In Cerebellar Atrophy of 12-Month-Old ATM-Null Mice, Transcriptome Upregulations Concern Most Neurotransmission and Neuropeptide Pathways, While Downregulations Affect Prominently Itpr1, Usp2 and Non-Coding RNA

Abstract

The autosomal recessive disorder Ataxia-Telangiectasia is caused by a dysfunction of the stress response protein, ATM. In the nucleus of proliferating cells, ATM senses DNA double-strand breaks and coordinates their repair. This role explains T-cell dysfunction and tumour risk. However, it remains unclear whether this function is relevant for postmitotic neurons and underlies cerebellar atrophy, since ATM is cytoplasmic in postmitotic neurons. Here, we used ATM-null mice that survived early immune deficits via bone-marrow transplantation, and that reached initial neurodegeneration stages at 12 months of age. Global cerebellar transcriptomics demonstrated that ATM depletion triggered upregulations in most neurotransmission and neuropeptide systems. Downregulated transcripts were found for the ATM interactome component Usp2, many non-coding RNAs, ataxia genes Itpr1, Grid2, immediate early genes and immunity factors. Allelic splice changes affected prominently the neuropeptide machinery, e.g., Oprm1. Validation experiments with stressors were performed in human neuroblastoma cells, where ATM was localised only to cytoplasm, similar to the brain. Effect confirmation in SH-SY5Y cells occurred after ATM depletion and osmotic stress better than nutrient/oxidative stress, but not after ATM kinase inhibition or DNA stressor bleomycin. Overall, we provide pioneer observations from a faithful A-T mouse model, which suggest general changes in synaptic and dense-core vesicle stress adaptation.

The ataxia-telangiectasia disease protein ATM controls vesicular protein secretion via CHGA and microtubule dynamics via CRMP5

“…1A, Supplementary Table S1). Knockdown efficiency and ATM cytoplasmic localization were confirmed previously (Reichlmeir et al, 2023).…”

“…Employing global proteomics, together with phosphoproteomics of direct ATM-dependent and additional indirect IMAC changes, we successfully defined many novel molecular targets of cytoplasmic ATM in human ATM-kd neuroblastoma, and filtered the initial events of pathogenesis in 8-month-old ATM-null mouse cerebellum. It is important to emphasize how these data show excellent overlap with (i) previously published proteome profiles of A-T patient cerebella (Lee et al, 2021), (ii) A-T patient cerebrospinal fluid (CSF) (Canet-Pons et al, 2018), (iii) 12-month-old ATM-null mouse cerebellar transcriptome profiles (Reichlmeir et al, 2023), (iv) reports on ATM association with vesicles and SYN1 (Li et al, 2009), (v) with β-adaptin / AP3B2 (Lim et al, 1998),…”

“…The stable ATM knockdown (kd) SH-SY5Y cell line (parental cell line ATCC: CRL-2266) was already published (Reichlmeir et al, 2023). In brief, stable ATM-kd cells were generated by lentiviral transduction of shRNA targeting ATM (hereafter referred to as shATM) compared to a control cell line targeting no known mammalian genes (non-target control, or NT CTRL).…”

“…S1C). The impact of the osmotic stressor CQ (Reichlmeir et al, 2023) caused significant DPYSL5 and TUBB3 transcript reduction, as opposed to SEMA3A transcript increase in shATM cells, while having no effect on MAP2 transcript (Fig. S2A).…”

“…As stated previously, ATM might serve as a platform for protein-protein interactions, in addition to functioning as kinase (Reichlmeir et al, 2023). Therefore, we performed global proteomics profiling in vitro in SH-SY5Y neuroblastoma cells with lentiviral transduction of either NT CTRL shRNA plasmid or shATM knockdown plasmid (Fig.…”