Waller Thomas J, Collins Catherine A, Dus Monica
Molecular, Cellular, and Developmental Biology Department, College of Literature, Science, and the Arts, The University of Michigan, Ann Arbor, MI, 48109, USA.
Molecular, Cellular, and Developmental Biology Department, College of Literature, Science, and the Arts, The University of Michigan, Ann Arbor, MI, 48109, USA; Department of Neurosciences, Case Western Reserve University, Cleveland, OH, 44106, USA.
Mol Metab. 2025 Jun 10;98:102187. doi: 10.1016/j.molmet.2025.102187.
Metabolic disruption is a central feature to many neurodegenerative diseases. Despite this, many gaps exist in our understanding of how these perturbations link to the mechanisms of neural disease. In this study, we sought to understand how genetically-controlled, cell-specific loss of pyruvate kinase (PyK) impacts motor neuron synaptic integrity and how the canonical neurodegenerative proteins DLK and SARM1 respond to this break in homeostasis.
This study made use of the genetically-tractable Drosophila melanogaster to cell-specifically express proteins (via the GAL4/UAS binary system), knockdown gene transcripts (via RNA interference), and knockout gene loci (via guide RNA-directed Cas9). Synaptic and axonal degeneration were measured through immunohistochemistry, microscopy, and blinded scoring of fly larvae at both early and later 3rd instar stages to test for progressive phenotypes. Nervous system injury through a physical nerve crush assay was used to assay functional outcomes of protective stress responses.
We found that knockdown or knockout of PyK results in progressive axonal and synaptic degeneration, dependent on signaling through DLK and SARM1. This degeneration is preceded by nuclear transcriptional activation by DLK and the downstream AP-1 transcription factor Fos. We also found evidence of a neuroprotective response through injury of PyK-deficient axons (before progressive degeneration has occurred), which results in delayed Wallerian degeneration. This delay shows dependence on DLK and Fos, and coincides with reduced axonal localization of SARM1 whose overexpression fully restores degeneration speed.
These data support a rheostat model of DLK signaling that both promotes and inhibits axon degeneration in response to metabolic disruption. This rheostat likely converges on regulation of SARM1, which is required for the progressive synapse loss following PyK, but also abolishes the protective delay in injury-induced Wallerian degeneration when overexpressed. Overall, we conclude that metabolic signaling through PyK is essential for the integrity of motor neuron axons and synapses, and that its disruption activates both neurodegenerative and neuroprotective mechanisms.
代谢紊乱是许多神经退行性疾病的核心特征。尽管如此，我们对这些扰动如何与神经疾病机制相关联的理解仍存在许多空白。在本研究中，我们试图了解丙酮酸激酶（PyK）的基因控制的细胞特异性缺失如何影响运动神经元突触完整性，以及经典神经退行性蛋白DLK和SARM1如何应对这种稳态破坏。
本研究利用基因易处理的黑腹果蝇，通过GAL4/UAS二元系统进行细胞特异性蛋白表达、通过RNA干扰敲低基因转录本以及通过引导RNA定向的Cas9敲除基因位点。通过免疫组织化学、显微镜检查以及对三龄幼虫早期和晚期的果蝇幼虫进行盲法评分来测量突触和轴突退化，以测试进展性表型。通过物理神经挤压试验造成神经系统损伤，以测定保护性应激反应的功能结果。
我们发现敲低或敲除PyK会导致进展性轴突和突触退化，这依赖于通过DLK和SARM1的信号传导。这种退化之前是DLK和下游AP-1转录因子Fos的核转录激活。我们还发现了通过PyK缺陷轴突损伤（在进展性退化发生之前）产生神经保护反应的证据，这导致沃勒变性延迟。这种延迟显示出对DLK和Fos的依赖性，并且与SARM1轴突定位减少相吻合，其过表达可完全恢复退化速度。
这些数据支持DLK信号的变阻器模型，该模型响应代谢紊乱既促进又抑制轴突退化。这种变阻器可能集中在对SARM1的调节上，SARM1是PyK后突触逐渐丧失所必需的，但过表达时也会消除损伤诱导的沃勒变性中的保护延迟。总体而言，我们得出结论，通过PyK的代谢信号传导对于运动神经元轴突和突触的完整性至关重要，并且其破坏会激活神经退行性和神经保护机制。
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