INTEGRATED SHORT- AND LONG-READ TRANSCRIPTOMICS UNCOVERS EXTENSIVE SPLICING ALTERATIONS IN AMYOTROPHIC LATERAL SCLEROSIS CEREBELLUM

TDP-43 protein inclusion pathology is observed in the primary sites of neurodegeneration (motor cortex and spinal cord) in 97% of amyotrophic lateral sclerosis (ALS) patients. For non-motor brain regions, the incidence of this hallmark pathology is variable. Dysregulation of RNA splicing is a key pathological mechanism in ALS, with loss of TDP-43 function leading to widespread “cryptic” splicing. We previously conducted a multi-region brain transcriptomic analysis of ALS to better understand the alterations occurring across brain regions variably affected by TDP-43 inclusions. Short-read RNA-seq was performed on five brain regions (motor cortex, prefrontal cortex, hippocampus, occipital cortex, cerebellum) each derived from 22 ALS cases and 11 controls. Intriguingly, despite the absence of TDP-43 inclusions, the cerebellum demonstrated the most ALS-specific alternative splicing events. To further explore this finding, we have generated parallel PacBio long-read and Illumina short-read RNA-seq for cerebellum tissue derived from 15 controls, 24 sporadic ALS cases and nine C9orf72-ALS cases (a genetic subgroup with non-TDP-43 cerebellar pathology). Bioinformatic analysis of the short-read RNA-seq using MAJIQ identified high levels of intron retention (sporadic ALS: 1,027 events [49% de novo], C9orf72-ALS: 1,375 events [61% de novo]) and 16 cryptic splicing events associated with TDP-43 loss of function in ALS cases. SQANTI3 classification of the FLAIR-generated long-read transcriptome identified 175,945 unique transcripts across our cerebellum samples, 43.8% of which were classified as novel emphasising the utility of long-read sequencing for comprehensive transcriptome characterisation. We hypothesise that identified cerebellar transcriptome alterations may represent early pathological events and/or neuroprotective mechanisms in ALS.