Temporal Changes in MicroRNA Function During Tau tangle Accumulation

描述（由申请人提供）：神经退行性tau病（阿尔茨海默病（AD）和额颞叶痴呆（FTD））的特征是细胞内含有tau的神经原纤维缠结（NFT）的积聚，进行性认知功能障碍和神经元死亡。对tau/tta-4510小鼠诱导性tau病模型的研究表明，在nft形成之前，中间tau物种的早期积累促进了导致神经元功能障碍和记忆丧失的事件。该模型与人类疾病的特征非常相似。mirna是一类广泛参与转录后调控的小rna，最近的研究数据表明，一些mirna在参与学习和记忆的神经元过程中具有特定的调控作用。本研究旨在验证tau诱导的tau/tta-4510模型中早期mirna失调导致这些小鼠观察到的认知缺陷的假设。这项研究背后的基本原理是基于我们令人兴奋的初步数据，该数据显示，与年龄匹配的对照组小鼠相比，在tau/tta小鼠中，多种mirna，包括miR-138（调节突触形成），在表达突变的tau转基因的tau/tta小鼠中失调，并表现出认知缺陷。此外，在最初的初步研究中发现的许多失调的mirna也在人类AD大脑中异常表达，并与缠结负荷显著相关。为了验证我们的假设，在Aim 1下，我们建议a)通过微阵列测量老年小鼠脑损伤模型中的miRNA水平，利用我们暂时抑制模型中转基因表达的能力来识别那些参与认知和神经变性的miRNA，而不是那些与NFT积累相关的miRNA， b)与人类FTD大脑中差异表达的miRNA数据集进行比较。c)鉴定物种间一致且具有临床相关性的mirna。在Aim 2中，Aim 1中定义的mirna将在早期tau聚集物种开始在小鼠大脑中积累的期间以及认知能力下降的开始（1个月，2.5个月和4个月）期间暂时测量，但在广泛的神经元损失和纠缠形成之前。然后，我们将使用LCM来证实，与对照组（PHF1阴性）相比，在缠结前神经元（PHF1阳性）中，暂时失调的mirna也发生了类似的改变。在Aim 3中，我们还将使用综合实验和计算方法确定暂时失调的mirna的功能mRNA靶点。在转染Pre-miR或anti-miR寡核苷酸后，在神经细胞系中发现失调的mrna，并通过SEED序列分析和指定的功能相关性预测为靶标，然后将在小鼠tau/tta皮质PHF1阳性和阴性神经元中作为miRNA调节的靶标进行验证。

DESCRIPTION (provided by applicant): The neurodegenerative tauopathies Alzheimer's disease (AD) and Frontotemporal dementia (FTD), are characterized by the intracellular build-up of tau-containing neurofibrillary tangles (NFT), and progressive cognitive dysfunction and neuron death. Studies of the tau/tta-4510 mouse inducible tauopathy model, which closely mimics features of the human diseases, have shown that early accumulation of intermediate-tau species, prior to the formation of NFTs, promote the events leading to neuronal dysfunction and memory loss. miRNAs are a widespread class of small RNAs involved in post-transcriptional regulation, and data from recent studies have illustrated a specific regulatory role for some miRNAs during the neuronal processes involved in learning and memory. This proposed study is designed to test the hypothesis that tau-induced early dysregulation of miRNAs in the tau/tta-4510 model results in the cognitive deficits observed in these mice. The rationale behind this proposed study is based on our exciting pilot data showing multiple miRNAs, including miR-138 (that regulates synapse formation) are dysregulated in tau/tta mice engineered to express the mutant tau transgene and that manifest with cognitive deficits, when compared to age-matched control mice. In addition, many of the dysregulated miRNAs found in the initial pilot study are also abnormally expressed in the human AD brain and are significantly correlated with tangle load. To test our hypothesis, Under Aim 1 we propose to a) Measure miRNA levels in the aged murine tauopathy model via microarrays, utilizing our ability to temporally suppress transgene expression in the model to identify those miRNAs involved in cognition and neurodegeneration rather than those which correlate with NFT accumulation, and b) Compare to datasets of differentially expressed miRNAs in human FTD brains, to c) Identify miRNAs that are concordant between the species and have clinical relevance. Under Aim 2, the miRNAs defined under Aim 1 will be measured temporally during the period that early tau aggregated species begin to accumulate in the murine brain, plus during the onset of cognitive decline (1 month, 2.5 months and 4 months), but prior to extensive neuron loss and tangle formation. We will then use LCM to confirm that temporally dysregulated miRNAs are similarly altered in pre-tangle neurons (PHF1 positive), when compared to controls (PHF1negative). Under Aim 3 we will also identify the functional mRNA targets of the temporally dysregulated miRNAs, using an integrated experimental and computational approach. mRNAs found to be dysregulated in a neuronal cell line, following transfection of Pre-miR or anti-miR oligonucleotides, and also predicted as targets via SEED sequence analysis and assigned functional relevance, will then be validated in the murine tau/tta cortical PHF1 positive and negative neurons as miRNA- regulated targets. PUBLIC HEALTH RELEVANCE: The successful completion of this exploratory study will lead to the identification of clinically relevant miRNAs that are dysregulated during the development of human tauopathy-associated neuropathology and contribute to the cognitive decline seen in human tauopathies such as FTD and AD. We will also identify the mRNA targets of these dysregulated miRNAs. Elucidating the potential role of miRNAs in tauopathy may ultimately allow for the development of medications that can delay, for example, the onset of FTD and AD. Similarly, preventing the diseases and/or their progression, may become a possibility following the successful identification of new targets to prevent miRNA dysregulation and the associated cognitive deficits, and is of exceptional relevance to the mission of the NIH.