中文 |

Research Progress

Scientists Reveal the Role of Autophagy in Regulating Axonal Development

Aug 30, 2017

"Autophagy", namely "self-eating", is a conserved physiological pathway to degrade and recycle senescent organelle and packaged endosome. The vesicle with multiple membranes called "autophagosome" is the most typical structure of autophagy. With packaging and delivery of autophagosome, cargos in endosome have access to lysosome for degradation.

Although neurons were applied in observing and identifying ultrastructure of autophagy early in 1970s, it is still elusive of how this critical pathway participates in regulation of neural development, neural degeneration and regeneration of injured axon. Basic and clinical research on neural autophagy as well as constructing autophagy family genes oriented animal model has become a hot topic in both neural science and medical science.

A recent study performed by researchers from Dr. ZHOU Yuxun's team at the Donghua University and Dr. QIU Zilong's Lab at the Institute of Neuroscience of Chinese Academy of Sciences demonstrated that Mir505-3p regulated axonal development by targeting Atg12 and autophagy pathway. They successfully constructed a mouse model deleting Mir505 gene based on CRISPR/Cas9 system, and showed that Mir505-3p negatively regulated autophagy to promote axonal development by using in utero electroporation and transmission electron microscopy. The study was published in Autophagy.

In a previous study, Dr. ZHOU's team discovered a quantitative trait loci which controls mouse puberty onset, and contains Mir505 gene, a potential regulator of this physiological event. Another study showed that the Mir505 located in a quantitative trait loci relative to human X-linked hypopituitarism. As the essence of puberty onset and hypopituitarism is neural development event, researchers attempted to check whether Mir505 influences neural development.

They first used cultured cortical neuron model to test whether Mir505 is involved in neural development. They found that the Mir505-3p, one of a mature form of Mir505 gene, specifically promoted polarity establishment, axonal growth and axonal branching with no obvious influence on dendrite. Another mature form of Mir505 gene was not responsible for these phenomena. Researchers observed the similar phenotypes in cortical neuron by adopting in utero electroporation.

By integrating results from bioinformatics, RNA sequencing and dual-luciferase assays, researchers screened out a direct target gene of Mir505-3p in neuron, Atg12. Atg12 was a key component of autophagy pathway that encoded ATG12 protein which interacted with ATG5 and ATG16L1, to form a protein complex and to regulate early initiation and elongation of autophagosome.

To investigate the role of Atg12 in regulating neuron development, researchers over-expressed Mir505-3p and Atg12 with lipofection or electroporation in cultured neurons. They found that Atg12 inhibited neuron polarity establishment, axonal growth and axonal branching, which were opposite to the effects of Mir505-3p. Besides, co-expression Mir505-3p rescued Atg12s effects on axonal development, indicating the inhibitory role of Atg12 in axonal development. 

Also, researchers observed that Atg12 rescued promotion effects on axonal growth and branching in both the ipsilateral side and the contralateral side of cortical plate caused by Mir505-3p, further demonstrating the inhibitory role of Atg12 on axonal development in vivo. 

To examine whether the Mir505-3p is required for mouse axonal development in vivo, they constructed Mir505 gene specific knock-out mice based on CRISPR/Cas9 system. Evidence of HE staining, toluidine blue staining and immunofluorescence staining showed that the density of axonal bundle was decreased in corpus callosum (cc), cingulum (cg) and fimbria (fi) regions in Mir505 KO mice, comparing to that of WT mice. However, no difference in the cell number of RBFOX3-positive neurons was observed, indicating that attenuation of axons resulted from defects in axonal development, rather than neuronal proliferation.

By utilizing this mouse model, researcher observed obvious alterations of autophagosome in both cultured cortical neuron and mouse cortex tissue. In Mir505 KO mice, the number and size of autophagosome were drastically decreased, whereas the number of mitochondria was increased. These results showed that deleting Mir505-3p led to general activation of autophagy pathway and Mir505-3p was required for maintaining normal autophagic flow.

The axonal development was a high-energy-demanding process, which required mitochondria that were available throughout the neuron, especially the axon, to provide a large amount of ATP. Local distribution and mobility of mitochondria in axons were critical to axonal growth and branching. The observation from transmission electron microscopy revealed that Mir505-3p finally influenced mitochondria number in axon via regulating Atg12/autophagy pathway, which resulted in promotion of axonal development. Finally, researchers showed that Mir505-3p generally regulated autophagy and mitochondria number by targeting Atg12 in primary MEF cells.

In this project, ZHOU's team and QIU's Lab established a Mir505 KO mouse model and revealed novel functions of Mir505-3p/Atg12/autophagy/mitochondria pathway on regulating axonal development both in vitro and in vivo, by using cultured cortical neuron, in utero electroporation and transmission electron microscopy. Taken together, this work provided another evidence supporting the role of autophagy in neural development.

Contact Us
  • 86-10-68597521 (day)

    86-10-68597289 (night)

  • 86-10-68511095 (day)

    86-10-68512458 (night)

  • cas_en@cas.cn

  • 52 Sanlihe Rd., Xicheng District,

    Beijing, China (100864)

Copyright © 2002 - Chinese Academy of Sciences