Improved detection of RNA foci in C9orf72 amyotrophic lateral sclerosis post-mortem tissue using BaseScope™ shows a lack of association with cognitive dysfunction.

Arpan R Mehta,Colin Smith,Bhuvaneish T Selvaraj,Samantha K Barton,Siddharthan Chandran,Karina Mcdade,Sharon Abrahams,Jenna M Gregory

doi:10.1093/braincomms/fcaa009

Abstract

The C9orf72 hexanucleotide repeat expansion is the commonest known genetic mutation in amyotrophic lateral sclerosis. A neuropathological hallmark is the intracellular accumulation of RNA foci. The role that RNA foci play in the pathogenesis of amyotrophic lateral sclerosis is widely debated. Historically, C9orf72 RNA foci have been identified using in situ hybridization. Here, we have implemented BaseScope™, a high-resolution modified in situ hybridization technique. We demonstrate that previous studies have underestimated the abundance of RNA foci in neurons and glia. This improved detection allowed us to investigate the abundance, regional distribution and cell type specificity of sense C9orf72 RNA foci in post-mortem brain and spinal cord tissue of six deeply clinically phenotyped C9orf72 patients and six age- and sex-matched controls. We find a correlation between RNA foci and the accumulation of transactive response DNA-binding protein of 43 kDa in spinal motor neurons (rs = 0.93; P = 0.008), but not in glia or cortical motor neurons. We also demonstrate that there is no correlation between the presence of RNA foci and the accumulation of transactive response DNA binding protein of 43 kDa in extra-motor brain regions. Furthermore, there is no association between the presence of RNA foci and cognitive indices. These results highlight the utility of BaseScope™ in the clinicopathological assessment of the role of sense RNA foci in C9orf72.

Highlights

A key post-mortem diagnostic hallmark of most amyotrophic lateral sclerosis (ALS) cases is the presence of intra-cytoplasmic transactive response DNA-binding protein of 43 kDa (TDP-43) aggregates in motor cortex and spinal cord motor neurons and glia, associated with anterior horn cell loss and gliosis (Arai et al, 2006; Neumann et al, 2006)
The mean number of foci per cell in motor brain and spinal cord regions was 2.6 for neurons and 1.1 for glia, and the mean number of foci per cell in extra-motor brain regions was 2.9 for neurons and 1.2 for glia. These data illustrate the substantially improved signal amplification and the sensitivity of this technique when compared with standard in situ hybridization (ISH) techniques (Aladesuyi Arogundade et al, 2019)
This improved detection is especially the case at low magnification, as illustrated in the low-magnification images shown in Supplementary Fig. 1, demonstrating clear staining of RNA foci without the need to examine a single nucleus at higher magnification

Summary

Introduction

A key post-mortem diagnostic hallmark of most amyotrophic lateral sclerosis (ALS) cases is the presence of intra-cytoplasmic transactive response DNA-binding protein of 43 kDa (TDP-43) aggregates in motor cortex and spinal cord motor neurons and glia, associated with anterior horn cell loss and gliosis (Arai et al, 2006; Neumann et al, 2006). Familial ALS (fALS) cases account for approximately 10% of all ALS cases; approximately 40% of fALS cases and a proportion of sporadic cases are caused by the C9orf hexanucleotide G4C2 repeat expansion, making it the commonest known ALS genetic mutation (DeJesusHernandez et al, 2011). Such patients typically present with motor symptoms; cognitive dysfunction (affecting all domains: fluency, language, executive and behavioural functions) is common, as is a neuropsychiatric presentation (Abrahams et al, 2000; Goldstein and Abrahams, 2013; Strong et al, 2017; Gregory et al, 2019). Their precise pathogenic role is widely debated (Vatsavayai et al, 2019); antisense RNA foci have been shown to exert a greater deleterious effect than sense RNA foci (Gendron et al, 2013; Zu et al, 2013; Cooper-Knock et al, 2015; Aladesuyi Arogundade et al, 2019) and antisense RNA foci, but not sense RNA foci, correlate with TDP-43 aggregation in C9orf motor neurons (Cooper-Knock et al, 2015; Aladesuyi Arogundade et al, 2019)

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Conclusion