利用AAV介導的CRISPR/Cas9技術在小鼠模型中探討Rtl1在大腦中的功能

Abstract

逆轉錄轉座子Gag樣基因1（Rtl1）是一個父源性表達的印跡基因，廣為人知其在胎盤及胎兒發育中的關鍵角色。令人意外的是，我們發現Rtl1在成鼠大腦中依然持續表達，特別集中於單胺能區域，例如腹側被蓋區（VTA）與藍斑核（LC）這兩個腦區對於情緒、動機與喚醒的調控至關重要。然而，Rtl1在發育完成後於這些神經迴路中所扮演的角色仍未明確。 為了釐清其功能，我們利用AAV-CRISPR/Cas9系統，選擇性敲落青少年期小鼠腹側被蓋區或藍斑核中的Rtl1表現。結果顯示，雖然在藍斑核中Rtl1的敲落對行為表現無明顯影響，但在腹側被蓋區中抑制Rtl1表達則導致探索行為增加、衝動控制改變以及輕微的運動障礙，顯示Rtl1在多巴胺系統中具有調節行為的作用。值得注意的是，這些行為變化並未伴隨腹側被蓋區內多巴胺含量的改變，但卻與週邊多巴胺濃度及酪胺酸羥化酶表現的變動有關。 我們亦將上述發現與早期Rtl1敲落模型進行比較。出生即缺乏Rtl1的小鼠表現出類似焦慮的行為，而在胚胎階段敲落Rtl1表現則導致明顯的過動行為。綜合而言，我們的研究結果指出，Rtl1在成鼠大腦中仍具功能性意義，並以區域性及時序性的方式參與行為調節。

Retrotransposon Gag like 1 (Rtl1) is a paternally expressed imprinted gene known for its essential role in placental and fetal development. Unexpectedly, we found that Rtl1 remains actively expressed in the adult brain, particularly in monoaminergic regions like the ventral tegmental area (VTA) and locus coeruleus (LC), which are critical for regulating emotion, motivation, and arousal. However, the functions of Rtl1 in these neural circuits beyond development have remained unclear. To investigate this, we used an AAV-mediated CRISPR/Cas9 approach to selectively knock down Rtl1 in either the adult VTA or LC. While Rtl1 knockdown in LC had no significant behavioral effects, its reduction in the VTA led to increased exploratory activity, impaired impulse control, and mild motor deficits, highlighting the role for Rtl1 in behavioral regulation via the dopaminergic system. Notably, these behavioral changes occurred without affecting dopamine content within the VTA itself but were associated with changes in peripheral dopamine levels and tyrosine hydroxylase expression. We further compared these effects to early-life Rtl1 knockdown models. Mice with Rtl1 loss from birth exhibited anxiety-like behaviors, while embryonic knockdown led to marked hyperactivity. Together, our findings suggest that Rtl1 continues to play a functional role in the adult brain and contributes to behavioral regulation in a manner dependent on both brain region and developmental stage.