Nsun2 Deficiency Promotes Tau Hyperphosphorylation and Neurodegeneration Through Epitranscriptomic Regulation of Mir-125B Yoon A
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bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448614; this version posted June 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 NSun2 deficiency promotes tau hyperphosphorylation and neurodegeneration through 2 epitranscriptomic regulation of miR-125b 3 Yoon A. Kim1, 2, Jennifer Blaze3,4, Tristan Winters1,2, Atul Kumar2, Ellen Tein1, Andrew A. 4 Sproul1, 2, Andrew F. Teich1,2, Francesca Bartolini2, Schahram Akbarian3,4, Gunnar Hargus1,2, 5 Ismael Santa-Maria1, 2, * 6 7 8 9 Affiliations: 10 1 Taub Institute for Research on Alzheimer's Disease & the Aging Brain, Columbia University, New 11 York, NY, USA. 12 2 Department of Pathology & Cell Biology, Columbia University, New York, NY, USA. 13 3 Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 14 4 Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, USA. 15 16 17 18 19 * Correspondence should be addressed to [email protected] 20 21 22 23 24 25 26 Keywords 27 Alzheimer’s disease, NSun2, neurodegeneration, Tau proteostasis, microRNA, methylation. 28 29 30 31 Author contributions 32 I.S.M and Y.K conceptualized the project and designed the study methodology. Y.K., J.B., T.W., A.K., E. 33 T and A.A.S., performed research; A.A.S., A.F.T, F.B., S.A. and G.H. contributed resources (study 34 materials, iPSC derived neuronal cultures, post-mortem brain samples). A.A.S., A.F.T, F.B., S.A., G.H., 35 and I.S.M supervised or managed the research. Y.K. and I.S.M. analyzed, interpreted and visualized the 36 data; Y.K. and I.S.M. wrote the initial manuscript paper; All authors provided critical feedback and 37 contributed to the final manuscript. 38 39 40 41 42 43 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448614; this version posted June 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 44 Abstract 45 Overproduction or suppression of certain microRNAs (miRNAs) in Alzheimer’s disease (AD) brains 46 promote alterations in tau proteostasis and neurodegeneration. However, the mechanisms governing how 47 specific miRNAs are dysregulated in AD brains are still under investigation. Epitranscriptomic regulation 48 adds a layer of post-transcriptional control to brain function during development and adulthood. NOP2/Sun 49 RNA methyltransferase 2 (NSun2) is one of the few known brain-enriched methyltransferases able to 50 modify mammalian non-coding RNAs and loss of function autosomal-recessive mutations in NSUN2 have 51 been associated with neurological abnormalities in humans. Here, we provide evidence that NSun2 is 52 expressed in adult human neurons in the hippocampal formation and prefrontal cortex. When we evaluated 53 NSun2 protein expression in post-mortem brain tissue from AD patients we find is dysregulated which was 54 also found in mice and human cellular AD models. To probe these observed alterations were unique to AD 55 we further evaluated brain tissue from other tauopathies, observing NSun2 protein levels were similar 56 between cases and controls. In a well-established Drosophila melanogaster model of tau-induced toxicity 57 we investigated the pathological role of NSun2 observing that reduction of NSun2 protein levels 58 exacerbated tau toxicity, while overexpression of NSun2 partially abrogated the toxic effects. We further 59 show using human induced pluripotent stem cell (iPSC) derived neuronal cultures that NSun2 deficiency 60 results in tau hyperphosphorylation and we found in primary hippocampal neuronal cultures NSun2 levels 61 decrease in response to amyloid-beta oligomers (AβO). Furthermore, in mice, we observed that NSun2 62 deficiency promotes aberrant levels of m6A methylated miR-125b and tau hyperphosphorylation. 63 Altogether, our study supports that neuronal NSun2 deficiency in AD promotes neurodegeneration by 64 altering tau phosphorylation and tau toxicity through an epitranscriptomic regulatory mechanism and 65 highlights a novel avenue for therapeutic targeting. 66 67 68 69 70 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448614; this version posted June 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 71 Introduction 72 MiRNAs, a class of non-coding small RNAs, are part of a vital regulatory mechanism that prevents the 73 deposition of tau protein. Several miRNAs, have been shown to regulate tau proteostasis by modulating tau 74 synthesis or post-translational modifications on tau, such as phosphorylation1, 2. However, mechanisms 75 governing how miRNAs are regulated in the brain or how they are dysregulated during the disease process 76 are poorly understood3-12. MiRNAs can be regulated at the transcriptional or post-transcriptional level13, 14. 77 One of the most frequent post-transcriptional modifications of RNA is methylation15-18. However, the 78 specific role that RNA methyltransferases play in neurodegeneration is poorly investigated. NSun2 is one 79 of the few brain-enriched methyltransferases known to facilitate methylation of non-coding RNAs, 80 including microRNAs19-23. Based on the previously reported putative neuroprotective role of NSun2 and its 81 constitutive expression in the mouse brain cortex and hippocampus19, 24, 25, we decided to explore the status 82 of NSun2 in AD models and human tissue. Here, we show that Nsun2 is downregulated in AD, modulates 83 tau toxicity in vivo, and regulates tau phosphorylation in part by promoting epitranscriptomic alterations in 84 miR-125b. 85 86 87 88 89 90 91 92 93 94 95 96 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448614; this version posted June 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 97 Results 98 NSun2 RNA methyltransferase is dysregulated in Alzheimer’s Disease brains. 99 Post-mortem examination of human control brains shows NSun2 positive immunostaining in neurons of 100 the hippocampal formation and the prefrontal cortex (Brodmann area 9). Immunolabeling of the nucleus 101 and dendrites shows for the first time that NSun2 protein is expressed in neurons in the adult human brain 102 (Supplementary Figure 1). To determine whether alterations in NSun2 are found in AD we next performed 103 NSun2 immunohistochemistry on brain sections from AD cases and controls (Supplementary Table 1). 104 Prefrontal cortex and hippocampal formation were analyzed as these are some of the most affected brain 105 regions in AD. The most salient feature was the neuronal nuclear decrease of NSun2 immunoreactivity in 106 both brain regions of AD cases compared to controls (Figure 1A). Apart from the decrease of nuclear 107 immunoreactivity, AD patients also show a decreased staining in the soma and neurites (right panels, 108 Figure 1A). 109 We then performed quantitative Western blot analysis to confirm whether levels of NSun2 are reduced in 110 AD brains. Immunoblots using antisera specifically recognizing human NSun226, 27 show a decrease in the 111 levels of NSun2 both in hippocampal formation and the prefrontal cortex, in comparison to samples from 112 control subjects (Figure 1 B, C). In this case, the reduction was more pronounced in the hippocampal 113 formation (53.18% decrease, P = 0.0009) than in the prefrontal cortex (41.36% decrease, P <0.0001) 114 (Figure 1B, C). Importantly, no significant difference was observed when comparing AD cases and 115 controls in the cerebellum, a brain area devoid of AD pathology (Figure 1D). Notably, we did not detect a 116 difference in the beta-III tubulin neuronal marker28 indicating that our observed differences might not be 117 secondary to neuronal loss. However, AD brains did show significantly higher levels of NSun2 mRNA in 118 the prefrontal cortex, although quantitative real-time PCR (QPCR) analysis did not show a significant 119 difference in the levels of NSun2 mRNA in the hippocampus, suggesting a compensatory mechanism 120 observable in the prefrontal cortex, a brain area that degenerates later in the disease process 121 (Supplementary Figure 2). Furthermore, we analyzed publicly available proteomic and transcriptomic 122 datasets29, 30 which showed results in agreement with our findings (Supplementary Figure 3). 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.16.448614; this version posted June 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 123 It is unlikely that NSun2 alone among methyltransferases has the ability to regulate microRNAs. Indeed, a 124 possible cooperative mechanism between NSun2 and Methyltransferase Like 3 (Mettl3) has been 125 proposed31. To investigate whether AD brains also show biochemical changes in Mettl3 we examined the 126 levels of Mettl3 expression in control and AD brains. Western blot analysis did not show a significant 127 decrease in Mettl3 levels (Supplementary Figure 4A, B). In addition, analysis of a publicly available 128 transcriptomic dataset did not show a significant change in Mettl3 mRNA levels corroborating our findings 129 (Supplementary Figure 4C). 130 Next, we sought to confirm in two distinctive AD models whether NSun2 downregulation is also observed 131 in these model systems. First, we analyzed the J20 mouse model of AD. This mouse model overexpresses 132 human Amyloid Precursor Protein (APP) with two mutations (APP KM670/671NL-V717F) linked to 133 familial AD32. J20 mice develop robust amyloid beta (A) pathology by five to seven months of age, 134 showing learning and memory deficits and changes in synaptic plasticity32.