Non-Methylation-Linked Mechanism of REST-Induced Neuroglobin Expression Impacts Mitochondrial Phenotypes in a Mouse Model of Amyotrophic Lateral Sclerosis

Abstract

Neuroglobin (Ngb) is a REST/NRSF-regulated protein, active in reactive oxygen species detoxification and cytochrome c inhibition, which provides a beneficial outcome in pathologies as Alzheimer's disease and strokes. Considering that oxidative stress and cell death are typical hallmarks of amyotrophic lateral sclerosis (ALS), we sought to explore Ngb's involvement along this disease progression. Ngb transcription was detected to be two-fold down-regulated in late-stage SOD^G93A mice, similarly as previously described for Alzheimer disease. Interestingly, in accordance with REST/NRSF transcription, Ngb expression is higher in spinal cords than in cortices. Hence, downstream REST/NRSF mechanisms were studied. A methylation cluster in Ngb's exon 1 (Chr12:87101763-87102586) was selected to assess methylation alterations, based on significantly altered positions in GEO DataSets of human c9orf72 and sporadic ALS cases. However, only the methylation percentage on position Chr12.87102586 was significantly increased in SOD^G93A mice. A larger impact can therefore be expected from the detected altered REST splicing; with levels of alternatively spliced, gene-activating REST4 to be lower than those of the gene-inhibitory full variant. To look further into the link between Ngb and ALS, we generated a double mutant Ngb^-/-SOD^G93A mouse model, which shows an earlier onset and severity of hind limb deficits. Mitochondria derived thereof showed an altered mean volume, granularity and Ca²⁺-induced swelling as compared to Ngb^Wt/WtSOD^G93A mice. These results indicate Ngb to be involved in and affected by the SOD1^G93A pathology, which could in part be attributed to its role in halting destabilizing events of mitochondrial swelling and phenotypes.

Keywords: REST(4)-splicing; calcium homeostasis; motor-neuron disease; neuroglobin; neuroprotection.