Abstract
The mechanisms underlying the selective degeneration of motor neurones in amyotrophic lateral sclerosis (ALS) are poorly understood. The aim of this study was to implement spatially resolved RNA sequencing in human post mortem cortical tissue from an ALS patient harbouring the C9orf72 hexanucleotide repeat expansion to identify dysregulated transcripts that may account for differential vulnerabilities of distinct (i) cell types and (ii) brain regions in the pathogenesis of ALS. Using spatial transcriptomics (ST) we analysed the transcriptome of post mortem brain tissue, with spatial resolution down to 100μm. Validation of these findings was then performed using BaseScope, an adapted, in situ hybridization technique with single-transcript single-cell-resolution, providing extensive regional and cell-type specific confirmation of these dysregulated transcripts. The validation cohort was then extended to include multiple post mortem brain regions and spinal cord tissue from an extended cohort of C9orf72, sporadic ALS (sALS) and SOD1 ALS cases. We identified sixteen dysregulated transcripts of proteins that have roles within six disease-related pathways. Furthermore, these complementary molecular pathology techniques converged to identify two spatially dysregulated transcripts, GRM3 and USP47, that are commonly dysregulated across sALS, SOD1 and C9orf72 cases alike. This study presents the first description of ST in human post mortem cortical tissue from an ALS patient harbouring the C9orf72 hexanucleotide repeat expansion. These data taken together highlight the importance of preserving spatial resolution, facilitating the identification of genes whose dysregulation may in part underlie regional susceptibilities to ALS, crucially highlighting potential therapeutic and diagnostic targets.
Highlights
Our experiments demonstrated specific spatial localization of regionally distinct transcripts (Figure S1)
This implies that spatial transcriptomics is able to detect and replicate differences found in whole-tissue experiments, it has the added benefit of being able to detect, more targeted, hypothesis driven and spatially resolved questions as implemented in our study
We were able to demonstrate differential dysregulation of transcripts across distinct central nervous system (CNS) regions, that may account for differential susceptibilities to the underlying disease process
Summary
Genetics can influence the development of amyotrophic lateral sclerosis (ALS), with both causative genes in. The additional benefit of examining the cerebellum is that, in ALS patients carrying the C9orf hexanucleotide repeat, there is a high burden of protein misfolding without the corresponding cell death that is seen in other brain areas [16] This is useful when examining transcriptional dysregulation because in brain areas with substantial cell death, for example in the motor cortex, there will be a number of transcripts whose dysregulation is related to cell death and is not related to active disease processes. Due to limitations in the spatial resolution of ST (100 lm), we implemented, in parallel, a complementary technique called BaseScope, as a targeted approach with singletranscript, single-cell resolution [17] Taken together these techniques have allowed us to interrogate the previously unexplored, spatial transcriptome of human post mortem ALS CNS tissue. The aim of this study was to detect and spatially resolve transcriptional differences between brain regions and their composite cell types in ALS patient neural tissue compared to control tissue, to identify underlying differential cell vulnerabilities to the disease process in ALS, to inform therapeutic target selection and biomarker development in this field
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