기억 형성에 관여하는 조절유전자 스크리닝 = Screening for Regulatory Genes Involved in Memory Formation

다국어 초록 (Multilingual Abstract)

Learning and memory found in both vertebrates and invertebrates have made critical contributions to the survival and evolution of these organisms. Degenerative brain diseases such as Alzheimer’s disease, in particular, cause severe impairments in learning and memory formation through extensive damage to neurons in the brain. Using Notch/APP-based reporters, which enable quantitative in vivo measurement of γ-secretase activity directly implicated in the onset of Alzheimer’s disease, we performed a genetic modifier screen and identified three genes: back seat driver (bsd), drongo, and starvin (stv). To determine whether these genes are involved in learning and memory formation, we first reduced the activity of each gene specifically in mushroom body neurons using RNA interference and then tested the aversive olfactory conditioning memory of these loss-of-function flies. Knockdown of bsd in the mushroom body led to reduced learning and long-term memory. In aged flies, however, no decrease in learning was observed, suggesting that age-dependent memory decline may be influenced at the level of bsd or at downstream regulatory steps of bsd. Knockdown of drongo in the mushroom body resulted in reduced learning and short-term memory in 4–6-day-old flies, but not in 8–10-day-old flies. Behavioral assays of stv knockdown flies indicated that the stv gene does not affect memory formation. In addition, we examined whether stv has a genetic interaction with amyloid-β peptides (Aβ40 and Aβ42), which are strongly associated with the pathogenesis of Alzheimer’s disease. In experiments involving expression of amyloid-β peptides, Aβ40 expression enhanced learning in 8–10-day-old flies, whereas Aβ42 expression showed no significant difference. Knockdown of stv combined with overexpression of Aβ40 induced learning deficits, while stv knockdown with Aβ42 overexpression did not yield significant differences. Specifically, in learning tests, expression of amyloid beta peptide and simultaneous knockdown of stv resulted in a decrease in learning performance compared to overexpression of Aβ40 or Aβ42 alone. Stv regulates heat shock protein activity, which is thought to prevent Aβ aggregation and thereby exert significant effects on learning. To demonstrate that these effects were not attributable to off-target effects of RNA interference-mediated gene downregulation, we are generating loss-of-function mutants. Using the CRISPR/Cas9 system, gRNA sequences were cloned into the pCFD6 vector for each gene, and injection services were commissioned to establish mutant fly lines.

목차 (Table of Contents)

• 그림 목록 ⅰ
• 표 목록 ⅱ
• Abstract ⅲ
• Ⅰ. 서 론 1
• Ⅱ. 재료 및 방법 6
• 노랑 초파리 라인 및 배양 환경 6
• UAS/GAL4 시스템 8
• 혐오 후각 조건화 기억력 실험 8
• CRISPR/Cas9 시스템을 활용한 돌연변이 초파리 생성 과정 10
• 통계 분석 방법 11
• Ⅲ. 결 과 13
• 버섯체에서 bsd 기능 상실이 기억에 미치는 영향 13
• 버섯체에서 drongo 기능 상실이 기억에 미치는 영향 13
• 버섯체에서 stv 기능 상실이 기억에 미치는 영향 14
• 버섯체에서 사람유래 Aβ 펩타이드 발현이 기억에 미치는 영향 15
• 버섯체에서 사람유래 Aβ 펩타이드 발현과 stv 기능 상실이 기억에 미치는 영향 16
• 버섯체에서 사람유래 Tau 펩타이드 발현이 기억에 미치는 영향 17
• CRISPR/Cas9 시스템으로 돌연변이를 만들기 위한 클로닝 17
• Ⅳ. 고 찰 19
• Ⅴ. 참고문헌 22
• FIGURES 27
• 국문초록 47