Abstract:
(1) Background: Recently, we showed aberrant nuclear/cytoplasmic boundaries/activity-dependent neuroprotective protein (ADNP) distribution in ADNP-mutated cells. This malformation was corrected upon neuronal differentiation by the ADNP-derived fragment drug candidate NAP (davunetide). Here, we investigated the mechanism of NAP nuclear protection. (2) Methods: CRISPR/Cas9 DNA-editing established N1E-115 neuroblastoma cell lines that express two different green fluorescent proteins (GFPs)-labeled mutated ADNP variants (p.Tyr718* and p.Ser403*). Cells were exposed to NAP conjugated to Cy5, followed by live imaging. Cells were further characterized using quantitative morphology/immunocytochemistry/RNA and protein quantifications. (3) Results: NAP rapidly distributed in the cytoplasm and was also seen in the nucleus. Furthermore, reduced microtubule content was observed in the ADNP-mutated cell lines. In parallel, disrupting microtubules by zinc or nocodazole intoxication mimicked ADNP mutation phenotypes and resulted in aberrant nuclear-cytoplasmic boundaries, which were rapidly corrected by NAP treatment. No NAP effects were noted on ADNP levels. Ketamine, used as a control, was ineffective, but both NAP and ketamine exhibited direct interactions with ADNP, as observed via in silico docking. (4) Conclusions: Through a microtubule-linked mechanism, NAP rapidly localized to the cytoplasmic and nuclear compartments, ameliorating mutated ADNP-related deficiencies. These novel findings explain previously published gene expression results and broaden NAP (davunetide) utilization in research and clinical development.
摘要:
(1)背景:最近,我们显示核/细胞质边界/活性依赖性神经保护蛋白(ADNP)在ADNP突变细胞中的异常分布。这种畸形在神经元分化后被ADNP衍生的片段药物候选物NAP(davunetide)纠正。这里,我们研究了NAP核保护的机制。(2)方法:CRISPR/Cas9DNA编辑建立的N1E-115神经母细胞瘤细胞系表达两种不同的绿色荧光蛋白(GFP)标记的突变ADNP变体(p。Tyr718*和p.Ser403*)。将细胞暴露于与Cy5缀合的NAP,随后进行活成像。使用定量形态学/免疫细胞化学/RNA和蛋白质定量进一步表征细胞。(3)结果:NAP在细胞质中迅速分布,在细胞核中也可见。此外,在ADNP突变的细胞系中观察到微管含量降低。并行,通过锌或诺考达唑中毒破坏微管模拟ADNP突变表型,并导致异常的核-细胞质边界,通过NAP治疗迅速纠正。没有注意到对ADNP水平的NAP效应。氯胺酮,用作控件,是无效的,但是NAP和氯胺酮都表现出与ADNP的直接相互作用,如通过硅对接所观察到的。(4)结论:通过微管连接机制,NAP迅速定位到细胞质和细胞核区室,改善突变的ADNP相关缺陷。这些新发现解释了先前发表的基因表达结果,并扩大了NAP(davunetide)在研究和临床开发中的利用。