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; Samantha K.  Barton; Karina McDade; Sharon  Abrahams; Siddharthan Chandran; Colin Smith; Jenna Gregory

doi:10.1093/braincomms/fcaa009

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, Samantha K. Barton, Karina McDade, Sharon Abrahams, Siddharthan Chandran, Colin Smith^* (Corresponding Author), Jenna Gregory^* (Corresponding Author)

^*Corresponding author for this work

Medical Sciences

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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.

Original language	English
Article number	fcaa009
Number of pages	10
Journal	Brain Communications
Volume	2
Issue number	1
Early online date	31 Jan 2020
DOIs	https://doi.org/10.1093/braincomms/fcaa009
Publication status	Published - Jan 2020

Bibliographical note

Acknowledgements
The authors would like to thank: (i) the Medical Research Council Edinburgh Brain Bank (ethics approval from East of Scotland Research Ethics Service, 16/ES/0084)) for supplying all post-mortem brain material and the Scottish Motor Neurone Disease Register (SMNDR)/Care Audit Research and Evaluation for Motor Neurone Disease (CARE-MND) consortium for all clinical and demographic data (ethical approval from Scotland A Research Ethics Committee 10/MRE00/78 and 15/SS/0216); (ii) The Scottish MND Clinical Specialist team for obtaining consent from patients with MND for inclusion in these studies; (iii) MND Scotland and the Sylvia Aitken Charitable Trust for funding CS to help to establish the MND Tissue Bank. The authors would also like to thank Advanced Cell Diagnostics for gifting the C9orf72 probe (BaseScopeTM Probe - BA-GGGGCCn-3zz-st, Cat Code: 704181), prior to it being made commercially
available.

Funding

A.R.M. is a Lady Edith Wolfson Clinical Fellow and is jointly funded by the Medical Research Council and the Motor Neurone Disease Association (MR/R001162/1). He also acknowledges support from the Rowling Scholars scheme, administered by the Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, Edinburgh, UK. The Chandran laboratory is supported by the Euan MacDonald Centre and the UK Dementia Research Institute, which
receives its funding from UK DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK. J.M.G. is funded by a starter grant for clinical lecturers from the Academy of Medical Sciences
(AMS: 210JMG 3102 R45620) and C.S. is supported by a Medical Research Council grant (MR/L016400/1).

Keywords

sense RNA foci
cognition
TDP-43
C9orf72
ALS

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VC The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
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Cite this

@article{4b623d1edfa54970bec0039688bcf7fc,

title = "Improved detection of RNA foci in C9orf72 amyotrophic lateral sclerosis post-mortem tissue using BaseScope{\texttrademark} shows a lack of association with cognitive dysfunction",

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{\texttrademark}, 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{\texttrademark} in the clinicopathological assessment of the role of sense RNA foci in C9orf72.",

keywords = "sense RNA foci, cognition, TDP-43, C9orf72, ALS",

author = "Mehta, {Arpan R.} and Barton, {Samantha K.} and Karina McDade and Sharon Abrahams and Siddharthan Chandran and Colin Smith and Jenna Gregory",

note = "Acknowledgements The authors would like to thank: (i) the Medical Research Council Edinburgh Brain Bank (ethics approval from East of Scotland Research Ethics Service, 16/ES/0084)) for supplying all post-mortem brain material and the Scottish Motor Neurone Disease Register (SMNDR)/Care Audit Research and Evaluation for Motor Neurone Disease (CARE-MND) consortium for all clinical and demographic data (ethical approval from Scotland A Research Ethics Committee 10/MRE00/78 and 15/SS/0216); (ii) The Scottish MND Clinical Specialist team for obtaining consent from patients with MND for inclusion in these studies; (iii) MND Scotland and the Sylvia Aitken Charitable Trust for funding CS to help to establish the MND Tissue Bank. The authors would also like to thank Advanced Cell Diagnostics for gifting the C9orf72 probe (BaseScopeTM Probe - BA-GGGGCCn-3zz-st, Cat Code: 704181), prior to it being made commercially available. Funding A.R.M. is a Lady Edith Wolfson Clinical Fellow and is jointly funded by the Medical Research Council and the Motor Neurone Disease Association (MR/R001162/1). He also acknowledges support from the Rowling Scholars scheme, administered by the Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, Edinburgh, UK. The Chandran laboratory is supported by the Euan MacDonald Centre and the UK Dementia Research Institute, which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council, Alzheimer{\textquoteright}s Society and Alzheimer{\textquoteright}s Research UK. J.M.G. is funded by a starter grant for clinical lecturers from the Academy of Medical Sciences (AMS: 210JMG 3102 R45620) and C.S. is supported by a Medical Research Council grant (MR/L016400/1).",

year = "2020",

month = jan,

doi = "10.1093/braincomms/fcaa009",

language = "English",

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journal = "Brain Communications",

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T1 - Improved detection of RNA foci in C9orf72 amyotrophic lateral sclerosis post-mortem tissue using BaseScope™ shows a lack of association with cognitive dysfunction

AU - Mehta, Arpan R.

AU - Barton, Samantha K.

AU - McDade, Karina

AU - Abrahams, Sharon

AU - Chandran, Siddharthan

AU - Smith, Colin

AU - Gregory, Jenna

N1 - Acknowledgements The authors would like to thank: (i) the Medical Research Council Edinburgh Brain Bank (ethics approval from East of Scotland Research Ethics Service, 16/ES/0084)) for supplying all post-mortem brain material and the Scottish Motor Neurone Disease Register (SMNDR)/Care Audit Research and Evaluation for Motor Neurone Disease (CARE-MND) consortium for all clinical and demographic data (ethical approval from Scotland A Research Ethics Committee 10/MRE00/78 and 15/SS/0216); (ii) The Scottish MND Clinical Specialist team for obtaining consent from patients with MND for inclusion in these studies; (iii) MND Scotland and the Sylvia Aitken Charitable Trust for funding CS to help to establish the MND Tissue Bank. The authors would also like to thank Advanced Cell Diagnostics for gifting the C9orf72 probe (BaseScopeTM Probe - BA-GGGGCCn-3zz-st, Cat Code: 704181), prior to it being made commercially available. Funding A.R.M. is a Lady Edith Wolfson Clinical Fellow and is jointly funded by the Medical Research Council and the Motor Neurone Disease Association (MR/R001162/1). He also acknowledges support from the Rowling Scholars scheme, administered by the Anne Rowling Regenerative Neurology Clinic, University of Edinburgh, Edinburgh, UK. The Chandran laboratory is supported by the Euan MacDonald Centre and the UK Dementia Research Institute, which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK. J.M.G. is funded by a starter grant for clinical lecturers from the Academy of Medical Sciences (AMS: 210JMG 3102 R45620) and C.S. is supported by a Medical Research Council grant (MR/L016400/1).

PY - 2020/1

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N2 - 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.

AB - 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.

KW - sense RNA foci

KW - cognition

KW - TDP-43

KW - C9orf72

KW - ALS

UR - http://dx.doi.org/10.1093/braincomms/fcaa009

U2 - 10.1093/braincomms/fcaa009

DO - 10.1093/braincomms/fcaa009

M3 - Article

SN - 2632-1297

VL - 2

JO - Brain Communications

JF - Brain Communications

IS - 1

M1 - fcaa009

ER -

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

Abstract

Bibliographical note

Keywords

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