Elsevier

Biomaterials

Volume 216, September 2019, 119248
Biomaterials

MnFe2O4 nanoparticles accelerate the clearance of mutant huntingtin selectively through ubiquitin-proteasome system

https://doi.org/10.1016/j.biomaterials.2019.119248Get rights and content

Abstract

Neurodegenerative disorders such as Huntington's disease (HD) are fundamentally caused by accumulation of misfolded aggregate-prone proteins. Previous investigations have shown that these toxic protein aggregates could be degraded through autophagy induced by small molecules as well as by nanomaterials. However, whether engineered nanomaterials have the capacity to degrade these protein aggregates via the ubiquitin-proteasome system (UPS), the other major pathway for intracellular protein turnover, was unknown. Herein, we have synthesized biocompatible MnFe2O4 nanoparticles (NPs) and demonstrated their unique effect in accelerating the clearance of mutant huntingtin (Htt) protein exhibiting 74 glutamine repeats [Htt(Q74)]. UPS, rather than autophagy, was responsible for the efficient Htt(Q74) degradation facilitated by MnFe2O4 NPs. Meanwhile, we demonstrated that MnFe2O4 NPs enhanced K48-linked ubiquitination of GFP-Htt(Q74). Moreover, ubiqinlin-1, but not p62/SQSTM1, served as the ubiquitin receptor that mediated the enhanced degradation of Htt(Q74) by MnFe2O4 NPs. Our findings may have implications for developing novel nanomedicine for the therapy of HD and other polyglutamine expansion diseases.

Introduction

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by misfolded huntingtin (Htt) protein exhibiting more than 35 CAG trinucleotide repeat expansion encoding polyglutamine (polyQ) in the N-terminal region [1]. Multiple lines of evidence implicate that accumulation of N-terminal aggregated mutant Htt fragments plays a crucial role in HD pathogenesis, represented by a toxic gain-of-function in neurons, thereby resulting in several symptoms including short-term memory damage, motor dysfunction, which eventually lead to death within 10–20 years [2]. Especially, the severity of HD is positively correlated with abnormal polyQ expansion, as earlier and more severe symptoms are commonly observed in patients with an increasing polyQ length [3]. Despite a tremendous effort, effective treatments for HD are still absent so far. Whereas, it is recently emphasized that decreasing the intracellular abundance of polyQ expansion, either through synthesis inhibition or clearance promotion, would be a promising therapeutic approach for HD [4,5].

The two main routes responsible for intracellular protein clearance in mammalian cells are the macroautophagy (hereafter simply referred to as autophagy)-lysosomal pathway (ALP) together with the ubiquitin (Ub)-proteasome system (UPS). They coordinately degrade damaged organelles, cytoplasmic or nuclear protein complexes, misfolded or unfolded proteins and also protein aggregates [6], depending on the ubiquitinated cargos waiting for degradation were conjugated with K48-linked or K63-linked poly-Ub chains [7]. Specifically, mutant Htt fragments with polyQ expansion can be degraded either by ALP or UPS. From the past decades, several small chemical compounds such as rapamycin, lithium, trehalose, rilmenidine, N10-substituted phenoxazine etc., have been proposed for polyQ clearance due to their autophagy-inducing abilities in neuronal cells [8]. Meanwhile, the GABAB receptor agonist Baclofen and the adenosine A2A receptor agonists Gastrodia elata, N6-(4-Hydroxybenzyl) and N6-(3-Indolylethyl) adenosines were reported to enhance the UPS function for reducing the protein levels of polyQ expansion [9,10].

During the past decades, with the bioengineering advancement and fine-tuning surface modifications, various kinds of nanomaterials have been potentially applied to medicine in preventing infection [11], facilitating rheumatoid arthritis treatment [12], targeted oncogene editing [13] and especially cancer imaging and therapy [[14], [15], [16], [17], [18]]. Recently, bioengineered nanocarriers have been applied for neurodegenerative diseases therapies based on stem cells such as neural stem cells (NSCs), a pivotal reservoir for self-renewing and differentiating into functional astrocytes, oligodendrocytes or neurons in response to specific stimuli [[19], [20], [21], [22]]. For instance, poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) were capable of facilitating the curcumin transport through the blood-brain barrier without compromising its integrity, consequently inducing neurogenesis and reversing learning and memory impairments [23]. Similarly, injectable gelatin-hydroxyphenylpropionic acid hydrogel and dextran sulfate/chitosan polyelectrolyte complex NPs were applied to load with stromal cell-derived factor-1α (SDF-1α) and effectively recruited endogenous NSCs for enhancing neural tissue regeneration [24]. Besides, the miR-124 loaded polymeric NPs containing perfluoro-1,5-crown ether (PFCE) and coated with protamine sulfate could be efficiently internalized by NSCs and neuroblasts, further promoted their neuronal commitment, maturation and eventually ameliorated motor symptoms [25]. Although nanocarriers have been applied to stem cells-mediated treatments of neurodegenerative diseases, the attempts to explore the intrinsic bioactivities of nanomaterials for effective HD therapy, were seldom reported [19]. Of the few investigations, CeO2 nanoparticles have been proven to activate ALP without inducing apoptotic cytotoxicity and could promote toxic aggregates clearance in disease cells [26]. Meanwhile, our group has revealed that europium hydroxide [EuIII(OH)3] nanorods and graphene oxide (GO) could accelerate the ALP-mediated clearance of mutant Htt through MEK/ERK1/2 signaling pathway activation [5,27,28]. As mentioned above, although mutant Htt could be degraded through ALP or UPS, no any nanomaterials have been addressed to facilitate mutant Htt clearance via UPS.

As an important superparamagnetic material, MnFe2O4 has already been shown to own excellent performance in biomedical applications including bioseparation, magnetic resonance imaging and localized hyperthermia induction [[29], [30], [31], [32], [33]]. Besides the traditional magnetic-based properties, the MnFe2O4 nanoparticles also have some intrinsic bioactivities, such as O2-evolving in tumor, warfarin detection in biofluids, as well as oxidase and DNA nuclease mimic activity [[34], [35], [36], [37]]. Inspired by these unique biological effects, it is of interest to explore the bioactivity of MnFe2O4 for other important biomedical applications such as Huntington's disease therapy.

In the present work, we have synthesized MnFe2O4 NPs by a simple method and demonstrated their excellent biocompatibility. Subsequently, we have introduced the neuronal cell line Neuro 2A stably expressing GFP-tagged mutant Htt aggregates with 74 polyQ tracts (mHtt-GFP-Q74) as an experimental model for assessment of the potential clearance efficacy of MnFe2O4 NPs. Later on, we focused on elucidating whether ALP or UPS has been involved in the clearance process. Finally, we attempted to explore the underlying mechanism by exploring the critical receptors for selective delivery of mHtt-GFP-Q74 to enter ALP or UPS (Fig. 1).

Section snippets

Materials

Ferric chloride hexahydrate, Manganese(II) chloride tetrahydrate and ammonium hydroxide were purchased from Sinopharm Chemical Reagent (Shanghai, China). Dextran, thiazolyl blue tetrazolium bromide (MTT, A600799) and G418 (B540723) were obtained from Sangon Biotech. (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM, 11995040), penicillin and streptomycin were purchased from Gibco (15140122, Thermo Fisher Scientific, Waltham, MA, USA), fetal bovine serum (FBS) was purchased from

Preparation and characterization of MnFe2O4 NPs

The obtained MnFe2O4 NPs were fully dispersed in water and displayed a brown appearance (Fig. 2a). According to the transmission electron microscope (TEM) image, the MnFe2O4 NPs presented irregular shapes, assembled by ultra-small nanocrystals which coated with dextran (Fig. 2b). The sizes of the NPs were mostly in the range of 30–150 nm revealed by the measurement of dynamic light scattering (Fig. 2c). The diffraction peak in X-ray diffraction (XRD) pattern was weak and broad, indicating the

Discussion

The traditional superparamagnetic MnFe2O4 nanoparticle has shown newly identified intrinsic bioactivities, such as O2-evolving in tumor and DNA nuclease mimic activity [34,37], promoting the further exploration of its biomedical application. In the present work, we have synthesized MnFe2O4 NPs by coating with dextran as surfactant (Fig. 2) and demonstrated their excellent biocompatibility (Fig. 3a, and b). Interestingly, we have found the MnFe2O4 NPs effectively enhanced the clearance of 74

Conclusion

In summary, MnFe2O4 NPs were successfully synthesized by a simple method and presented excellent biocompatibility. Importantly, MnFe2O4 NPs could accelerate the clearance of Htt protein exhibiting 74 CAG trinucleotide repeat expansion encoding polyglutamine [Htt(Q74)]. Although autophagy-lysosomal pathway (ALP) has been previously reported to be tightly linked with misfolded Htt(Q74) clearance, and MnFe2O4 NPs could induce autophagy in a dose- and time-dependent manner, however, the enhanced

Conflicts of interest

The authors declare no competing financial interest.

Data availability

The data that support this study are available within the article and its Supplementary data files or available from the corresponding authors upon request.

Author contributions

J. X., C.-Z. L. and L.-P. W. conceived the idea and supervised the project. L. Z., P.-F. W. and Y.-H. S. planned and performed the experiments, collected and analyzed the data. L. D., Y.-D. W, Z.-Y. H., S. F., S. T. and J.-L. M. assisted with the experiments and characterizations. L. Z., P.-F. W., Y. L., J. X., C.-Z. L. and L.-P. W. co-wrote the manuscript. All authors discussed the results and commented on the manuscript.

Acknowledgements

The authors gratefully acknowledge Prof. David C. Rubinsztein (Cambridge Institute for Medical Research, University of Cambridge, UK) for providing GFP-Htt(Q74) plasmid. The presented research was financially supported by the National Natural Science Foundation of China (81401518, 31430028, 81601600, 81630019, 21701165, 51572067, 21501039, 81701823), the China Postdoctoral Science Foundation (2016M590576, 2017T100455, 2017M612079), Scientific Research Fundation of the Institute for

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