Novel antibodies reveal presynaptic localization of C9orf72 protein and reduced protein levels in C9orf72 mutation carriers.
Novel antibodies reveal presynaptic localization of C9orf72 protein and reduced protein levels in C9orf72 mutation carriers.
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DOI：
10.1186/s40478-018-0579-0
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发表时间：
2018-08-03
影响因子：
7.1
通讯作者：
Neumann M
中科院分区：
文献类型：
--
作者：
Frick P;Sellier C;Mackenzie IRA;Cheng CY;Tahraoui-Bories J;Martinat C;Pasterkamp RJ;Prudlo J;Edbauer D;Oulad-Abdelghani M;Feederle R;Charlet-Berguerand N;Neumann M
C9 orf 72中的六核苷酸重复序列扩增是额颞叶痴呆和肌萎缩侧索硬化症最常见的遗传原因，但该突变的致病机制尚未解决。单倍不足已被提出作为一个潜在的机制。然而，降低的C9 orf 72蛋白水平是否以及如何有助于疾病发病机制的见解仍然有限，因为C9 orf 72在中枢神经系统（CNS）中的表达、定位和功能是不确定的，部分原因是目前可用的C9 orf 72抗体的特异性差。在这里，我们产生并表征了新的敲除验证的抗C9 orf 72的单克隆大鼠和小鼠抗体。我们发现C9 orf 72是一种低丰度、细胞质、高度可溶性的蛋白质，其长481个氨基酸的同种型是小鼠组织和人脑中主要（如果不是唯一）表达的蛋白质同种型。作为C9 orf 72重复扩增的结果，与对照组（n = 26）相比，我们的C9 orf 72突变携带者系列（n = 17）中小脑中的C9 orf 72蛋白水平降低至80%。然而，小脑蛋白水平和临床表型之间没有相关性。最后，通过利用互补的免疫组织化学和生物化学方法，包括对人iPSC衍生的运动神经元的分析，我们鉴定了C9 orf 72，除了其与溶酶体的缔合之外，还定位于突触前体并且能够与RAB 3蛋白家族的所有成员相互作用，提示C9 orf 72通过潜在地充当RAB 3蛋白的鸟嘌呤核苷酸交换因子而在调节突触囊泡功能中的作用。总之，我们的研究结果提供了进一步的证据证明单倍不足的潜在机制C9 orf 72的发病机制，通过展示C9 orf 72突变载体和重要的新见解C9 orf 72在中枢神经系统中的生理作用的蛋白水平降低。此外，所描述的新型单克隆C9 orf 72抗体将是进一步剖析C9 orf 72的细胞和分子功能的有用工具。本文的在线版本（10.1186/s40478-018-0579-0）包含补充材料，可供授权用户使用。
Hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis, but the pathogenic mechanism of this mutation remains unresolved. Haploinsufficiency has been proposed as one potential mechanism. However, insights if and how reduced C9orf72 proteins levels might contribute to disease pathogenesis are still limited because C9orf72 expression, localization and functions in the central nervous system (CNS) are uncertain, in part due to the poor specificity of currently available C9orf72 antibodies. Here, we generated and characterized novel knock-out validated monoclonal rat and mouse antibodies against C9orf72. We found that C9orf72 is a low abundant, cytoplasmic, highly soluble protein with the long 481 amino acid isoform being the predominant, if not exclusively, expressed protein isoform in mouse tissues and human brain. As consequence of the C9orf72 repeat expansion, C9orf72 protein levels in the cerebellum were reduced to 80% in our series of C9orf72 mutation carriers (n = 17) compared to controls (n = 26). However, no associations between cerebellar protein levels and clinical phenotypes were seen. Finally, by utilizing complementary immunohistochemical and biochemical approaches including analysis of human iPSC derived motor neurons, we identified C9orf72, in addition to its association to lysosomes, to be localized to the presynapses and able to interact with all members of the RAB3 protein family, suggestive of a role for C9orf72 in regulating synaptic vesicle functions by potentially acting as guanine nucleotide exchange factor for RAB3 proteins. In conclusion, our findings provide further evidence for haploinsufficiency as potential mechanism in C9orf72 pathogenesis by demonstrating reduced protein levels in C9orf72 mutation carriers and important novel insights into the physiological role of C9orf72 in the CNS. Moreover, the described novel monoclonal C9orf72 antibodies will be useful tools to further dissect the cellular and molecular functions of C9orf72. The online version of this article (10.1186/s40478-018-0579-0) contains supplementary material, which is available to authorized users.
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影响因子：
16.2
作者：
Ash PE;Bieniek KF;Gendron TF;Caulfield T;Lin WL;Dejesus-Hernandez M;van Blitterswijk MM;Jansen-West K;Paul JW 3rd;Rademakers R;Boylan KB;Dickson DW;Petrucelli L
通讯作者：
Petrucelli L
影响因子：
3.4
作者：
Guillemin, I;Becker, M;Nothwang, HG
通讯作者：
Nothwang, HG
影响因子：
46.9
作者：
Maury, Yves;Come, Julien;Nedelec, Stephane
通讯作者：
Nedelec, Stephane
DOI：
10.1093/brain/awu120
发表时间：
2014-07
期刊：
Brain : a journal of neurology
影响因子：
--
作者：
Cooper-Knock J;Walsh MJ;Higginbottom A;Robin Highley J;Dickman MJ;Edbauer D;Ince PG;Wharton SB;Wilson SA;Kirby J;Hautbergue GM;Shaw PJ
通讯作者：
Shaw PJ
影响因子：
12.7
作者：
Mackenzie, Ian R.;Arzberger, Thomas;Neumann, Manuela
通讯作者：
Neumann, Manuela