DLK-dependent protein network regulates hippocampal glutamatergic neuron degeneration. DLK-dependent protein network regulates hippocampal glutamatergic neuron degeneration

The conserved MAPKKK DLK plays many roles in neuronal development, axon injury, and neuronal stress responses. The outcomes of activating or inhibiting DLK signaling depend on cell-type and cellular contexts. Emerging evidence has implicated DLK signaling in several neurodegenerative diseases. However, our understanding of the DLK-dependent cellular network in the central nervous system remains limited. Here, we investigated roles of DLK in hippocampal glutamatergic neurons, using conditional knockout and overexpression mice. We find that dorsal CA1 and dentate gyrus neurons are particularly vulnerable to elevated DLK activity. We performed RiboTRAP-seq analysis and identified the DLK dependent translatome, majority of which are involved in neuronal developmental processes, neuronal stress responses, and synapse formation and function. Increasing DLK signaling is associated with disruptions of microtubules, potentially involving Stmn4. We also show that in primary hippocampal neurons DLK regulates neurite outgrowth, axon specification, and synapse formation. This study broadens our understanding of both conserved and cell-type specific effects of DLK signaling. The identification of translational targets of DLK in glutamatergic neurons has relevance to our understanding of neurodegenerative disease. Overall design: To investigate DLK roles in hippocampal glutamatergic neurons we generated mice with conditional overexpression or conditional knockout of DLK in glutamatergic neurons using Vglut1-Cre mice. Mice were crossed to Ribotag mice and actively translated mRNA from hippocampal glutamatergic neurons from the respective genotypes was sequenced. All samples are from the P15 timepoint. Three or four biological replicates for overexpression or knockout and respective sibling controls were used.

保守型丝裂原活化蛋白激酶激酶激酶（MAPKKK）DLK在神经元发育、轴突损伤及神经元应激反应中发挥多种功能。激活或抑制DLK信号通路的效应取决于细胞类型与细胞所处的微环境。越来越多的研究证据表明，DLK信号通路与多种神经退行性疾病密切相关。然而，目前我们对中枢神经系统中DLK依赖型细胞网络的认知仍十分有限。
本研究通过构建条件性基因敲除与过表达小鼠模型，探究了DLK在海马谷氨酸能神经元中的功能。研究发现，背侧CA1区与齿状回神经元对DLK活性升高尤为敏感。我们开展了RiboTRAP-seq（核糖体亲和纯化测序）分析，鉴定得到DLK依赖型翻译组，其中多数基因参与神经元发育过程、神经元应激反应以及突触形成与功能调控。DLK信号通路的增强与微管结构破坏相关，这一过程可能涉及Stmn4。此外，我们在原代海马神经元中证实，DLK可调控神经元突起生长、轴突特化以及突触形成。
本研究加深了我们对DLK信号通路保守性功能与细胞类型特异性效应的认知。鉴定谷氨酸能神经元中DLK的翻译调控靶点，对于我们理解神经退行性疾病具有重要参考价值。
实验整体设计：为探究DLK在海马谷氨酸能神经元中的功能，我们借助Vglut1-Cre工具小鼠，构建了在谷氨酸能神经元中条件性过表达或条件性敲除DLK的小鼠模型。将上述小鼠与RiboTag（RiboTag）工具小鼠杂交，随后对不同基因型小鼠海马谷氨酸能神经元中的活跃翻译mRNA进行测序。所有实验样本均取自出生后第15天（P15）的小鼠。过表达组、敲除组及其对应的同窝对照样本均设置3~4个生物学重复。