Neuroprotective effects of inhibitors of Acid-Sensing ion channels (ASICs) in optic nerve crush model in rodents

ABSTRACT

Purpose: The purpose of the current study was to assess the potential involvement of acid-sensing ion channel 1 (ASIC1) in retinal ganglion cell (RGC) death and investigate the neuroprotective effects of inhibitors of ASICs in promoting RGC survival following optic nerve crush (ONC).

Results: ASIC1 protein was significantly increased in optic nerve extracts at day 7 following ONC in rats. Activated calpain-1 increased at 2 and 7 days following ONC as evidenced by increased degradation of α-fodrin, known substrate of calpain. Glial fibrillary acidic protein levels increased significantly at 2 and 7 days post-injury. By contrast, glutamine synthetase increased at 2 days while decreased at 7 days. The inhibition of ASICs with amiloride and psalmotoxin-1 significantly increased RGC survival in rats following ONC (p < 0.05, one-way ANOVA). The mean number of surviving RGCs in rats (n = 6) treated with amiloride (100 µM) following ONC was 1477 ± 98 cells/mm² compared with ONC (1126 ± 101 cells/mm²), where psalmotoxin-1 (1 μM) treated rats (n = 6) and subjected to ONC had 1441 ± 63 RGCs/mm² compared with ONC (1065 ± 76 RGCs/mm²). Average number of RGCs in control rats (n = 12) was 2092 ± 46 cells/mm². Blocking of ASICs also significantly increased RGC survival from ischemic-like insult from 473 ± 80 to 842 ± 49 RGCs/mm² (for psalmotoxin-1) and from 628 ± 53 RGCs/mm² to 890 ± 55 RGCs/mm² (for amiloride) with p ≤ 0.05, using one-way ANOVA. Acidification (a known activator of ASIC1) increased intracellular Ca²⁺ ([Ca²⁺]_i) in rat primary RGCs, which was statistically blocked by pretreatment with 100 nM psalmotoxin-1.

Conclusions: ASIC1 up-regulation-induced influx of extracellular calcium may be responsible for activation of calcium-sensitive calpain-1 in the retina. Calpain-1 induced degradation of α-fodrin and leads to morphological changes and eventually neuronal death. Therefore, blockers of ASIC1 can be used as potential therapeutics in the treatment of optic nerve degeneration.

Abbreviations: 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF); acid-sensing ion channels (ASICs); analysis of variance (ANOVA); bicinchoninic acid (BCA); brain-derived neurotrophic factor (BDNF); central nervous system (CNS); ciliary neurotrophic factor (CNTF); dimethyl sulfoxide (DMSO); endoplasmic reticulum (ER); ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA); ethylenediaminetetraacetic acid (EDTA); Food and Drug Administration (FDA); glial fibrillary acidic protein (GFAP); glutamine synthetase (GS); intraocular pressure (IOP); kilodalton (kDa); Krebs-Ringer Buffer (KRB); optic nerve crush (ONC); phosphate-buffered saline (PBS); plasma membrane (PM); polymerase chain reaction (PCR); retinal ganglion cell (RGC); RNA Binding Protein With Multiple Splicing (RBPMS); room temperature (RT); standard error of the mean (SEM).

KEYWORDS:

• Acid-sensing ion channels (ASICs)
• optic nerve degeneration
• neuroprotection
• retinal ganglion cells

Acknowledgments

This publication was supported in part by grants from Brightfocus Foundation and intramural grant from UNTHSC to A.D. We thank Dr He who assisted in the proof-reading of the manuscript.

Funding

This work was supported by the BrightFocus Foundation [G2015163] and an intramural grant from UNTHSC to A.D.

Additional information

Funding

This work was supported by the BrightFocus Foundation [G2015163] and an intramural grant from UNTHSC to A.D.