撰文 | 十一月
责编 | 兮
今年4月，中科院神经科学研究所杨辉团队在Cell上发文，运用CRISPR-CasRx系统在小鼠中实现了高效且特异地将纹状体中星形胶质细胞转分化成为多巴胺神经元用以该改善小鼠中帕金森疾病的症状【1】（专家点评Cell丨杨辉/周海波等利用CasRx在神经性疾病治疗领域取得重大进展），为众多神经退行性疾病的治疗提供了新的可能。该项研究通过运用最新开发的RNA靶向CRISPR系统CasRx特异性地在视网膜穆勒胶质细胞中敲低Ptbp1基因的表达，首次在成体中实现了视神经节细胞的再生，并且恢复了永久性视力损伤模型小鼠的视力。同时，该研究还证明了这项技术可以非常高效且特异地将纹状体内的星形胶质细胞转分化成多巴胺神经元，并且基本消除了帕金森疾病的症状。该研究将为未来根治以上两种严重的神经退行性疾病提供了可能，同时也为众多神经退行性疾病的治疗提供一个新的途径。
在Cell文章见刊之际，美国加州大学圣地亚哥分校付向东研究组在预印本网站bioRxiv上线了在动物模型中敲低PTB蛋白治疗帕金森综合征的文章（https://www.biorxiv.org/content/10.1101/2020.04.06.028084v1.article-metrics）。2020年6月24日，付向东研究组的文章正式发表在Nature上，题为Reversing a model of Parkinson’s disease with in situ converted nigral neurons，（注：该文章于2017年12月开始投稿，后于2018年11月转投Nature）实现了通过一步敲低PTB的方式在小鼠帕金森动物模型中区域特异性将星形胶质细胞转分化形成神经元的方式来改善帕金森症状。
帕金森症的特征是黑质中多巴胺神经元的缺失【2】，而再生医学与为治疗以细胞缺失为特征的疾病带来了巨大的希望。考虑到某些体细胞存在一定的可塑性【3】，通过转分化在原位改变细胞命运从而避免免疫识别的方法获得了广泛关注【4】。在小鼠的大脑中，星形胶质细胞的可塑性被证明可以用来产生新的神经元，而在疾病模型中这些神经元可以改善病症【5】。但是目前相关的研究还很有限，转分化产生的神经元是否能够重建内源神经环路的功能也很不清楚。
付向东研究组此前的研究发现RNA结合蛋白PTB以及其神经元类似物nPTB在控制神经元诱导和成熟过程中的作用，而且证明了通过敲低这些RNA结合蛋白可以在体外将不同类型的细胞有效的转化为功能性神经元（先后发表在Cell和Nature Neuroscience杂志，薛愿超博士为第一作者）【6,7】。因此，作者们希望用比较简单的策略将黑质中的神经胶质细胞直接转化为多巴胺能神经元。
星形胶质细胞为大脑的体内转分化提供了几个优势，首先这些非神经元细胞数量丰富，在损伤时增殖，其次星形胶质细胞具有高度可塑性【8】。顺序性下调PTB和nPTB可以将成纤维细胞转分化形成神经元【7】。为了进一步增加该信号通路在星形胶质细胞中转分化形成神经元中的作用，作者们使用了小鼠大脑皮层和中脑以及人胎儿大脑皮层的星形胶质细胞进行检测。作者们发现，通过单独敲低PTB就能够实现在小鼠和人中将星形胶质细胞转分化为神经元。
为了对转分化形成的神经元进行功能方面的检测，作者们使用慢病毒表达的PTB的shRNA表达在小鼠皮层的星形胶质细胞之中。在四周之后，50-80%的shPTB转入的细胞中出现神经元的形态并且神经元相关细胞标记物可以对其进行标记。通过转分化前后RNA-seq的结果对比，作者们发现转化过程中典型的星形胶质细胞相关的基因表达被抑制而神经元相关的基因表达则上升。另外，通电压门控钠离子和钾离子通道电流以及重复的动作电位放电，作者们确认了这些转分化而来的神经元具有神经活性。进一步地，作者们在小鼠体内使用腺病毒相关载体敲低PTB，在小鼠中脑中随着时间逐渐产生星形胶质细胞到神经元的转化，而且新产生的这些多巴胺神经元会逐渐成熟，形成突触连接（图1）。
图1 在小鼠中注射AAV-shPTB促进星形胶质细胞转分化形成神经元
在成功地产生多巴胺神经元后，作者们希望探究这些神经元具有在损伤后重建黑质信号通路的潜能。药物处理的帕金森小鼠模型与人类帕金森症相似，多巴胺神经元会大量减少。在帕金森小鼠模型中，表达AAV-shPTB能够从内源中脑星形胶质细胞中诱导新的神经元的产生，部分恢复丢失的多巴胺神经元以及其在黑质多巴胺信号通路中的轴突。除此之外，通过高效液相色谱实验，作者们确认了帕金森小鼠模型中敲低PTB产生的新的神经元能够恢复多巴胺的分泌，同时能够逆转帕金森相关的运动表型。
另外，由于反义寡核苷酸（Antisense oligonucleotides, ASOs）在调节多种大脑相关的疾病中发挥的新兴力量，作者们合成并筛选了包含硫代磷酸酯骨架以及荧光素的PTB ASOs。其中的三个PTB ASOs能够在转染到小鼠星形胶质细胞后显著降低PTB的表达量，同时促进神经元相关标记物的表达。并且这些新产生的神经元能够分泌多巴胺以及神经元活性。再对选择性表达在星形胶质细胞的转基因小鼠进行PTB ASOs的注射后，与对照相比能够促进神经元的产生。膜片钳记录显示这些在存活转化的神经元中显示出功能性神经生理特性。另外，在帕金森小鼠模型中PTB ASOs的注射能够挽救疾病相关的运动表型。
星形胶质细胞转分化为神经元的模式图。图片引自Ernest Arenas教授在Nature上配发的News & View评论文章。
总的来说，付向东研究组的工作介绍了一种一步转分化产生功能性神经元的策略，利用神经元重编程的区域特异性，可以有效地将中脑星形胶质细胞转化为黑质纹状体多巴胺通路的功能性多巴胺神经元，逆转帕金森小鼠模型中的运动表型，该工作将为多种神经退行性疾病提供可能的治疗策略。
值得一提的是，这篇Nature论文的审稿人之一Ernest Arenas教授（卡罗林斯卡学，Karolinska Institute，帕金森疾病领域的专家）还撰写了评论文章，对付向东组的工作和杨辉组此前发表的工作同时进行了点评，高度评价了两项殊途同归的重要研究成果，特别是其在临床转化上的重大意义。由于两项工作采用的方法不同，具体到实验细节部分也存在一些显著差异之处，因此Ernest Arenas对两篇文章也进行了比较，提出了一些有待进一步解决的建设性问题。
此外，针对付向东lab的这篇Nature工作，国外科学媒体也采访了几位神经生物学领域的专家，原文如下：
1. NEW COMMENT Dr Tilo Kunath, Group Leader in the Centre for Regenerative Medicine, University of Edinburgh, said:
“This is a remarkable study that describes the production of new dopamine-releasing nerve cells in the mouse brain by the suppression of a single gene, PTBP1.  This simple, one-step procedure uncovers surprising biology and therapeutic potential of non-nerve cells known as astrocytes in the brain.  If this gene therapy is shown to work in humans, it will have significant implications for regenerative medicine for Parkinson’s disease and other neurodegenerative conditions – but we are not at that stage yet.
2. NEW COMMENT Prof Robert Howard, Professor of Old Age Psychiatry, UCL, said:
“That astrocytes can be converted to functioning neurons through the silencing of a single gene in the mouse brain and the observation that this can lead to new neurons that resemble those lost in a mouse model of Parkinson’s disease, with improvements in motor symptoms, is an extraordinary scientific discovery.  This opens up a completely novel avenue for development of treatments to "rebuild" damaged brains in Alzheimer’s and Parkinson’s diseases.
“We will, one day, have effective treatments for these devastating degenerative brain diseases and a specific discovery like this will turn out to have been a pivotal step on the journey – it’s important to bear in mind, however, that this kind of advance rarely translates into safe and effective treatments.  Consider the failure of stem cell technologies to help neurological disorders, for example.”
3. Prof Tara Spires-Jones, UK Dementia Research Institute at the University of Edinburgh & Deputy Director, Centre for Discovery Brain Sciences, University of Edinburgh, said:
“This paper by Fu and team presents some fascinating data showing the potential to convert one type of “supporting” brain cell, called astrocytes, into functional neurons that wire up in mouse brain and are able to rescue some function in a model of Parkinson’s disease.  While the principle of this study is remarkable and promising, it is important to note that it was conducted in mice with group sizes from 3-8 and there is a long way to go to translate this into a treatment for people.”
4. Prof John Hardy, Professor of Neuroscience, UCL, said:
“I think this is a very interesting piece of work in which glial cells in mice in the substantia nigra (the site where cells are lost in Parkinson’s disease) are directly converted into dopamine neurons.  This is a very exciting and (to me) an unexpected result from a theoretical point of view.  As a piece of basic science, it is really exciting.  Whether it will help in the development of therapies for Parkinson’s disease is much less clear… but exiting work.”
5. Prof Bart De Strooper, Director of the UK Dementia Research Institute, UCL, said:
“This is a very complete, in-depth and promising study in the search for treatments of Parkinson’s disease.
“Using cellular and animal models, the authors have shown that suppressing just one protein is sufficient to convert supporting cells in the brain into neurons.  These converted cells functionally replace dopaminergic neurons lost in Parkinson’s and can restore deficits in an animal model of disease.  The authors have used multiple approaches, of which one or two may be feasible in humans.
“As with all proof-of-concept, preclinical studies, there are still several hurdles to overcome before we see this in the clinic and providing real benefits for those living with the condition.  The main question now, as discussed by the authors, is whether this treatment will be effective in older people where the brain has lost some plasticity with age.
“That said, this is an extremely refreshing study in the neurodegeneration field and opens up avenues for further research exploring the restoration of brain circuitry in Parkinson’s disease.  It may even be possible to apply the same approach to other neurodegenerative disorders, as well as brain trauma.”
原文链接：
https://doi.org/10.1038/s41586-020-2388-4
制版人：老翅膀
参考文献
1.Zhou, H. et al. Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease in Mice. Cell 181, 590-603 e516, doi:10.1016/j.cell.2020.03.024 (2020).
2.Poewe, W. et al. Parkinson disease. Nature reviews. Disease primers 3, 17013, doi:10.1038/nrdp.2017.13 (2017).
3.Cohen, D. E. & Melton, D. Turning straw into gold: directing cell fate for regenerative medicine. Nature reviews. Genetics 12, 243-252, doi:10.1038/nrg2938 (2011).
4.Barker, R. A., Gotz, M. & Parmar, M. New approaches for brain repair-from rescue to reprogramming. Nature 557, 329-334, doi:10.1038/s41586-018-0087-1 (2018).
5.Rivetti di Val Cervo, P. et al. Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson's disease model. Nature biotechnology 35, 444-452, doi:10.1038/nbt.3835 (2017).
6.Xue, Y. et al. Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits. Cell 152, 82-96, doi:10.1016/j.cell.2012.11.045 (2013).
7.Xue, Y. et al. Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells. Nature neuroscience 19, 807-815, doi:10.1038/nn.4297 (2016).
8.Yu, X., Nagai, J. & Khakh, B. S. Improved tools to study astrocytes. Nature reviews. Neuroscience 21, 121-138, doi:10.1038/s41583-020-0264-8 (2020).
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