Study on the characteristics of adult neural stem cells in the subcallosal zone
뇌량하영역 성체신경줄기세포 특성 연구
- 주제(키워드) Subcallosal zone , adult neural stem cell , traumatic brain injury
- 발행기관 고려대학교 대학원
- 지도교수 선웅
- 발행년도 2013
- 학위수여년월 2013. 2
- 학위구분 박사
- 학과 일반대학원 의학과
- 세부전공 분자인체유전학전공
- 원문페이지 113 p
- 실제URI http://www.dcollection.net/handler/korea/000000039330
- 본문언어 영어
- 제출원본 000045795131
초록/요약
Neural stem cells (NSCs) produce most cells composing the nervous system during development, and remain in adulthood. Adult neurogenesis, defined as a process of generating new neurons in adult brain, occurs throughout life spatially restricted at least two defined “neurogenic” adult brain regions; the subventricular zone (SVZ) of anterior lateral ventricle and the subgranular zone (SGZ) of dentate gyrus. The existence of NSCs in the adult brain raises the possibility of repair or regeneration in pathological conditions, such as brain injury and neurodegenerative diseases. Previous reports indicate differential responses of newly produced neuroblasts depending on the brain injury site, suggesting that different ability of NSCs depending on their localization. Therefore, it is important to identify and characterize a novel neurogenic region, and examine its function and respond of aNSCs in normal or injury condition. Recently, subcallosal zone (SCZ) was newly discovered as a novel adult neurogenic region containing NSCs. Adult NSCs in the SCZ exhibit self-renewal and differentiation into multiple lineages including neurons, oligodendrocytes, and astrocytes, as other conventional neurogenic regions in vitro. Although adult NSCs in the SCZ can generate to neuroblast, newly generated neuroblasts fail to differentiate into mature neurons in vivo, and disappear by Bax-dependent cell death. In human, aNSCs exist in the subcortical white matter as a counterpart of the mouse SCZ. Because human has well-developed white matter, the SCZ may be important source for adult neurogenesis, and more knowledge about the SCZ may be beneficial for developing regeneration strategies after brain damage in human. In the present study, I characterized the molecular signatures of the SCZ aNSCs, and explored their response after traumatic bin injury (TBI). In chapter 1, I addressed the characteristics of aNSCs derived from the SCZ. To examine the capacity of proliferation and differentiation, I used SVZ aNSCs as comparative control to understand their potency. SCZ aNSCs yielded less number of neurospheres that were formed at passage 0 in vitro, but their efficiency of expansion was similar to that of SVZ-derived neurospheres. When two aNSCs were cultured for 6 days in differentiation condition, significantly less proportion of neurons was produced from the SCZ aNSCs comparing to the SVZ aNSCs. Because these difference of the neuronal differentiation capacity may be partly due to different gene expression profiles, microarray analysis were performed to compare the variation of gene expression between two populations. Gene expression signature of SCZ aNSCs were greatly similar to SVZ aNSCs with approximately 99.7% identity, and only 83 genes exhibited more than 1.5-fold difference. Among these differentially expressed genes, the expression of CRBP1 gene in SCZ aNSCs was markedly low comparing to the SVZ aNSCs. Interestingly, suppression of CRBP1 in SVZ NSCs inhibited neuronal differentiation in vivo and in vitro. Because CRBP1 is a key regulator of retinoic acid-production, effect of RA treatment on neuronal differentiation was examined. Treatment with RA consistently augmented neuronal differentiation in both SCZ and SVZ aNSCs. These results indicate that RA signaling contributes to the neuronal differentiation of aNSCs and may determine the capacity of neuronal differentiation. In chapter 2, I examined the regenerative potential of SCZ aNSCs in the brain injury model. Previously, it is known that the number of SCZ-derived neuroblasts increases in the injured brain and these newly generated neuroblasts also migrates toward injury site. Thus, I further explored their fate in vivo. Although SCZ-derived cells differentiated into astrocytes or oligodendrocytes following TBI, neuroblasts failed to mature into neurons and underwent Bax-dependent apoptosis. On the other hand, infection with anti-apoptotic gene Bcl-xL expressing retrovirus the SCZ markedly increased the survival neuroblasts. While most of survived neuroblasts failed to mature and atrophied, a subset of cells exhibited mature neuronal marker, NeuN. These results demonstrate the potential and limitation of anti-apoptotic treatment using endogenous neurogenesis for brain repair.
more목차
ABSTRACT 1
CONTENTS 4
LIST OF FIGURES 8
Background and Purpose 9
1. Neural stem cells (NSCs) in the adult brain 10
2. Adult neural stem cells in the subcallosal zone 12
2-1. Anatomy of the SCZ 13
2-2. Development of the SCZ 15
3. Purpose 26
CHAPTER 1.
The characteristics of adult neural stem cells in the subcallosal zone
ABSTRACT 29
INTRODUCTION 30
MATERIALS and METHODS 32
1. Adult neural stem cell culture 32
2. Animals and surgery 33
3. Microarray 33
4. Plasmid and retrovirus preparation 34
5. Immunostainings 35
6. Western blot 36
7. Polymerase Chain Reaction (PCR) 37
RESULTS
1. Comparison of the proliferating potential of aNSCs derived from the SCZ and SVZ 38
2. Comparison of the differentiation potential of NSCs from the SCZ and SVZ 38
3. Comparison of gene expression profiles of SCZ and SVZ NSCs 39
4. Validation of microarray with RT-PCR 40
5. CRBP1 expression in SCZ and SVZ aNSCs 40
6. Suppression of CRBP1 expression inhibits neuronal differentiation in vitro 41
7. Changes in the cell fate by suppression of CRBP1 expression in vivo 41
8. Effect of retinoic acid on the proliferation of NSCs 42
9. Influence of RA on the differentiation of NSCs 42
DISCUSSION
1. The differential gene expression in regional specificity 62
2. CRBP1-mediated RA signaling contributes to neuronal differentiation. 64
CHAPTER 2.
Effects of the overexpression of Bcl-xL on the injury-induced neurogenesis from adult subcallosal zone
ABSTRACT 68
INTRODUCTION 70
MATERIALS and METHODS 72
1. Animals and treatments 72
2. Stereotaxic surgery 72
3. Virus production 73
4. Histology 73
5. Statistical analysis 74
RESULTS
1. Changes in the number of neuroblasts in the cerebral cortex after cryogenic traumatic brain injury (cTBI) 75
2. Complete PCD of newly formed neuroblasts in the injured cerebral cortex 75
3. Overexpression of anti-apoptotic gene Bcl-xL increased the number of neuroblasts in the injured cerebral cortex 76
4. Failure of neuronal maturation of SCZ-derived neuroblasts following Bcl-xL overexpression 77
5. Atrophy of neuroblasts after Bcl-xL overexpression 78
DISCUSSION
1. Increased neurogenesis in the SCZ by traumatic brain injury 92
2. Prevention the cell death of SCZ-derived neuroblasts 94
3. Limitation of neuronal maturation 95
REFERENCES 97
국문초록 106
Acknowledgment 109
