## 4 Discussion
### The Alternative Splice Profile of ATM-Null Mouse Cerebellum at 12 Months of Age  
Previous studies of ATM dysfunction demonstrated its impact on alternative splicing [53], and of course the stimulus-/stress-dependent changes in phosphorylation cascades would first impact the splice apparatus before topoisomerase-dependent immediate-early reactions and more cumbersome chromatin unpackaging events permit the subsequent transcription adaptations. Overall, our interrogation of the transcriptome for factors with strong exon-splicing-index effects confirmed significant enrichments in three pathways. Neurotransmission (_Sclc17a6_, _Sclc18a2_, _Sclc7a3_, _Sclc5a7_, _Sclc1a4_, _Scn3b_, _Kcnq5_, _Gabra_, _Gla1_, _Gla3_, _Dbh_, _Vink1_, _Car9_, _Micu3_), neuropeptides/neurorophins (_Oprm1_, _Baip3_, _Gnas_, _Dlk1_, _Dlg2_, _Rasgrf2_, _Ngef_, _Cec11_, _Dgkk_, _Ccl27a_, _Pcs85_, _Cpne6_, _Ptrpd_, _Gfn1_) and synaptic adhesion (_Fxyd5_, _Cbln4_, _Cbln2_, _Nrip2_) modulation was prominent. _Baip3_ splice changes could contribute to impaired biogenesis of secretory vesicles, with consequences for the Ca2+ stimulated release of neurotransmitters and neuropeptides [197]. While cerebellar tissue has a broad expression profile, a neural cell culture model would express only a small subset of these molecules, so further validation experiments were focused on _Oprm1_, which exhibited an exceptional exon-splicing index of 39.7, and which acts to dampen glutamatergic neurotoxicity in the contacts between cerebellar granule neuron projections (parallel fibres) and Purkinje neuron dendrites [198; 199; 200], which is the cerebellar site most vulnerable to ataxia pathogenesis [201; 202]. With a prominent negative exon-splicing index of \(-5.03\) (\(p=0.005\)), _Scl7a6_ (encoding VGLUT2) also displayed evident adaptation of its exon structure, providing additional evidence that also glutamatergic climbing fibre-signalling is modulated by ATM. In contrast, _Sclc17a7_ (VGLUT1 in parallel fibres) showed a change only for its 3\({}^{\prime}\)-exon with nominal significance and an exon-splicing index of 1.6. While the ClariomD microarray has oligonucleotides to detect sequences within most exons, the Taqman RT-qPCR assays in contrast are optimised to detect exon-exon-boundaries, so a validation experiment by RT-qPCR can only confirm the dysregulation of a specific mRNA overall, and may detect whether it disappears for a specific exon, but will not quantify the selective inclusion/exclusion of an exon. Overall, it is important to be cautious regarding the value of this splicing profile, because dysregulations of a complete mRNA may not be equally represented by every oligonucleotide, mimicking true alternative splice changes, and because experimental quantitative validation across species is cumbersome. Thus, we consider these data as preliminary screen. Differential Detergent Fractionation of Adult Mouse Cerebellum and SH-SY5Y Neuroblastoma Cells Detects ATM Mainly in Cytosol  
For validation of these mouse findings in the human species, a knockdown of ATM in neuroblastoma SH-SY5Y cells was employed, taking into account the previous usefulness of such human in vitro modelling projects in autophagy and chemoresistance studies of ATM [203; 204]. Neuroblastoma cell lines are known to represent a mixed population, termed N-type (neural) and S-type (substrate-adherent, epithelial-like) cells. While N-type cells are neuroblast-like with little cytoplasm and few neuritic processes, the S-type cells have a bigger cytoplasm and flattened morphology with strong attachment to the substrate [205; 206; 207; 208]. Unexpectedly, _ATM_-KD SH-SY5Y cells displayed gross alterations in appearance, with larger and flattened cell bodies without processes, while the non-targeting control (NT CTRL) cells retained an overall neuroblast-like appearance and displayed short neuritic processes (Figure S2). Thus, flattened cells without processes that are predominant for _ATM_-KD cells could reflect a shift in neuroblastoma cell populations towards S-type cells.