Protease-resistant SOD1 aggregates in amyotrophic lateral sclerosis demonstrated by paraffin-embedded tissue (PET) blot.

Petra Steinacker,Johannes Attems,Albert C Ludolph,Christian Berner,Markus Otto,Dietmar R Thal

doi:10.1186/s40478-014-0130-x

Abstract

ObjectivesThe 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.MethodsFormalin-fixed and paraffin-embedded brain and spinal cord tissue from SOD1G93A 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 SOD1E100G mutation.ResultsMouse 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 SOD1G93A 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.DiscussionApplicability 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.

Highlights

Neurodegenerative diseases are characterized by the accumulation of proteins with aberrant conformation in the affected tissues
Mouse and human paraffin-embedded brain and spinal cord tissue can be blotted to membranes and stained with anti-superoxide dismutase 1 (SOD1) antibodies
Applicability of the paraffin-embedded tissue (PET) blot technique to demonstrate SOD1 aggregates in Amyotrophic lateral sclerosis (ALS) tissue associated with mutations in the SOD1 gene offers a new approach to examine potential spreading of aggregates in the course of ALS

Summary

Introduction

Neurodegenerative diseases are characterized by the accumulation of proteins with aberrant conformation in the affected tissues. It was recognized that template assisted misfolding or seeded polymerization contributes to the burden of protein aggregates in a number of proteinopathies, comparable to Amyotrophic lateral sclerosis (ALS) is a phenotypically heterogeneous disease characterized by degeneration of upper and lower motoneurons. Mutated SOD1 forms aggregates of fibrillar structure in the affected tissues [7,8] and recent data suggest that there is a propensity for aggregation of posttranslationally modified wild type SOD1 in a subset of sporadic ALS cases [9]. Misfolding and aggregation of SOD1 in the spinal cord and the motor cortex is observed in mice transgenic for disease associated mutated human SOD1 representing the most established model for ALS [10]. Neuronal inclusions are positive for ubiquitin and p62 [11,12]

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