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Abstract：
BACKGROUND: Helsmoortel-Van der Aa syndrome is a neurodevelopmental disorder in which patients present with autism, intellectual disability, and frequent extra-neurological features such as feeding and gastrointestinal problems, visual impairments, and cardiac abnormalities. All patients exhibit heterozygous de novo nonsense or frameshift stop mutations in the Activity-Dependent Neuroprotective Protein (ADNP) gene, accounting for a prevalence of 0.2% of all autism cases worldwide. ADNP fulfills an essential chromatin remodeling function during brain development. In this study, we investigated the cerebellum of a died 6-year-old male patient with the c.1676dupA/p.His559Glnfs*3 ADNP mutation.
RESULTS: The clinical presentation of the patient was representative of the Helsmoortel-Van der Aa syndrome. During his lifespan, he underwent two liver transplantations after which the child died because of multiple organ failure. An autopsy was performed, and various tissue samples were taken for further analysis. We performed a molecular characterization of the cerebellum, a brain region involved in motor coordination, known for its highest ADNP expression and compared it to an age-matched control subject. Importantly, epigenome-wide analysis of the ADNP cerebellum identified CpG methylation differences and expression of multiple pathways causing neurodevelopmental delay. Interestingly, transcription factor motif enrichment analysis of differentially methylated genes showed that the ADNP binding motif was the most significantly enriched. RNA sequencing of the autopsy brain further identified downregulation of the WNT signaling pathway and autophagy defects as possible causes of neurodevelopmental delay. Ultimately, label-free quantification mass spectrometry identified differentially expressed proteins involved in mitochondrial stress and sirtuin signaling pathways amongst others. Protein-protein interaction analysis further revealed a network including chromatin remodelers (ADNP, SMARCC2, HDAC2 and YY1), autophagy-related proteins (LAMP1, BECN1 and LC3) as well as a key histone deacetylating enzyme SIRT1, involved in mitochondrial energy metabolism. The protein interaction of ADNP with SIRT1 was further biochemically validated through the microtubule-end binding proteins EB1/EB3 by direct co-immunoprecipitation in mouse cerebellum, suggesting important mito-epigenetic crosstalk between chromatin remodeling and mitochondrial energy metabolism linked to autophagy stress responses. This is further supported by mitochondrial activity assays and stainings in patient-derived fibroblasts which suggest mitochondrial dysfunctions in the ADNP deficient human brain.
CONCLUSIONS: This study forms the baseline clinical and molecular characterization of an ADNP autopsy cerebellum, providing novel insights in the disease mechanisms of the Helsmoortel-Van der Aa syndrome. By combining multi-omic and biochemical approaches, we identified a novel SIRT1-EB1/EB3-ADNP protein complex which may contribute to autophagic flux alterations and impaired mitochondrial metabolism in the Helsmoortel-Van der Aa syndrome and holds promise as a new therapeutic target.
RESULTS: The clinical presentation of the patient was representative of the Helsmoortel-Van der Aa syndrome. During his lifespan, he underwent two liver transplantations after which the child died because of multiple organ failure. An autopsy was performed, and various tissue samples were taken for further analysis. We performed a molecular characterization of the cerebellum, a brain region involved in motor coordination, known for its highest ADNP expression and compared it to an age-matched control subject. Importantly, epigenome-wide analysis of the ADNP cerebellum identified CpG methylation differences and expression of multiple pathways causing neurodevelopmental delay. Interestingly, transcription factor motif enrichment analysis of differentially methylated genes showed that the ADNP binding motif was the most significantly enriched. RNA sequencing of the autopsy brain further identified downregulation of the WNT signaling pathway and autophagy defects as possible causes of neurodevelopmental delay. Ultimately, label-free quantification mass spectrometry identified differentially expressed proteins involved in mitochondrial stress and sirtuin signaling pathways amongst others. Protein-protein interaction analysis further revealed a network including chromatin remodelers (ADNP, SMARCC2, HDAC2 and YY1), autophagy-related proteins (LAMP1, BECN1 and LC3) as well as a key histone deacetylating enzyme SIRT1, involved in mitochondrial energy metabolism. The protein interaction of ADNP with SIRT1 was further biochemically validated through the microtubule-end binding proteins EB1/EB3 by direct co-immunoprecipitation in mouse cerebellum, suggesting important mito-epigenetic crosstalk between chromatin remodeling and mitochondrial energy metabolism linked to autophagy stress responses. This is further supported by mitochondrial activity assays and stainings in patient-derived fibroblasts which suggest mitochondrial dysfunctions in the ADNP deficient human brain.
CONCLUSIONS: This study forms the baseline clinical and molecular characterization of an ADNP autopsy cerebellum, providing novel insights in the disease mechanisms of the Helsmoortel-Van der Aa syndrome. By combining multi-omic and biochemical approaches, we identified a novel SIRT1-EB1/EB3-ADNP protein complex which may contribute to autophagic flux alterations and impaired mitochondrial metabolism in the Helsmoortel-Van der Aa syndrome and holds promise as a new therapeutic target.
摘要：
背景：Helsmoortel-VanderAa综合征是一种神经发育障碍，智力残疾,和常见的神经外特征，如喂养和胃肠道问题，视觉障碍，和心脏异常。所有患者在活动依赖性神经保护蛋白（ADNP）基因中表现出杂合的从头无义或移码停止突变，占全球所有自闭症病例的0.2%。ADNP在大脑发育过程中具有重要的染色质重塑功能。在这项研究中,我们调查了一名死亡的6岁男性患者的小脑c.1676dupA/p。His559Glnfs*3ADNP突变。
结果：患者的临床表现为具有代表性的Helsmoortel-VanderAa综合征。在他的一生中，他接受了两次肝移植手术，之后孩子因多器官衰竭而死亡。进行了尸检，和各种组织样本进行进一步分析。我们对小脑进行了分子表征，参与运动协调的大脑区域，以其最高的ADNP表达而闻名，并将其与年龄匹配的对照受试者进行了比较。重要的是,对ADNP小脑的全基因组分析确定了CpG甲基化差异和导致神经发育延迟的多种途径的表达。有趣的是,差异甲基化基因的转录因子基序富集分析表明，ADNP结合基序富集最显著。尸检大脑的RNA测序进一步确定了WNT信号通路的下调和自噬缺陷可能是神经发育迟缓的原因。最终,无标记定量质谱鉴定了参与线粒体应激和沉默调节蛋白信号通路等的差异表达蛋白。蛋白质-蛋白质相互作用分析进一步揭示了一个包括染色质重塑剂(ADNP，SMARCC2、HDAC2和YY1），自噬相关蛋白（LAMP1，BECN1和LC3）以及参与线粒体能量代谢的关键组蛋白去乙酰化酶SIRT1。通过小鼠小脑中的直接共免疫沉淀，通过微管末端结合蛋白EB1/EB3进一步生化验证了ADNP与SIRT1的蛋白质相互作用，表明染色质重塑和线粒体能量代谢之间重要的线粒体表观遗传串扰与自噬应激反应有关。线粒体活性测定和患者来源的成纤维细胞的染色进一步支持了这一点，这表明ADNP缺陷人脑中的线粒体功能障碍。
结论：这项研究形成了ADNP尸检小脑的基线临床和分子特征，为Helsmoortel-VanderAa综合征的疾病机制提供新的见解。通过结合多维和生化方法，我们发现了一种新的SIRT1-EB1/EB3-ADNP蛋白复合物,该复合物可能导致Helsmoortel-VanderAa综合征的自噬通量改变和线粒体代谢受损,有望成为新的治疗靶点.
结果：患者的临床表现为具有代表性的Helsmoortel-VanderAa综合征。在他的一生中，他接受了两次肝移植手术，之后孩子因多器官衰竭而死亡。进行了尸检，和各种组织样本进行进一步分析。我们对小脑进行了分子表征，参与运动协调的大脑区域，以其最高的ADNP表达而闻名，并将其与年龄匹配的对照受试者进行了比较。重要的是,对ADNP小脑的全基因组分析确定了CpG甲基化差异和导致神经发育延迟的多种途径的表达。有趣的是,差异甲基化基因的转录因子基序富集分析表明，ADNP结合基序富集最显著。尸检大脑的RNA测序进一步确定了WNT信号通路的下调和自噬缺陷可能是神经发育迟缓的原因。最终,无标记定量质谱鉴定了参与线粒体应激和沉默调节蛋白信号通路等的差异表达蛋白。蛋白质-蛋白质相互作用分析进一步揭示了一个包括染色质重塑剂(ADNP，SMARCC2、HDAC2和YY1），自噬相关蛋白（LAMP1，BECN1和LC3）以及参与线粒体能量代谢的关键组蛋白去乙酰化酶SIRT1。通过小鼠小脑中的直接共免疫沉淀，通过微管末端结合蛋白EB1/EB3进一步生化验证了ADNP与SIRT1的蛋白质相互作用，表明染色质重塑和线粒体能量代谢之间重要的线粒体表观遗传串扰与自噬应激反应有关。线粒体活性测定和患者来源的成纤维细胞的染色进一步支持了这一点，这表明ADNP缺陷人脑中的线粒体功能障碍。
结论：这项研究形成了ADNP尸检小脑的基线临床和分子特征，为Helsmoortel-VanderAa综合征的疾病机制提供新的见解。通过结合多维和生化方法，我们发现了一种新的SIRT1-EB1/EB3-ADNP蛋白复合物,该复合物可能导致Helsmoortel-VanderAa综合征的自噬通量改变和线粒体代谢受损,有望成为新的治疗靶点.
