DYRK1A磷酸化MEF2D的分子机制及其对神经细胞生理功能的影响

Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) has been shown to be involved in learning and memory impairments in Alzheimer's disease; however, the function and regulatory mechanism of DYRK1A in nervous system remain unclear. Our recent study showed DYRK1A can be regulated by REST in a negative feedback loop. Myocyte enhancer factor 2 (MEF2D) plays vital roles in neuronal survival and spine remodeling. Studies have shown that the dynamic phosphorylation of MEF2D regulated its protein stability and transcriptional activity. As both DYRK1A and MEF2D were highly expressed in the brain, we hypothesized that there is a close crosstalk between DYRK1A and MEF2D. Our preliminary studies have shown that MEF2D activated DYRK1A gene transcription through a MEF2 responsive element in -262-248bp of DYRK1A promoter. Our results also showed that DYRK1A and MEF2D were co-localized in nucleus and coordinately expressed in the mice brain during development. DYRK1A increased both endogenous and exogenous MEF2D protein levels through its phosphorylation of MEF2D. Specifically we propose: 1. To further study the molecular mechanism underlying the increasing effect of DYRK1A on MEF2D expression. i）To study the protein-protein interactions of DYRK1A and MEF2D. Co-IP assay will be applied to study the interactions of DYRK1A and MEF2D as well as its mutants. Con-focal microscopy will be used to examine their co-localizations. ii）To examine how phosphorylation by DYRK1A interacts with MEF2D protein degradation pathways. Cycloheximide-pulse chase will be applied to investigate if DYRK1A phosphorylation will interrupt MEF2D degradation pathways. Specific inhibitors such as Z-DEVD-FMK, lactacystin and chloroquine will be used to block three degradation pathways respectively, thereafter, effect of DYRK1A on MEF2D will be examined. 2. To investigate the physiological effects of MEF2D phosphorylation by DYRK1A. i) To investigate the effect on MEF2D transcriptional activity. Reporter construct pMREluc and EMSA will be used to inspect the transcriptional activity of MEF2D with DYRK1A overexpression or knockdown. RT-PCR of MEF2D target genes such as Nur77 will also be used to reflect MEF2D activity. ii) To investigate the effect on neuronal viability. Since MEF2D is a major neuronal survival factor, DYRK1A may affect neuronal viability via its regulation of MEF2D. MTT, TUNEL and caspase activation status will be measured to examine neuronal viability. iii) To investigate if the effect on spine remodeling. Primary neurons will be cultured and transfected with DYRK1A and MEF2D. The synapses will be stained with Synapsin-1 and PSD-95. mEPSCs will be recorded to reflect the synapse activity. As MEF2D is a critical factor for neuronal viability and spine remodeling, regulation of MEF2D by DYRK1A further suggested involvement of DYRK1A in AD pathogenesis.

研究显示双特异性酪氨酸磷酸化调节激酶1A（DYRK1A）参与了老年痴呆症的记忆与学习障碍，但DYRK1A在神经系统中的功能及调节机制仍然未知。我们最近的文章显示DYRK1A可以被REST调控，而且DYRK1A失衡降低了REST蛋白水平。肌细胞增强因子2D（MEF2D）在神经细胞的分化存活中有重要功能。由于DYRK1A和MEF2D 在神经组织中都高度表达且都参与了神经发育过程，我们假设二者存在相互作用。我们的预实验显示MEF2D可以激活DYRK1A的基因转录，DYRK1A可以在S180和S231位点磷酸化MEF2D并显著升高MEF2D蛋白水平。在此我们将继续研究DYRK1A对MEF2D磷酸化的分子机制，研究DYRK1A磷酸化MEF2D对其生理功能，包括转录活性、神经元存活率以及神经突触可塑性的影响。DYRK1A对MEF2D的功能调节进一步揭示了DYRK1A在AD发病中的作用。

双特异性酪氨酸磷酸化调节激酶1A（DYRK1A）位于21号染色体唐氏综合征关键区域（DSCR，Down syndrome critical region）。DYRK1A与唐氏征（DS）及阿尔兹海默氏症（AD）的认知障碍密切相关，对细胞的增殖和脑部发育具有重要作用，因此DYRK1A是研究该类疾病重要的候选基因。MEF2D属于肌细胞特异性增强因子2转录因子家族成员，研究发现其参与调控神经细胞分化及发育。我们的研究首次证实，DYRK1A异构体5启动子区-254bp到-268bp区域存在一个有效的MEF2D反应元件，MEF2D可以结合在该反应元件，增强DYRK1A的启动子活性，上调DYRK1A的基因表达，并进一步增加DYRK1A的激酶活性。研究还发现，DYRK1A与MEF2D在小鼠脑部发育过程中的表达呈正相关性，提示两者在脑部发育中可能存在协同作用。因此，我们的研究结果为进一步研究DYRK1A与MEF2D在神经发育中的作用提供理论支持。