초록 ▼

신경교세포는 뇌의 발달 과정에서 불필요한 시냅스를 청소하여 뇌의 항상성을 유지하는 핵심적인 역할을 한다. 하지만 꼭 보존 되어야할 시냅스의 포식을 억제하는 기작이 무엇인지는 아직 규명된 바가 없다. 본 연구에서는 면역 체계에서 면역억제 신호로 알려진 PD-1 과 PD-L1 이 신경교세포의 시냅스 포식을 억제하는 것을 연구하였다. PD-1 단백질이 뇌의 발달 시기에 해마에 있는 별아교세포와 미세아교세포에서 발현되는 것을 확인하였다. 그리고 PD-1 의 리간드인 PD-L1 은 뇌세포의 시냅스, 특히 흥분성과 억제성 프리시냅스와 흥분성

신경교세포는 뇌의 발달 과정에서 불필요한 시냅스를 청소하여 뇌의 항상성을 유지하는 핵심적인 역할을 한다. 하지만 꼭 보존 되어야할 시냅스의 포식을 억제하는 기작이 무엇인지는 아직 규명된 바가 없다. 본 연구에서는 면역 체계에서 면역억제 신호로 알려진 PD-1 과 PD-L1 이 신경교세포의 시냅스 포식을 억제하는 것을 연구하였다. PD-1 단백질이 뇌의 발달 시기에 해마에 있는 별아교세포와 미세아교세포에서 발현되는 것을 확인하였다. 그리고 PD-1 의 리간드인 PD-L1 은 뇌세포의 시냅스, 특히 흥분성과 억제성 프리시냅스와 흥분성 포스트시냅스에 발현되는 것을 밝혔다. PD-1 억제 쥐 모델의 뇌에서 PD-L1 단백질의 발현이 프리시냅스에서 유의미하게 증가하는 것을 관찰하였고, 이것이 신경교세포의 활성화와 연관이 없는 것을 확인하였다. 또한, 살아있는 별아교세포에서 시냅토좀 포식을 정량하여 PD-1-PD-L1 상호작용이 포식 작용을 억제하는 것을 확인하였다. 본 연구 결과를 통해 PD-1-PD-L1 신호가 별아교세포의 시냅스 포식을 억제하는 작용을 함으로써 뇌의 정상적인 발달에 중요한 역할을 한다는 것을 밝혔다.

(출처 : 요약 2p)

Abstract ▼

Introduction
The regulation of synaptic connections between neurons is critical for ensuring proper brain function. Majority of the neuronal synapse formation and elimination occur early in life, and especially during the postnatal period.
Importantly, impairment in synapse elimination during

Introduction
The regulation of synaptic connections between neurons is critical for ensuring proper brain function. Majority of the neuronal synapse formation and elimination occur early in life, and especially during the postnatal period.
Importantly, impairment in synapse elimination during this critical period is reported as the leading cause of developmental neuropsychiatric diseases, such as autism or schizophrenia (Fig 1A). In recent decades, it has been found that the glial cells in the brain play significant roles in this synapse elimination. Both astrocytes andmicroglia, which are different types of glia, display phagocytic receptors that recognize “eat-me” signals on redundant synapses and selectively engulf them (Fig 1B).While the mechanism for glia’s synapse engulfment has been extensively studied, however, the inhibitory mechanism that suppresses synapse elimination has not yet been found. Therefore, this study aims to find the immune checkpoint that can inhibit glial phagocytosis of neuronal synapses.

Mounting evidence suggests similarities between immune cells and glial cells, especially in regard to activation and phagocytosis. Studies have demonstrated that the immune molecules, CD47 and SIRPα, which are known as effective inhibitor of immune cells, has been found to act as “don’t eat me” signal by microglia.4 However, another known effective immune checkpoint, a programmed cell death protein (PD-1) has not yet been investigated in glial cells. In the immune system, PD-1 acts by inhibiting the T-cell receptor, mainly by recruiting a tyrosine phosphatase called SHP-2 to dephosphorylate T-cell receptor’s immunoreceptor tyrosine-based activation (ITAM) motif (Fig 2A). This effectively blocks downstream signaling by ZAP70, a member of Syk family kinase. In the brain, the astrocytes express Multiple EGF Like Domains 10 (MEGF10) receptor, which also contain ITAM motif that needs to be phosphorylated by Syk family to activate downstream pathway (Fig 2B). It has been demonstrated that inhibition of Syk leads to reduction in engulfment. However, the function of PD-1 in the braindevelopmental period has not yet been explored. Given the similarity of T-cell and astrocyte’s signaling pathway and the effective ability of PD-1 in suppressing the immune function, we hypothesized that PD-1 may also act as“don’t eat me” signal in the brain.

In this study, we test the hypothesis that PD-1 serves as the immune checkpoint of astrocytes and that PD-L1 acts as “don’t eat me” signal by the neuronal synapses. Using immunohistochemistry, we report for the first time that PD-1 is expressed in astrocytes and microglia, and that the expression peaks in the hippocampus during the period of robust pruning in the early brain development. On the other hand, PD-L1 was found to be expressed on both pre and post synapses of the mouse brain during postnatal period. Based on this finding, we tested whether PD-1 affects astrocyte’s phagocytic ability at the in vitro level. We found that the number of synaptosomes phagocytosed by in vitro astrocytes decreased when PD-1 and PD-L1 interaction occurs. Finally, at the in vivo level, we assessed whether PD-1 knockout (KO) will cause changes in the neuronal synapses. We found that PDL1 expression increases significantly on pre-synapses in the PD-1 KO mouse compared to wildtype (WT), but that these changes are not due to activation of glial cells, which may occur when PD-1 is absent. Taken together, these data demonstrate that PD-1 may act as “don’t eat me” signal by astrocytes and neurons to inhibit engulfment of synapses during brain developmental period.

(출처 : 본문 Introduction 3p)

목차 Contents

• COVER ... 1
• 석박사모험연구사업 최종 보고서 ... 2
• Introduction ... 3
• Results ... 5
• 1. PD-1 expression peaks in the hippocampus during early developmental period ... 5
• 2. PD-1 is expressed in microglia and astrocytes ... 7
• 3. PD-L1 is expressed on the synapses of neurons ... 9
• 4. PD-1 KO mouse have increased PD-L1 expression on pre-synapses ... 11
• 5. Glial cells were not activated in the early developmental period in PD-1 KO mouse ... 13
• 6. PD-1-PD-L1 binding reduces phagocytosis of synaptosome-pHrodo by in vitro astrocytes ... 15
• Discussion ... 17
• Summary of results ... 17
• Suggestions to improve current study ... 17
• Suggested future studies ... 18
• Bibliography ... 20
• End of Page ... 20