成體神經幹細胞衰老過程中蛋白質恆定之角色探討

Abstract

成體神經幹細胞（neural stem cells, NSCs）的衰退與認知功能障礙和老化相關的神經退化性疾病有關。最近的研究顯示在成年哺乳動物的大腦中，主要有再生能力的成體神經幹細胞位於側腦室的腦室下區（subventricular zone, SVZ）以及海馬迴的齒狀回（dentate gyrus, DG）。隨著老化的進程，蛋白質體平衡（proteostasis）的失衡增加了異常蛋白聚集的風險，同時也是老化和許多神經退行性疾病的標誌。儘管目前有關於中年老鼠（12個月大）和年輕成年NSCs（2個月大）蛋白質體的研究，但尚未有專門針對高齡老鼠（20-21個月大）和年輕老鼠（2個月大）進行得比較。 在與博士後研究員林文淵博士的合作下，我們對21種不同的NSC培養基進行了神經球（neurosphere）形成效能測試。其中包括8種先前已發表的培養基和13種由三種基礎培養基和兩種常用於神經細胞培養的添加物組合而成的培養基。我們發現其中一種先前被用於將幹細胞分化為神經細胞的培養基（M12），展現了最佳的神經球形成能力。在相同的條件下，它形成了最多且最大的神經球，揭示了其在神經球培養中的重要性和應用性。此外，在13種未被發表過的培養基中，我們的結果顯示其中3種具有形成神經球的能力。在本研究中，我們使用M12培養由年輕和高齡小鼠SVZ細胞形成的神經球做為研究模型。 關於老年老鼠的SVZ細胞形成神經球的能力是否下降一直存在爭議，因此我們比較了老年和年輕老鼠的SVZ細胞形成神經球的能力。我們的結果支持SVZ細胞形成神經球的能力隨年齡下降的論點，顯示老年老鼠的SVZ細胞形成的神經球明顯較年輕的少，並且其增殖能力也顯著降低。此外，與先前的研究一致，經過兩次繼代後，這種老年神經球形成能力下降的特性不復存在。我們的培養系統保留了老年神經幹細胞增殖能力降低的特性，這也與已知的大腦內成年神經新生（neurogenesis）在老化過程中下降的情況一致。 鑒於蛋白質N端乙醯化（N-terminal acetylation，NAc）在老化期間對蛋白質體平衡的重要性，我們還分析了年輕和老年神經球內蛋白質N端乙醯化比例的變化。使用高鹼性逆相層析分離（High pH reversed-phase liquid chromatography, RPLC）搭配液相層析串聯式質譜儀（LC-MS/MS），我們識別出N端乙醯化程度受年齡調控的蛋白。其中，TMSB10（Thymosin beta-10）、PPIA（Peptidyl-prolyl cis-trans isomerase A）、SLC6A11（Sodium- and chloride-dependent GABA transporter 3）和 PSMD1（26S proteasome non-ATPase regulatory subunit1）的N端乙醯化程度在高齡神經球中顯著提高，而DDT（D-dopachrome decarboxylase）則顯著降低。此外我們發現老年神經球中未修飾的蛋白質N端程度降低，暗示其蛋白質N端乙醯化提高的可能，且未修飾的蛋白質N端程度在SVZ和DG中呈現組織特異性。這些發現提供了成年神經幹細胞老化的潛在調節機制，並有助於更深入探討成體神經幹細胞老化的原因。

The decline of adult neural stem cell (NSC) has been linked to cognitive impairment and aging-associated neurodegenerative diseases. Recent studies have identified regenerative-capable NSCs in the adult mammalian brain primarily localized within the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus (DG) of the hippocampus. During aging, the loss of proteostasis increases the risk of abnormal protein aggregates, marking a hallmark of aging and many neurodegenerative diseases. Despite current studies on the proteome of middle-aged mice (12 mo.) and young adult NSCs (2 mo.), there has been no comparison specifically for aged mice (20-21 mo.) and young mice (2 mo.). In collaboration with postdoc Wen-Yuan Lin, we conducted neurosphere-forming efficiency tests on 21 different NSC culture media. These included 8 media previously published and 13 media formulated by combining three basal mediums and two supplements commonly used for neural cell culture. We found that one of the culture media (M12), previously used for differentiating stem cells into neural cells, exhibited the best neurosphere-forming ability. Under the same conditions, it formed the most and largest neurospheres, revealing its importance and applicability in neurosphere culture. Additionally, among the 13 unpublished culture media, we identified three with neurosphere-forming capabilities. Therefore, we used M12 culture medium to cultivate neurospheres formed by young and aged mouse SVZ cells as our research model. Due to controversies in previous research regarding whether the neurosphere-forming ability of SVZ cells in aged mice declines, we initially compared the neurosphere-forming capabilities of SVZ cells from aged and young mice. Supporting the viewpoint that the neurosphere-forming ability of SVZ cells decreases with age, our results showed that SVZ cells from aged mice notably formed fewer neurospheres, with their proliferation capacity decreased. Furthermore, in alignment with previous studies, the age-dependent decline in neurosphere-forming ability ceased after two passages. Our culture system retained the characteristic of decreased proliferation in aged NSCs, consistent with the known in vivo decline in adult neurogenesis with aging. Additionally, owing to the importance of protein N-terminal acetylation (NAc) in proteostasis during aging, we also analyzed the change in protein N-terminal acetylation ratio within young and old neurospheres. We conducted a comprehensive analysis using high pH reversed-phase liquid chromatography (RPLC) coupled with LC-MS/MS. The proteomic analysis identified proteins with a change in the N-terminal acetylation ratio associated with age. In the aged neurospheres, the N-terminal acetylation levels of TMSB10 (Thymosin beta-10), PPIA (Peptidyl-prolyl cis-trans isomerase A), SLC6A11 (Sodium- and chloride-dependent GABA transporter 3), and PSMD1 (26S proteasome non-ATPase regulatory subunit1) were significantly increased, while DDT (D-dopachrome decarboxylase) exhibited a significant decrease. Besides, our study unveiled elevated levels of N-terminal acetylation in aged neurospheres, displaying tissue-specific patterns in DG and SVZ. These findings provide potential regulatory mechanisms underlying the aging process of adult NSCs and contribute to a deeper understanding of the reasons behind it.