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Distinct patterns of microstructural changes in mouse models of ALS/FTD with mutations in Tardbp, Fus and C9orf72

Aurea Martins Bach¹, Cristiana Tisca¹, Mohamed Tachrount¹, Carmelo Milioto^2,3, Mireia Carcolé^2,3, Shoshana Spring⁴, Remya R. Nair^5,6, Thomas J. Cunningham^6,7, Elizabeth M. C. Fisher⁸, Adrian M. Isaacs^2,3, Brian J. Nieman⁴, Jason Lerch¹, and Karla Miller¹
¹Wellcome Centre for Integrative Neuroimaging, FMRIB, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom, ²UK Dementia Research Institute at UCL, Faculty of Brain Sciences, University College London, London, United Kingdom, ³Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, University College London, London, United Kingdom, ⁴Mouse Imaging Centre, The Hospital for Sick Children, Toronto, ON, Canada, ⁵Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom, ⁶Mammalian Genetics Unit, MRC Harwell Institute, Oxford, United Kingdom, ⁷MRC Prion Unit and Institute of Prion diseases, University College London, London, United Kingdom, ⁸Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London, United Kingdom

Synopsis

Keywords: Neurodegeneration, Preclinical

Amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD) form a disease spectrum with shared clinical, pathological and genetic features. The most common mutations in ALS/FTD are in the genes C9Orf72, TARDBP and FUS. We used post-mortem diffusion kurtosis imaging to assess microstructure imaging phenotypes in mouse models of ALS/FTD with mutations in these genes. While mice with mutation in Tardbp presented reduced FA and MO in various white matter tracts, no difference could be detected in mice with mutation in Fus, and increased MO in a portion of the corpus callosum was observed in mice with mutation in C9orf72.

Introduction

Amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD) form a disease spectrum with shared clinical, pathological and molecular features¹. ALS patients present mainly motor defects, while FTD patients have changes in executive function, language and behaviour. Approximately 50% of ALS patients present behavioural and cognitive impairment and ~40% of FTD patients develop motor dysfunction². More than 30 genes are causative for ALS¹, of which at least 17 genes are linked to ALS and FTD². The most common genetic mutations in ALS and FDT are in C9Orf72, TARDBP, and FUS genes².
The disease mechanisms in ALS/FTD are not fully understood and these diseases lack a cure or treatment. Animal models that recapitulate features of ALS/FTD are essential to better understand disease mechanisms and assess potential therapeutics. Due to the heterogeneous nature of ALS/FTD, a range of animal models may be needed to better represent the ALS/FTD subgroups³.
MRI can provide biomarkers of ALS/FTD and contribute to the understanding of disease mechanisms and the development of candidate therapies. Imaging signatures of FTD/ALS include cortical thinning⁴, atrophy of subcortical grey matter⁵, and altered diffusion-MRI metrics⁶, varying according to the gene involved⁷. Despite the numerous animal models of ALS/FTD, most imaging studies focus on the SOD1 mouse, which is only representative of 2% of ALS cases⁸. In this study, we explore post-mortem diffusion MRI phenotypes in a broader range of mouse models of ALS/FTD with mutations in the genes Tardbp, Fus, and C9orf72.

Materials and methods

In total, 59 mice divided into three cohorts were studied. Mice had mutations in Tardbp (TDP-M323K⁹: 8 TDP-M323K homozygous mutants, 8 wild-type (WT) littermates, 12 month-old), Fus (homozygous FUSDelta14^10,11: 7 FUSDelta14 homozygous mutants, 10 WT littermates, 3-month-old), or C9orf72 (9 C9orf72-(PR)400 expressing PR dipeptide repeats¹², 7 C9orf72-(GR)400 expressing GR dipeptide repeats¹², and 10 WT littermates, 20 month-old). Ages were selected to reflect advanced stages of the disease in each model. Mice were perfused with paraformaldehyde 4% in 0.1 M PBS under anaesthesia. Perfusion solutions contained 2 mM of Gd-contrast agent (Gd-CA, Gadovist, Bayer Vital GmbH, Leverkusen, Germany). Heads were removed and dissected, keeping brains in the skull. Brains were immersed in 4% paraformaldehyde with 2 mM Gd-CA for 24h at 4^oC, and then kept in PBS with 2 mM Gd-CA and 0.05% azide at 4^oC until scanned.
Diffusion MRI was performed on a 7.0 tesla MRI scanner using a receive-only 4-channels surface cryoprobe and a volume transmit resonator (Bruker Biosystems, Etlingen, Germany). Diffusion-weighted images were acquired in 30 diffusion directions distributed across the sphere for 2 shells (b=2,500/10,000s/mm²), with four interleaved b=0 volumes (segmented EPI, TE/TR=30/500ms, 12 segments, 100μm isotropic resolution, scan time~12h). A separated b=0 volume with reversed phase encoding was acquired prior to the diffusion-weighted images and allowed to correct off-resonance effects using the package eddy¹³ (after Gibbs ringing correction¹⁴). Signal was fitted with the diffusion kurtosis model using FSL¹⁵ dtifit with the --kurtdir option to estimate fractional anisotropy (FA), mean diffusivity (MD), mode of anisotropy¹⁶ (MO), and kurtosis along parallel and perpendicular directions. Parallel and perpendicular kurtosis were then used to extract mean kurtosis (MK), and fractional anisotropy of kurtosis (FAK) (Figure 1, A-E).
Data was analysed separately for each cohort (TDP-M323K, FUSDelta14 and C9orf72). The b=0 images from each animal were averaged and the average b0 images were registered to a common space using pydpiper¹⁷. The same transforms were used to register all parametric maps to the common space for each cohort. FA maps were averaged, thresholded to include only voxels with FA above 0.3, and then skeletonised using FSL-TBSS¹⁸ to create a skeletonised white matter mask (Figure 1, F). For each parametric map, voxels extracted with this mask were compared between mutant mice and age-matched littermates using FSL-randomise¹⁹. Differences were considered significant for p<0.05 after family-wise error (FWE) correction for multiple comparisons in each metric.

Results

Multiple white matter tracts presented reduced FA and reduced MO in TDP-M323K mice (Figure 2). No differences could be detected in MD, MK, or FAK.
In this study, no differences could be detected in white matter for any of the diffusion MRI metrics assessed in homozygous FUSDelta14 mice.
Finally, both C9orf72-(GR)400 and C9orf72-(PR)400 mice presented reduced MO in the anterior portion of the corpus callosum (Figure 3). No further alteration could be detected for FA, MD, MK or FAK in these mice.

Discussion and conclusions

We have previously described that the pattern of volumetric changes is different in TDP-M323K, FUSDelta14 and C9Orf72-PR(400)/-GR(400) mice¹² (Figure 4). In this study, we showed that in addition to widespread volumetric changes²⁰, TDP-M323K mice also present reduced FA and MO in several white matter tracts. In contrast, FUSDelta14-hom mice, which present volumetric changes affecting various brain regions¹¹, did not show any detectable alteration in white matter microstructure in the current study. Finally, C9orf72-(PR)400 and C9orf72-(GR)400 mice presented subtle changes in MO in the anterior portion of the corpus callosum. This region is close to the taenia tecta, the only structure where altered volume was observed in these animals¹². These results expand the range of mouse models of ALS/FTD with microstructural alterations assessed with MRI and reinforce the complementarity of structural and diffusion MRI.

Acknowledgements

This work was supported by the Wellcome Trust (grant 202788/Z/16/Z), MRC and Harwell funding. The Wellcome Centre for Integrative Neuroimaging is supported by core funding from the Wellcome Trust (grant 203139/Z/16/Z).

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Figures

Figure 1. Example of the parametric maps derived from diffusion kurtosis imaging. A. Fractional anisotropy (FA, windowed 0-1). B. Fractional anisotropy of kurtosis (FAK, windowed 0-1). C. Mean diffusivity (MD, windowed 0-0.001). D. Mean kurtosis (MK, windowed 0-1). E. Mode of anisotropy (MO, windowed -1 - +1). F. Skeletonised white matter mask for voxel-wise analyses.

Figure 2. Reduced fractional anisotropy (FA) and mode of anisotropy (MO) in TDP-M323K mice. Both alterations point to the loss of integrity of white matter tracts in this mouse model. Voxels marked in blue/aqua had reduced FA (A) or MO (B) in TDP-M323K mice when compared to WT littermates at FWE 5%, and voxels marked in red had increased MO in TDP-M323K mice (B). In (A), lighter blue reflects smaller p-values. Tracts involved include the corpus callosum, external capsule, internal capsule, fimbria, fasciculus retroflexus, and optic tract.

Figure 3. Mode of anisotropy (MO) is increased in C9orf72-(GR)400 and C9orf72-(PR)400 in the anterior portion of the corpus callosum. MO reflects the shape of the diffusion tensor: higher MO is observed in regions where the diffusion tensor is shaped like a cigar, while lower MO is observed in regions where the tensor has a planar shape¹⁶. Increased MO in the corpus callosum of mice with mutation in C9orf72 may point to selective degeneration of fibres in a region of crossing fibres, leading to the predominancy of one fibre population.

Figure 4. Diffusion and structural MRI reveal distinct patterns of volumetric and microstructural alterations in mouse models of ALS/FTD with mutation in Tardbp, Fus, and C9orf72. (A). Mice with mutation in Tardbp have reduced FA and MO, and (B) widespread changes in relative volume. (C). Mice with mutation in Fus did not present any detectable alteration in diffusion MRI, despite (D) multiple foci of relative volumetric changes. (E) Mutation in C9orf72 led to increased MO in the anterior corpus callosum and (F) increased volume in a nearby region of the olfactory cortex.

Proc. Intl. Soc. Mag. Reson. Med. 31 (2023)

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DOI: https://doi.org/10.58530/2023/0586