SOD1 Lysine 123 Acetylation in the Adult Central Nervous System.

Michael Kaliszewski,Blaise Bossy,Wenjun Song,Shelby L Blaes,Robert S Shaffer,Ting-Ting Huang,Ella Bossy-Wetzel,Henry A Hauser,Austin K Kennedy,Andrew B Knott

doi:10.3389/fncel.2016.00287

Abstract

Superoxide dismutase 1 (SOD1) knockout (Sod1−/−) mice exhibit an accelerated aging phenotype. In humans, SOD1 mutations are linked to familial amyotrophic lateral sclerosis (ALS), and post-translational modification (PTM) of wild-type SOD1 has been associated with sporadic ALS. Reversible acetylation regulates many enzymes and proteomic studies have identified SOD1 acetylation at lysine 123 (K123). The function and distribution of K123-acetylated SOD1 (Ac-K123 SOD1) in the nervous system is unknown. Here, we generated polyclonal rabbit antibodies against Ac-K123 SOD1. Sod1 deletion in Sod1−/− mice, K123 mutation or preabsorption with Ac-K123 peptide all abolished antibody binding. Using immunohistochemistry, we assessed Ac-K123 SOD1 distribution in the normal adult mouse nervous system. In the cerebellum, Ac-K123 SOD1 staining was prominent in cell bodies of the granular cell layer (GCL) and Purkinje cell dendrites and interneurons of the molecular cell layer. In the hippocampus, Ac-K123 SOD1 staining was strong in the fimbria, subiculum, pyramidal cells and Schaffer collateral fibers of the cornus ammonis field 1 (CA1) region and granule and neuronal progenitor cells of the dentate gyrus. In addition, labeling was observed in the choroid plexus (CP) and the ependyma of the brain ventricles and central canal of the spinal cord. In the olfactory bulb, Ac-K123 SOD1 staining was prominent in axons of sensory neurons, in cell bodies of interneurons and neurites of the mitral and tufted cells. In the retina, labeling was strong in the retinal ganglion cell layer (RGCL) and axons of retinal ganglion cells (RGCs), the inner nuclear layer (INL) and cone photoreceptors of the outer nuclear layer (ONL). In summary, our findings describe Ac-K123 SOD1 distribution to distinct regions and cell types of the normal nervous system.

Highlights

Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that converts superoxide radicals to hydrogen peroxide and oxygen (McCord and Fridovich, 1969)
To test whether the antibodies were specific for SOD1 and lacked cross-reactivity with unrelated proteins, brain tissue lysates of Sod1+/+ and Sod1−/− mice were separated by sodium dodecyl sulfate (SDS)-PAGE and western blot membranes were probed with primary antibodies for Ac-lysine 123 (K123) SOD1, total SOD1 or α-tubulin
The results indicate that our new antibody is specific to SOD1 and lacks cross-reactivity with unrelated proteins using western blotting techniques

Summary

Introduction

Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that converts superoxide radicals to hydrogen peroxide and oxygen (McCord and Fridovich, 1969). SOD1 is a predominantly cytoplasmic enzyme that is ubiquitously expressed, conserved across species and abundant in most cell types of the nervous system (Crapo et al, 1992; Pardo et al, 1995). Oxidation of SOD1 has been linked to sporadic ALS (Rakhit et al, 2002), indicating that specific post-translational modifications (PTMs) of the wild-type protein may contribute to late-onset disease. K123 maps to the electrostatic loop of SOD1, a region required for copper binding, protein folding and substrate recruitment (Crapo et al, 1992; Pardo et al, 1995). Lysine acetylation is a reversible PTM that regulates the interaction, subcellular localization, folding and activity of many proteins (Kouzarides, 2000). Dysregulation of lysine acetylation has been reported in numerous disorders including neurodegeneration and cancer (Drazic et al, 2016)

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