성인뇌 및 배아암종세포의 초기분화단계에서 만성적 알코올 노출에 의해 유도된 유전자의 특성

Abstract

에탄올은 전형적인 기형 유발 물질로 발달장애, 기형 및 신경장애 등의 다양한 문제를 야기한다. 임신 중 과잉 알코올 섭취는 태아에 다양한 기형과 장애를 야기하는데 이를 통틀어 태아 알코올 스펙트럼 장애라 한다. 태아 알코올 증후군으로 알려진 이 장애는 안면 기형, 중추신경계 발달 장애 (주로 소뇌증), 저신장, 소안구증, 심장 장애 그리고 행동 및 정신장애 등의 증상을 포함하고 있다. 본 학위논문은 만성 알코올 섭취가 초기 배아 분화 때 유전자 발현에 주는 신경기형유발 역할(neuroteratogenic role) 을 다루고 있다. 또한 알코올 섭취와 관련된 뇌 부분의 약리유전체적 변형을 통한 날트렉손의 메커니즘을 규명하고자 하였다. 첫번째 연구에서, 임신 초기 단계 (pre-gestational stage)와 흡사한 배아 분화 초기 단계에서의 알코올 장기 노출의 역할을 조사하였다. 결과적으로 알코올이 세포 형태, 세포 주기 진행에 영향을 주었고 배아 암세포가 배상체 (embryoid body)로 분화될 때 필요한 줄기 마커 (stemness markers)의 저해를 또한 늦추게 한다. 유전자 발현 프로파일링과 신호전달 통로 조사를 통해 에탄올은 초기 배발생에 관여하는 유전자와 신호전달 경로를 저해하고 있다. 후속연구에서 배상체에 에탄올을 처리하면 형성체 특이 유전자인 goosecoid (GSC)의 발현을 야기시키는 것이 밝혀졌다. GSC 는 Nodal 신호전달체계의 타깃 유전자로, 이 전달체계를 통해 배아 분화 초기 단계 내 에탄올에 의한 GSC 변화에 영향을 줄 수 있다. 추가로 우리는 에탄올이 Nodal 신호전달체계에 관련된 다양한 전사 조절 인자의 상향조절을 야기한다는 것을 알아냈다. 이러한 결과들을 볼 때, 에탄올에 의한 GSC 발현 증가는 Nodal 신호체계에 의해 조절되며 이는 FASD 환자 내 에탄올의 최기성의 분자적 메커니즘 분석에 새로운 활로를 제공한다. 또한 에탄올 처리로 인해 배아 구조 형성, 신경체계 발달, 그리고 태반 및 배아 혈관 신생에 관련된 몇몇 유전자를 변하게 되는데, 이들 모두가 공통적으로 FASD 에 의해 지장을 받는다는 결과를 보고한 바가 있다. 특히 배상체에 에탄올이 처리되면 ALDOC, ENO2, 그리고 CDH1 의 발현이 저해되었고, 이에 비해 PTCH1, EGLN1, VEGFA 및 DEC2 의 발현은 증가했다. 또한 현 연구를 통해 폴산이 에탄올 노출에 의한 중요 유전자 발현 변화를 대다수 원상태로 복구할 수 있음을 밝혔다. 두 번째 연구에서, 우리는 만성 에탄올 노출이 인간 배아 암세포 내 초기 신경 분화 때의 유전자 발현에 미치는 효과를 분석하였다. NCCIT 세포는 레티노산에 의한 확정 부착 배양액에서 에탄올과 폴산을 처리한 상태 또는 처리하지 않은 상태에서 신경 전구체/계통으로 분화되었다. 분화 초기에 일어나는 유전자 발현 프로파일은 상보적 DNA 마이크로어레이로 분석하였다. 타깃 유전자 간의 직접적인 관계성을 찾기 위해 통합 경로 분석을 수행하였다. 위 실험을 통해 에탄올이 초기 뇌 발달에 관여하는 유전자와 신호전달체계의 조절을 방해하는 것을 볼 수 있었다. 유효성 확인 연구를 통해 에탄올에 노출되면 중추신경계 발달, 시냅스 형성 및 전달, 신경전달물질 수용체 활성과 관련된 PCDHB14, GABRB1, CTNND2, NAV3, RALDH1, 그리고 OPN5 과 같은 중요 유전자의 발현이 억제되는 것을 확인할 수 있었다. 추가로, 초기 신경 분화 때 CHRNB4 의 발현이 에탄올 처리 후 증가했음을 보고한 바 있다. GeneGo 경로 분석으로 확인했을 때 에탄올에 의해 조절이 변화하는 유전자들은 FASD 와 관련된 신경증, 뇌전증 및 알코올 관련 장애와 같은 질병 경로에 위치하는 것으로 밝혀졌다. 이를 볼 때 초기 두뇌 발달 때 에탄올에 노출되면 신경 최기성 반응이 일어남을 알 수 있다. 이번 발견을 통해 보면, 에탄올에 의한 신경계 장애 또는 불충분한 시냅스 형성이 FASD 환자에게 일어나는 신경발달 장애의 근본적인 원인으로써 작용한다고 생각한다. 세번째 연구는 알코올 의존 환자의 치료를 위한 (in order to cure alcohol dependent patients) 날트렉손의 작용원리를 밝히기 위해 알코올 섭취에 관련된 뇌 영역의 약리유전체적 변형을 조사하였다. 에탄올 섭취 후 날트렉손을 처리한 C57BL/6 쥐의 해마와 전전두엽에서 일어나는 유전자 발현 프로파일은 상보적 DNA 마이크로어레이로 분석하였다. 에탄올을 섭취한 쥐에게 4 주 동안 날트렉손 200 μl (16 mg/kg)을 복강 투여하였을 때 해마 (394 개) 와 전전두엽 (566 개) 내 상당 수의 유전자 발현에 변화가 일어났다. Ingenuity Pathway Analysis (IPA) 을 사용하여 전전두엽과 해마에서 날트렉손 처리 후 변화가 있었던 후보 유전자 집합 내 생물경로 및 상호관계를 분석하였다. 우리는 세포 형태, 사멸, 신경계 발달 및 기능, 그리고 신경계 질환과 관련된 유전자 네트워크들을 발견했다. 확인 실험에서는 전전두엽 내 트렌스티레틴 (transthyretin; Ttr) 과 단백질인산화 효소 Cγ (protein kinase C [PKC] γ)의 발현이 증가했지만 해마에서는 증가하지 않는 것을 밝혀냈다. 끝으로 현 연구는 에탄올에 노출된 쥐의 뇌 내 날트렉손의 약리유전체적 반응을 보여주며 이는 알코올 관련 질병의 근본원인 및 가능성 있는 치료법에 대한 이해를 도울 수 있을 것이다. 요컨대, 이 학위논문에 나온 연구는 배아 발달 초기에서의 에탄올에 의한 신경기형유발에 대한 분자적 메커니즘의 새로운 시각을 제시하며, 에탄올을 섭취한 쥐의 특정 뇌 영역 내 날트렉손의 메커니즘을 보여주고 있다.|Ethanol is a classical teratogen that is capable of inducing a wide range of developmental, morphological and neurological defects. Excess ethanol consumption during pregnancy can lead to a spectrum of deficiencies and abnormalities in the developing fetus, collectively known as fetal alcohol spectrum disorder (FASD). The strongest manifestation of FASD, also known as fetal alcohol syndrome (FAS), can include: craniofacial malformations, abnormal central nervous system development (typically microencephaly), short stature, icrophthalmia, heart defects, and behavioral and psychological problems. In this dissertation, I investigated the neuroteratogenic role that chronic ethanol exposure plays in gene expression changes during early embryonic differentiation. Additionally, I examined the underlying mechanisms of naltrexone by investigating the pharmacogenomic variations in the brain regions associated with alcohol consumption. Firstly, I examined the role of chronic ethanol exposure during early stage of embryonic differentiation (resembling pre-gestational stage). I demonstrated that ethanol affected the cell morphology, cell cycle progression and also delayed the down-regulation of stemness markers during differentiation of embryonic carcinoma (EC) cells to embryoid body (EB). Gene expression profiling and pathway analysis demonstrated that ethanol deregulates many genes and pathways that are involved in early embryogenesis. Follow-up analyses revealed that ethanol exposure to EBs induced the expression of an organizer specific gene, goosecoid (GSC) in comparison to controls. GSC is a target gene of Nodal signaling pathway, which can mediate the ethanol induced alteration of GSC during early stage of embryonic differentiation. In further analysis, I demonstrated that ethanol induces the up-regulation of different transcriptional regulators involved in Nodal signaling pathway. I also confirmed the involvement of Nodal signaling in the regulation of ethanol induced-GSC by blocking of nodal like signals. In addition, I presented a list of possible target genes to which ethanol-induced GSC may function and causes embryo malformation. Together, these results suggest that ethanol-induced GSC expression is mediated by Nodal signaling, which may provide a new avenue for analyzing the molecular mechanisms behind EtOH teratogenicity in FASD individuals. Moreover, I reported that ethanol treatment altered several important genes that are involved in embryonic structure formation, nervous system development, and placental and embryonic vascularization, which are all common processes that FASD can disrupt. Specifically, ethanol treatment let to a reduction in ALDOC, ENO2, and CDH1 expression, whereas EtOH treatment induced the expression of PTCH1, EGLN1, VEGFA, and DEC2 in treated EBs. The present study also demonstrated that folic acid (FA) treatment was able to correct the expression of the majority of genes deregulated by ethanol exposure during early embryo development. In second study, I examined the effects of chronic ethanol exposure on gene expression during early neuronal differentiation of human EC cells. NCCIT cells were differentiated into neuronal precursors/lineages in defined adherent culture by retinoic acid (RA) in the presence or absence of ethanol and folic acid. A complementary DNA microarray analysis was used to profile gene expression changes during early neuronal differentiation. Integrated pathway enrichment analysis was performed to find the direct relationships between genes of interest. Gene expression profiling and pathway analysis demonstrated that ethanol deregulates many genes and pathways that are involved in early brain development. Validation studies revealed that ethanol exposure down-regulated several important genes, such as PCDHB14, GABRB1, CTNND2, NAV3, RALDH1, and OPN5, which are involved in central nervous systems (CNS) development, synapse assembly, synaptic transmission, and neurotransmitter receptor activity. Moreover, I reported that ethanol treatment increased the expression of CHRNB4 during early neuronal differentiation. GeneGo pathway analysis revealed that the ethanol-deregulated genes mapped to disease pathways that were relevant to FASD (such as neurotic disorders, epilepsy, and alcohol-related disorders). Together these results indicated a neuroteratological response to ethanol exposure during early brain development. These findings suggest that the impairment of the neurological system or suboptimal synapse formation resulting from ethanol exposure could underlie the eurodevelopmental disorders in individuals with FASD. Finally, in order to cure alcohol dependent patients, I examined the underlying mechanisms of naltrexone by investigating the pharmacogenomic variations in the brain regions associated with alcohol consumption. A complementary DNA microarray analysis was used to profile gene expression changes in the hippocampus and prefrontal cortex (PFC) of C57BL/6 mice injected with naltrexone following ethanol treatment. Intraperitoneal administration of 200 μl (16 mg/kg) of naltrexone for four weeks caused alterations in the expression of a wide range of hippocampal (394) and PFC (566) genes in ethanol-treated mice. Ingenuity pathway analysis (IPA) software was used to search for biological pathways and interrelationships between gene networks in the subsets of candidate genes that were altered in the naltrexone-treated PFC and ippocampus. I found gene networks associated with cell morphology, cell death, nervous system development and function, and neurological disease. Confirmation studies revealed that the expression of transthyretin (Ttr) and protein kinase C (PKC) γ were increased in the PFC but not in the hippocampus of naltrexone-treated mice. Finally, the present study demonstrates a pharmacogenomic response to naltrexone in the brains of ethanol-consuming mice, and may enhance our understanding of the underlying causes and possible treatments of alcohol use disorders. In summary, the works presented in this dissertation provides a new insight into the molecular mechanisms of ethanol induced neuroteratogenicity during early embryonic development, and also presents a protected mechanism of naltrexone in the specific brain regions of ethanol-consuming mice.; Ethanol is a classical teratogen that is capable of inducing a wide range of developmental, morphological and neurological defects. Excess ethanol consumption during pregnancy can lead to a spectrum of deficiencies and abnormalities in the developing fetus, collectively known as fetal alcohol spectrum disorder (FASD). The strongest manifestation of FASD, also known as fetal alcohol syndrome (FAS), can include: craniofacial malformations, abnormal central nervous system development (typically microencephaly), short stature, icrophthalmia, heart defects, and behavioral and psychological problems. In this dissertation, I investigated the neuroteratogenic role that chronic ethanol exposure plays in gene expression changes during early embryonic differentiation. Additionally, I examined the underlying mechanisms of naltrexone by investigating the pharmacogenomic variations in the brain regions associated with alcohol consumption. Firstly, I examined the role of chronic ethanol exposure during early stage of embryonic differentiation (resembling pre-gestational stage). I demonstrated that ethanol affected the cell morphology, cell cycle progression and also delayed the down-regulation of stemness markers during differentiation of embryonic carcinoma (EC) cells to embryoid body (EB). Gene expression profiling and pathway analysis demonstrated that ethanol deregulates many genes and pathways that are involved in early embryogenesis. Follow-up analyses revealed that ethanol exposure to EBs induced the expression of an organizer specific gene, goosecoid (GSC) in comparison to controls. GSC is a target gene of Nodal signaling pathway, which can mediate the ethanol induced alteration of GSC during early stage of embryonic differentiation. In further analysis, I demonstrated that ethanol induces the up-regulation of different transcriptional regulators involved in Nodal signaling pathway. I also confirmed the involvement of Nodal signaling in the regulation of ethanol induced-GSC by blocking of nodal like signals. In addition, I presented a list of possible target genes to which ethanol-induced GSC may function and causes embryo malformation. Together, these results suggest that ethanol-induced GSC expression is mediated by Nodal signaling, which may provide a new avenue for analyzing the molecular mechanisms behind EtOH teratogenicity in FASD individuals. Moreover, I reported that ethanol treatment altered several important genes that are involved in embryonic structure formation, nervous system development, and placental and embryonic vascularization, which are all common processes that FASD can disrupt. Specifically, ethanol treatment let to a reduction in ALDOC, ENO2, and CDH1 expression, whereas EtOH treatment induced the expression of PTCH1, EGLN1, VEGFA, and DEC2 in treated EBs. The present study also demonstrated that folic acid (FA) treatment was able to correct the expression of the majority of genes deregulated by ethanol exposure during early embryo development. In second study, I examined the effects of chronic ethanol exposure on gene expression during early neuronal differentiation of human EC cells. NCCIT cells were differentiated into neuronal precursors/lineages in defined adherent culture by retinoic acid (RA) in the presence or absence of ethanol and folic acid. A complementary DNA microarray analysis was used to profile gene expression changes during early neuronal differentiation. Integrated pathway enrichment analysis was performed to find the direct relationships between genes of interest. Gene expression profiling and pathway analysis demonstrated that ethanol deregulates many genes and pathways that are involved in early brain development. Validation studies revealed that ethanol exposure down-regulated several important genes, such as PCDHB14, GABRB1, CTNND2, NAV3, RALDH1, and OPN5, which are involved in central nervous systems (CNS) development, synapse assembly, synaptic transmission, and neurotransmitter receptor activity. Moreover, I reported that ethanol treatment increased the expression of CHRNB4 during early neuronal differentiation. GeneGo pathway analysis revealed that the ethanol-deregulated genes mapped to disease pathways that were relevant to FASD (such as neurotic disorders, epilepsy, and alcohol-related disorders). Together these results indicated a neuroteratological response to ethanol exposure during early brain development. These findings suggest that the impairment of the neurological system or suboptimal synapse formation resulting from ethanol exposure could underlie the eurodevelopmental disorders in individuals with FASD. Finally, in order to cure alcohol dependent patients, I examined the underlying mechanisms of naltrexone by investigating the pharmacogenomic variations in the brain regions associated with alcohol consumption. A complementary DNA microarray analysis was used to profile gene expression changes in the hippocampus and prefrontal cortex (PFC) of C57BL/6 mice injected with naltrexone following ethanol treatment. Intraperitoneal administration of 200 μl (16 mg/kg) of naltrexone for four weeks caused alterations in the expression of a wide range of hippocampal (394) and PFC (566) genes in ethanol-treated mice. Ingenuity pathway analysis (IPA) software was used to search for biological pathways and interrelationships between gene networks in the subsets of candidate genes that were altered in the naltrexone-treated PFC and ippocampus. I found gene networks associated with cell morphology, cell death, nervous system development and function, and neurological disease. Confirmation studies revealed that the expression of transthyretin (Ttr) and protein kinase C (PKC) γ were increased in the PFC but not in the hippocampus of naltrexone-treated mice. Finally, the present study demonstrates a pharmacogenomic response to naltrexone in the brains of ethanol-consuming mice, and may enhance our understanding of the underlying causes and possible treatments of alcohol use disorders. In summary, the works presented in this dissertation provides a new insight into the molecular mechanisms of ethanol induced neuroteratogenicity during early embryonic development, and also presents a protected mechanism of naltrexone in the specific brain regions of ethanol-consuming mice.