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RNA methyltransferase NSun2 deficiency promotes neurodegeneration through epitranscriptomic regulation of tau phosphorylation - PubMed

RNA methyltransferase NSun2 deficiency promotes neurodegeneration through epitranscriptomic regulation of tau phosphorylation

Yoon A Kim et al. Acta Neuropathol. 2023 Jan.

Abstract

Epitranscriptomic regulation adds a layer of post-transcriptional control to brain function during development and adulthood. The identification of RNA-modifying enzymes has opened the possibility of investigating the role epitranscriptomic changes play in the disease process. NOP2/Sun RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to methylate mammalian non-coding RNAs. NSun2 loss of function due to autosomal-recessive mutations has been associated with neurological abnormalities in humans. Here, we show NSun2 is expressed in adult human neurons in the hippocampal formation and prefrontal cortex. Strikingly, we unravel decreased NSun2 protein expression and an increased ratio of pTau/NSun2 in the brains of patients with Alzheimer's disease (AD) as demonstrated by Western blotting and immunostaining, respectively. In a well-established Drosophila melanogaster model of tau-induced toxicity, reduction of NSun2 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. Conditional ablation of NSun2 in the mouse brain promoted a decrease in the miR-125b m6A levels and tau hyperphosphorylation. Utilizing human induced pluripotent stem cell (iPSC)-derived neuronal cultures, we confirmed NSun2 deficiency results in tau hyperphosphorylation. We also found that neuronal NSun2 levels decrease in response to amyloid-beta oligomers (AβO). Notably, AβO-induced tau phosphorylation and cell toxicity in human neurons could be rescued by overexpression of NSun2. Altogether, these results indicate that neuronal NSun2 deficiency promotes dysregulation of miR-125b and tau phosphorylation in AD and highlights a novel avenue for therapeutic targeting.

Keywords: Alzheimer’s disease; Methylation; MicroRNA; NSun2; Neurodegeneration; Tau phosphorylation.

© 2022. The Author(s).

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Figures

Fig. 1
Fig. 1

NSun2 RNA methyltransferase is downregulated in Alzheimer’s disease. a Representative NSun2 immunohistochemistry images of the hippocampal formation (CA1) and prefrontal cortex (Brodmann area 9) of controls and Alzheimer’s disease (AD) human brains. Quantifications of the intensity of NSuns2 in cells within the prefrontal cortex (b) and CA1 (c). Western blots for NSun2, β-actin and β-III-tubulin on the prefrontal cortex (d, f, g) and hippocampus (e, h, i) with densitometry quantification of NSun2 protein normalized to β-actin (f, h) and β-III-tubulin (g, i). Scale bar in a = 50 μm. *p < 0.05, **p < 0.01, ****p < 0.0001 by Mann–Whitney U test. Data represent mean ± SEM

Fig. 2
Fig. 2

NSun2 protein levels in human primary tauopathies. Western blots for NSun2 and β-actin were performed on postmortem brain tissue from patients with Primary Age-Related Tauopathy (PART) with dementia (a–c), Progressive Supranuclear Palsy (PSP) (d–g) and control individuals (a–g). The Western blots for NSun2 and β-actin were performed on the prefrontal cortex (a, d), hippocampus (b, e), cerebellum (c, g) and globus pallidus (f). Densitometry quantification of NSun2 protein normalized to β-actin did not reveal significant differences in the levels of NSun2 protein between control, PART and PSP groups. Mann–Whitney U test was applied. Data represent mean ± SEM

Fig. 3
Fig. 3

Neuronal NSun2 deficiency is associated with increased phospho-tau expression in Alzheimer’s disease brains. Representative images of immunostainings for AT8 (red) and NSun2 (green) of a the prefrontal cortex and e the hippocampus (CA1) in control individuals and patients with AD with quantifications of the intensity of NSuns2 (b, f) and AT8 (c, g) and their ratio (d, h) in cells within the prefrontal cortex (b–d) and CA1 (f–h). Note that the y-axis is presented as log10 scale. Scale bars in a and e = 50 μm. ***p < 0.001, ****p < 0.0001 by Mann–Whitney U test. Data represent mean ± SEM

Fig. 4
Fig. 4

NSun2 deficiency promotes tau hyperphosphorylation and tau toxicity. a Representative immunofluorescence images of human iPSC-derived neurons transduced with shNSun2 or scramble control immunostained using NSun2 (green), phospho-serine 214-tau (pSer-214-tau; red) and MAP2 (white) antibodies. b Quantification of the intensity of NSun2 and pSer214-tau expression in shNSun2 or scramble control neurons presented as ratio. c Representative Western blots with indicated antibodies demonstrating the effects of shNSun2 on the levels of phosphorylated forms of tau. d Quantification of phosphorylated tau in neurons transduced with shNSun2, plotted relative to the levels of total tau (after normalization of total tau with β-actin levels). *p < 0.05 by Student’s t test. Data represent mean ± SEM. e, f Regulation of tau toxicity by NSun2 in drosophila tau models. e Co-expression of NSun2-RNAi (n = 26) with human tau exacerbated the rough eye phenotype compared with that observed in the GFP-RNAi control (n = 29). f Co-expression of NSun2 (n = 18) partially suppressed the human tau–induced rough eye phenotype compared with that seen in the GFP control (n = 28). The yellow marked area shows the degenerated part of eyes. Scale bars, 200 μm. Histograms show quantitative assessment of eye phenotypes. ****p ≤ 0.0001 by Mann–Whitney U test. Data represent mean ± SEM

Fig. 5
Fig. 5

NSun2 deficiency promotes epitranscriptomic alterations in miR-125b. a Diagram showing that RNA was isolated from the forebrain of NSun2 conditional KO mice and non-transgenic controls and was subjected to immunoprecipitation analysis using an anti-m6A antibody or IgG control. b, c The presence of miR-125b in the Input (n = 3) m6A (n = 3) and IgG (n = 3; used as negative control) materials was analyzed by QPCR. Histograms show quantification of miR-125b levels with respect to control in Input (b) and m6A (c) materials. *p < 0.05 by Student’s t test. Data represent mean ± SEM. Representative images (d, f, h) and quantifications (e, g, i) of immunohistochemistry on the frontal cortex of NSun2 conditional KO and non-transgenic control mice with phospho-Serine 202-phospho-Threonine 205 (AT8) (d, e), phospho-Serine 214-tau (f, g) and phospho-Serine 262-tau (h, i) antibodies showing a marked increase in phospho-tau immunostaining in NSun2 conditional KO animals. Note that the y-axis is presented as log10 scale. Scale bars, 100 μm. j, k After site directed mutagenesis, transduction of human iPSC-derived neurons with miR-125b vector induced a significant increase of pSer-214-tau levels when compared to scrambled miR control, while cells transduced with 125b mutant vector (miR-125b∆m6A) exhibited significant higher levels of pSer-214-tau. *p < 0.05 and **p < 0.01 by one-way ANOVA with Bonferroni post hoc test

Fig. 6
Fig. 6

NSun2 is downregulated in response to amyloid-β oligomers. a Western blot quantification of NSun2 protein levels in rat primary hippocampal neurons untreated (control) or treated with 300 nM AβO for the indicated times. Top portion of the panel shows representative Western blots. Histograms show densitometry quantification of NSun2 protein levels (bottom of the panel). NSun2 values are normalized against β-actin. Data represent mean ± SEM. *p < 0.05 by One-way ANOVA with Bonferroni post hoc test. b QPCR analysis of NSun2 mRNA levels in rat primary hippocampal neurons untreated (control) or treated with 300 nM AβO for the indicated times. No significant changes in the levels of NSun2 mRNA are found. c Western blot quantification of phospho-tau and total tau levels in rat primary hippocampal neurons untreated (control) or treated with 300 nM AβO for 24 h. Phospho-tau levels are plotted relative to the levels of total tau (after normalization of total tau with β-actin levels; also shown here). d Co-expression of NSun2 (n = 16) partially suppressed the Aβ42-induced rough eye phenotype compared with that seen in the GFP control (n = 16). The marked area shows the degenerated part of eyes. Histograms show quantitative assessment of eye phenotypes. ****p ≤ 0.0001 by Mann–Whitney U test. Data represent mean ± SEM. e Human iPSC-derived neurons were treated with AβO and protein lysates were collected 24 h later for analysis. Representative Western blots with antibodies directed against pSer214-tau and total tau demonstrate the effects of AβO treatment and rescue by NSun2 overexpression in human neurons. f Quantification of phospho-tau in AβO-challenged neurons in the presence of absence of NSun2 demonstrating significant reduction of tau phosphorylation after overexpression of NSun2. g Cell viability assay demonstrating partial rescue of cell viability in AβO-challenged neurons after overexpression of NSun2. *p < 0.05, **p < 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001 One-way ANOVA with Bonferroni post hoc test. Data represent mean ± SEM

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