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Anatomical and microstructural brain alterations in the TDP-M323K mouse model of amyotrophic lateral sclerosis
Aurea B Martins-Bach1, Mohamed Tachrount1, Cristiana Tisca1, Lily Qiu1, Shoshana Spring2, Jacob Ellegood2, Brian J Nieman2, John G. Sled2, Remya Raghavan-Nair 3, Elizabeth Fisher4, Thomas Cunningham3, Jason Lerch1, and Karla Miller1
1Wellcome Centre for Integrative Neuroimaging, University of Oxford, Oxford, United Kingdom, 2Mouse Imaging Centre, The Hospital for Sick Children, Toronto, ON, Canada, 3Mammalian Genetics Unit, MRC Harwell Institute, Oxford, United Kingdom, 4Department of Neuromuscular Diseases, Institute of Neurology, University College London, London, United Kingdom

Synopsis

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease, characterized by aggregates of TDP-43 protein in the brain of most patients. The TDP-M323K mouse has a mutation in the gene encoding the Tdp-43 protein, and presents progressive motor and neurological phenotypes. In this study, we assessed structural and microstructural alterations in the brain of TDP-M323K mice with preclinical MRI (7 tesla). High resolution images showed brain atrophy, but relative volume changes included hypertrophy in the cortex and hippocampus. Diffusion MRI revealed alterations compatible with neurodegeneration in the white matter and striatum. TDP-M323K mice recapitulate brain imaging phenotypes observed in ALS patients.

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that leads to death usually 3-4 years after onset. More than 30 genes have already been related to ALS, and patients show variable clinical alterations1. Despite this clinical and genetic variability, more than 98% of ALS patients present TDP-43 aggregates in the brain2. TDP-43 is an RNA-binding protein encoded by the gene TARDBP with a role on RNA processing and maturation, and mutations in TARDP account for ~5% of familial ALS3. However, the pathogenic mechanisms in ALS and how TDP-43 contributes to it are still unknown.
Due to the heterogeneous nature of ALS, a range of animal models might better represent the ALS subgroups, potentially helping to better understand the disease mechanisms and in the assessment of candidate therapeutical approaches4. The TDP-M323K mouse is a model of ALS with a missense mutation in the Tardp gene (p.M323K) induced by ENU mutagenesis. Homozygous TDP-M323K mice present altered splicing in target mRNAs and altered regulation of the expression of multiple genes. These mice develop progressive neurological and motor phenotypes, with grip strength loss at 12 months of age, p62 and ubiquitin inclusions in the spinal cord and brainstem at 18 months of age, and reduction in motor units and motor neuron count in the spinal cord at 24 months of age5. However, it is not clear if they develop brain alterations similar to those observed in human ALS patients6,7.
In this study, we aimed to identify brain phenotypes in 12-month-old homozygous TDP-M323K mice using post-mortem MRI. Volumetric and microstructural changes were assessed with high-resolution structural T2w images and diffusion MRI.

Materials and Methods

Eight homozygous TDP-M323K mice and 8 wild-type (WT) littermates (females, 12-month-old, C57BL/6J-DBA/2J background) were studied (one sample excluded from the dMRI analysis due to image artefacts). Mice were anesthetized with ketamine/xylazine and intracardially perfused with PBS followed by formalin-4%, both containing a Gd-contrast agent (Gd-CA, 2mM; Gadovist, Bayer Vital GmbH, Leverkusen, Germany). Brains were kept in the skull, immersed overnight in formalin-4%/Gd-CA (2mM), and stored at 4°C in PBS/Gd-CA (2mM)/Na-azide (0.05%) until scanned.
Structural MRI (Figure 1a) was performed on a 7.0 tesla MRI scanner (Agilent Inc., Palo Alto, CA), using sixteen custom-built solenoid coils in parallel8 (T2-weighted 3D-FSE, TEeff/TR=30/350ms, 6 echoes/TR, 4 averages, 40μm isotropic resolution, cylindrical acquisition of k-space9, scan time~14h). Images were registered using MBM.py10. Volumes were estimated from the Jacobian determinants and modelled as a function of genotype (mouse weight as a covariate, FDR correction). We considered both local volume changes, where a first affine registration was used to remove global scaling differences before calculating the relative Jacobian determinant, and global volume changes, where the absolute Jacobian determinant includes these global scaling differences.
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~14h). A separated b=0 volume with reversed phase encoding allowed to correct off-resonance effects using the package eddy11 (after Gibbs ringing correction12). Signal was fitted with diffusion kurtosis13 (tensor and mean kurtosis) and NODDI models14 (Figure 1b-f). The median value in regions of interest (Table 1) was extracted from the registered maps and compared between genotypes (t-tests, Bonferroni correction).

Results and Discussion

Total brain volume was 17.9% smaller in TDP-M323K mice (TDP-M323K: 382.2±7.5 mm3, WT 466.9±6.7 mm3, p=1.5e-12). However, TDP-M323K mice weighted 17.4% more than WT (TDP: 41.2±5.6 g, WT: 35.1±3.2 g, p=0.02, Figure 2), possibly recapitulating the overeating behaviour observed in ALS patients15.
In the voxel-wise analysis, almost all brain regions were smaller in TDP-M323K when comparing global volumes calculated from the absolute Jacobians (Figure 3a). Multiple white matter regions presented atrophy in the relative comparison (Figure 3b). Interestingly, TDP-M323K showed increased relative volume, mainly in the cortex and hippocampus. This contrasts with the cortical thinning generally observed in ALS patients6, but is in agreement with increased grey matter volume in ALS patients without cognitive impairment7. It may reflect earlier stages of the disease, where pathological alterations such as inflammation would precede neuronal loss and atrophy, or compensatory mechanisms within the motor and extra-motor network.
FA was reduced in the cingulum bundle, dorsal fornix and striatum of TDP-M323K mice. ICVF was also reduced in the cingulum bundle and OD was increased in the striatum (Figure 4). These alterations are in agreement with similar observations in ALS patients7 and mice16,17, and point to microstructural changes compatible with neurodegeneration and inflammation. Contrarily, TDP-M323K mice presented increased FA/reduced OD in the the optic tract. This apparently paradoxical increased FA has been already observed in neurodegeneration18, and can be associated with differential involvement of tracts in regions of crossing fibres or developmental differences.

Conclusions

TDP-M323K mice recapitulate brain imaging phenotypes observed in ALS patients, with volumetric and microstructural alterations detected with structural and diffusion MRI in 12-month-old mice. At this stage of the disease, a mild reduction in grip strength had been previously reported, indicating that earlier brain phenotypes could be detected with MRI in this mouse model.

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. Representative images and diffusion MRI maps. (a) T2w structural image for volumetric analysis. b-d: Parametric maps extracted from diffusion MRI after modelling the signal to the diffusion kurtosis model: FA: fractional anisotropy (b), MD: mean diffusivity (c), and MK: mean kurtosis (d). e,f: Parametric maps extracted using the NODDI model: OD: orientation dispersion (e) and ICVF: intracellular volume fraction (f). Scale bar: 5 mm.

Table 1. Parameters estimated with diffusion kurtosis and NODDI modelling. Median values were extracted from ROIs using masks from atlas-based segmentation19. White matter masks from individual tracts were skeletonized (multiplied by a skeletonized white matter mask generated using TBSS20) to avoid partial volume effects. Diffusion MRI results in ventral brain regions, including the corticospinal tract, were not evaluated due to lower SNR and poorer fitting quality.

Figure 2. Brain volume is reduced in TDP-M323K mice, despite TDP-M323K mice weighting more than WT littermates. There was no correlation between brain volume and mouse weight in each genotype.

Figure 3. Effect of genotype in brain volume in TDP-M323K mice. (a) When compared directly to WT mice, TDP-M323K mice showed smaller volumes in almost all regions of the brain. (b) When volumes relative to the whole brain size were compared, TDP-M323K mice showed atrophy mainly in the white matter, while regions in the cortex and hippocampus had relatively increased volume. Most of the spatial patterns in t-statistics maps are bilaterally symmetric. Atrophic regions in TDP-M323K mice are show in blue and hypertrophic regions are show in red/yellow. False discovery rate of 5%.


Figure 4. Diffusin MRI reveals microstructural alterations in white and grey matter in TDP-M323K mice.

Proc. Intl. Soc. Mag. Reson. Med. 29 (2021)
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