摘要
第一部分: LINGO-1与WNK1分子相互作用在调节Nogo抑制轴突延长中的功能研究
LINGO-1是一个三元复合受体的一个组分,这个三元受体复合物作为三种髓鞘相关抑制因子的共有受体介导跨膜信号转导,导致生长锥塌陷及抑制神经突起延长。尽管对LINGO-1分子的功能已进行了广泛研究,但其下游信号转导仍未明确。本研究通过酵母双杂交筛选发现LINGO-1可与一种丝苏氨酸激酶WNK1分子发生结合。并通过活细胞内FRET和脑组织裂解液免疫共沉淀检测进一步得到验证,且这种结合在Nogo66处理时会增强。形态学结果提示WNK1与LINGO-1共存于皮层神经元内。进一步研究发现,通过RNA干扰抑制WNK1表达或过表达WNK1(123-510)均可削弱Nogo66引起的突起延长抑制并抑制RhoA激活。此外,通过免疫共沉淀发现WNK1与RhoGDI在脑组织内结合,且这种结合在Nogo66处理时会减弱,进一步提示其对RhoA激活的调节作用。
综上,我们的研究结果提示WNK1是一个新的参与调制LINGO-1介导的神经突起延长的信号转导分子。这为深入探讨LINGO-1的生物学功能及其机制提供了新思路。
第二部分WNK3激酶受LINGO-1的负性调控介导Nogo诱导的神经元的凋亡摘要
神经元的凋亡不论对神经系统生理发育还是在损伤后或神经退行性疾病过程中的病理变化方面都发挥着举足轻重的作用。神经系统特异性跨膜蛋白LINGO-1,已经被充分证明在调节突起延伸和再生中发挥负性调控作用,最近又提出它还在神经元的凋亡中发挥着作用,但是其确切的功能和内在机制仍旧不明。在此我们提出LINGO-1与丝苏氨酸激酶WNK3在神经系统内相互作用,而后者在体外和在体试验中表现出对神经元的凋亡发挥着保护作用,呈激酶依赖方式。值得关注的是,LINGO-1的激活,不论在配体水平还是在受体水平,都足以启动神经元的凋亡,而且同时WNK3的磷酸化也会受到抑制,因此提出了MAIFs诱导神经元的凋亡的另一种新的机制——LINGO-1的激活介导细胞凋亡发生的增加是由于抑制了WNK3激酶的活性。
Part One LINGO-1 interacts with WNK1 to regulate Nogo-induced inhibition of neurite extension
LINGO-1 is a component of the tripartite receptor complexes, which act as a convergent mediator of the intracellular signaling in response to myelin-associated inhibitors and lead to collapse of growth cone and inhibition of neurite extension. Although the function of LINGO-1 has been intensively studied, its downstream signaling remains elusive. In the present study, a novel interaction between LINGO-1 and a serine-threonine kinase WNK1 was identified by yeast two-hybrid screen. The interaction was further validated by fluorescence resonance energy transfer and co-immunoprecipitation, and this interaction was intensified by Nogo66 treatment. Morphological evidences showed that WNK1 and LINGO-1 were co-localized in cortical neurons. Furthermore, either suppressing WNK1 expression by RNA interference or overexpression of WNK1(123-510) attenuated Nogo66-induced inhibition of neurite extension and inhibited the activation of RhoA. Moreover, WNK1 was identified to interact with Rho-GDI1, and this interaction was attenuated by Nogo66 treatment, further indicating its regulatory effect on RhoA activation.
Taken together, our results suggest that WNK1 is a novel signaling molecule involved in regulation of LINGO-1 mediated inhibition of neurite extension.
Part Two WNK3 kinase was negatively regulated by LINGO-1in Nogo-induced neuronal apoptosis
Neuronal apoptosis has an impactful role in neuronal system physiology during development and pathology after injury or during degenerative diseases. The neuronal system specific transmembrane protein LINGO-1, which has been well documented in negative regulating in neurite extension and regeneration, nowadays was proposed also has an role in neuronal apoptosis, however, its certain role and the underlied mechanism is still vague. Here we proposed LINGO-1 interacts with the serine-threonine kinase WNK3 in neuronal system, and the latter exhibits a protective role in neuronal apoptosis in vitro and in vivo during development in a kinase-dependent manner. In particular, LINGO-1 activation, either at a ligand level or an receptor level, would be sufficient to initiate neuronal apoptosis and at the same time WNK3 phosphoralation is also down-regulated, therefore provid an alternative mechanism for LINGO-1 activation-mediated elevation in apoptotic cell death, that is, by inhibition of WNK3 kinase activity. .
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