Identification of new kinase clusters required for neurite outgrowth and retraction by a loss-of-function RNA interference screen
Identification of new kinase clusters required for neurite outgrowth and retraction by a loss-of-function RNA interference screen
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DOI：
10.1038/sj.cdd.4402258
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发表时间：
2008-02-01
影响因子：
12.4
通讯作者：
Nicotera, P.
中科院分区：
文献类型：
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作者：
Loh, S. H. Y.;Francescut, L.;Nicotera, P.
突触完整性的破坏、连接性的丧失和轴突变性是神经变性的早期和基本的组成部分。虽然神经细胞死亡机制已被彻底研究，但对轴突损伤所涉及的信号和再生过程知之甚少。在这里，我们进行了基于全基因组RNA干扰的正向遗传筛查，使用针对所有人类激酶的小干扰RNA，并确定了对生长锥体塌陷、轴突回缩和轴突生长至关重要的激酶家族簇。在59个被确认为神经突起生长的正调控因子中，近50%属于TK/TKL受体亚群，这突显了细胞外配体在这一过程中的重要性。66个其他激酶抑制轴突生长，其中没有一个是TK/TKL成员，而79个激酶抑制溶血磷脂酸诱导的轴突回缩。这两个抑制过程中都有20个激酶参与，这表明它们有共同的机制。在这组20个激酶中，一些(ULK1、PDK1、MAP4K4)以前已经与轴突事件有关，而另一些(MAST2、FASTK、CKM和DGUOK)则没有。对于一组激酶，在大鼠小脑原代培养中验证了对轴突生长的影响。在轴突损伤模型中，使用原代大鼠中脑培养物进一步测试了影响再生的能力。最后，我们证明了AGC激酶亚群中两个成员ROCK1和PKN1的单倍性不足能够抑制III类常染色体显性遗传性视网膜色素变性果蝇模型的视网膜退化。
Disruption of synaptic integrity, loss of connectivity and axodendritic degeneration are early and essential components of neurodegeneration. Although neuronal cell death mechanisms have been thoroughly investigated, less is known about the signals involved in axodendritic damage and the processes involved in regeneration. Here we conducted a genome-wide RNA interference-based forward genetic screen, using small interfering RNA targeting all human kinases, and identified clusters of kinases families essential for growth cone collapse, neurite retraction and neurite outgrowth. Of 59 kinases identified as positive regulators of neurite outgrowth, almost 50% were in the tyrosine kinase/tyrosine kinase-like (TK/TKL) receptor subgroups, underlining the importance of extracellular ligands in this process. Neurite outgrowth was inhibited by 66 other kinases, none of which were TK/TKL members, whereas 79 kinases inhibited lysophosphatidic acid-induced neurite retraction. Twenty kinases were involved in both inhibitory processes suggesting shared mechanisms. Within this group of 20 kinases, some (ULK1, PDK1, MAP4K4) have been implicated previously in axonal events, but others (MAST2, FASTK, CKM and DGUOK) have not. For a subset of kinases, the effect on neurite outgrowth was validated in rat primary cerebellar cultures. The ability to affect regeneration was further tested in a model of axodendritic lesion using primary rat midbrain cultures. Finally, we demonstrated that haploinsufficiency of two members of the AGC kinase subgroup, ROCK1 and PKN1, was able to suppress retinal degeneration in Drosophila model of class III Autosomal Dominant Retinitis Pigmentosa.