Table_1_Tracking cell turnover in human brain using 15N-thymidine imaging mass spectrometry.docx

Microcephaly is often caused by an impairment of the generation of neurons in the brain, a process referred to as neurogenesis. While most neurogenesis in mammals occurs during brain development, it thought to continue to take place through adulthood in selected regions of the mammalian brain, notably the hippocampus. However, the generality of neurogenesis in the adult brain has been controversial. While studies in mice and rats have provided compelling evidence for neurogenesis occurring in the adult rodent hippocampus, the lack of applicability in humans of key methods to demonstrate neurogenesis has led to an intense debate about the existence and, in particular, the magnitude of neurogenesis in the adult human brain. Here, we demonstrate the applicability of a powerful method to address this debate, that is, the in vivo labeling of adult human patients with 15N-thymidine, a non-hazardous form of thymidine, an approach without any clinical harm or ethical concerns. 15N-thymidine incorporation into newly synthesized DNA of specific cells was quantified at the single-cell level with subcellular resolution by Multiple-isotype imaging mass spectrometry (MIMS) of brain tissue resected for medical reasons. Two adult human patients, a glioblastoma patient and a patient with drug-refractory right temporal lobe epilepsy, were infused for 24 h with 15N-thymidine. Detection of 15N-positive leukocyte nuclei in blood samples from these patients confirmed previous findings by others and demonstrated the appropriateness of this approach to search for the generation of new cells in the adult human brain. 15N-positive neural cells were easily identified in the glioblastoma tissue sample, and the range of the 15N signal suggested that cells that underwent S-phase fully or partially during the 24 h in vivo labeling period, as well as cells generated therefrom, were detected. In contrast, within the hippocampus tissue resected from the epilepsy patient, none of the 2,000 dentate gyrus neurons analyzed was positive for 15N-thymidine uptake, consistent with the notion that the rate of neurogenesis in the adult human hippocampus is rather low. Of note, the likelihood of detecting neurogenesis was reduced because of (i) the low number of cells analyzed, (ii) the fact that hippocampal tissue was explored that may have had reduced neurogenesis due to epilepsy, and (iii) the labeling period of 24 h which may have been too short to capture quiescent neural stem cells. Yet, overall, our approach to enrich NeuN-labeled neuronal nuclei by FACS prior to MIMS analysis provides a promising strategy to quantify even low rates of neurogenesis in the adult human hippocampus after in vivo15N-thymidine infusion. From a general point of view and regarding future perspectives, the in vivo labeling of humans with 15N-thymidine followed by MIMS analysis of brain tissue constitutes a novel approach to study mitotically active cells and their progeny in the brain, and thus allows a broad spectrum of studies of brain physiology and pathology, including microcephaly.

小头畸形（Microcephaly）通常由大脑神经元生成障碍所致，这一过程被称为神经发生（neurogenesis）。多数哺乳动物的神经发生发生于脑发育阶段，但此前认为在哺乳动物大脑的特定区域，尤其是海马体（hippocampus）中，神经发生会持续至成年时期。然而，成体大脑中神经发生的普遍性一直存在争议。针对小鼠和大鼠的研究已为成年啮齿类海马体存在神经发生提供了极具说服力的证据，但由于缺乏适用于人类的关键神经发生检测方法，学界围绕成人脑海马体神经发生的存在性，尤其是其程度展开了激烈争论。本研究证明了一种可用于解决这一争议的高效方法的适用性：对成年人类患者进行15N-胸苷（15N-thymidine，一种无危害的胸苷形式）的体内标记，该方法无任何临床危害或伦理顾虑。通过多同位素成像质谱法（Multiple-isotype imaging mass spectrometry, MIMS）对因医学原因切除的脑组织进行分析，可在单细胞水平、亚细胞分辨率下对特定细胞新合成DNA中掺入的15N-胸苷进行定量。两名成年人类患者，一名为胶质母细胞瘤（glioblastoma）患者，另一名为难治性右侧颞叶癫痫患者，接受了为期24小时的15N-胸苷输注。对两名患者血液样本中15N阳性白细胞细胞核的检测，验证了其他研究者此前的发现，同时证明了该方法可用于探索成体人脑中新细胞的生成。在胶质母细胞瘤组织样本中，15N阳性神经细胞可被轻易识别，且15N信号的强度范围表明，我们检测到了在24小时体内标记期间完全或部分经历S期（S-phase）的细胞，以及由此产生的子代细胞。与之形成对比的是，在该癫痫患者切除的海马体组织中，所分析的2000个齿状回（dentate gyrus）神经元均未出现15N-胸苷摄取阳性，这与"成人脑海马体神经发生速率极低"的观点相符。值得注意的是，本次研究检测神经发生的可能性有所降低，原因在于：（i）分析的细胞数量较少；（ii）所探究的海马体组织可能因癫痫而出现神经发生水平下降；（iii）24小时的标记时长可能过短，无法捕捉处于静息状态的神经干细胞（quiescent neural stem cells）。但总体而言，我们在多同位素成像质谱分析前通过流式细胞术（Fluorescence-Activated Cell Sorting, FACS）富集NeuN标记的神经元细胞核的方法，为定量成人脑海马体中即使速率极低的神经发生提供了一种颇具前景的策略。从通用视角及未来展望来看，对人类进行15N-胸苷体内标记，随后对脑组织进行多同位素成像质谱分析，是一种研究大脑中有丝分裂活跃细胞及其子代细胞的全新方法，因此可广泛应用于脑生理学与病理学的各类研究，包括小头畸形相关研究。