Autophagy-mediated Clearance Of Ubiquitinated Mutant Huntingtin By Graphene Oxide

Many of the neurodegenerative disorders such as Huntington’s disease (HD) are caused by the accμmulation of intracytoplasmic aggregate-prone proteins. Metabolic system in eukaryocyte is composed of ubiquitin-proteasome system and autophagy-lysosomal pathway. As the protein aggregates in neurodegenerative disorder cells are too large to gain access to the proteasome cavities. So these aggregate-prone proteins are mainly degraded by autophagy, thus elevating the autophagy level to enhance degradation of these proteins represents an emerging viable approach for the treatment of neurodegenerative diseases. C60 fullerene based nanoparticles can elicite autophagy. combining its antioxidative activities, this nanoparticles could protect cells from Aβ damage. Otherwise, it was reported that single-walled carbon nanotubes (SWNT) could restored the impaired autophagy levels in Alzheimer’s disease (AD) cells, and thus facilitate the degradation of autophagic substrates. As one of the carbon-based nano-materials, graphene oxide have been extensively used for its advantages, such as biocompatibility, ultrahigh surface to volμme ratio, abundant surface groups and special photothermal effect. In this report we showed that graphene oxide (GO) could effectively enhanced the clearance of mutant huntingtin, the aggregate-prone protein underlying the pathogenesis of HD.Firstly, a modified Hummers method was used to prepare GO from natural graphite and use a series of method to characterizate in detail, such as, atomic force microscopy (AFM), Ultraviolet-Visible (UV-VIS) spectroscopy and FTIR spectra. As reported prevously, GO have little toxity to cells. Next western blot assay and fluorescence microscopy image all prove graphene oxide (GO) could effectively enhanced the clearance of mutant huntingtin aggregate. Besides we found that, this enhancing effect of GO was autophagy-mediated and proteasome-independent, as blocking autophagy by chemical inhibitors at either the autophagosome formation stage or the autophagosome-lysosome fusion stage, or more specifically by knocking-down an essential autophagy gene, led to a significant reduction in the ability of GO to enhance huntingtin protein degradation, but proteasome inhibitor could not effect this degradation. Furthermore, we detected the autophagy caused by GO comprehensively and systematacially. Interestingly, the autophagy induced by GO had the normal capacity to degrade its cargo including LC3-Ⅱ, GFP-LC3-Ⅱ and huntingtin proteins, but did not cause degradation of p62/SQSTM1, a well-known autophagic substrate and an often-used marker for assessing the cargo-degrading capacity of autophagy. However, the increase of free GFP and autophagosome certified that, autophagy flux caused by GO was complete. Moreover we illustrated the mechanism of autophagy triggered by GO. We detected that as another molecular in beclinel complex, PtdIns3K possessed the same impotant role as Beclinel.Also, this autophay pathway was not mTOR mediated. In addition, we discovered the phosphorylation level of ERKwas rised obviously, and its inhibitor could also inhibite autophagy induced by GO. From the above, GO induced autophagy was in a mTOR-independent and ERK-dependent pathway.Finally, we explored the mechanism of this phenomenon caused by graphene oxide. We found that graphene oxide could elevate ubiquitin level in the treated cells. Immunoprecipitation and binding assay showed that ubiquitinated huntingtin protein preferentially binds to GO, the same with HD protein aggragates. The appearances of abundant GO in autophagosomes and autolysosomes, raised the possibility that GO may directly deliver the bound protein to autophagosomes for degradation. Our results revealed a novel biological function of GO and may have implications for developing nanomaterial-based therapeutics for neurodegenerative diseases.