## 4 Discussion
### The Alternative Splice Profile of ATM-Null Mouse Cerebellum at 12 Months of Age
The remodelling of Ca2+ signalling was already demonstrated to be altered in S-type cells [206]. Given that SH-SY5Y cells were already shown to require ATM mediated phosphorylation of CREB protein at serine 133, to enable retinoic acid induced differentiation [209], this change of gross morphology in _ATM_-KD cells may represent a loss of differentiation. Indeed, dysregulation of retinoic acid dependent differentiation regulators was also evident in the 12-month-old ATM-null mouse cerebellar transcriptome, where most components of this well characterised pathway [201; 202; 203] were strongly dysregulated,displaying increased _Nrip2_ (Figure 2a), _Nrip3_, _Rorb_, _Creb5_, _Crebl2_ and _Zflx3_ mRNA levels versus decreased expression of _Tcf4_, _Crtc2_, _Foxo3_ and the ataxia disease gene _Rora_, as well as downstream _Itpr1_[213; 214]. A more proliferative state of the _ATM_-KD cells may result in reduced expression of many neurotransmission factors, potentially explaining the drastic downregulation of _OPRM1_ in _ATM_-KD neuroblastoma cells, as opposed to increased _Oprm1_ in the old ATM-null cerebellum (Figure 6d). Although some dysregulations of neuropeptide signalling were massive in the old ATM-null mouse cerebellum and were very relevant for phenotypes of A-T--e.g., the increases in mRNA of growth hormone inhibitor somatostatin (_Sst_), vasodilator preprotachykinin (_Tac1_) and the tachykinin receptor (_Tac1_), as well as the glutamate-excitability factor VGLUT2 (encoded by _Scl17a6_)--the SH-SYSY neuroblastoma line did not express these genes. It is also important to consider that glutamate availability is very restricted for excitable neurons in vivo, but provided constantly in overdose to cultured neuroblastoma cells. Thus, our in vitro model had very limited value to model and study the ATM-dependent stress-adaptations of neurotransmitter- and neuropeptide-containing vesicles, but seemed quite helpful for the study of upstream factors in the interactance of ATM and of immediate-early responses in the nucleus. The use of differential detergents to achieve subcellular fractionation of the old adult mouse cerebellum and of SH-SYSY neuroblastoma line, localised ATM mainly to the cytosol, both in unstressed and in stressed conditions, in WT cells and _ATM_-KD cells (Figure 3, Figure S3). The absence of ATM from the nucleus is in excellent agreement with subsequent findings that characteristic ATM-null cerebellar transcriptome profile anomalies such as _USP2_ and _PER1_ downregulations could not be elicited robustly by the DNA DSB stressor bleomycin in vitro, but were mirrored best by osmotic stress instead (Figure 4). Our observations that ATM is almost exclusively found within cytoplasmic fractions of the cerebellum and SH-SYSY cells are in excellent agreement with a previous immunohistochemical study that localised ATM in the cytosol of cerebellar tissue Purkinje neurons from a mouse mutant [32], but they contrast with human reports and with the immunohistochemical observation of ATM in the nucleus of cerebellar Purkinje and granule neurons, once they are dissected and kept in organotypic slice cultures [215; 216]. While nuclear ATM clearly has a role for DNA repair in proliferating cells, these fractionation findings emphasise the urgent need to understand what the functions of cytosolic ATM in postmitotic neurons are, and how impaired stress adaptation there might trigger a neurodegenerative process. Given the inefficiency of ATM kinase inhibition by KU-55933 to reproduce transcript changes observed in cerebellar tissue and upon _ATM_-KD in vitro, we consider the possibility that cytoplasmic ATM acts as a protein scaffold and interaction platform, rather than a kinase in cytoplasmic signalling. This was already proposed when cytoplasmic ATM was demonstrated to serve as a docking site for PP2A to dephosphorylate AKT and thereby regulate cell death upon ER-stress via an ATM-GSK3\(\beta\)-\(\alpha\)NAC/\(\gamma\)TX signalling axis [217].