## 3 Results
### The Cerebellar Transcriptome Profile of ATM-Null Mice at 12 Months of Age
The ATM-Null Cerebellar mRNA Dysregulations Are Mimicked in SH-SH5Y Cells by ATM Knockdown Rather Than ATM Kinase Antagonism, and by CQ Better Than Trophic/Oxidative/ Genotoxic Stress  
To understand whether the cerebellar dysregulations of old ATM-null mice are due to ATM absence as a platform for protein complex formation, or absent ATM kinase activity, we assessed if they are recapitulated after stress in neuroblastoma cells upon KD of _ATM_ mRNA, or after treatment with KU-55933 (KU) as a pharmacological inhibitor of ATM-mediated phosphorylation (scheme and control of _ATM_ mRNA levels in Figure 4a,b). As representative transcripts under control of ATM, we chose upstream effectors such as _USP2_  
Figure 3: ATM localisation in subcellular fractions by differential detergents. The workflow scheme is shown on the left side. On the right side, samples represent cell fractions of cytoplasm (cyto), mitochondria (mito) and nucleus (nuc). The purity of each aliquot was assessed by the markers GAPDH for cytoplasm, HSP60 for mitochondria, and LAMIN-A/C for nucleus. The size markers in kilodalons (kDa) on the right margin of each gel confirm the expected molecular weight of each protein studied. (**a**) Immunoblot detecting ATM in WT versus _Atm_-KO mouse cerebella from 3.5-month-old mice. (**b**) Immunoblot detecting ATM in non-target shRNA transduced control (NT CTRL) versus shATM transduced mutant SH-SYSY neuroblastoma cells. in view of its role within the ATM-interactome, and _PERI_ as immediate-early transcript modulated by phosphorylation cascades (Figure 4c-f). Figure 4c shows the expected significant downregulation of _USP2_ after CQ administration in the _ATM_-KD cells (to 65% of control after CQ, and further reduction to 48% and 38% in shATM cells with and without CQ-stress), while in KU-treated cells downregulation of _USP2_ was only generated by CQ-treatment but not the kinase inhibition. Similarly, Figure 4d shows a significant ATM-dependent CQ stressor effect, with the significant 1.5-fold induction of _PERI_ by CQ stressor being abolished to control levels in shATM cells. Again, this effect was not reproduced in KU-treated cells. For _USP2_ (Figure 4e), genotoxic and oxidative stress were unable to trigger the downregulation; only trophic stress via treatment with the PI3K-inhibitor LY-294002 resulted in a significant ATM-dependent reduction. For _PERI_ (Figure 4f), all other stressors were ineffective. Exploiting the availability of a specific and sensitive anti-PERI antibody, a reduction of PERI protein was found in neuroblastoma cells with stable _ATM_-KD even before the application of acute stress (Figure 4g). The administration of CQ resulted in a PERI reduction in NT CTRL cells, but a converse PERI protein induction in shATM cells. Thus, a combination of _ATM_-KD with CQ-stress appeared to represent the best in vitro modelling approach in SH-SYSY neuroblastoma cells, to investigate the roles of cerebellar mRNA dysregulations in aged ATM-null cerebellum. Also in Human Cells, ATM-Deficiency Impacts Key Pathomechanism Factors Like Interactor ATMIN, Immediate-Early mRNA FOXO3, Osmotic Regulator RRAGD, Vasoconstriction Regulator ECEL1, and Ataxia Transcripts GRID2, ITPR1 and MME  
Although the global transcriptome profile of an old ATM-null cerebellum identified many novel pathogenesis events, it remained unclear to what degree these findings are conserved in humans, and whether they can be explained by osmotic stress. Therefore, validation experiments were conducted with RT-qPCR and quantitative immunoblots to assess key factors in human SH-SYSY cells with _ATM_-KD, unstressed or after CQ administration. For validation of individual dysregulations, we selected crucial effectors of ATM function and critical determinants of the phenotypes that characterise A-T.