## 3 Results
### The Cerebellar Transcriptome Profile of ATM-Null Mice at 12 Months of Age
Asterisks reflect significance: * = \(p\)\(\leq\) 0.05, ** = \(p\)\(\leq\) 0.01, *** = \(p\)\(\leq\) 0.001, *** = \(p\)\(\leq\) 0.001, *** = \(p\)\(\leq\) 0.0001, ns = non-significant. Data are displayed as bar plots with data points, mean \(\pm\) SEM. Figure 6: Genome-wide survey of alternative splicing in cerebellum from 12-month-old ATM-null mice reveals enrichment for neuropeptide signalling pathways and neurotransmission. (**a**) Table of all  
transcripts that display excessive alternative splicing (Filter criteria: Exon Splicing Index >/\(<\) +/\(-\)5, Exon _p_-value < 0.05, Exon FDR _p_-value < 0.25 and Group: Multiple Complex and Coding). Increased exon-splicing index is highlighted in red, decreased exon-splicing index in green. (**b**) STRING functional connection networks ([[https://string-db.org/](https://string-db.org/)]([https://string-db.org/](https://string-db.org/)), accessed on 6 April 2023) of these alternatively spliced transcripts. Red buttons belong to the neuropeptide signaling pathway, blue buttons are implicated in regulation of neurotransmitter levels, and green buttons exert functions during synapse organization. (**c**) Structure view of the _Oprn1_ transcript structure with splicing indexes displayed in line plots for WT (grey) and _Atm_-KO (purple) cerebellum. Validation experiments in (**d**) cerebellum of 12-month-old _Atm_-KO vs. WT (4 vs. 4) mice and (**e**) SH-SYSY _ATM_-KD cells under CQ-stress. Asterisks reflect significance: * = \(p\) \(\leq\) 0.05, ** = \(p\) \(\leq\) 0.01, *** = \(p\) \(\leq\) 0.001, ns = non-significant. _Oprn1_ stood out with an overall exon-splicing index of 39.17, which is summarised graphically (Figure 6c) by the Transcriptome Analysis Console (Applied Biosystems) for each oligonucleotide probe along the transcript structure. Extrapolating probes within individual exons or at the junction between known exons or cryptic exons from these data in mouse onto known facts in human, we tested the credibility of these _Oprn1_ splice changes by RT-qPCR in human neuroblastoma cells. It should also be taken into account that an oligonucleotide probe within an exon may show a differential increase or decrease, while a Taqman assay is usually quantifying the amplification product at the junction between two adjacent exons, so a splice change may be detectable by a specific RT-qPCR assay but not by the neighbouring assay, and the effects may not be conserved between species, with differing exon number nomenclature. As shown in Figure 6d, the exon 2-3 boundary of _Oprn1_ exhibited a 15-fold signal increase in old ATM-null cerebellum, while the exon 5-6 junction signal was unaltered. In the human SH-SYSY cells, the RT-qPCR results of exon 1-2 and exon 3-4 boundaries in _Oprn1_ transcript also showed a massive dysregulation upon ATM deficiency (reduction to 5%), with no significant change after osmotic stress (Figure 6e). This downregulation was not detected upon ATM kinase inhibitor treatment (Figure S4c), where _Oprn1_ transcript levels remained stable. _Oprn1_ transcript downregulation exclusively in the _ATM_-KD condition was robustly reproduced also in experiments with BLEO, NaARS and LY-294002 (Figure S4c), reflecting stressor-independent effects of ATM deficiency itself. Thus, although the splicing details may differ between species and cell type, with cerebellar tissue even revealing opposite effects than cultured neuroblastoma cells, the mouse microarray data and validation experiments by RT-qPCR in mouse cerebellum and human neuroblastoma cells clearly identified the opioid mu receptor as mRNA under control of ATM.