Neural Diseases in a Dish: Drug Testing on Multielectrode Arrays

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，这种死亡会离散地和可预测地影响大脑的不同区域，海马和内嗅皮层主要受到影响。这些大规模的变化与细胞水平上的功能网络连通性的扰动是平行和先于的，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。研究表明，Ab可能驱动初始的高兴奋性，进而引发繁殖性神经病理学，而致病性tau可能介导细胞沉默和死亡等后期效应（Busche et al., 2019）。事实上，在阿尔茨海默病小鼠模型中，tau蛋白已被证明是长期增强能力下降的先决条件（Shipton et al., 2011）。然而，到目前为止，还没有研究描述Ab和tau在AD进展模型中对网络连通性的相互作用。相反，大脑的体内记录和细胞培养或大脑切片的体外分析通常只能提供这种动态的快照。多电极阵列（MEAs）允许在体外网络电信号的空间和时间分析。与其他一些电生理技术不同，mea对细胞没有侵入性，可以作为一个培养平台，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进展过程中的网络干扰提供了机会。此外，mea对药物应用具有高度适应性，这意味着可以研究药物干预的机会。最初的工作将在来自AppNL-G-F敲入（KI）小鼠和野生型（WT）小鼠的初级海马神经元上进行，后者将作为对照。AppNL-G-F模型包含人类APP基因的KI，具有三个家族ad相关突变，分别导致Ab前体蛋白中的瑞典（KN670/671NL）、北极（E693G）和伊比利亚（I716F）氨基酸替换（Saito et al., 2014）。神经元将在mea上培养，以促进对形成的网络的研究。将使用常规突发分析（Cotterill等人，2016）、图理论测量（Schröter等人，2015）和先进的多维交叉相关分析收集和分析自发和受刺激网络活动的测量。此外，原代神经元将在盖层上培养，以促进双光子Ca2+成像和全细胞电生理实验，分别获得细胞类型特异性和突触活动的见解。免疫组织化学、Western blot和RT-PCR或RNA-Seq分析可能用于评估补充神经病理学，如Ab和tau负荷，以及与AD和神经元兴奋性相关的蛋白质的基因表达变化。在这项工作之后，在生理相关水平上表达3R和4R tau亚型的人源化MAPT KI小鼠系（Saito等人，2019）将与AppNL-G-F KI小鼠杂交，产生AppNL-G-F/MAPT dKI小鼠，并重复上述实验。单独从MAPT KI小鼠系中提取的海马神经元将作为对照。在所有情况下，诸如临床用于治疗焦虑和惊厥性癫痫发作的gaba能激动剂苯二氮卓类药物可用于评估这些海马模型中增加的抑制性信号传导是否可以影响ad样表型的发展。陈晓明，陈晓明，陈晓明，等。（2019）神经科学进展22(1):57-64。张建军，张建军，张建军，等。（2016）神经物理学报，32(2):306-321。Saito， T., et .（2014）神经科学学报17(5):661-663。Saito， T.等。（2019）生物化学学报，294(34):12754-12765。Schröter， M. S.等。（2015）神经科学杂志35(14):5459-5470。Shipton， O. A.等（2011）神经科学杂志31(5):1688-1692

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