## 4 Discussion
### The Alternative Splice Profile of ATM-Null Mouse Cerebellum at 12 Months of Age
Further research is necessary to elucidate the potential functions of ATM as a protein scaffold in the cytoplasm. Validation Work in SH-SYSY Cells Shows ATM-Deficiency to Impair the CQ-Triggered Regulation of Postsynaptic Calcium Release Channel ITPR1, in Parallel to Immediate-Early Transcripts PER1/NR4A1  
The mechanistic validation experiments in this study (Figures 4, 5 and S4) focused on A-T-phenotype-related factors, documenting consistent reductions for ITPR1 levels upon ATM deficiency. Given that ATM is a member of the phosphatidylinositol 3' kinase-like kinase (PIKK) enzyme family, it may have functional interaction with the inositol-1,4,5-trisphosphate-receptor IP3R, so this decrease of _ITPR1_ mRNA and IP3R abundance may also represent a crucial primary loss-of-function event in autosomal recessive A-T. Genetic loss-of-function of IP3R has a profound impact on the calcium-dependent excitability of Purkinje neurons and was repeatedly observed as sufficient to cause hereditary progressive cerebellar neurodegeneration, with deletion of one _ITPR1_ gene copy via haploinsufficiency triggering ataxia inheritance in autosomal dominant manner [218; 219]. Thus, our observation of IP3R abundance reduction below 50% in _ATM_-KD neuroblastoma cells (Figure 5b)emerges from the transcriptome profile validation work as arguably the most important molecular event, which might explain the preferential infection of cerebellar neurons [57]. In this context, it is important to note that parallel loss of IP3R together with two more ataxia-responsible proteins was previously demonstrated in cerebellar tissue of A-T patients [66], namely the calcium homeostasis factor INPP5A (also known as Type I Inositol 1,4,5-Trishophate 5-Phosphatase) [220], and CA8 (also known as Carbonic Anhydrase 8) [221; 222]. Furthermore, a protein complex of ITPR1 as endoplasmic reticulum Ca\({}^{2+}\) homeostasis regulator with the mitochondrial HSP70-family member/chaperone GRP75 (=HSPA9) and the mitochondrial voltage-gated Ca\({}^{2+}\) homeostasis channel VDAC1 was previously shown to mediate ATM dysfunction in bronchial cells after nutrient stress [30]. A very similar expression alteration in the ATM-null mouse cerebellar transcriptome may therefore be relevant, where downregulation of the ataxia gene _Itpr1_ occurred in parallel with downregulation of the HSP70-family member/chaperone _Hspa12a_ and several voltage-gated Ca\({}^{2+}\) homeostasis channels in the plasma membrane (_Cacna1a_, _Cacna1c_, _Cacna1d_, _Cacna1g_). Cytosolic HSPA12A protein has a very specific role, modulating its interactor SORL1 with downstream GFRA1/2 [223; 224], all of which showed downregulated mRNA levels in our ATM-null mouse cerebellar transcriptome profiling study, suggesting that the sorting of trophic signalling receptors is abnormally regulated. In addition, the receptor tyrosine kinase ERBB2 is regulated both by SORL1 and by USP2 [225; 226], and this may underlie the reduced cerebellar levels of _Erbb2 ip_ as a factor responsible for the surface localisation of glutamate receptors [227]. In analogy to the impact of nutrient deficits as stressors via ATM on endoplasmic reticulum and mitochondria homeostasis as previously published [30], the transcriptional dysregulation of ITPR1, HSPA12A (instead of HSPA9/GRP75) and its interactors SORL1 and ERBB2 in ATM-null cerebellum may therefore constitute the primary pathogenesis pathway and explain why, when neurotransmitter receptors/transporters/neuropeptide modulators are not in the right position in polarised processes, trophic signalling deficits ensue, and tissue shrinkage ensues over time. Given that SORL1, GFRA1/2, ERBB2, are poorly or not expressed in SH-SYSY neuroblastoma cells according to the Human Protein Atlas (last accessed on 12 September 2023), we made no attempt to model this trophic pathogenesis cascade in vitro. The reduction of IP3R would also mediate a postsynaptic excitability deficit, and contribute to the diminished transcriptional response of immediate-early transcripts such as _PER1_ and _NR4A1_.