Oral Presentation 47th Lorne Genome Conference 2026

Integrated short- and long-read transcriptomics uncovers extensive splicing alterations in motor neuron disease cerebellum (133275)

Natalie Grima 1 , Andrew N Smith 1 , Claire E Shepherd 2 3 , Lyndal Henden 1 , Dominic B Rowe 1 , Matthew C Kiernan 3 4 5 , Ian P Blair 1 , Kelly L Williams 1
  1. Motor Neuron Disease Research Centre, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia
  2. School of Biomedical Science, Faculty of Medicine and Health, UNSW, Sydney, NSW, Australia
  3. Neuroscience Research Australia, Randwick, NSW, Australia
  4. Brain and Mind Centre, The University of Sydney, Sydney, NSW, Australia
  5. Department of Neurology, Royal Prince Alfred Hospital, Sydney, NSW, Australia

TDP-43 protein inclusion pathology is observed in the primary sites of neurodegeneration (motor cortex and spinal cord) in 97% of motor neuron disease (MND) patients. For non-motor brain regions, some (e.g. prefrontal cortex, hippocampus) demonstrate variable incidence of TDP-43 inclusions across MND patients while others (e.g. occipital cortex, cerebellum) are consistently spared of this pathology and neurodegeneration. Dysregulation of RNA splicing is a key pathological mechanism in MND, with loss of TDP-43 function leading to widespread “cryptic” splicing. We previously conducted a multi-region brain transcriptomic analysis of MND to better understand the alterations occurring across brain regions variably affected by TDP-43 inclusions. Short-read RNA-seq was performed on five regions (motor cortex, prefrontal cortex, hippocampus, occipital cortex, cerebellum) each derived from 22 MND cases and 11 controls. Intriguingly, despite the absence of TDP-43 inclusions, the cerebellum demonstrated the most MND-specific alternative splicing events. This included a switch in POLDIP3 transcript usage, an established marker of TDP-43 loss of function.

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 MND cases and nine C9orf72-MND cases. A pathological feature of C9orf72-MND is abundant RNA foci and TDP-43-negative dipeptide-repeat protein inclusions in the cerebellum. Bioinformatic analysis of the short-read RNA-seq using a combination of MAJIQ and LeafCutter identified high levels of intron retention (sporadic MND: 1,027 events [49% de novo], C9orf72-MND: 1,375 events [61% de novo]) and 16 cryptic splicing events associated with TDP-43 loss of function in MND 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 MND.