Effect of estrogen on autophagic response after stroke in mice

국문 초록 (Abstract)

폐경이 지난 여성에서 뇌졸중 발생률이 크게 증가하게 된다. 폐경이 지난 여성의 체내에는 에스트로겐이 부족하고 이것이 여성의 뇌졸중 발생률, 사망률, 재발률, 부작용의 발생 증가로 이어지게 된다. 뇌졸중 발생 시 뉴런에서는 다양한 작용이 중 하나인 autophagy가 일어나고 뇌졸중과 같은 스트레스에서 다양한 정도로 활성화되어 신경세포를 보호하는 역할을 한다. 따라서 본 연구에서는 여성에서 높게 발병되는 뇌졸중의 원인을 autophagy 연구를 통해 규명하고자 한다.
뇌졸중 후 에스트로겐(E2)에 의한 autophagy 반응을 작용을 연구하기 위해, aged and OVX mouse에 tMCAO를 수행하였다. 뇌경색의 개선 여부 및 뇌 조직 Western blot과 Immunofluorescence, qPCR를 통해 autophagy marker들의(LC3B, p62, LAMP1, CTSD, Transcription factor EB (TFEB), me-TFEB)의 단백질 및 mRNA 활성을 확인했다.
본 실험 결과를 통해서 E2 level 및 뇌졸중의 중증도에 따라 autophagy 반응이 다르게 나타나는 것을 알 수 있었다. 우선, 성별에 따른 뇌경색을 비교하면, female의 뇌경색 볼륨이 male보다 작았다. 뇌졸중의 중증도에 따라 비교하면, mild condition에서는 E2의 뇌경색의 개선된 것을 볼 수 있었던 반면, severe condition에서는 E2가 뇌경색이 악화되었다. young(3M) mice에서는 E2가 autophagy를 억제하는 반면, 체내에 E2 level이 적다고 생각되는 Aged (1Y) mice에서는 E2가 autophagy를 유도하는 것을 확인했다. 이것을 조직면역학적 관점으로 확인했을 때, LC3B와 LAMP1의 colocalization은 young female에서 E2 처리 시 감소하는 반면, aged female에서는 stroke 후 감소한 colocalization이 E2 처리 후 증가하는 경향을 발견했다.
여러 autophagy marker들 중 autophagy master regulator인 TFEB을 중점적으로 보았고, female에서 나이가 들수록 뇌에서 TFEB mRNA 발현이 감소했다. 또한 N2a cell의 hypoxia 그룹에서 E2 처리 시 TFEB의 mRNA 발현이 증가했다. Young female에서 TFEB 및 me-TFEB의 레벨을 확인한 결과 mild condition과 severe conition 모두에서 E2 처리 시 me-TFEB의 발현량이 증가했다. 하지만 Aged tMCAO mice에서는 TFEB과 me-TFEB의 발현 차이가 없었다. me-TFEB의 발현량을 성별에 따라 보았을 때, 조직학적으로 발현량이 다르며, striatum에서 female의 TFEB 발현량이 male보다 더 높았다.
E2와 TFEB의 interaction을 밝히기 위해 estrogen receptor(ERs)와 TFEB을 이용해 진행한 ICC 및 PLA에서 다수의 colocalization 및 red signal을 확인할 수 있었다. 이는 ER과 TFEB이 같은 프로모터에 위치해 interaction을 함을 시사한다.
E2에 의해 영향받는 자가포식 단계를 확인하기 위해 chloroquine(CQ)을 신경세포에 처리하였다. E2 처리 시 cell viability가 증가하고 immunoblot에서 E2와 CQ가 처리된 group에 LC3B가 증가한 것으로 보아 E2가 stroke 시 brain 자가포식을 촉진하고 E2가 자가포식 upstream 조절자임을 시사합니다.
따라서 E2 및 중증에 따른 TFEB의 증감으로 autophagy가 조절되고 그로 인해 뇌졸중에서의 E2에 의한 autophagy 반응을 정확히 규명하는 증거를 제공하고자 한다.

다국어 초록 (Multilingual Abstract)

The incidence of stroke is significantly higher in postmenopausal women. Decreased estrogen levels in postmenopausal women leads to increased risk of stroke and mortality, stroke recurrence, and stroke-related complications. Autophagy is a function of neurons that protects the nerve cells. Autophagic response is effected by neurons to varying extent in stress conditions such as stroke. Therefore, this study aims to identify the etiology of stroke, which has a high incidence in women, by assessing autophagic response.
To investigate autophagic response to estrogen (E2) treatment after stroke, tMCAO was performed on aged and OVX mice. Resolution of brain infarction and the protein and mRNA activities of autophagy markers (LC3, p62, LAMP1, CTSD, transcription factor EB (TFEB), and methylated-TFEB) were assessed through Western blot, IHC staining, and qPCR of brain tissue.
The results of this experiment show that autophagic response varies by E2 level and the severity of stroke. First, when comparing cerebral infarction by sex, females had lower infarct volumes. Further comparison by severity of stroke revealed that, in mild stroke conditions, E2 treatment resolves cerebral infarction, whereas in severe stroke conditions, E2 treatment exacerbated cerebral infarction. Our results confirm that E2 inhibits autophagy in young (3 months old) mice but promotes autophagy in aged (1 year old) mice. This difference is likely attributable to varying E2 levels in the body between these age groups. Immunohistochemistry analysis confirmed that the colocalization of LC3B and LAMP1 decreased during E2 treatment in young females, whereas the decrease in colocalization after stroke tended to increase with E2 treatment in aged females.
Among several autophagy markers, we focused on TFEB, an autophagy master regulator, and found that TFEB mRNA expression in the brain decreases with age in females. Furthermore, TFEB mRNA expression increased upon E2 treatment in the hypoxia group of N2a cells. On investigating TFEB and methylated-TFEB levels in young female mice, we observed an increase in methylated-TFEB levels with E2 treatment in both mild and severe stroke conditions. However, TFEB and methylated-TFEB expression did not differ in aged tMCAO mice. Analysis of methylated-TFEB expression by sex, showed histochemical differences, and TFEB expression was higher in females than in males in the striatum.
To investigate the interaction between E2 and TFEB, numerous colocalizations and red PLA signals were measured in ICC, and PLA was performed using estrogen receptors (ERs) and TFEB. Results show that ER and TFEB are located in the same promoter region and interact.
To determine which stage of autophagy is affected by E2, neurons were treated with chloroquine (CQ). Cell viability increased with E2 treatment and LC3B levels increased in the E2 and CQ-treated group in immunoblot analyses. These findings suggest that E2 promotes autophagy in the brain after stroke and acts as an upstream regulator of autophagy.
We aim to provide evidence that autophagy is regulated by increases or decreases in TFEB expression based on E2 level and stroke severity, which aids in better understanding of the autophagic response mediated by E2 in stroke.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION = 1
• Ⅱ. MATERIALS AND METHODS = 8
• 2.1. Animals = 8
• 2.2 Cell culture = 8
• 2.3. Transient middle cerebral artery occlusion ischemic stroke model (tMCAO) = 9
• 2.4. Ovariectomized mouse model (OVX) = 10
• 2.5. Oxygen-glucose deprivation (OGD) = 11
• 2.6. Drug administration = 11
• 2.7. Neurological severity score (NSS) = 13
• 2.8. Measurement of infarct volume by cresyl violet and 2,3,5-Triphenyltetrazolium chloride (TTC) staining = 15
• 2.9. Cell viability assay = 16
• 2.10. Immunofluorescense = 16
• 2.11. Proximity ligation assay (PLA) = 17
• 2.12. RNA extraction and Quantitative polymerase chain reaction (qPCR) = 18
• 2.13. Western Blot Analysis = 20
• 2.14. Statistical analysis = 21
• Ⅲ. RESULTS = 22
• 3.1. Sex showed different infarction after tMCAO in mice = 22
• 3.2. Aging or severity of ischemic damage gave different infarction after tMCAO in mice = 26
• 3.3. Sex differences showed different autophagic response = 28
• 3.4. Different E2 level by OVX or aging showed different autophagic response = 31
• 3.5. TFEB mRNA expressions were changed depending on sex and aging = 38
• 3.6. Protein levels of TFEB and its modification in male and female after tMCAO = 40
• 3.7. CARM1 increased in the nucleus after tMCAO = 51
• 3.8. Interaction between TFEB and E2 was found out colocalization and proximity in vivo and in vitro = 53
• 3.9. Role of E2 in stage of autophagic responses in vitro = 55
• Ⅳ. DISCUSSION = 59
• Ⅴ. REFERENCE = 64