Neuroprotection by acrolein sequestration through exogenously applied scavengers and endogenous enzymatic enabling strategies in mouse EAE model

Abstract

We have previously shown that the pro-oxidative aldehyde acrolein is a critical factor in MS pathology. In this study, we found that the acrolein scavenger hydralazine (HZ), when applied from the day of induction, can suppress acrolein and alleviate motor and sensory deficits in a mouse experimental autoimmune encephalomyelitis (EAE) model. Furthermore, we also demonstrated that HZ can alleviate motor deficits when applied after the emergence of MS symptoms, making potential anti-acrolein treatment a more clinically relevant strategy. In addition, HZ can reduce both acrolein and MPO, suggesting a connection between acrolein and inflammation. We also found that in addition to HZ, phenelzine (PZ), a structurally distinct acrolein scavenger, can mitigate motor deficits in EAE when applied from the day of induction. This suggests that the likely chief factor of neuroprotection offered by these two structurally distinct acrolein scavengers in EAE is their common feature of acrolein neutralization. Finally, up-and-down regulation of the function of aldehyde dehydrogenase 2 (ALDH2) in EAE mice using either a pharmacological or genetic strategy led to correspondent motor and sensory changes. This data indicates a potential key role of ALDH2 in influencing acrolein levels, oxidative stress, inflammation, and behavior in EAE. These findings further consolidate the critical role of aldehydes in the pathology of EAE and its mechanisms of regulation. This is expected to reinforce and expand the possible therapeutic targets of anti-aldehyde treatment to achieve neuroprotection through both endogenous and exogenous manners.

Figure 1

EAE motor symptom scores of EAE mice with and without hydralazine treatment commencing at the induction or symptom emergence. (A) Symptom scores over time for hydralazine and sham treatment starting at the induction in EAE mice. Hydralazine treatment significantly reduced the severity of symptoms starting on day 13, contributing to both later apparent onset and lower peak severity (comparison between EAE and EAE + HZ). (B) Hydralazine treatment delayed the average day of onset, defined as the day upon which the motor score reached 1.0, from 9.2 ± 0.73 days post induction to 18.2 ± 0.66 days post induction. (C) Hydralazine treatment reduced peak severity of motor impairment from 3.1 ± 0.37 to 0.9 ± 0.1. (D) Delaying hydralazine treatment until the appearance of motor symptoms significantly reduced disease severity starting on the fourth day of treatment and continuing until the end of the study (comparison between EAE and EAE + HZ). (E) Delayed treatment significantly reduced peak motor symptom scores from 3.54 ± to 0.24 to 2.38 ± 0.32. For A-E, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2

EAE motor symptom scores of EAE mice with and without phenelzine treatment commencing at the induction. (A) Symptom scores over time for phenelzine and sham treated EAE mice. Phenelzine treatment significantly reduced the severity of symptoms starting on day 16, contributing to both later apparent onset and lower peak severity (comparison between EAE and EAE + PZ). (B) Phenelzine treatment delayed the average day of onset, defined as the day upon which the motor score reached 1.0, from 13.14 ± 1.03 days post induction to 23.5 ± 2.92 days post induction. (C) Phenelzine treatment reduced peak severity of motor impairment from 3.43 ± 0.43 to 1.33 ± 0.67. For A-C, *p < 0.05, **p < 0.01.

Figure 3

Acrolein scavengers alleviated hypersensitivity and demonstrated a tendency to mitigate elevated expression of TRPA1 on day 10 post induction in EAE mice. (A) Effect of hydralazine treatment (initiated at the induction) on mechanical hyperreflexia assessed on day 10 post induction. EAE mice exhibited significantly lower hind paw withdrawal thresholds at 0.45 ± 0.05 g compared to healthy controls, which showed an average withdrawal threshold of 2.11 ± 0.15. Hydralazine treatment partially alleviated this to 0.76 ± 0.09 g, which is improved compared to untreated EAE mice, but still exhibited higher sensitivity than the control group. (B) Relative expression of TRPA1 on day 10 post induction. TRPA1 expression in EAE mice was elevated compared to the control group, with a 10.25 ± 4.4 fold increase in expression compared to the control group (1.54 ± 0.58 fold). Hydralazine-treated (initiated at the induction) animals trended towards lower TRPA1 expression when compared to untreated EAE mice, but this difference was not statistically significant due to high variance in both groups. For (A, B), *p < 0.05, ***p < 0.001.

Figure 4

Immunostaining of FDP-Lysine in the spinal cords of EAE and hydralazine-treated mice. FDP-Lysine, an acrolein adduct, was significantly elevated in the spinal cord of EAE mice compared to controls on day 10 post induction. Treatment with hydralazine, a carbonyl scavenger, reduced the presence of FDP-Lysine compared to untreated EAE mice. No significant differences were observed in levels of FDP-Lysine between hydralazine treated animals and control. *p < 0.05.

Figure 5

Immunostaining of myeloperoxidase in the spinal cords of EAE and hydralazine-treated mice. MPO was significantly elevated in the spinal cord of EAE mice compared to controls on day 10 post induction. Hydralazine treatment reduced the presence of MPO compared to untreated mice. No significant differences were observed between hydralazine treated mice and control animals. * p < 0.05.

Figure 6

The effect of functional regulation of ALDH2 on the level of acrolein and MPO in spinal cord. (A) The comparison of the level of FDP-Lysine in the spinal cords of various conditions assessed on day 20 post induction. FDP-Lysine was significantly elevated in the spinal cord of wild-type EAE mice compared to controls. However, Alda-1 treatment in wild-type EAE mice suppressed levels of FDP-Lysine compared to untreated mice. Furthermore, ALDH2*2 mutant EAE mice exhibited increased FDP-lysine compared to wild-type EAE mice. No significant differences were observed between controls and Alda-1 treated wild-type mice. *p < 0.05. (B) The comparison of the level of myeloperoxidase in the spinal cord of various conditions assessed on day 20 post induction. MPO was significantly elevated in the spinal cord of wild-type EAE mice compared to controls. However, Alda-1 treatment in wild-type EAE mice suppressed levels of MPO compared to untreated mice. Furthermore, ALDH2*2 mutant EAE mice exhibited increased MPO compared to wild-type EAE mice. No significant differences were observed between controls and Alda-1-treated wild-type mice. *p < 0.05 compared to wild-type EAE. (C) Correlation of FDP-Lysine with myeloperoxidase. Linear regression of levels of FDP-Lysine and myeloperoxidase was performed to examine the relationship between the elevation of myeloperoxidase and acrolein generation. Regression analysis demonstrated a positive relationship with a significant correlation between levels of acrolein and myeloperoxidase (R² = 0.7419, p < 0.05).

Figure 7

Modulation of ALDH2 activity resulted in correspondent changes in motor and sensory behavioral deficits. (A) Comparison of motor function of various conditions in wild-type and ALDH2*2 mice. While control mice displayed no functional deficits, wild-type EAE mice display typical motor deficits (black). The treatment of Alda-1, a drug which enhances ALDH2 activity, in wild-type EAE mice (green) commenced immediately after induction alleviated motor deficits on multiple days starting from day 12 post induction (*p < 0.05 compared to EAE). Conversely, ALDH2*2 knock-in mice (red), which have a deficient form of the enzyme, exhibited significantly more severe motor deficits when compared to wild-type EAE mice starting from day 13 post induction (*p < 0.05 compared to EAE). (B) Comparison of sensory hypersensitivity of various conditions in wild-type and ALDH2*2 mice. Wild-type EAE mice display significant hypersensitivity based on mechanical withdrawal thresholds at day 10 post induction compared to the control group. However, this hypersensitivity was significantly alleviated in Alda-1 treated animals, when commenced immediately post induction. While there was a tendency of worsening hypersensitivity in ALDH2*2 mice compared to wild-type EAE, no significant difference was observed between them. *p < 0.05.