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. 2021 Dec 10:12:780588.
doi: 10.3389/fphar.2021.780588. eCollection 2021.

Sigma-1 Receptor is a Pharmacological Target to Promote Neuroprotection in the SOD1G93A ALS Mice

Núria Gaja-Capdevila  1   2 , Neus Hernández  1   2 , Xavier Navarro  1   2 , Mireia Herrando-Grabulosa  1   2
Affiliations

Sigma-1 Receptor is a Pharmacological Target to Promote Neuroprotection in the SOD1G93A ALS Mice

Núria Gaja-Capdevila et al. Front Pharmacol. .

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by the death of motoneurons (MNs) with a poor prognosis. There is no available cure, thus, novel therapeutic targets are urgently needed. Sigma-1 receptor (Sig-1R) has been reported as a target to treat experimental models of degenerative diseases and, importantly, mutations in the Sig-1R gene cause several types of motoneuron disease (MND). In this study we compared the potential therapeutic effect of three Sig-1R ligands, the agonists PRE-084 and SA4503 and the antagonist BD1063, in the SOD1G93A mouse model of ALS. Pharmacological administration was from 8 to 16 weeks of age, and the neuromuscular function and disease progression were evaluated using nerve conduction and rotarod tests. At the end of follow up (16 weeks), samples were harvested for histological and molecular analyses. The results showed that PRE-084, as well as BD1063 treatment was able to preserve neuromuscular function of the hindlimbs and increased the number of surviving MNs in the treated female SOD1G93A mice. SA4503 tended to improve motor function and preserved neuromuscular junctions (NMJ), but did not improve MN survival. Western blot analyses revealed that the autophagic flux and the endoplasmic reticulum stress, two pathways implicated in the physiopathology of ALS, were not modified with Sig-1R treatments in SOD1G93A mice. In conclusion, Sig-1R ligands are promising tools for ALS treatment, although more research is needed to ascertain their mechanisms of action.

Keywords: SOD1 G93A transgenic; amyotrophic lateral sclerosis; motoneuron; neurodegenerative disease; sigma-1 receptor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
PRE-084, BD1063 and SA4503 prevent MN death under chronic excitotoxicity in vitro (A-C) Plots showing the percentage of SMI-32 positive cells in the ventral horn of spinal cord hemislices under excitotoxicity by THA and the addition of Sig-1R ligands (mean ± SEM; n = 16–24 hemisections per treatment). One-way ANOVA followed with Bonferroni’s post hoc test: ****p < 0.0001, ***p < 0.001; **p < 0.01, *p < 0.05 vs THA alone condition (D) Microphotograph of a SCOC slice labeled with SMI-32 antibody, and representative images of the ventral horn at 28 DIV of all tested conditions. Scale bar 100 μm.
FIGURE 2
FIGURE 2
Treatment with PRE-084, BD1063 and SA4503 enhances neuromuscular function in SOD1G93A female mice (A) Body weight of mice was monitored weekly during the 8 weeks of the follow-up (B-C) Values of the amplitude of compound muscle action potentials (CMAP) of tibialis anterior (TA) and plantar interosseus muscles (D) Graph showing the effect of the different Sig-1R treatment on functional outcome assessed with the rotarod test (n = 15 WT; n = 15 SOD1 saline; n = 12 SOD1 BD1063; n = 7 SOD1 SA4503 0.25 mg/kg; n = 7 SOD1 SA4503 1 mg/kg, and n = 14 SOD1 PRE-084 mice) (E) Probability of clinical onset of disease evaluated by the fall in the rotarod test. Some Sig-1R ligands delayed the onset of locomotion deficits but without significant differences (F) Plot of the percentage of innervated NMJ (overlap of signals) in the different experimental groups (n = four to six mice per group) (G) Representative confocal images of GA NMJs at 16 weeks of age. The maximum projection images shown were generated from 1.3 µm z projections. Scale bar 100 μm. Data are mean ± SEM, analyzed by One-way (F) and Two-way (A-D) ANOVA with Bonferroni’s multiple comparisons test. *p < 0.05 vs SOD1G93A saline mice.
FIGURE 3
FIGURE 3
PRE-084 and BD1063 reduce spinal MN degeneration and decrease microgliosis in SOD1G93A female mice at 16 weeks of age (A) Quantification of surviving MNs (mean number of MNs per section ±SEM) in the lumbar spinal cord, showing improved MN preservation with Sig-1R ligands PRE-084 and BD1063. (B) Representative spinal cord images of MNs stained with cresyl violet. Scale bar 100 μm. (C, E) Representative images of immunoreactivity for Iba1 (C), a marker for microglia, and GFAP (E), a marker of astrocytes, in the ventral horn of WT and SOD1G93A mice with or without Sig-1R treatments. Scale bar 50 μm. D, F) Graphs showing the quantification of percentage of Iba-1 (D) and GFAP (F) immunolabeling in ventral horn of spinal cord (n = 10 WT, n = 10 SOD1 saline, n = 8 SOD1 BD1063, n = 7 SOD1 SA4503 0.25 mg/kg, n = 7 SOD1 SA4503 1 mg/kg and n = 10 SOD1 PRE-084 mice). *p < 0.05 vs SOD1G93A saline mice.
FIGURE 4
FIGURE 4
Protein levels of autophagic flux markers in SOD1G93A female mice. Representative blots and protein level quantification of the autophagic markers Beclin-1 and LC3-II at 8 (A, B) and 16 (C, D) weeks of age. Quantification of the p62/SQSTM1 immunolabeling in the MNs (E) and in the glia (whole image without MNs) (F). (G) Representative images of p62/SQSTM1 (red), GFAP (cyan) and FluoroNissl (green) immunofluorescence in the ventral horn of lumbar spinal cord of WT and SOD1G93A mice at 16 weeks. White arrows show examples of MNs with cytosolic p62 accumulation and asteriscs show p62 immunolabeling in the astroglia. Scale bar 50 μm. Data is mean ± SEM; n = three to five mice per group. One-way ANOVA followed with Bonferroni’s post hoc test for multiple comparison. *p < 0.05 vs SOD1G93A saline, #p < 0.05 vs WT.
FIGURE 5
FIGURE 5
Protein levels of ER stress markers in SOD1G93A female mice. Representative blots of ER stress markers at 8 (A) and 16 (B) weeks of age. Quantification of protein levels of Sig-1R, BiP, ratio p-IRE1/IRE1, ratio XBP1s/XBP1unspliced and CHOP at 8 (C) and 16 (D) weeks of age. Data is mean ± SEM; n = three to five mice per group. One-way ANOVA followed with Bonferroni’s post hoc test for multiple comparison. *p < 0.05 vs SOD1G93A saline, #p < 0.05 vs WT.
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