Temporal Changes in MicroRNA Function During Tau tangle Accumulation

描述(申请人提供)：神经退行性疾病阿尔茨海默病(AD)和额颞部痴呆(FTD)的特征是细胞内含有tau的神经原纤维缠结(NFT)，以及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NFTs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。MiRNAs是一类广泛参与转录后调控的小RNAs，最近的研究数据表明，一些miRNAs在参与学习和记忆的神经元过程中具有特定的调控作用。这项拟议的研究旨在检验这样一种假设，即tau导致tau/tta-4510模型中miRNAs的早期调节失调导致在这些小鼠中观察到的认知缺陷。这项拟议研究背后的基本原理是基于我们令人兴奋的试点数据，该数据显示，与年龄匹配的对照组小鼠相比，包括miR-138(调节突触形成的miR-138)在内的多个miRNAs在tau/tta小鼠中受到失调，这些miRNAs表达突变的tau转基因基因，并表现出认知缺陷。此外，在最初的先导研究中发现的许多失调的miRNAs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，在目标1下，我们建议a)通过微阵列测量老年小鼠共济失调模型中的miRNA水平，利用我们在模型中暂时抑制转基因表达的能力来识别那些参与认知和神经退化的miRNAs，而不是那些与NFT积累相关的miRNAs，以及b)与人类FTD大脑中差异表达的miRNAs数据集进行比较，c)确定物种之间一致且具有临床相关性的miRNAs。在目标2下，在目标1下定义的miRNAs将在早期tau聚集物种开始在小鼠大脑中积累的期间，以及在认知功能下降开始期间(1个月、2.5个月和4个月)，但在广泛的神经元丢失和缠结形成之前进行临时测量。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，时间上失调的miRNAs也发生了类似的变化。在目标3下，我们还将使用一种综合的实验和计算方法来确定暂时失调的miRNAs的功能mRNA靶标。在前miR或反miR寡核苷酸转染后，在神经细胞系中发现的mRNAs是失调的，也通过种子序列分析和指定的功能相关性预测为靶点，然后将在小鼠tau/tta皮质PHF1阳性和阴性神经元中验证为miRNA调节靶点。 公共卫生相关性：这项探索性研究的成功完成将导致识别出临床上相关的miRNAs，这些miRNAs在人类脊椎病相关神经病理学的发展过程中处于失调状态，并导致人类脊椎病(如FTD和AD)的认知能力下降。我们还将确定这些调控失调的miRNAs的mRNA靶标。阐明miRNAs在肌萎缩侧索硬化症中的潜在作用最终可能有助于药物的开发，例如延缓FTD和AD的发生。同样，在成功确定新的目标以防止miRNA失调和相关的认知缺陷之后，预防疾病和/或其进展可能成为一种可能性，并且与国家卫生研究院的任务具有特殊的相关性。

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.