探討鋅離子活化 MTF-1 對初代培養大鼠胚胎皮質神經元自噬、發炎及基因表現之調控角色

Abstract

鋅離子恆定對神經元功能、壓力適應及存活至關重要，而細胞內鋅離子失衡已被證實與神經退化病理機制有關。金屬反應轉錄因子1（metal-responsive transcription factor-1, MTF-1）是一種鋅敏感性轉錄因子，已知參與金屬離子恆定與壓力反應，但其在glutamate誘發之神經壓力下的角色尚未清楚。本研究以初代培養大鼠胚胎皮質神經元為主要系統，PC12細胞為輔助模型，探討鋅依賴性MTF-1核內定位、自噬體累積及轉錄變化之關係。藉由ImageJ及R語言進行MTF-1免疫螢光影像分析，結果顯示，在PC12細胞中，內源性MTF-1於基礎狀態下主要位於細胞質；在MTF-1過度表現條件下，dopamine與ZnCl₂刺激可促進MTF-1核內累積。相較之下，神經元中的內源性MTF-1於基礎狀態下即呈現較高核內定位；glutamate單獨刺激未進一步增加MTF-1核內比例，但鋅螯合劑TPEN可降低神經元中MTF-1的核內定位，且在glutamate刺激下更為明顯，顯示神經元中MTF-1核內定位可能依賴細胞內鋅可用性維持。以GFP-LC3 puncta影像分析評估自噬體累積顯示，PC12細胞中MTF-1過度表現對LC3 puncta形成的影響具時間與刺激條件依賴性；在神經元中，glutamate誘發時間依賴性的LC3 puncta增加，且MTF-1過度表現可進一步增強持續性glutamate壓力下的LC3 puncta累積。RNA-seq分析顯示，短時間glutamate刺激可誘發時間序列轉錄反應，並影響與神經元壓力、發炎及神經訊號調節相關的基因表現。透過表現模式篩選，本研究鑑定出候選鋅依賴性glutamate反應基因，其表現受TPEN前處理削弱；其中誘導基因主要包含metallothioneins、發炎、免疫及自噬相關候選基因，而鋅依賴性下調基因則與NMDA受體訊號、離子通道活性及突觸功能相關。綜上所述，本研究顯示細胞內鋅可用性與MTF-1核內定位及glutamate誘發之轉錄重塑有關，並支持MTF-1可能作為鋅敏感性壓力反應網絡的一環，連結glutamate刺激、自噬體累積、發炎基因表現與神經訊號轉錄調節。

Zinc homeostasis is critical for neuronal function, stress adaptation, and survival, and zinc dysregulation is linked to neurodegenerative pathology. Metal-responsive transcription factor-1 (MTF-1) is a zinc-sensitive transcription factor involved in metal homeostasis and stress responses, but its role in glutamate-stressed neurons remains unclear. This study used primary-cultured rat embryonic cortical neurons as the main model and PC12 cells as a complementary system to examine MTF-1 nuclear localization, autophagosome accumulation, and zinc-dependent transcriptional changes. ImageJ- and R-based analysis showed that endogenous MTF-1 was mainly cytoplasmic in PC12 cells, whereas dopamine and ZnCl₂ promoted nuclear accumulation under MTF-1 overexpression. In neurons, endogenous MTF-1 was nuclear-enriched; glutamate alone did not increase nuclear MTF-1, but TPEN reduced neuronal nuclear localization, particularly with glutamate, suggesting zinc-dependent maintenance. GFP-LC3 imaging showed time- and stimulus-dependent effects of MTF-1 overexpression on LC3 puncta in PC12 cells. In neurons, glutamate increased LC3 puncta, and MTF-1 overexpression enhanced puncta accumulation under sustained stress. RNA-seq revealed organized glutamate-responsive changes related to neuronal stress, inflammation, and neuronal signaling. Pattern-based screening identified candidate zinc-dependent glutamate-responsive genes attenuated by TPEN pretreatment: induced candidates included metallothioneins and inflammation-, immune-, or autophagy-related genes, whereas downregulated candidates were associated with NMDA receptor signaling, ion channel activity, and synaptic function. Together, these findings suggest that MTF-1 may link glutamate stress to autophagosome accumulation, inflammatory gene expression, and neuronal signaling through a zinc-sensitive stress network.