## 3 Results
### The Cerebellar Transcriptome Profile of ATM-Null Mice at 12 Months of Age
For the human IP3R protein encoded by _ITPR1_ transcript, these experiments confirmed a strong reduction of abundance (to about 35%) upon ATM-deficiency (Figure 5b). In contrast, the quantitative immunoblots indicated the protein levels of USP2 to be unchanged by CQ and by _ATM_-KD (Figure 5c), so apparently the significant _USP_2 mRNA reduction after CQ and _ATM_-KD demonstrated in Figure 4c does not rapidly impact steady-state immunoreactivity, and it may be that posttranslational control of USP2 and its MDM4/HDMX-MRN-complex-association [86; 108; 109] are more decisive for short-term USP2 activity regulation than its resynthesis. Still, our mRNA findings confirm USP2 as a very consistently ATM-dependent factor whose expression is modified by cytosolic ATM in neural cells, and which functions very upstream in the ATM-dependent stress response pathways--so it might be a useful target of novel preventive therapies. ATM-Null Mouse Cerebellum Alternatively Spliced mRNAs Are Enriched for Neurotransmission and Neuropeptide Signalling Factors  
For rapidity, stress responses are conveyed through the cytoplasm by phosphorylation signals to adapt transcription of immediate-early genes in the nuclear chromatin, but even more directly stress signalling readjusts the alternative splicing of existing transcripts in the nucleus and the editing of actively translated transcripts in the cytosol. Since the ClariomD microarrays employed for cerebellar transcriptome profiling represent almost all exons, we also assessed splice dysregulations in this dataset from aged ATM-null mice, employing the \"Alt Splice View\" function in the Transcriptome Analysis Console from AppliedBiosystems. Interestingly, upon filtering for significant changes (_p_-value < 0.05, FDR _p_-value < 0.25) with strong fold-change (Exon Splicing Index >/\(<\) +/-5), a set of 40 alternatively spliced transcripts was identified. Of these, 31 displayed an overall increased exon-splicing index, while 9 showed a reduced exon-splicing index (Figure 6a). More detailed assessment revealed that most of them represent altered quantities of a single exon within a dysregulated transcript, and because of concerns that not all oligonucleotide probes within an mRNA can be expected to exhibit parallel linear signal changes and might therefore mimic alternative splicing artificially, we decided to annotate such observations for the main candidates (Table S1, second datasheet). Again, the neuropeptide signaling pathway (FDR q = 1.16 \(\times\) 10\({}^{-6}\)), regulation of neurotransmitter levels and synapse organization were prominent, as identified on the STRING Web browser (Figure 6b). Figure 5: The stressor chloroquine provides the most effective model in SH-SYSY _ATM_-KD cells, for representative strong dysregulations in different pathways, which were previously documented in aged ATM-null cerebella. (**a**) _ATMIN_ mRNA (n = 6) was analysed as interactome component of ATM. _NR4A1_ (n = 6) was analysed as immediate early gene. _RRADG_ (n = 3) transcript levels were analysed as positive controls for osmotic stress elicited by CQ. _ECEL1_ (n = 3) and _MME_ (n = 3) were analysed for the group of neuropeptide endopeptidaseases. mRNAs for _GRID2_ (n = 3), _ITPRI_ (n = 3) and _RORA_ (n = 3) were analysed as known ataxia disease genes. (**b**) The protein Inositol-1,4,5-Trisphosphate Receptor (IP3R, encoded by _ITPRI_) was also significantly reduced in shATM cells compared to NT CTRL cells as determined by quantitative immunoblots, while induction by CQ stressor treatment did not reach significance (n = 3). The double band around 315 kDa was quantified by densitometry. Tubulin A (TUBA) was used as sample loading control and normaliser, in view of its high abundance similar to IP3R. (**c**) USP2 protein appeared unchanged in quantitative immunoblots (n = 3) of shATM cells compared to NT CTRL cells, despite the transcript induction shown in Figure 3e. For statistical trends, the precise \(p\)-value was shown.