Antisense peptide nucleic acid targeting GluR3 delays disease onset and progression in the SOD1 G93A mouse model of familial ALS

Abstract

Glutamate excitotoxicity is strongly implicated as a major contributing factor in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Excitotoxicity results from elevated intracellular calcium ion (Ca²⁺) levels, which in turn recruit cell death signaling pathways. Recent evidence suggests that α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit (GluR) stoichiometry is a dominant factor leading to excess Ca²⁺ loading in neurodegeneration. In particular, the Ca²⁺ permeable glutamate receptor subunit 3 (GluR3) has been implicated in several neurologic conditions such as bipolar disorder and epilepsy. Recent proteomic analysis within our group on the copper zinc superoxide dismutase (SOD1)^G93A transgenic mouse model of familial ALS (FALS) reveals a potentially deleterious upregulation of GluR3 in spinal cord compared to that in wild-type littermates. Based on this finding we designed a 12mer antisense peptide nucleic acid (PNA) directed against GluR3. This sequence significantly reduced levels of GluR3 protein and protected neuroblastoma × spinal cord (NSC-34) cells against death induced by the AMPA receptor-specific agonist (S)-5-fluorowillardiine. We subsequently treated SOD1^G93A mice thrice weekly with intraperitoneal injections of the antisense PNA (2.5 mg/kg) commencing at postnatal day 50. Mice treated with the antisense sequence had significantly extended survival compared to mice injected with a nonsense sequence. Western blot analysis, however, did not reveal a significant reduction in GluR3 protein levels in whole extracts of the lumbar spinal cord. These results suggest that interference with the GluR3 component of the AMPA receptor assembly may be a novel strategy for controlling excitotoxic destruction of motor neurons and may lead to new therapeutic opportunities for the treatment of human ALS.