Baxter Paul S, Dando Owen, Hardingham Giles E
Edinburgh Medical School, UK Dementia Research Institute, The University of Edinburgh, Edinburgh, United Kingdom.
Centre for Discovery Brain Sciences, Edinburgh Medical School, University of Edinburgh, Edinburgh, United Kingdom.
Front Mol Neurosci. 2023 Jul 3;16:1214439. doi: 10.3389/fnmol.2023.1214439. eCollection 2023.
A variety of proteins can be encoded by a single gene via the differential splicing of exons. In neurons this form of alternative splicing can be controlled by activity-dependent calcium signaling, leading to the properties of proteins being altered, including ion channels, neurotransmitter receptors and synaptic cell adhesion molecules. The pre-synaptic cell adhesion molecule Neurexin 1 () is alternatively spliced at splice-site 4 (SS4) which governs exon 22 inclusion (SS4) and consequently postsynaptic NMDA receptor responses. Nrxn1 was reported to be subject to a delayed-onset shift in SS4 splicing resulting in increased exon 22 inclusion, involving epigenetic mechanisms which, if disrupted, reduce memory stability. Exon inclusion at SS4 represented one of hundreds of exons reported to be subject to a genome-wide shift in fractional exon inclusion following membrane depolarization with high extracellular K that was delayed in onset. We report that high K does not increase the SS4/SS4 ratio in cortical neurons, but does induce a delayed-onset NMDA receptor-dependent neuronal death. In mixed neuronal/astrocyte cultures this neuronal death results in an increase in the astrocyte: neuron ratio, and a misleading increase in SS4/SS4 ratio attributable to astrocytes having a far higher SS4/SS4 ratio than neurons, rather than any change in the neurons themselves. We reassessed the previously reported genome-wide delayed-onset shift in fractional exon inclusion after high K exposure. This revealed that the reported changes correlated strongly with differences in exon inclusion level between astrocytes and neurons, and was accompanied by a strong decrease in the ratio of neuron-specific: astrocyte-specific gene expression. As such, these changes can be explained by the neurotoxic nature of the stimulation paradigm, underlining the importance of NMDA receptor blockade when using high K depolarizing stimuli.
单个基因可通过外显子的差异剪接编码多种蛋白质。在神经元中，这种选择性剪接形式可由活性依赖的钙信号传导控制，导致蛋白质特性发生改变，包括离子通道、神经递质受体和突触细胞粘附分子。突触前细胞粘附分子Neurexin 1（）在剪接位点4（SS4）处进行选择性剪接，该位点控制外显子22的包含（SS4），从而影响突触后NMDA受体反应。据报道，Nrxn1在SS4剪接中存在延迟发生的转变，导致外显子22的包含增加，这涉及表观遗传机制，若该机制被破坏，会降低记忆稳定性。SS4处的外显子包含是据报道在高细胞外钾膜去极化后分数外显子包含在全基因组范围内发生转变的数百个外显子之一，且这种转变延迟发生。我们报告称，高钾并不会增加皮质神经元中的SS4/SS4比率，但确实会诱导延迟发生的NMDA受体依赖性神经元死亡。在混合神经元/星形胶质细胞培养物中，这种神经元死亡会导致星形胶质细胞与神经元的比率增加，以及SS4/SS4比率出现误导性增加，这是因为星形胶质细胞的SS4/SS4比率远高于神经元，而不是神经元自身发生了任何变化。我们重新评估了先前报道的高钾暴露后全基因组范围内分数外显子包含的延迟发生转变。这表明所报道的变化与星形胶质细胞和神经元之间外显子包含水平的差异密切相关，并且伴随着神经元特异性与星形胶质细胞特异性基因表达比率的大幅下降。因此，这些变化可以通过刺激模式的神经毒性本质来解释，这凸显了在使用高钾去极化刺激时阻断NMDA受体的重要性。
