3D patient-derived organoids: A viable and complementary model to study neural system dysfunction in Alzheimer's Disease

阿尔茨海默病（AD）是一种毁灭性的疾病，有效的治疗方法尚未确定。我们实验室最近的证据表明，神经回路功能障碍在阿尔茨海默病中很早就出现，在认知缺陷之前形成，并影响随后的疾病进展，最终导致神经退行性变和痴呆。阿尔茨海默病的神经回路表现出早期的神经兴奋性，在疾病后期转变为低活性，这分别是淀粉样蛋白和tau（阿尔茨海默病中积累的标志蛋白）的病理作用的结果。小鼠模型对于以高时空分辨率解剖这些电路水平的变化至关重要，同时为潜在的病理机制和候选治疗靶点提供了非凡的见解。然而，到目前为止，临床前模型中有希望的治疗方法的临床转化（例如，减少/去除β淀粉样蛋白）已被证明是不可行的，这突出了动物模型的局限性以及消除人类疾病相关病理改变方面的挑战。目前研究阿尔茨海默病中神经回路功能变化的研究通常是在携带突变/转基因或表现出再现人类疾病中观察到的表型的蛋白质病变的小鼠中进行的。这类实验（通常是中等严重程度）是高度复杂、侵入性和低通量的，因为通常需要进行手术和植入颅骨装置，并注射生物传感器/植入电极以实现高分辨率的体内记录。这些挑战强调需要识别和验证补充和替代模型，这些模型更忠实地概括了阿尔茨海默病中的人类大脑回路，并有利于在阿尔茨海默病研究中使用动物模型。最近出现了患者来源的诱导多能干细胞（iPSC）方法的技术进步，提供了一种辅助方法来模拟人类疾病，可以在严格控制的实验条件下进行研究和调节。值得注意的是，我们的合作者，UCL的Selina Wray教授，在NC3Rs资助的工作（通过2013年的crackit在Untangle项目上，https://nc3rs.org.uk/crackit/untangle）的基础上，成功地从家族性AD和tau病患者中生成了长期可行的同质脑类器官，这些器官形成了复杂的神经回路，并允许研究与人类相关的神经成分中与疾病相关的回路水平的变化。在这里，我们建议将这一专业知识与Busche实验室最先进的结构和功能记录技术相结合，以研究AD中的神经元回路（即，Neuropixels多通道电生理学，双光子钙成像，3D光片显微镜）。在此过程中，我们的目标是利用和结合两个团队建立的尖端方法，建立一个全新的平台，以获得对AD电路功能的变革性见解，促进临床转化，并显著减少对动物模型的依赖。患者来源的iPSC培养将具有完整的疾病相关遗传图谱，提出的高分辨率记录技术将允许在相关细胞类型和覆盖电路中详细调查疾病机制，并为询问导致临床AD发展的早期细胞和分子变化提供新的机会。随着人们越来越认识到阿尔茨海默病早期神经回路功能障碍的重要性，以及世界上越来越多的实验室利用动物模型来了解阿尔茨海默病神经回路的机制基础，我们预计，我们的类器官模型的验证将导致现有动物工作的25%的实质性替代，不仅在我们的实验室，而且随后，全世界约20个实验室（相当于每年约300只动物）研究AD的电路功能障碍。

Alzheimer's Disease (AD) is a devastating disorder for which effective disease-modifying treatments are yet to be identified. Recent evidence from our laboratory indicates that neural circuit dysfunction emerges very early in AD, preceding cognitive deficits and shaping subsequent disease progression, ultimately leading to neurodegeneration and dementia. Neural circuits in AD exhibit early neuronal hyperexcitability which transitions to hypoactivity in later disease, as a result of the pathological effects of Amyloid beta and tau (the hallmark proteins that accumulate in AD), respectively. Mouse models have been crucial to dissecting these circuit-level changes with high spatiotemporal resolution while providing remarkable insights into potential pathological mechanisms and candidate therapeutic targets. Nevertheless, clinical translation of promising therapies in preclinical models (e.g., reduction/removal of Amyloid beta) has so far proven unviable, highlighting the limitations of animal-models as well as the challenge in disambiguating human-disease relevant pathological alterations. Current research investigating changes in neural circuit function in AD is typically conducted in mice that carry mutations/transgenes or manifest proteinopathies which reproduce phenotypes observed in human disorders. Such experiments (often of moderate severity) are highly complex, invasive, and of low-throughput, as it is often necessary to perform surgeries and implant cranial apparatus and inject biosensors/implant electrodes to enable high-resolution in-vivo recordings. These challenges emphasise the need to identify and validate complimentary and alternative models that more faithfully recapitulate human brain circuits in AD and beneficially address the use of animal models in AD research.Technical advances in patient-derived induced pluripotent stem cell (iPSC) methodologies have recently emerged that provide an auxiliary approach with which to model human disease that can be studied and modulated under tightly controlled experimental conditions. Notably, our collaborator on this proposal, Prof. Selina Wray at UCL, building on work funded by the NC3Rs (through CRACK-IT in 2013 on the Untangle project, https://nc3rs.org.uk/crackit/untangle), has successfully generated long-term viable and homogeneous cerebral organoids from familial AD and tauopathy patients which form complex neuronal circuits and permit the study of disease-relevant circuit-level changes in human-associated neural components. Here, we propose to uniquely integrate this expertise with that of the Busche Laboratory's in state-of-art structural and functional recording techniques to study neuronal circuits in AD (i.e., Neuropixels multi-channel electrophysiology, two-photon calcium imaging, 3D lightsheet microscopy). In so doing, we aim to leverage and combine the cutting-edge methods established by both groups to establish a radically novel platform to gain transformative insights into AD circuit function, promote clinical translation, and significantly reduce reliance on animal models. Patient-derived iPSC cultures will have the complete disease-relevant genetic profile, and the proposed high-resolution recording techniques will permit the detailed investigation of disease mechanisms in relevant cell types and overlying circuits, and provide a novel opportunity to interrogate the early cellular and molecular changes that lead to the development of clinical AD. With the increasing recognition of the importance of early neural circuit dysfunction in AD, and a growing number of laboratories worldwide utilising animal models to understand the mechanistic underpinnings of neuronal circuitry in AD, we anticipate that validation of our organoid model will lead to a substantial replacement of 25% of existing animal work, not only in our laboratory but also, subsequently, in the ~20 laboratories worldwide (equating to ~300 animals/year) which study circuit dysfunction in AD.