School of Health Sciences, Purdue University, West Lafayette, IN 47907, USA.
Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, West Lafayette, IN 47907, USA.
Int J Mol Sci. 2022 Aug 31;23(17):9888. doi: 10.3390/ijms23179888.
The subventricular zone (SVZ) in lateral ventricles is the largest neurogenic region in adult brain containing high amounts of copper (Cu). This study aims to define the role of Cu in adult neurogenesis by chelating labile Cu ions using a well-established Cu chelator D-Penicillamine (D-Pen). A neurosphere model derived from adult mouse SVZ tissues was established and characterized for its functionality with regards to neural stem/progenitor cells (NSPCs). Applying D-Pen in cultured neurospheres significantly reduced intracellular Cu levels and reversed the Cu-induced suppression of NSPC’s differentiation and migration. An in vivo intracerebroventricular (ICV) infusion model was subsequently established to infuse D-Pen directly into the lateral ventricle. Metal analyses revealed a selective reduction of Cu in SVZ by 13.1% (p = 0.19) and 21.4% (p < 0.05) following D-Pen infusions at low (0.075 μg/h) and high (0.75 μg/h) doses for 28 days, respectively, compared to saline-infused controls. Immunohistochemical studies revealed that the 7-day, low-dose D-Pen infusion significantly increased Ki67(+)/Nestin(+) cell counts in SVZ by 28% (p < 0.05). Quantification of BrdU(+)/doublecortin (DCX)(+) newborn neuroblasts in the rostral migration stream (RMS) and olfactory bulb (OB) further revealed that the short-term, low-dose D-Pen infusion, as compared with saline-infused controls, resulted in more newborn neuroblasts in OB, while the high-dose D-Pen infusion showed fewer newborn neuroblasts in OB but with more arrested in the RMS. Long-term (28-day) infusion revealed similar outcomes. The qPCR data from neurosphere experiments revealed altered expressions of mRNAs encoding key proteins known to regulate SVZ adult neurogenesis, including, but not limited to, Shh, Dlx2, and Slit1, in response to the changed Cu level in neurospheres. Further immunohistochemical data indicated that Cu chelation also altered the expression of high-affinity copper uptake protein 1 (CTR1) and metallothionein-3 (MT3) in the SVZ as well as CTR1 in the choroid plexus, a tissue regulating brain Cu homeostasis. Taken together, this study provides first-hand evidence that a high Cu level in SVZ appears likely to maintain the stability of adult neurogenesis in this neurogenic zone.
侧脑室的室下区 (SVZ) 是成年人大脑中最大的神经发生区域，含有大量的铜 (Cu)。本研究旨在通过使用一种成熟的 Cu 螯合剂 D-青霉胺 (D-Pen) 螯合不稳定的 Cu 离子，来定义 Cu 在成年神经发生中的作用。建立了源自成年小鼠 SVZ 组织的神经球模型，并对其作为神经干细胞/祖细胞 (NSPC) 的功能进行了表征。在培养的神经球中应用 D-Pen 可显著降低细胞内 Cu 水平，并逆转 Cu 诱导的 NSPC 分化和迁移抑制。随后建立了一种脑室内 (ICV) 输注模型，直接将 D-Pen 输注到侧脑室。金属分析显示，与盐水输注对照组相比，D-Pen 以低 (0.075 μg/h) 和高 (0.75 μg/h) 剂量分别输注 28 天，SVZ 中的 Cu 分别选择性减少 13.1%（p = 0.19）和 21.4%（p < 0.05）。免疫组织化学研究显示，与盐水输注对照组相比，7 天、低剂量 D-Pen 输注可使 SVZ 中的 Ki67(+) / Nestin(+) 细胞计数增加 28%（p < 0.05）。对穹窿迁移流 (RMS) 和嗅球 (OB) 中 BrdU(+) / 双皮质素 (DCX)(+) 新生神经母细胞的定量分析进一步表明，与盐水输注对照组相比，短期、低剂量 D-Pen 输注导致 OB 中更多的新生神经母细胞，而高剂量 D-Pen 输注则导致 OB 中更少的新生神经母细胞，但更多的新生神经母细胞在 RMS 中被阻滞。长期（28 天）输注显示出类似的结果。神经球实验的 qPCR 数据显示，在神经球中，与 Cu 水平变化相对应，编码已知调节 SVZ 成年神经发生的关键蛋白的 mRNAs 的表达发生改变，包括但不限于 Shh、Dlx2 和 Slit1。进一步的免疫组织化学数据表明，Cu 螯合也改变了 SVZ 中高亲和力铜摄取蛋白 1 (CTR1) 和金属硫蛋白 3 (MT3) 的表达，以及脉络丛中调节大脑 Cu 稳态的 CTR1 的表达。总之，本研究提供了第一手证据，表明 SVZ 中的高 Cu 水平可能有助于维持该神经发生区成年神经发生的稳定性。
