Reversal of neurodegeneration through pTau clearance by chimeric scavenger receptor monocytes

项目概要/摘要 阿尔茨海默病（AD）是一种无法治愈的神经退行性疾病，将继续成为全球性疾病。 随着全世界社会老龄化，我们面临着健康挑战，但我们完全缺乏能够减缓或逆转这种挑战的疗法。 疾病进展。过度磷酸化的Tau（pTau）缠结是AD的病理学标志，并且与β-Tau缠结不同， 淀粉样蛋白，与临床认知功能减退高度相关。小胶质细胞占主导地位的髓腔 在中枢神经系统（CNS）内，但清除pTau的能力有限， 进步。这些细胞响应pTau同时分泌神经毒性促炎细胞因子 摄取加速神经变性并加速疾病进展。目前，不存在 可以有意义地减少高毒性的pTau种类，寡聚pTau（opTau），而不触发这种 有害的神经炎症因此，对于催化opTau的新型治疗剂存在未满足的需求。 降解而不触发CNS骨髓细胞释放炎性细胞因子。在肿瘤免疫中，我们 已经观察到清道夫受体FcγRIIb是由骨髓基质表达的抑制性受体， 其通过网格蛋白介导的内吞作用内在化，并可起到抑制针对多种肿瘤的细胞毒性的作用。 我们提出了一个新的概念，即清道夫受体的抗炎特性可以被利用 来改变AD中对opTau的骨髓反应。与小胶质细胞相反，外周单核细胞保留了 清除pTau组装，但确实分泌神经毒性促炎细胞因子。我们假设单核细胞 通过抗体重定向的嵌合清道夫受体（CSR）结合和内化opTau的工程将停止 通过保护神经元免受opTau介导的神经毒性，同时抑制炎症， 细胞因子释放我们已经成功地设计了一种外周巨噬细胞/单核细胞细胞系， 设计的CSR构建体，其由FcγRIIb支架和opTau单链可变区片段组成 在一个实施方案中，本发明提供了基于抗opTau抗体的单链抗体（ScFv），其限制AD小鼠模型中的神经变性。一是 测量表达Fc γ RIIb CSR的单核细胞/巨噬细胞细胞系对opTau的摄取，并确定 无论内化依赖于吞噬作用还是网格蛋白介导的内吞作用。接下来，我们将调查 通过这些表达CSR的细胞降解opTau的效率，以及确定这是否 降解是溶酶体依赖性的。为了确定CSR单核细胞是否能够保护原发性 为了使原代神经元免受opTau毒性和它们自身的炎性爆发，我们将原代神经元与 单独的opTau，与亲本单核细胞，或与表达CSR的单核细胞，并测量树突完整性 相对于碎片化。最后，我们将进行CSR单核细胞到Tau中的心室内输注。 p301 S转基因小鼠从6月龄开始，并评估其预防或延迟两种疾病发作的能力 认知衰退、CNS神经变性和病理性CNS炎症。虽然风险很高，但我们 初步数据表明该方法具有保护神经元免受opTau毒性的潜力。

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) is an incurable neurodegenerative disease that will continue to grow as a global health challenge as societies age worldwide, yet we entirely lack therapies capable of slowing or reversing disease progression. Hyperphosphorylated Tau (pTau) tangles are pathological hallmarks of AD and, unlike β- Amyloid, highly correlate with clinical cognitive deterioration. Microglia dominate the myeloid compartment within the central nervous system (CNS), but have a limited capacity to clear pTau that declines as AD progresses. Concomitant secretion of neurotoxic proinflammatory cytokines by these cells in response to pTau uptake hastens neurodegeneration and accelerates disease progression. At present, no therapies exist which can meaningfully diminish the highly toxic pTau species, oligomeric pTau (opTau), without triggering this deleterious neuroinflammation. Thus, there is an unmet need for a novel therapeutic that catalyzes opTau degradation without triggering inflammatory cytokine release from CNS myeloid cells. In tumor immunity, we have observed that the scavenger receptor, FcγRIIb, is an inhibitory receptor expressed by the myeloid stroma that internalizes via clathrin-mediated endocytosis and can act to dampen cytotoxicity against diverse tumors. We propose the novel concept that the anti-inflammatory properties of scavenger receptors can be harnessed to alter the myeloid response to opTau in AD. In contrast to microglia, peripheral monocytes retain the ability to clear pTau assemblies, but do secrete neurotoxic proinflammatory cytokines. We hypothesize that monocytes engineered to bind and internalize opTau via antibody-redirected chimeric scavenger receptors (CSR) will halt the progression of AD by protecting neurons from opTau-mediated neurotoxicity while dampening inflammatory cytokine release. We have successfully engineered a peripheral macrophage/monocyte cell line to express our designed CSR construct which consists of an FcγRIIb scaffold and an opTau single-chain variable fragment (ScFv) based on an anti-opTau antibody which limits neurodegeneration in AD mouse models. First, we will measure uptake of opTau by a monocyte/macrophage cell line expressing the FcyRIIb CSR, and determine whether internalization relies on phagocytosis or clathrin-mediated endocytosis. Next, we will investigate the efficiency of degradation of opTau by these CSR expressing cells, as well as determine whether this degradation is lysosome-dependent. To ascertain whether CSR monocytes are capable of protecting primary neurons from opTau toxicity and from their own inflammatory burst, we will co-culture primary neurons with opTau alone, with parental monocytes, or with CSR expressing monocytes and measure dendrite integrity versus fragmentation. Finally, we will perform intra-cerebroventricular infusion of CSR monocytes into Tau p301S transgenic mice starting at 6 months of age and assess their capacity to prevent or delay onset of both cognitive decline, CNS neurodegeneration, and pathologic CNS inflammation. Although high risk, our preliminary data suggests this approach has potential to protect neurons from opTau toxicity.