Kouwenhoven Willemieke M, Robinson Edward J, Hamberg Daniek, von Oerthel Lars, Smidt Marten P, van der Heide Lars P
Swammerdam Institute for Life Sciences, Molecular Neuroscience Lab, University of Amsterdam, Amsterdam, The Netherlands.
Cell Death Dis. 2025 Apr 1;16(1):230. doi: 10.1038/s41419-025-07552-w.
Mesodiencephalic dopamine neurons (mdDA) of the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) play critical roles in regulating movement and motivation. Pitx3 is an essential transcription factor required for proper embryonic development and terminal differentiation of mdDA neurons. Although Pitx3 is expressed in every mdDA neuron, its ablation results only in the absence of the SNc, not the VTA. The developmental stage at which the loss of SNc first becomes apparent, as well as the underlying mechanism, remains elusive. Here, we demonstrate, using a Pitx3 knockout GFP knock-in mouse model, that this loss does not occur during embryogenesis but rather postnatally. Quantification of GFP expression revealed a significant reduction in the total number of dopamine neurons at postnatal day 3, but not at embryonic day 14.5, 155, and 18.5. Mechanistically this reduction is accompanied by an increase in the number of cleaved caspase 3-positive GFP neurons, suggesting apoptosis. In addition, RT-PCR performed on isolated GFP neurons, one day before the loss of dopamine neurons revealed a notable elevation in the expression of the pro-apoptotic BH3-only factor Noxa. Overexpression of Noxa in dopaminergic MN9D cells dose-dependently increases the level of cleaved caspase 3 and the number of propidium iodide-positive cells, indicating that Noxa expression is sufficient to induce cell death in dopamine cells. Additionally, Noxa expression in MN9D cells, combined with a Bax-inhibiting peptide, reduces the number of cleaved caspase 3-positive and propidium iodide-positive cells, further supporting apoptosis as the mechanistic form of cell death. Overall, our study provides insights into the cell death machinery implicated in the loss of dopamine neurons, which may hold relevance for diseases affected by the loss of dopamine neurons such as Parkinson's disease, where this is a hallmark feature.
黑质致密部（SNc）和腹侧被盖区（VTA）的中脑多巴胺能神经元（mdDA）在调节运动和动机方面发挥着关键作用。Pitx3是mdDA神经元正常胚胎发育和终末分化所必需的转录因子。尽管Pitx3在每个mdDA神经元中都有表达，但其缺失仅导致SNc的缺失，而不是VTA的缺失。SNc缺失首次变得明显的发育阶段以及潜在机制仍然不清楚。在这里，我们使用Pitx3基因敲除绿色荧光蛋白敲入小鼠模型证明，这种缺失不是在胚胎发生期间发生的，而是在出生后发生的。对绿色荧光蛋白表达的定量分析显示，出生后第3天多巴胺能神经元的总数显著减少，但在胚胎第14.5、15.5和18.5天没有减少。从机制上讲，这种减少伴随着裂解的半胱天冬酶3阳性绿色荧光蛋白神经元数量的增加，表明细胞凋亡。此外，在多巴胺能神经元缺失前一天对分离的绿色荧光蛋白神经元进行的逆转录聚合酶链反应（RT-PCR）显示，仅含BH3结构域的促凋亡因子Noxa的表达显著升高。Noxa在多巴胺能MN9D细胞中的过表达剂量依赖性地增加了裂解的半胱天冬酶3的水平和碘化丙啶阳性细胞的数量，表明Noxa的表达足以诱导多巴胺能细胞死亡。此外，MN9D细胞中Noxa的表达与Bax抑制肽相结合，减少了裂解的半胱天冬酶3阳性和碘化丙啶阳性细胞的数量，进一步支持细胞凋亡是细胞死亡的机制形式。总的来说，我们的研究为与多巴胺能神经元缺失相关的细胞死亡机制提供了见解，这可能与受多巴胺能神经元缺失影响的疾病如帕金森病有关，而多巴胺能神经元缺失是帕金森病的一个标志性特征。
