## 3 Results
### The Cerebellar Transcriptome Profile of ATM-Null Mice at 12 Months of Age
Extreme upregulations of several factors that are selectively expressed in the choroid plexus, whose presence in the cerebellar samples was not controlled, were interpreted as artefacts. Figure 1: Workflow and quality control for genome-wide cerebellar transcriptome analysis from 12-month-old _Atm_-KO mice. (**a**) Schematic representation of the workflow performed in age and sex-matched _Atm_\({}^{*/+}\) versus _Atm_\({}^{-/-}\) mice (n = 4 vs. 4, two males and two females each, mutant selection based on reduced cerebellar weight). mRNA was extracted from cerebella of these animals using Trizol, and ClariomD microarray hybridisation was then performed. Data were analysed using the TAC Software provided by Affymetrix, and by PANTHER Overrepresentation analysis of pathway enrichments. (**b**) Mouse genotype validation via RT-qPCR, detecting the quantity of WT _Atm_ transcript versus its reduction due to exon deletion and nonsense-mediated RNA decay in the _Atm_-KO samples, using _Tbp_ transcript as normaliser (n = 4). (**c**) Mouse genotype validation via quantitative immunoblots, regarding ATM protein absence (3–8% Tris-acetate gels, lane 4 empty) versus beta-actin (ACTB) (4–20% Tris-glycine gel) as loading control (WT vs. _Atm_-KO, 3 vs. 4). Asterisks reflect significance: *** = \(p\)\(\leq\) 0.001. Data are displayed as mean \(\pm\) SEM. A bioinformatics survey of gene ontology terms in biological processes and molecular functions by PANTHER software indicated prominent deficits in corticotropin-dependent stress responses, as well as presynaptic machinery and vesicle priming (Figure 2c), versus prominent excess transcripts for neurotransmitter loading and channel activity (Figure 2d). The upregulations of neurotransmission components occurred without selectivity for any cell type, involving glutamate, GABA, glycin, muscarinic and nicotinic acetylcholine, as well as dopamine receptor transcripts. Notably, the neuropeptide signaling pathway (GO:0007218) was the 17 th most enriched term among upregulates (FDR \(p\) = 2.02 \(\times\) 10\({}^{-5}\)) and also showed a non-selective pattern in general, involving somatostatin, tachycardia, neurotensin, endothelin, vasobibin, encephalitis, opioid mu and kappa3 signalling components. Furthermore, detailed bioinformatics studies showed significant enrichment on the STRING webplatform for ataxia genes, vesicular factors, calcium homeostasis factors, and immediate-early genes. The factors involved in these enrichments are annotated in Table S1, together with all ATM protein interactome components and the ATM kinase target proteins known at present. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**ID**} & \multicolumn{2}{c}{**Cerebellum 12 Months Avg (log2)**} & \multirow{2}{*}{**Fold Change**} & \multirow{2}{*}{**FDR _p_-Val**} & \multirow{2}{*}{**Gene Symbol**} \\ \cline{2-2} \cline{5-6}  & **KO** & & & & \\ \hline TC1100002122.mm.1 & 10.58 & 9.71 & 1.83 & 0.0081 & Neft \\ TC140000965.mm.1 & 13.04  
Figure 2: Genome-wide survey of transcript levels in cerebella from 12-month-old ATM-null mice. (**a**) Global transcriptome documentation via Clarion D microarrays, visualised as a volcano plot with  
symmetry due to logarithmic scales, where the \(X\)-axis shows down- versus up-regulations (in a green versus red colour, respectively) while the \(Y\)-axis shows the significance of changes via false detection rates (FDR), identifying factors with relevance for pathway enrichments and for follow-up studies by their gene symbols. (**b**) Total amount of detected transcripts (65,956) and ratio of transcripts that passed the filter criteria (8257, 12.52%). Of these, 43.42% (3585) were upregulated and 56.58% (4672) were downregulated. The upregulation and downregulation were further classified into different transcript categories, namely ribosomal, coding, precursor micro-RNA, pseudogene, small RNA, non-coding, tRNA and multiple complex, highlighting a prominent downregulation of non-coding RNAs.