Roles of Cdk5 in neurodevelopment and neurodegeneration

我们在神经退行性变方面工作的一个主要目标是剖析各种病理机制之间的关系。在过去的一年里，我们在这方面取得了重大进展(发表在三篇论文中)。 在过去的一年里，我们开发了一种通过定量表达谱来测量个体或群体的生理年龄的方法，并使用这种方法来表明我们的苍蝇模型中退化的一个机制是加速衰老的开始。(自那以后，我们开发的方法已被大量实验室在衰老领域使用，其原理也应用于蛋白质组和代谢组学数据集。)鉴于这为我们提供了一种定量解释衰老对退化的贡献的方法，它也使我们能够从我们的退化突变体的表达谱中减去这种影响，从而分离出退化的非衰老成分。这揭示了存在与衰老正交的第二条途径，通过该途径抑制自噬导致先天性免疫反应的神经毒性激活，并且其结果是免疫效应蛋白的过度表达，如抗菌肽，实际上导致敏感神经元群体(多巴胺神经元；在Shukla，2019a和b中报道)的死亡。 在鉴定自噬/免疫退变途径的同时，我们还鉴定了第三条途径。在表征我们的神经变性突变体(编码CDK5激活亚单位p35的基因的零突变)的表型时，我们先前证明该突变导致与细胞退化相关的轴突细胞骨架的严重破坏，特别是轴突起始片段(AIS)的严重破坏。自从我们发表这篇文章以来，其他实验室已经表明，在其他退行性疾病模型中也观察到了同样的相关性，例如小鼠的肌萎缩症模型。然而，他们的工作和我们之前的工作都不能提供证据表明AIS缺陷是导致退化的原因，还是简单地相关。在我们的新工作中，我们通过表达核化AIS的Ankyrin Paralog的显性负形式直接扰乱了AIS的结构，并发现这足以导致轴突碎裂和神经元丢失。我们进一步表明，锚蛋白诱导的AIS丢失机制与依赖于CDK5/p35的AIS破坏机制在基因上是分开的。因此，这些数据提供了强有力的证据，表明轴突细胞骨架的破坏以及通过AIS的失调而导致的神经元兴奋性，确实是我们的果蝇tautation模型中神经退行性变的第三个、可区分的原因路径(SPurrier，等人)。2019年)。 我们现在计划扩大我们对衰老、免疫和变性三者关系的分析；调查线粒体功能障碍和突触丢失如何符合我们已经形成的变性图景，并开始努力筛选能够调节这种疾病遗传易感性的果蝇变性开始的突变体。

A primary goal of our work in neurodegeneration has been to dissect the relationships among the various mechanisms of pathology. In the past year, we have made significant advances in this effort (published in three papers). In the previous year, we had developed a method for measuring the physiological age of an individual or population by quantitative expression profiling and used this method to show that one mechanism of degeneration in our fly model is acceleration of the onset of aging. (The method we developed has since been used by a large number of labs in the aging field, and its principles have been applied to proteomic and metabolomic datasets, as well.) Given that this provided us with a way to account quantitatively for the contribution of aging to degeneration, it also gave us the ability to subtract that effect from expression profiles of our degeneration mutant and thus isolate non-aging components of degeneration. This revealed the existence of a second pathway, orthogonal to aging, by which inhibition of autophagy causes neurotoxic activation of the innate immune response, and that it is the resulting hyperexpression of immune effector proteins, such as antimicrobial peptides, that actually cause the death of a sensitive neuronal population (dopamine neurons; reported in Shukla, 2019 a and b). In parallel to our identification of an autophagy/immunity pathway of degeneration, we also identified yet a third pathway. In characterizing the phenotype of our neurodegeneration mutant (a null mutant of the gene encoding the Cdk5 activating subunit, p35), we previously demonstrated that the mutation causes severe disruption of the axonal cytoskeleton, and in particular of the axon initial segment (AIS), that is correlated with cell degeneration. Since we published this, other labs have shown that the same correlation is observed in other models of degeneration, such as a tauopathy model in the mouse. However, neither their work nor our previous work could provide evidence as to whether AIS defects are causal for degeneration, or simply correlated. In our new work we disrupted AIS structure directly, by expression of a dominant-negative form of the Ankyrin paralog that nucleates the AIS, and found that this was sufficient to cause axon fragmentation and neuron loss. We further showed that the mechanism of ankyrin-induced AIS loss is genetically separate from the mechanism of Cdk5/p35-dependent AIS disruption. Together, these data therefore provide strong evidence that disruption of the axonal cytoskeleton, and of neuronal excitability through dysregulation of the AIS, are indeed a third, distinguishable, causal pathway of neurodegeneration in our tauopathy model in the fly (Spurrier, et al. 2019). We now plan to extend our analysis of the three-way relationship of aging, immunity and degeneration; investigate how mitochondrial dysfunction and synapse loss fit into the picture of degeneration that we have developed, and begin efforts to screen for mutants that can modulate the onset of degeneration in flies that are genetically predisposed to this pathology.