Research Article: Tyrosine-mutated AAV2-mediated shRNA silencing of PTEN promotes axon regeneration of adult optic nerve

Date Published: March 21, 2017

Publisher: Public Library of Science

Author(s): ZhengRu Huang, ZiZhong Hu, Ping Xie, QingHuai Liu, Steven Barnes.

http://doi.org/10.1371/journal.pone.0174096

Abstract

Activating PI3K/AKT/mTOR signaling pathway via deleting phosphatase and tensin homolog (PTEN) has been confirmed to enhance intrinsic growth capacity of neurons to facilitate the axons regeneration of central nervous system after injury. Considering conditional gene deletion is currently not available in clinical practice, we exploited capsid residue tyrosine 444 to phenylalanine mutated single-stranded adeno-associated virus serotype 2 (AAV2) as a vector delivering short hairpin RNA to silence PTEN to promote retinal ganglion cells (RGCs) survival and axons regeneration in adult rat optic nerve axotomy paradigm. We found that mutant AAV2 displayed higher infection efficiency to RGCs and Müller cells by intravitreal injection, mediated PTEN suppression, resulted in much more RGCs survival and more robust axons regeneration compared with wild type AAV2, due to the different extent of the mTOR complex-1 activation and glutamate aspartate transporter (GLAST) regulation. These results suggest that high efficiency AAV2-mediated PTEN knockdown represents a practicable therapeutic strategy for optic neuropathy.

Partial Text

Like other mammalian mature central nerve system (CNS) neurons, retinal ganglion cells (RGCs) are normally unable to regenerate axons spontaneously after optic nerve injury, which causes irreversible vision loss. The failure of axons regeneration has been attributed to the apoptosis of RGCs, insufficient intrinsic growth capacity of mature neurons, lack of suitable stimuli, and inhibitory extracellular environment [1–2]. In the past decade, many evidences have supported that activating the intrinsic growth capacity is able to induce a robust regenerative response in mature axotomized RGCs [3–4]. Deletion of phosphatase and tensin homolog (PTEN), which is a negative regulator of mammalian target of rapamycin (mTOR), have been demonstrated to wake up the regenerative ability of adult corticospinal neurons and RGCs [5–6]. However, conditional gene deletion is currently impossible to translate to clinical practice, while therapies based on small-interfering RNA (siRNA) to knockdown target genes may be the most useful strategy for the treatment of optic neuropathy [7].

In this study, we demonstrated the much higher transduction efficiency of Y444F ssAAV2 to RGCs, especially to Müller cells compared to Wt ssAAV2. With Y444F ssAAV2 mediated shRNA targeting PTEN, RGCs survival and axons regeneration was significantly induced through activating PTEN/mTOR pathway and regulating the expression of GLAST after ONA in rats.

 

Source:

http://doi.org/10.1371/journal.pone.0174096

 

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