBI exposure and liraglutide/twincretin treatment do not impact the overall condition of the mice, as assessed in the elevated plus maze at both 7 and 30 days post trauma. Anxiety-like behavior assessment at 7 (A) and 30 (B) days post injury. The time spent in open arms of the maze was recorded and compared between groups. One-way ANOVA analysis showed that there were no differences between groups and that all groups of mice spent equivalent times in the open arms at both 7 and 30 days post mTBI [A: F(3,56) = 0.064; NS and B: F(3,51) = 0.374; NS]. Values are mean ± SEM. Locomotor activity at 7 (C) and 30 (D) days post injury. The number of total entrances to the arms of the maze was recorded and compared between groups. One-way ANOVA analysis demonstrated that there were no differences between the groups and all groups of mice had an approximately equal number of entrances to the arms at both 7 and 30 days post mTBI [C: F(3,56) = 0.830; NS, and D: F(3,51) = 0.218; NS]. Values are mean ± SEM.

FIGURE 3

Liraglutide and twincretin treatments lead to improvement in mTBI-induced cognitive impairments at both 7 and 30 days post trauma. Visual memory was assessed using the novel object recognition (NOR) paradigm at 7 days (A) and 30 days (B) post injury. Preference index was used to represent the relative time that animals spend exploring a novel object compared to a familiar one, which reflects visual memory. One-way ANOVA test followed by Fisher’s LSD post hoc analysis revealed that the preference index of mTBI group was significantly lower than all other groups at both time points [A: F(3,60) = 17.135; p < 0.001 and B: F(3,47) = 23.22; p < 0.001, ^∗∗p < 0.01, ^∗∗∗p < 0.001). The preference index of the liraglutide treated group was significantly lower than sham and twincretin treated group at 7 days (#p < 0.05 vs. sham and §p < 0.05 vs. mTBI + liraglutide), while only lower than twincretin treated group at 30 days (§p < 0.05 vs. mTBI + liraglutide). Values are mean ± SEM. Spatial memory was evaluated using the Y-maze paradigm at 7 (C) and 30 days (D) post injury. Preference index was used to represent the relative time that animals spent exploring a novel arm of the maze compared to a familiar one, reflecting spatial memory. One-way ANOVA followed by Fisher’s LSD post hoc analysis revealed that the preference index of mTBI group was significantly lower than all other groups at both time points [C: F(3,75) = 15.851; p < 0.001 and D: F(3,61) = 9.811; p < 0.001, ^∗∗∗p < 0.001]. Treatment with either liraglutide or twincretin led to similar normalization. Values are mean ± SEM.

FIGURE 4

Liraglutide and twincretin treatments mitigate mTBI-induced neurodegeneration, 72 h post trauma. Graphs showing the quantification of degenerating neurons as the ratio of FJB/NeuN in the temporal cortex (A), CA3 region (B), and the dentate gyrus (C). One-way ANOVA test followed by Fisher’s LSD post hoc analysis demonstrated that mTBI induction led to significant elevation in the ratio of degenerating neurons compared to sham tissues in all regions tested [A: F(3,16) = 26.695; p < 0.001, B: F(3,16) = 48.130; p < 0.001, and C: F(3,15) = 73.021; p < 0.001, ^∗∗∗p < 0.001]. Treatment with either liraglutide or twincretin caused a significantly lower FJB/NeuN ratio compared to mTBI (^∗∗∗p < 0.001), suggesting a reduction in the development of neurodegeneration following the injury. Values are mean ± SEM. (D) Representative images of immunohistochemical staining in the dentate gyrus are presented. NeuN positive cells are shown in red and FJB positive cells are shown in green; merged images show the overlap of FJB/NeuN positive cells. The scale bars are 50 μm.

FIGURE 5

Liraglutide and twincretin treatments do not affect the mTBI-induced elevation in reactive astrocyte expression, 72 h post trauma. Graphs presenting quantification of the total surface labeled with GFAP in the temporal cortex (A), CA3 region (B), and the dentate gyrus (C). One-way ANOVA followed by Fisher’s LSD post hoc analysis revealed that mTBI induction resulted in a significant elevation in GFAP expressing cells compared to sham tissues in all regions tested [A: F(3,14) = 3.917; p < 0.05, B: F(3,15) = 3.785; p < 0.05, and C: F(3,15) = 4.178; p < 0.05, ^∗∗p < 0.01]. Treatment with neither liraglutide nor twincretin affected this elevation as compared to mTBI. Liraglutide treatment following mTBI did not prevent the elevation in GFAP expression as compared to sham mice in the CA3 region (^∗p < 0.05); GFAP expression in the twincretin treatment group was significantly higher than control in all three regions (^∗p < 0.05). Values are mean ± SEM. (D) Representative images of immunohistochemical staining in the CA3 region are presented. GFAP positive cells are shown in red and DAPI positive cells are shown in blue. The scale bars are 75 μm.

FIGURE 6

Liraglutide and twincretin treatments reduce the mTBI-induced elevation in activated microglia expression, 72 h post trauma. Graphs presenting quantification of the total surface labeled with Iba-1 in the temporal cortex (A), CA3 region (B), and the dentate gyrus (C). One-way ANOVA followed by Fisher’s LSD post hoc analysis demonstrated that mTBI induction led to a significant elevation in Iba-1 expressing cells compared to sham tissues in all regions tested [A: F(3,14) = 5.117; p < 0.05, B: F(3,14) = 4.060; p < 0.05, and C: F(3,14) = 11.102; p < 0.01, ^∗p < 0.05 in the cortex and CA3 and ^∗∗p < 0.01 in the dentate gyrus]. Treatment with either liraglutide or twincretin following mTBI induction attenuated this elevation as compared to mTBI with no treatment. Values are mean ± SEM. (D) Representative images of immunohistochemical staining in the temporal cortex are presented. Iba-1 positive cells are shown in green and DAPI positive cells are shown in blue. The scale bars are 75 μm.

FIGURE 7

mTBI results in a decline in p-PKA levels in ipsilateral cortex. Graphs presenting p-PKA expression in ipsilateral/right (A) and contralateral/left (C) cortex and ipsilateral (E) and contralateral (G) hippocampus in sham and 1 h to 1 week following mTBI. One-way ANOVA followed by Fisher’s LSD post hoc analysis revealed a significant reduction in p-PKA expression in cortex right 24 h to 1 week following mTBI [A: F(5,36) = 12.060; p < 0.001, ^∗∗∗p < 0.001 sham vs. 24, 48 h and ^∗p < 0.05 sham vs. 72 h, 1 w], ^##p < 0.01, ^###p < 0.001 mTBI 1 h vs. mTBI 24 h and vs. 48 h, respectively, when the maximal reduction present in 48 h was followed by an elevation in the levels of PKA (72 h–1 week, §p < 0.05 vs. 48 h). Blots from all other regions remained unchanged by the injury [C: F(5,36) = 0.809; NS, E: F(5,36) = 0.585; NS, G: F(5,35) = 1.373; NS]. Values are mean ± SEM. (B,D,F,H) Representative images of gel electrophoresis followed by immunoblot analysis using antibodies against p-PKA and α-tubulin in ipsilateral cortex, contralateral cortex, ipsilateral hippocampus, and contralateral hippocampus, respectively. A crop of the original image was performed in 7F at loading site number 2 of the gel due to an abnormal value of a control sample that was therefore excluded from the statistical analysis.

FIGURE 8

mTBI challenge does not affect the p-PI3K levels in the cortex and hippocampus. Graphs showing p-PI3K expression in ipsilateral/right (A) and contralateral/left (C) cortex and ipsilateral (E) and contralateral (G) hippocampus in sham and 1 h to 1 week following mTBI. One-way ANOVA analysis showed no significant differences between the groups in all brain regions, indicating that mTBI induction does not affect the p-PI3K levels at the times tested [A: F(5,24) = 0.538; NS, C: F(5,24) = 0.365; NS, E: F(5,24) = 1.236; NS, G: F(5,23) = 0.813; NS]. Values are mean ± SEM. (B,D,F,H) Representative images of gel electrophoresis followed by immunoblot analysis using antibodies against p-PI3K and α-tubulin in ipsilateral cortex, contralateral cortex, ipsilateral hippocampus, and contralateral hippocampus, respectively.

FIGURE 9

Liraglutide and twincretin treatments block mTBI-induced reduction in p-PKA expression in the ipsilateral cortex. (A) Graph showing the p-PKA expression levels in ipsilateral/right cortex 48 h following mTBI. One-way ANOVA followed by Fisher’s LSD post hoc analysis revealed a significant reduction in p-PKA expression in ipsilateral injured cortex 48 h following mTBI as compared to sham [F(5,28) = 12.603; p < 0.001, ^∗∗p < 0.01]. Treatment with either liraglutide or twincretin reduced the decline observed in mTBI affected brains (^∗∗p < 0.01, respectively). Values are mean ± SEM. (B) Representative images of gel electrophoresis followed by immunoblot analysis using antibodies against p-PKA and α-tubulin in the ipsilateral cortex. A crop of the original image was performed, and sample number 3 was replaced by sample number 8, since the sample of mTBI + liraglutide was on loading site number 8 of the gel.