The Primate Corticospinal Connectome and Transcriptome - Supplement

摘要 我们最近发表的一份报告表明，值得注意的是，小鼠的成年皮质脊髓神经元 脊髓损伤（SCI）后的胚胎转录状态（Poplawski et al. Nature 2020）。该状态 持续两周，然后恢复到一个成年的转录状态，在这个状态下，神经元对 再生疗法这项拨款旨在确定皮质脊髓神经元的胚胎逆转是否也 发生在脊髓损伤后的灵长类皮质脊髓运动神经元，如果是这样，这种状态持续多久。这一点至关重要 计划人类临床试验的信息，因为修复受损脊髓的努力最有可能 在皮质脊髓处于胚胎状态并且最能够再生的时间段内有效， 对促生长疗法有反应。 迄今为止，我们的研究结果表明，灵长类皮质脊髓神经元的胚胎逆转确实似乎 发生.我们在SCI后3、10和20天的时间点取样。我们发现胚胎逆转存在于 第10天，几乎在第20天消失。我们现在寻求在本研究中增加一个额外的时间点，即15天。如果 SCI后15天仍存在胚胎逆转，可以延长潜在患者的持续时间， 招募进入临床试验，这是增加合格人数的重要进步 进行促再生试验这些信息不仅对我们的干细胞项目很重要， 翻译，但对于任何人类的促再生治疗试验。 我们将使用迄今为止已成功应用于该补助金的技术。我们将执行RNAseq， 使用表达HA-和FLAG-标记的逆转录AAV载体在恒河猴中的皮质脊髓神经元 多核糖体然后，我们使用翻译核糖体亲和纯化（TRAP）来获得和测序 皮质脊髓特异性mRNA种类。我们将在当前的时间轴中增加一个损伤后15天组（n= 3）， 再增加一只猴（n=1）至我们的10天后顶部，以加强统计功效并实现批次校正。

Abstract We recently published a report demonstrating that, remarkably, adult corticospinal neurons in the mouse revert to an embryonic transcriptional state after spinal cord injury (SCI) (Poplawski et al. Nature 2020). This state persists for two weeks, then reverts to an adult transcriptional state in which neurons are more refractory to regenerative therapies. This grant seeks to determine whether embryonic reversion of corticospinal neurons also occurs in primate corticospinal motor neurons after SCI, and if so, how long this state endures. This is critical information for planning human clinical trials because efforts to repair the injured spinal cord are most likely to be effective in the time period that corticospinal are in an embryonic state and most able to regenerate and respond to pro-growth therapies. Our results to date have shown that embryonic reversion of primate corticospinal neurons indeed appears to occur. We sampled time points of 3, 10 and 20 days after SCI. We find that embryonic reversion is present at Day 10 and is nearly gone by Day 20. We now seek to add an additional time point, 15 days, to this study. If embryonic reversion is still present 15 days after SCI, one could extend the duration of potential patient recruitment into clinical trials, representing an important improvement in increase the number of people eligible for pro-regenerative trials. This information is important not only for our stem cell program that we aim to clinically translate, but for any pro-regenerative treatment trial in humans. We will use techniques that have been successfully employed in this grant to date. We will perform RNAseq of corticospinal neurons in rhesus monkeys using retro-AAV vectors expressing HA- and FLAG- tagged polyribosomes. We then use translational Ribosomal Affinity Purification (TRAP) to obtain and sequence corticospinal-specific mRNA species. We will add to our current timeline a 15 days post injury group (n= 3) and one more monkey (n=1) to our 10 day post top strengthen statistical power and enable batch correction.