Neural Diseases in a Dish: Drug Testing on Multielectrode Arrays

培养皿中的神经疾病：多电极阵列上的药物测试

• 批准号: 2274268
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2274268
• 关键词: Neural; Diseases; Dish; Drug; Testing

Project Background Alzheimer's disease (AD) is characterised by progressive and irreversible neuronal cell death which discretely and predictably affects different brain regions, with the hippocampus and entorhinal cortex being primarily affected. These large-scale changes are paralleled and preceded by perturbations to functional network connectivity at the cellular level with the AD-pathogenic proteins Amyloid b (Ab) and tau possibly having distinct roles in this regard.It is suggested that Ab may drive an initial hyperexcitability which may in turn instigate propagative neuropathology, whereas pathogenic tau may mediate later effects such as cell silencing and death (Busche et al., 2019). Indeed, tau has previously been shown to be a prerequisite for the decreases in long-term potentiation seen in mouse models of AD (Shipton et al., 2011). However, to date, no study has characterised the interplay of Ab and tau on network connectivity in models of AD progression. On the contrary, in vivo recordings from the brain and in vitro analyses of cell cultures or brain slices usually only provide a snapshot of this dynamic.Multi-Electrode Arrays (MEAs) allow for spatial and temporal analyses of electrical signalling in in vitro networks. Unlike some other electrophysiological techniques, MEAs are non-invasive to cells and act as a culture platform which allows for regular, long-term analyses of integrated network activity. MEAs therefore provide an opportunity for the investigation of network disturbances during early AD progression. Furthermore, MEAs are highly amenable to drug application meaning that the opportunities for pharmacological intervention can be investigated.Project Approach Initial work will be performed on primary hippocampal neurones derived from AppNL-G-F knock-in (KI) mice and wild-type (WT) mice which will act as a control. The AppNL-G-F model contains a KI of the human APP gene with three familial-AD-relevant mutations which cause the Swedish (KN670/671NL), Arctic (E693G) and Iberian (I716F) amino acid substitutions in the Ab precursor protein respectively (Saito et al., 2014).Neurones will be cultured on MEAs to facilitate investigations of the networks formed. Measures of spontaneous and stimulated network activity will be collected and analysed using conventional burst analysis (Cotterill et al., 2016), graph theoretical measures (Schröter et al., 2015), and advanced multidimensional cross-correlational analysis. Additionally, primary neurones will be cultured on coverslips to facilitate two-photon Ca2+ imaging and whole-cell electrophysiological experiments, gaining insights into cell-type-specific and synaptic activities respectively. Immunohistochemical, Western blot and RT-PCR or RNA-Seq analyses are likely to be used to assess supplementary neuropathology such as Ab and tau burden, and changes in gene expression for the proteins implicated in AD and neuronal excitability.Following this work, the humanised MAPT KI mouse line, which expresses both 3R and 4R tau isoforms at physiologically-relevant levels (Saito et al., 2019), will be cross-bred with the AppNL-G-F KI mice to produce AppNL-G-F/MAPT dKI mice and the aforementioned experiments repeated. Hippocampal neurones derived from the MAPT KI mouse line alone will be used as a control. In all circumstances pharmaceuticals such as the GABAergic agonist benzodiazepine, used clinically in the treatment of anxiety and convulsive epileptic seizures, may be used to assess whether increased inhibitory signalling can influence the development of AD-like phenotypes in these hippocampal models.Busche, M. A., et al. (2019) Nat Neurosci 22(1):57-64.Cotterill, E., et al. (2016) J Neurophysiol 116(2):306-321.Saito, T., et al. (2014) Nat Neurosci 17(5):661-663.Saito, T., et al. (2019) J Biol Chem 294(34):12754-12765.Schröter, M. S., et al. (2015) J Neurosci 35(14):5459-5470.Shipton, O. A., et al. (2011) J Neurosci 31(5):1688-1692

阿尔茨海默病（AD）的特征是进行性和不可逆的神经元细胞死亡，其离散地和可预测地影响不同的脑区域，海马和内嗅皮层主要受到影响。这些大规模的变化是由在细胞水平上的功能网络连接的扰动引起的，AD致病蛋白淀粉样蛋白B（Ab）和tau可能在这方面具有不同的作用。这表明Ab可能驱动初始的过度兴奋，这反过来可能引起传播性神经病理学，而致病性tau可能介导后期效应，如细胞沉默和死亡（Busche et al.，2019年）。事实上，tau先前已被证明是AD小鼠模型中所见的长时程增强降低的先决条件（Shipton et al.，2011年）。然而，到目前为止，还没有研究描述了Ab和tau在AD进展模型中对网络连接的相互作用。相反，在体内记录从大脑和体外分析的细胞培养物或脑切片通常只提供一个快照，这种dynamic.Multi-Electrode阵列（MEA）允许在体外网络的电信号的空间和时间分析。与其他一些电生理学技术不同，MEA对细胞是非侵入性的，并作为一个培养平台，允许对综合网络活动进行定期、长期的分析。因此，MEA为研究早期AD进展期间的网络干扰提供了机会。此外，多边环境协定是非常适合药物应用的意思是，药理干预的机会，可以investigated.Project方法的初步工作将进行原代海马神经元来自AppNL-G-F基因敲入（KI）小鼠和野生型（WT）小鼠将作为对照。AppNL-G-F模型包含具有三个家族性AD相关突变的人APP基因的KI，所述突变分别导致Ab前体蛋白中的瑞典（KN 670/671 NL）、北极（E693 G）和伊比利亚（I716 F）氨基酸取代（Saito et al.，2014).将在多边环境协定上培养神经元，以促进对所形成的网络的研究。将收集自发和受激网络活动的量度，并使用常规爆发分析（Cotterill等人，2016），图论测量（Schröter等人，2015年），和先进的多维交叉相关分析。此外，原代神经元将在盖玻片上培养，以促进双光子Ca 2+成像和全细胞电生理实验，分别深入了解细胞类型特异性和突触活动。免疫组织化学、蛋白质印迹和RT-PCR或RNA-Seq分析可能用于评估补充的神经病理学，例如Ab和tau负荷，以及与AD和神经元兴奋性有关的蛋白质的基因表达的变化。2019），将与AppNL-G-F KI小鼠杂交以产生AppNL-G-F/MAPT dKI小鼠，并重复上述实验。将单独使用来自MAPT KI小鼠系的海马神经元作为对照。在所有情况下，临床上用于治疗焦虑和惊厥性癫痫发作的药物如GABA能激动剂苯二氮杂，可用于评估增加的抑制性信号传导是否可影响这些海马模型中AD样表型的发展。一、等（2019）Nat Neurosci 22（1）：57- 64。等（2016）J Neurophysiol 116（2）：306- 321. Saito，T.，等（2014）Nat Neurosci 17（5）：661- 663. Saito，T.，等（2019）J Biol Chem 294（34）：12754- 12765.美国，等（2015）J Neurosci 35（14）：5459- 5470.一、等人（2011）J Neurosci 31（5）：1688-1692