Dyrk1a triplication in Down's syndrome and its overexpression in Alzheimer's disease suggest a role for increased DYRK1A activity in the abnormal metabolism of APP. Transport defects are early phenotypes in the progression of Alzheimer's disease, which lead to APP processing impairments. However, whether DYRK1A regulates the intracellular transport and delivery of APP in human neurons remains unknown. From a proteomic dataset of human cerebral organoids treated with harmine, a DYRK1A inhibitor, we found expression changes in protein clusters associated with the control of microtubule-based transport and in close interaction with the APP vesicle. Live imaging of APP axonal transport in human-derived neurons treated with harmine or overexpressing a dominant negative DYRK1A revealed a reduction in APP vesicle density and enhanced the stochastic behavior of retrograde vesicle transport. Moreover, harmine increased the fraction of slow segmental velocities and changed speed transitions supporting a DYRK1A-mediated effect in the exchange of active motor configuration. Contrarily, the overexpression of DYRK1A in human polarized neurons increased the axonal density of APP vesicles and enhanced the processivity of retrograde APP. In addition, increased DYRK1A activity induced faster retrograde segmental velocities together with significant changes in slow to fast anterograde and retrograde speed transitions, suggesting the facilitation of the active motor configuration. Our results highlight DYRK1A as a modulator of the axonal transport machinery driving APP intracellular distribution in neurons, and stress DYRK1A inhibition as a putative therapeutic intervention to restore APP axonal transport in Down's syndrome and Alzheimer's disease.

SIGNIFICANCE STATEMENT Axonal transport defects are early events in the progression of neurodegenerative diseases, such as Alzheimer's disease. However, the molecular mechanisms underlying transport defects remain elusive. Dyrk1a kinase is triplicated in Down's syndrome and overexpressed in Alzheimer's disease, suggesting that DYRK1A dysfunction affects molecular pathways leading to early-onset neurodegeneration. Here, we show by live imaging of human-derived neurons that DYRK1A activity differentially regulates the intracellular trafficking of APP. Further, single-particle analysis revealed DYRK1A as a modulator of axonal transport and the configuration of active motors within the APP vesicle. Our work highlights DYRK1A as a regulator of APP axonal transport and metabolism, supporting DYRK1A inhibition as a therapeutic strategy to restore intracellular dynamics in Alzheimer's disease.

唐氏综合症中的Dyrk1a三倍体及其在阿尔茨海默氏病中的过度表达表明 DYRK1A 活性增加在 APP 异常代谢中发挥作用。转运缺陷是阿尔茨海默病进展的早期表型，会导致 APP 处理障碍。然而，DYRK1A 是否调节人类神经元中 APP 的细胞内运输和递送仍不清楚。从用 DYRK1A 抑制剂去氢骆驼蓬碱处理的人类大脑类器官的蛋白质组数据集中，我们发现蛋白质簇的表达变化与基于微管的运输的控制相关，并与 APP 囊泡密切相互作用。用去氢骆驼蓬碱处理或过表达显性失活 DYRK1A 的人源神经元中 APP 轴突运输的实时成像显示 APP 囊泡密度降低并增强逆行囊泡运输的随机行为。此外，去氢骆驼蓬碱增加了慢节段速度的比例并改变了速度转换，支持 DYRK1A 介导的主动运动配置交换效应。相反，DYRK1A 在人极化神经元中的过度表达增加了 APP 囊泡的轴突密度，并增强了逆行 APP 的持续加工能力。此外，DYRK1A 活性的增加诱导了更快的逆行节段速度，以及从慢到快的顺行和逆行速度转换的显着变化，表明主动运动配置的促进。我们的结果强调 DYRK1A 作为轴突运输机制的调节剂，驱动神经元中 APP 的细胞内分布，并强调 DYRK1A 抑制作为一种推定的治疗干预措施，可恢复唐氏综合症和阿尔茨海默氏病的 APP 轴突运输。

意义陈述轴突运输缺陷是神经退行性疾病（例如阿尔茨海默病）进展的早期事件。然而，运输缺陷背后的分子机制仍然难以捉摸。 Dyrk1a激酶在唐氏综合症中出现三次，并在阿尔茨海默病中过度表达，这表明 DYRK1A 功能障碍会影响导致早发性神经退行性变的分子途径。在这里，我们通过人源神经元的实时成像表明 DYRK1A 活性差异调节 APP 的细胞内运输。此外，单颗粒分析揭示 DYRK1A 作为轴突运输的调节剂和 APP 囊泡内主动马达的配置。我们的工作强调 DYRK1A 作为 APP 轴突运输和代谢的调节剂，支持 DYRK1A 抑制作为恢复阿尔茨海默病细胞内动力学的治疗策略。