## 3 Results
### The Cerebellar Transcriptome Profile of ATM-Null Mice at 12 Months of Age
These experiments focused on ATM interactors, ataxia genes, neurotransmitter loading factors, glutamate receptors, immediate early response components, and neuropeptide signalling molecules (Figure 2e). The RT-qPCR validation of these selected dysregulations was extended to cerebellar tissue from 1.5-3-month-old ATM-null versus age-/sex-matched WT mice, showing similar dysregulations to occur early on for _Nr4a1_, _Nr4a2_, _Oprm1_, and _Tacr1_ (Figure S1). Furthermore, the 12-month-old ATM-null cerebellar transcriptome confirmed previous RT-qPCR results in an ATM-null cerebellum at the age of 2 months [67] regarding the downregulations of _Itrp1_, _Atp2b2_ and _Grin2c_, versus the upregulations of _Grin2b_ and _Cyp4a1_ mRNA levels. In Human Neural Cells with Stable ATM-K knockdown, Cerebellar Hallmark Dysregulations Are Recapitulated Best after Osmotic Stress, and Partially after Trophic Stress  
ATM-deficiency was studied further in cell culture, to assess the reproducibility of these findings in humans, and to identify the most suitable stressor in vitro that mirrors such age-associated dysregulations, while enabling us to generate unlimited samples for mechanistic studies. We used the human SH-SYSY neuroblastoma cell line, introduced various ATM shRNAs via lentiviral transduction, and produced a stable _ATM_-KD cell line that achieved high ATM protein- and mRNA-reduction for further analysis. The most efficient KD was produced by shRNA#2 (hereafter be referred to as shATM), triggering obvious changes in cellular morphology (Figure S2a), and reductions of _ATM_ transcript to 36% (Figure S2b) and protein to 9.5% (Figure S2c), compared to the non-target shRNA control (NT CTRL) condition. In this human neural cell line, the application of the osmotic stressor chloroquine (CQ) did not alter the abundance of ATM protein, but induced phosphorylation at ATM residue S1981 (1.8-fold, with \(p=0.1408\) in three biological replicates), an expected event for DDR-triggered autophosphorylation/activation of this stress sensor molecule (Figure S2d). To assess whether SH-SYSY neuroblastoma cells have a similar distribution of ATM in subcellular fractions as adult cerebellar tissue, differential detergent isolation of nuclear, mitochondrial, and cytoplasmic fractions was performed firstly in cerebella from WT versus ATM-null mice at the age of 3.5 months (Figure 3a), and secondly in SH-SYSY NT CTRL cells compared to shATM cells (Figure 3b). Even though there was some leakage from the GAPDH-immunopositive cytoplasmic (cyto) fraction to the HSP60-positive mitochondrial (mito) and the LAMIIN-A/C-positive nuclear (nuc) fraction, ATM was clearly located in the cytoplasmic rather than the nuclear fraction in the mouse cerebellum (Figure 3a). This finding is novel, since previous analyses until maximal cerebellar age of 6 weeks, after completion of Purkinje neuron maturation and granule cell precursor migration [96; 97; 98], had observed ATM more in nuclear than in cytosolic fractions. A localisation in the cytosolic fraction was also clearly observed for ATM in SH-SYSY cells, although the gels exhibited some leakage of the nuclear fraction to the mitochondrial fraction (Figure 3b). Importantly, this cytoplasmic localisation of ATM in vitro was not altered by administration of the osmotic stressor CQ or the genotoxic stressor bleomycin (BLEO) in several independent experiments (Figure S3a,b). One experiment with LY stress, and one experiment with NaARS stress, also failed to detect an ATM localisation change. These results indicate that the _ATM_ knockdown in SH-SYSY neuroblastoma cells can be used as useful in vitro models for neural _Atm_-deficiency, regarding transcript and protein levels, stress induction and subcellular fractionation.