Overk C R, Rockenstein E, Florio J, Cheng Q, Masliah E
Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, United States.
Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093, United States; Department of Pathology, University of California, San Diego, La Jolla, CA 92093, United States.
Neuroscience. 2015 Dec 3;310:549-60. doi: 10.1016/j.neuroscience.2015.08.068. Epub 2015 Sep 2.
Abnormal accumulation of amyloid β (Aβ), α-synuclein (α-syn), and microtubule-associated protein tau (tau) have been implicated in neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and Pick's disease (PiD). The mechanisms through which aggregated versions of α-syn, Aβ, and tau may lead to neurodegeneration are not entirely clear, however, there is emerging evidence that neuronal calcium dysregulation is at play. Two-photon microscopy is a powerful tool that can be used to measure in vivo alterations of calcium transients using animal models of neurodegeneration, and when coupled with statistical methods to characterize functional signals, can reveal features that identify and discern between distinct mouse types. We studied four mouse models of neurodegenerative diseases, wild-type (WT) α-syn, E57K α-syn, amyloid precursor protein (APP), and triple-repeat (3R)-Tau and Non-transgenic (tg) littermates using two-photon microscopy. We found that for calcium transients, simple measures such as area under the curve (AUC) and peak width in the 1-Hz whisker pad stimulation paradigm, were significantly increased for WT α-syn, E57K α-syn and APP mice across all cortical depths compared to Non-tg mice. A similar result was found in the 3-Hz paradigm in E57K α-syn mice. Spontaneous calcium transient AUC was significantly higher in WT α-syn mice and lower for APP and 3R Tau mice at 150-μm depth. Going beyond simple measure differences such as group means for AUC, signal peak width, and spontaneous calcium activity counts, we built statistical classifiers to characterize neuronal calcium signals to identify and discern, with quantified measures of confidence, all mouse types. We tested our classifier with FK506, which regulates mitochondrial calcium and found that this drug modulated the WT α-syn calcium transients to such an extent that the classifier easily identified the calcium transients as belonging to Non-tg mice. The coupling of two-photon microscopy data and statistical classifiers serves to effectively create a bioassay where the number of animals and scientific resources can be reduced without compromising the results of the experiment.
淀粉样β蛋白（Aβ）、α-突触核蛋白（α-syn）和微管相关蛋白tau（tau）的异常积累与包括阿尔茨海默病（AD）、帕金森病（PD）和皮克病（PiD）在内的神经退行性疾病有关。然而，α-syn、Aβ和tau的聚集形式可能导致神经退行性变的机制尚不完全清楚，不过，越来越多的证据表明神经元钙调节异常在其中起作用。双光子显微镜是一种强大的工具，可用于使用神经退行性疾病动物模型测量体内钙瞬变的变化，并且当与统计方法相结合以表征功能信号时，可以揭示识别和区分不同小鼠类型的特征。我们使用双光子显微镜研究了四种神经退行性疾病小鼠模型，野生型（WT）α-syn、E57K α-syn、淀粉样前体蛋白（APP）和三重复（3R）-Tau以及非转基因（tg）同窝小鼠。我们发现，对于钙瞬变，在1-Hz触须垫刺激范式下，与非tg小鼠相比，WT α-syn、E57K α-syn和APP小鼠在所有皮质深度下，诸如曲线下面积（AUC）和峰值宽度等简单测量值均显著增加。在E57K α-syn小鼠的3-Hz范式中也发现了类似结果。在150-μm深度处，WT α-syn小鼠的自发钙瞬变AUC显著更高，而APP和3R Tau小鼠则更低。除了诸如AUC的组均值、信号峰值宽度和自发钙活性计数等简单测量差异之外，我们构建了统计分类器来表征神经元钙信号，以便通过量化的置信度测量来识别和区分所有小鼠类型。我们用调节线粒体钙的FK506测试了我们的分类器，发现这种药物对WT α-syn钙瞬变的调节程度使得分类器能够轻松地将钙瞬变识别为属于非tg小鼠。双光子显微镜数据与统计分类器的结合有效地创建了一种生物测定方法，在不影响实验结果的情况下，可以减少动物数量和科学资源。
