Protease-resistant SOD1 aggregates in amyotrophic lateral sclerosis demonstrated by Paraffin Embedded Tissue (PET) blot

Abstract

Objectives: The paraffin-embedded tissue (PET) blot technique followed by limited protease digestion has been established to detect protein aggregates in prion diseases, alpha-synucleopathies, and tauopathies. We analyzed whether the scope of the method can be extended to analyze aggregates in mouse and human tissue with amyotrophic lateral sclerosis (ALS) associated with superoxide dismutase 1 (SOD1) mutation.Methods: Formalin-fixed and paraffin-embedded brain and spinal cord tissue from SOD1 G93A mice was first analyzed for the expression of SOD1, aggregated SOD1, ubiquitin, and p62 by convential immunohistochemistry and then used to establish the PET blot technique, limited protease digest, and immunodetection of SOD1 aggregates. The method was then transferred to spinal cord from an ALS patient with SOD1 E100G mutation.Results: Mouse and human paraffin-embedded brain and spinal cord tissue can be blotted to membranes and stained with anti-SOD1 antibodies. The SOD1 labelling is abolished after limited proteolytic digest in controls, whereas under identical conditions SOD1 aggregates are detected the SOD1 G93A mouse model of ALS and in human familial ALS. The most prominent areas where aggregates could be detected are the brainstem and the anterior horn of the spinal cord.Discussion: Applicability of the PET blot technique to demonstrate SOD1 aggregates in ALS tissue associated with mutations in the SOD1 gene offers a new approach to examine potential spreading of aggregates in the course of ALS.

Commentary: alpha-synuclein interacts with SOD1 and promotes its oligomerization

“…Nishiyama et al reported that alphasynuclein aggregates in the substantia nigra and locus coeruleus of five PD patients were immuno-positive for SOD1 11 . However, these findings should be interpreted with caution because cells without alpha-synuclein inclusions in the substantia nigra and locus coeruleus did not show SOD1 immunoreactivity in this study although SOD1 is known to be expressed in the substantia nigra which has been confirmed by Steinacker et al 12 . A systematic study with post-mortem material from a large number of patients is needed to determine the frequency and relevance of alpha-synuclein-SOD1 co-localization in protein deposits.…”

Satellite Glial Cells of the Dorsal Root Ganglion: A New “Guest/Physiopathological Target” in ALS

“…In addition to the motor cortex and spinal cord, the abnormal accumulation of mutant SOD1 has also been reported in other brain regions, including the temporal cortex, hippocampus, and cerebellum (Steinacker et al, 2014 ) as well as DRG neurons in individuals with ALS (Sábado et al, 2014 ). Importantly, to the best of our knowledge, this is the first study suggesting SGCs as a potential non-motor target in ALS.…”

Loss of charge at solvent exposed Lys residues does not induce the aggregation of superoxide dismutase 1

“…Mutant SOD1 of fALS is generally viewed as being more prone to misfold and aggregate [14,[19][20][21][22][23]. SOD1 immuno-reactive inclusions in surviving spinal motor neurons is a common, but not uniformly found, pathologic feature of SOD1-linked fALS [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]. Notably, the SOD1 inclusions found in patients appear to lack the features of amyloid (Thioflavin and Congo Red negative) [24,40].…”