1. Nanoparticles loaded with proteasomes, protein complexes that degrade defective proteins, were nontoxic and readily taken up by cells in vitro.
2. Delivered proteasomes promoted targeted degradation of toxic tau proteins implicated in Alzheimer’s disease.
Evidence Rating Level: 3 (Average)
Study Rundown: In Alzheimer’s disease and other neurodegenerative conditions, reduced proteasome activity in neurons is linked to the toxic accumulation of proteins such as tau. Reducing the buildup of such toxic proteins may serve as a way to decrease proteotoxic stress and lesion formations associated with Alzheimers. This study demonstrated the feasibility of introducing proteasomes into cells as a means to decrease levels of tau protein and tau protein aggregates. Silica nanoparticles were designed with surface pores large enough to each contain a proteasome molecule. Proteasome-loaded nanoparticles were nontoxic and taken up by cells via endocytosis, a standard pathway of molecule transport into the cell. Proteasomes retained function when bound to nanoparticles, carrying out reactions to break down other molecules at a comparable rate to free proteasomes. Interestingly, the proteasomes specifically degraded tau that was overexpressed at aberrant levels in the cell, but did not act on protein targets present at normal levels, including tau when not overexpressed. Cells under tau stress treated with the complexes had significantly reduced tau aggregation and tau-induced cytotoxicity.
The nanoparticle delivery system described in this study represented a novel method of transporting large, complex proteins into the cell. This effective strategy of targeting tau made use of cellular degradation machinery; however, the proteasomes used for delivery were purified from cells and the limitations of such an involved process will need to be addressed before the technology can be widely implemented. With further development and testing beyond cell culture models, nanoparticle delivery of proteasomes has potential as a therapy to treat Alzheimer’s disease and other conditions associated with protein aggregation.
In-Depth [in vitro study]: Nonantigenic silica nanoparticles were synthesized with an approximate diameter of 200nm and average pore size of 28nm. Proteasomes were purified from a HEK293 human cell line and loaded into the nanoparticle pores. Proteasome loading reached saturation at a 1:50 proteasome-to-nanoparticle mass ratio, which corresponded to approximately 37 proteasome molecules per nanoparticle. As measured by the Michaelis constant for enzyme kinetics, the degradation activity of nanoparticle-bound proteasomes (KM=44.4μM) was similar to that of free proteasomes (KM=40.2). Cell uptake of the loaded nanoparticles, measured using microscopy and flow cytometry of fluorescently labeled particles, was mediated by endocytosis. The proteasome-nanoparticle complex did not affect cell viability.
The ability of the delivered proteasomes to promote tau degradation was assessed in a HEK239-derived cell line. In the specific cell line used, overexpression of tau protein was induced by doxycycline. Such overexpression was associated with the formation of tau protein aggregates. Treatment with proteasome-loaded nanoparticles decreased tau levels in a dose-dependent fashion, and degraded tau significantly more than treatment with nanoparticles alone (p<0.01). Testing of various proteasome targets and expression levels showed that delivered proteasomes specifically degraded overexpressed proteins. Degradation activity was not observed for cells with normal levels of Nrf2, p53, or tau. In contrast, degradation activity was observed for cells with overexpressed levels of GFPu and GFP-cODC.
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