Newly Synthesized Indolylacetic Derivatives Reduce Tumor Necrosis Factor-Mediated Neuroinflammation and Prolong Survival in Amyotrophic Lateral Sclerosis Mice

Abstract

The debilitating neurodegenerative disease known as amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of motor neurons (MNs) in the brain, spinal cord, and motor cortex. The ALS neuroinflammatory component is being characterized and includes the overexpression of mediators, such as inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α). Currently, there are no effective treatments for ALS. Indeed, riluzole, an N-methyl-D-aspartate (NMDA) glutamate receptor blocker, and edaravone, a reactive oxygen species (ROS) scavenger, are currently the sole two medications approved for ALS treatment. However, their efficacy in extending life expectancy typically amounts to only a few months. In order to improve the medicaments for the treatment of neurodegenerative diseases, preferably ALS, novel substituted 2-methyl-3-indolylacetic derivatives (compounds II-IV) were developed by combining the essential parts of two small molecules, namely, the opioids containing a 4-piperidinyl ring with indomethacin, previously shown to be efficacious in different experimental models of neuroinflammation. The synthesized compounds were evaluated for their potential capability of slowing down neurodegeneration associated with ALS progression in preclinical models of the disease in vitro and in vivo. Notably, we produced data to demonstrate that the treatment with the newly synthesized compound III: (1) prevented the upregulation of TNF-α observed in BV-2 microglial cells exposed to the toxin lipopolysaccharides (LPS), (2) preserved SHSY-5Y cell survival exposed to β-N-methylamino-l-alanine (L-BMAA) neurotoxin, and (3) mitigated motor symptoms and improved survival rate of SOD1G93A ALS mice. In conclusion, the findings of the present work support the potential of the synthesized indolylacetic derivatives II-IV in ALS treatment. Indeed, in the attempt to realize an association between two active molecules, we assumed that the combination of the indispensable moieties of two small molecules (the opioids containing a 4-piperidinyl ring with the FANS indomethacin) might lead to new medicaments potentially useful for the treatment of amyotrophic lateral sclerosis.

Figure 1

Chemical structures of compound II (R = H) and compound III (R = CH₂CH₃).

Figure 2

Chemical structure of compound IV.

Scheme 1. Synthetic Route for Compounds II–IV

Figure 3

Survival of SHSY-5Y cells exposed to L-BMAA neurotoxin induced by compounds II, III, and IV. The MTT assay was performed 24 h after the last exposure to compounds and L-BMAA. Compounds II, III, and IV were added 20 min before each L-BMAA-exposure. Each data point is the mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison test was used for statistical analysis. n = at least 3 for group. *p < 0.05 versus control.

Figure 4

Effects of compounds II–IV on iNOS expression induced by LPS exposure in microglia cell cultures. Representative Western blots and densitometric quantification of iNOS levels in BV-2 microglial cell cultures. Data were normalized on the β-actin levels. n = at least 3 for column. Each data point is the mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison test was used for statistical analysis. *p < 0.05 versus control.

Figure 5

Effect of compound III on NO generation induced by LPS in BV-2 microglia cell cultures. Each data point is the mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison test was used for statistical analysis. n = at least 3 for column. ****p < 0.0001 vs control group.

Figure 6

Effects of compounds II–IV on LPS-induced TNF-α expression in microglia cells. Representative Western blot and densitometric quantification of TNF-α in BV-2 microglial cells. Data were normalized on the β-actin levels. n = at least 3 for column. Each data point is the mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison test was used for statistical analysis. *p < 0.05 versus control; ^p < 0.05 versus LPS 100 ng/mL.

Figure 7

Effects of compounds II–IV on LPS-induced COX2 expression in microglia cells. Representative Western blot and densitometric quantification of COX2 in BV-2 microglial cells. Data were normalized on the β-actin levels. n = at least 3 for column. Each data point is the mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison test was used for statistical analysis.

Figure 8

Effect of prolonged compound III treatment on motor symptoms and survival rates in ALS-affected mice (SOD1 G93A).