Increased processing of SINE B2 non coding RNAs unveils a novel type of transcriptome de-regulation underlying amyloid beta neuro-pathology. Increased processing of SINE B2 non coding RNAs unveils a novel type of transcriptome de-regulation underlying amyloid beta neuro-pathology

More than 97% of the mammalian genome is non-protein coding, and repetitive elements account for more than 50% of noncoding space. However, the functional importance of many non-coding RNAs generated by these elements and their connection with pathologic processes remains elusive. We have previously shown that B2 RNAs, a class of non-coding RNAs that belong to the B2 family of SINE repeats, mediate the transcriptional activation of stress response genes (SRGs) upon application of a stimulus. Notably, B2 RNAs bind RNA Polymerase II (RNA Pol II) and suppress SRG transcription during pro-stimulation state. Upon application of a stimulus, B2 RNAs are processed into fragments and degraded, which in turn releases RNA Pol II from suppression and upregulates SRGs. Here, we demonstrate a novel role for B2 RNAs in transcriptome response to amyloid beta toxicity and pathology in mouse hippocampus. In healthy hippocampi, activation of SRGs is followed by a transient upregulation of pro-apoptotic factors, such as p53 and miRNA-34c, which target SRGs creating a negative feedback loop that facilitates return to the pro-stimulation state. Using an integrative RNA genomics approach, we show that in mouse hippocampi with amyloid pathology and in an in vitro cell culture model of amyloid beta toxicity, this regulatory loop is dysfunctional due to increased levels of B2 RNA processing, constitutively elevated SRG expression and high p53 levels. Evidence indicates that Hsf1, a master regulator of stress response, mediates B2 RNA processing in cells, and is upregulated during amyloid toxicity accelerating the processing of SINE RNAs and SRG hyper-activation. This data attributes a role to SINE RNA processing in a pathological process as well as a new function to Hsf1 that is independent of its transcription factor activity. Our study reveals that in mouse, SINE RNAs constitute a novel pathway deregulated in amyloid beta pathology, with potential implications for similar cases in the human brain, such as Alzheimer’s disease (AD). Overall design: In this study, we employed a transgenic mouse model of amyloid pathology, APP NL−G−F (APP) (Saito et al., 2014) and the respective wild type control C57BL/6J (WT). We have tested three different mouse ages that correspond to different phases of amyloid beta pathology: i) pre-symptomatic stage with undetectable (very low) amyloid plaque load, (3 months - 3m/ APP: 3x mice, WT:2x mice), ii) stage of symptom manifestation (that coincides with the active neurodegeneration phase and appearance of amyloid plaques (6 months - 6m/ APP: 3x mice, WT:3x mice), and iii) terminal stage of the pathology (12 months - 3m/ APP: 3x mice, WT:3x mice). Whole hippocampi, (hippocampi separated from both the left (L) and right (R) hemishere of each mouse and pooled together), represented as hippocampus L+R, were isolated from the above mice, and the extracted RNA from each hippocampus was separated into two samples: one enriched in short-RNAs (sR) and one in long-RNAs (LR). Each sample was then subjected to next-generation sequencing (short-RNA-seq and long-RNA-seq, respectively). We also employed the hippocampal HT-22 cell line (Davis & Maher, 1994). We treated HT22 cells with either an LNA against Hsf1 (anti-Hsf1) or a scramble LNA (ctrl) followed by incubation with amyloid beta peptides (42/ 1-42 aa) or a control peptide with an inverted amino acid sequence (R/ reverse 42-1). Biological replicates were as follows: 42/Ctrl: x4, R/Ctrl: x4, 42/Anti-Hsf1:x3, R/Anti-Hsf1:x3 Subsequently RNA was extracted, separated into short-RNA and long-RNA samples as above, and subjected to next generation sequencing (short-RNA-seq and long-RNA-seq, respectively).哺乳动物基因组中超过97%为非编码序列，重复元件占非编码区域的50%以上。然而，由这些元件产生的众多非编码RNA的功能重要性，及其与病理过程的关联仍不明晰。我们此前的研究表明，B2 RNA作为一类属于短散在核元件（SINE, Short Interspersed Nuclear Element）B2家族的非编码RNA，可介导外界刺激下应激反应基因（SRGs, Stress Response Genes）的转录激活。值得注意的是，B2 RNA可结合RNA聚合酶II（RNA Pol II），并在促刺激状态下抑制应激反应基因的转录。当受到外界刺激时，B2 RNA会被切割为片段并降解，从而解除RNA Pol II的抑制，上调应激反应基因的表达。 本研究揭示了B2 RNA在小鼠海马体应对淀粉样β蛋白毒性与病理变化的转录组应答中的全新作用。在健康海马体中，应激反应基因激活后，促凋亡因子（如p53与靶向应激反应基因的miRNA-34c）会出现瞬时上调，形成负反馈回路，帮助恢复至促刺激状态。通过整合RNA基因组学方法，我们发现，在存在淀粉样病理的小鼠海马体以及淀粉样β蛋白毒性的体外细胞模型中，该调控回路出现功能异常：表现为B2 RNA加工水平升高、应激反应基因组成型高表达以及p53水平升高。 研究证据显示，热休克因子1（Hsf1, Heat Shock Factor 1）作为应激反应的主调控因子，可介导细胞内B2 RNA的加工，且在淀粉样蛋白毒性过程中被上调，加速短散在核元件RNA的加工与应激反应基因的过度激活。本研究数据赋予了短散在核元件RNA加工在病理过程中的作用，同时也揭示了热休克因子1不依赖其转录因子活性的全新功能。 我们的研究表明，在小鼠体内，短散在核元件RNA构成了一条在淀粉样β蛋白病理中失调的全新通路，这一发现对人类大脑中类似的病理过程（如阿尔茨海默病，AD, Alzheimer’s Disease）具有潜在的借鉴意义。 整体实验设计：本研究采用淀粉样病理转基因小鼠模型APP NL−G−F（简称APP）（Saito等，2014）及其对应的野生型对照C57BL/6J（简称WT）。我们选取了对应淀粉样β蛋白病理不同阶段的三个小鼠年龄组：i) 症状前阶段：淀粉样斑块负荷无法检测（水平极低），（3月龄——3m：APP组3只，WT组2只）；ii) 症状显现阶段：对应活跃的神经变性阶段与淀粉样斑块出现时期（6月龄——6m：APP组3只，WT组3只）；iii) 病理终末期（12月龄——3m/ APP：APP组3只，WT组3只）。从上述小鼠中分离完整海马体（将每只小鼠左右半球的海马体分离后合并，记为海马体L+R），从每个海马体中提取的RNA被分为两份：一份富集短链RNA（sR），另一份富集长链RNA（LR）。随后分别对两份样本进行下一代测序（短链RNA测序与长链RNA测序）。 我们同时采用了海马体HT-22细胞系（Davis & Maher，1994）。将HT22细胞分别用靶向Hsf1的锁核酸（LNA, Locked Nucleic Acid）（anti-Hsf1）或乱序锁核酸（ctrl，对照）处理，随后用淀粉样β肽（42/1-42氨基酸）或氨基酸序列倒置的对照肽（R/反向42-1）进行孵育。生物学重复设置如下：42/Ctrl组4例，R/Ctrl组4例，42/Anti-Hsf1组3例，R/Anti-Hsf1组3例。随后按照上述方法提取RNA，分为短链RNA与长链RNA样本，并分别进行下一代测序（短链RNA测序与长链RNA测序）。