Mid-Gestation lethality of Atxn2l-Ablated Mice

Abstract

Depletion of yeast/fly Ataxin-2 rescues TDP-43 overexpression toxicity. In mouse models of Amyotrophic Lateral Sclerosis via TDP-43 overexpression, depletion of its ortholog ATXN2 mitigated motor neuron degeneration and extended lifespan from 25 days to >300 days. There is another ortholog in mammals, named ATXN2L (Ataxin-2-like), which is almost uncharacterized but also functions in RNA surveillance at stress granules. We generated mice with Crispr/Cas9-mediated deletion of Atxn2l exons 5-8, studying homozygotes prenatally and heterozygotes during aging. Our novel findings indicate that ATXN2L absence triggers mid-gestational embryonic lethality, affecting female animals more strongly. Weight and development stages of homozygous mutants were reduced. Placenta phenotypes were not apparent, but brain histology showed lamination defects and apoptosis. Aged heterozygotes showed no locomotor deficits or weight loss over 12 months. Null mutants in vivo displayed compensatory efforts to maximize Atxn2l expression, which were prevented upon nutrient abundance in vitro. Mouse embryonal fibroblast cells revealed more multinucleated giant cells upon ATXN2L deficiency. In addition, in human neural cells, transcript levels of ATXN2L were induced upon starvation and glucose and amino acids exposure, but this induction was partially prevented by serum or low cholesterol administration. Neither ATXN2L depletion triggered dysregulation of ATXN2, nor a converse effect was observed. Overall, this essential role of ATXN2L for embryogenesis raises questions about its role in neurodegenerative diseases and neuroprotective therapies.

Keywords: RNA chaperone; SCA2; Spinocerebellar ataxia type 2; fronto-temporal lobar dementia; nutrient endocytosis; poly(A)-tail; tauopathy; tyrosine kinase receptor signaling.

Figure 1

Scheme of genetic ablation within Atxn2l, with its splicing and translation effects. Ataxin-2-like (A) at the DNA level with neighbor genes Tufm and Sh2b1, indicating the deletion region in letters and lines of magenta color (also showing alternative translation start codons (ATG), structure from exon 1 to exon 22 with introns, alternative translation STOP codons, start of poly(A) tail); (B) at the mRNA level with alternatively spliced isoforms in separate red boxes; (C) at the protein level with relevant known sequence motifs (pro-rich refers to proline-rich-domain, MPL motif was reported by Meunier et al. 2002), highlighting the phylogenetically conserved Lsm, LsmAD and PAM2 motifs in a red box (deletion and frameshift are limiting translation to an N-terminal fragment until magenta line).

Figure 2

(A) Protein abundance of 3 different epitopes spanning ATXN2L in control Atxn2l^+/+ and homozygous Atxn2l^−/− MEF (n = 4vs4). The protein was completely absent in three assessed epitopes from residue 150 until the most C-terminal region (aa: amino acid). (B) Protein abundance of ATXN2L in heterozygous Atxn2l^+/− cerebellum (cb) compared to wildtype littermates, or in heterozygous Atxn2l^+/− cortex (ctx), in comparison to one wildtype Atxn2l^+/+ MEF line. MEFs exhibit two specific bands of ATXN2L, in contrast to brain tissue where the larger band dominates. The overall ATXN2L levels were decreased to approx. 50% in heterozygous tissues (n = 2). Detection was done for the epitope aa 712-1050. (C) In Atxn2l^−/− MEF (upper row, n = 4vs4), RT-qPCR showed that mRNA expression of Atxn2l was absent at the boundary between exons 7-8 that was genetically deleted, and significantly reduced at the boundaries of exons 1-2 and 10-11, possibly due to nonsense-mediated RNA decay. In tissue from fore-/hind-limbs [lower row, 3 wildtype (Atxn2l^+/+, white bars), 4 heterozygous (Atxn2l^+/−, grey bars), 2 Atxn2l-null embryos (Atxn2^−/−, black bars)], the Atxn2l mRNA expression measured at the boundary of exons 7-8 was again absent in Atxn2l^−/− and reduced by 50% in Atxn2l^−/+ mice, whereas 5′-upstream and 3′-downstream from the deletion the increased Atxn2l expression level of null mutants suggested transcript upregulation efforts either via the Atxn2l promoter or via altered mRNA stabilization/degradation to compensate the loss of protein function. The exon 1c-2 assay detects an alternatively spliced isoform. Asterisks represent significance (p < 0.01 **, p < 0.0001 ****).

Figure 3

Comparison of Atxn2l^−/− embryos with their littermates, at different gestational stages. Embryonal days reflect approximate values. (A) The only Atxn2l^−/− mouse (male) identified at E20 in comparison to a male WT brother. (B) An Atxn2l^−/− mouse (male) reaching E16, exhibiting retarded development, and decreased weight, in comparison to the heterozygous Atxn2l^+/− female sibling. The petechial bleeds were not seen in other null mutants. (C) An Atxn2l^−/− mouse (male) at E15-16 with liver size and blood filling similar to male WT brother. (D) An Atxn2l^−/− female embryo at E13 with retarded growth and development and its male WT sibling.

Figure 4

Evident growth/weight phenotype: Atxn2l^+/+ embryos with Atxn2l^−/− littermates (WT above, homozygote mutants below, E13 on left side, E14 in center, E20 on right side).

Figure 5

H&E histology at E14 showed good separation of neuron layers in the cortex of Atxn2l^+/+ brain (left); in littermate Atxn2l^−/− mice (right), less lamina definition and many neurons were observed with nuclear condensation, which that is indicative of apoptosis; the diameter of brain cortex was also thinner.

Figure 6

(A) Cell culture images of Atxn2l^+/+ and Atxn2l^−/− MEF illustrating the increased number of giant multinucleated cells in the absence of Atxn2l. (B) Quantification of giant cells for 4 different age- matched littermate lines with 3 technical replicates each. The diagram on the left side shows the increase with consistency for the individual MEF lines 1-4, while the diagram on the right side represents the overall increase across all lines. Statistical testing was done by two-way ANOVA and t-test with Welch’s correction, respectively, significance levels were illustrated by ** for p < 0.01, by T for 0.05 < p <0.1.

Figure 7

(A) The mRNA expression of ATXN2L in human SH-SY5Y neuroblastoma cells was induced by nutrient deprivation in HBSS medium over 48 h with low glucose/no amino acids, in the absence of lipids normally supplied via FCS, similar to the expression of (B) ATXN2 mRNA. (C) For the induction peak at 24 h, the expression upregulation of ATXN2L was partially prevented upon supplementation of 10% FCS, while glucose or amino acids alone had no rescue effect; (D) FCS addition also diminished ATXN2 expression, while glucose and amino acid administration enhanced the expression further; (E) The HBSS induction of ATXN2L was reduced at least as much by low cholesterol concentrations as by FCS, while higher dosage of cholesterol elicited even stronger upregulations. (F) This effect was similarly observed for ATXN2. A-D: n = 3; E/F: n = 8. Statistical testing was done by one-way ANOVA, significance levels were illustrated by asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Comparison was always in respect to nutrient abundant control condition, if not stated otherwise.