Ralf P. Friedrich

EGCG remodels mature α-synuclein and amyloid-β fibrils and reduces cellular toxicity

Protein misfolding and formation of β-sheet-rich amyloid fibrils or aggregates is related to cellular toxicity and decay in various human disorders including Alzheimer’s and Parkinson’s disease. Recently, we demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibits α-synuclein and amyloid-β fibrillogenesis. It associates with natively unfolded polypeptides and promotes the self-assembly of unstructured oligomers of a new type. Whether EGCG disassembles preformed amyloid fibrils, however, remained unclear. Here, we show that EGCG has the ability to convert large, mature α-synuclein and amyloid-β fibrils into smaller, amorphous protein aggregates that are nontoxic to mammalian cells. Mechanistic studies revealed that the compound directly binds to β-sheet-rich aggregates and mediates the conformational change without their disassembly into monomers or small diffusible oligomers. These findings suggest that EGCG is a potent remodeling agent of mature amyloid fibrils.

Small-molecule conversion of toxic oligomers to nontoxic β-sheet–rich amyloid fibrils

Several lines of evidence indicate that prefibrillar assemblies of amyloid-β (Aβ) polypeptides, such as soluble oligomers or protofibrils, rather than mature, end-stage amyloid fibrils cause neuronal dysfunction and memory impairment in Alzheimers disease. These findings suggest that reducing the prevalence of transient intermediates by small molecule-mediated stimulation of amyloid polymerization might decrease toxicity. Here we demonstrate the acceleration of Aβ fibrillogenesis through the action of the orcein-related small molecule O4, which directly binds to hydrophobic amino acid residues in Aβ peptides and stabilizes the self-assembly of seeding-competent, β-sheet-rich protofibrils and fibrils. Notably, the O4-mediated acceleration of amyloid fibril formation efficiently decreases the concentration of small, toxic Aβ oligomers in complex, heterogeneous aggregation reactions. In addition, O4 treatment suppresses inhibition of long-term potentiation by Aβ oligomers in hippocampal brain slices. These results support the hypothesis that small, diffusible prefibrillar amyloid species rather than mature fibrillar aggregates are toxic for mammalian cells.

Transcription factors Sox8 and Sox10 perform non-equivalent roles during oligodendrocyte development despite functional redundancy

Development of myelin-forming oligodendrocytes in the central nervous system is dependent on at least two members of the Sox family of high-mobility-group-containing transcription factors. Sox9 is involved in oligodendrocyte specification, whereas Sox10 is required for terminal differentiation. We show that oligodendrocytes in the spinal cord additionally express the highly related Sox8. In Sox8-deficient mice, oligodendrocyte development proceeded normally until birth. However, terminal differentiation of oligodendrocytes was transiently delayed at early postnatal times. Sox8-deficient mice thus exhibited a similar, but less severe phenotype than did Sox10-deficient mice. Terminal oligodendrocyte differentiation was dramatically delayed in Sox8-deficient mice with only a single functional Sox10 allele hinting at redundancy between both Sox proteins. This redundancy was also evident from the fact that Sox8 bound to naturally occurring Sox10 response elements, was able to form DNA-dependent heterodimers with Sox10 and activated Sox10-specific oligodendrocytic target genes in a manner similar to Sox10. However, Sox8 expression levels were significantly lower than those for Sox10. Resulting differences in protein amounts might be a main reason for the weaker impact of Sox8 on oligodendrocyte development and for unidirectional compensation of the Sox8 loss by Sox10.

Development of a lauric acid/albumin hybrid iron oxide nanoparticle system with improved biocompatibility

The promising potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various nanomedical applications has been frequently reported. However, although many different synthesis methods, coatings, and functionalization techniques have been described, not many core-shell SPION drug delivery systems are available for clinicians at the moment. Here, bovine serum albumin was adsorbed onto lauric acid-stabilized SPIONs. The agglomeration behavior, zeta potential, and their dependence on the synthesis conditions were characterized with dynamic light scattering. The existence and composition of the core-shell-matrix structure was investigated by transmission electron microscopy, Fourier transform infrared spectroscopy, and zeta potential measurements. We showed that the iron oxide cores form agglomerates in the range of 80 nm. Moreover, despite their remarkably low tendency to aggregate even in a complex media like whole blood, the SPIONs still maintained their magnetic properties and were well attractable with a magnet. The magnetic properties were quantified by vibrating sample magnetometry and a superconducting quantum interference device. Using flow cytometry, we further investigated the effects of the different types of nanoparticle coating on morphology, viability, and DNA integrity of Jurkat cells. We showed that by addition of bovine serum albumin, the toxicity of nanoparticles is greatly reduced. We also investigated the effect of the particles on the growth of primary human endothelial cells to further demonstrate the biocompatibility of the particles. As proof of principle, we showed that the hybrid-coated particles are able to carry payloads of up to 800 μg/mL of the cytostatic drug mitoxantrone while still staying colloidally stable. The drug-loaded system exhibited excellent therapeutic potential in vitro, exceeding that of free mitoxantrone. In conclusion, we have synthesized a biocompatible ferrofluid that shows great potential for clinical application. The synthesis is straightforward and reproducible and thus easily translatable into a good manufacturing practice environment.

The Class III POU Domain Protein Brn-1 Can Fully Replace the Related Oct-6 during Schwann Cell Development and Myelination

ABSTRACT For differentiation, Schwann cells rely on the class III POU domain transcription factor Oct-6, which is expressed transiently when Schwann cells have established a one-to-one relation with axons but have not yet started to myelinate. Loss of Oct-6 leads to a transient arrest in this promyelinating stage and a delay in myelination. Although the closely related POU domain protein Brn-2 is coexpressed with Oct-6 in Schwann cells, its loss has only mild consequences. Combined loss of both POU domain proteins, in contrast, dramatically increases the myelination delay, raising the question of how related POU domain proteins compare to each other in their activities. Here, we have replaced Oct-6 expression in the mouse with expression of the class III POU domain protein Brn-1. Although this protein is not normally expressed in Schwann cells, Brn-1 was capable of fully replacing Oct-6. Brn-1 efficiently induced Krox-20 expression as a prerequisite for myelination. Onset and extent of myelination were also indistinguishable from that of the wild type in mice that carried only Brn-1 instead of Oct-6 alleles. Similar to Oct-6, Brn-1 down-regulated its own expression at later stages of myelination. Thus, class III POU domain proteins can fully replace each other in Schwann cell development.

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEONLA) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEONLA with an additional protein corona formed by bovine serum albumin (SEONLA-BSA) and commercially available Rienso® particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products.

670 nm Laser Light and EGCG Complementarily Reduce Amyloid-β Aggregates in Human Neuroblastoma Cells: Basis for Treatment of Alzheimer's Disease?

OBJECTIVE The aim of the present study is to present the results of in vitro experiments with possible relevance in the treatment of Alzheimers disease (AD). BACKGROUND DATA Despite intensive research efforts, there is no treatment for AD. One root cause of AD is the extra- and intracellular deposition of amyloid-beta (Aβ) fibrils in the brain. Recently, it was shown that extracellular Aβ can enter brain cells, resulting in neurotoxicity. METHODS After internalization of Aβ(42) into human neuroblastoma (SH-EP) cells, they were irradiated with moderately intense 670-nm laser light (1000 Wm(-2)) and/or treated with epigallocatechin gallate (EGCG). RESULTS In irradiated cells, Aβ(42) aggregate amounts were significantly lower than in nonirradiated cells. Likewise, in EGCG-treated cells, Aβ(42) aggregate amounts were significantly lower than in non-EGCG-treated cells. Except for the cells simultaneously laden with Aβ(42) and EGCG, there was a significant increase in cell numbers in response to laser irradiation. EGCG alone had no effect on cell proliferation. Laser irradiation significantly increased ATP levels in Aβ(42)-free cells, when compared to nonirradiated cells. Laser-induced clearance of Aβ(42) aggregates occurred at the expense of cellular ATP. CONCLUSIONS Irradiation with moderate levels of 670-nm light and EGCG supplementation complementarily reduces Aβ aggregates in SH-EP cells. Transcranial penetration of moderate levels of red to near-infrared (NIR) light has already been amply exploited in the treatment of patients with acute stroke; the blood-brain barrier (BBB) penetration of EGCG has been demonstrated in animals. We hope that our approach will inspire a practical therapy for AD.

Nanoparticles size-dependently initiate self-limiting NETosis-driven inflammation

Significance The current widespread exposure of humans to natural as well as man-made nanomaterials due to the deployment of nanoparticles (NPs) as food additives, as vaccine- or drug-delivery vehicles, and in diagnostic procedures encourages the evaluation of their interaction with the innate immune system. Understanding how organisms cope with hydrophobic and chemically inert particulate matter, which is excluded from metabolic processing, is of major importance for interpreting the responses associated with the use of NPs in the biosphere. The containment of NPs within neutrophil-derived aggregates locally orchestrates the resolution of inflammation. Overriding this mechanism bears the risk of inducing chronic inflammation and causing tissue damage. The critical size for strong interaction of hydrophobic particles with phospholipid bilayers has been predicted to be 10 nm. Because of the wide spreading of nonpolar nanoparticles (NPs) in the environment, we aimed to reveal the ability of living organisms to entrap NPs via formation of neutrophil extracellular traps (NETs). Upon interaction with various cell types and tissues, 10- to 40-nm-sized NPs induce fast (<20 min) damage of plasma membranes and instability of the lysosomal compartment, leading to the immediate formation of NETs. In contrast, particles sized 100–1,000 nm behaved rather inertly. Resulting NET formation (NETosis) was accompanied by an inflammatory reaction intrinsically endowed with its own resolution, demonstrated in lungs and air pouches of mice. Persistence of small NPs in joints caused unremitting arthritis and bone remodeling. Small NPs coinjected with antigen exerted adjuvant-like activity. This report demonstrates a cellular mechanism that explains how small NPs activate the NETosis pathway and drive their entrapping and resolution of the initial inflammatory response.

Pharmaceutical formulation of HSA hybrid coated iron oxide nanoparticles for magnetic drug targeting

In this work we present a new formulation of superparamagnetic iron oxide nanoparticles (SPIONs) for magnetic drug targeting. The particles were reproducibly synthesized from current good manufacturing practice (cGMP) - grade substances. They were surface coated using fatty acids as anchoring molecules for human serum albumin. We comprehensively characterized the physicochemical core-shell structure of the particles using sophisticated methods. We investigated biocompatibility and cellular uptake of the particles using an established flow cytometric method in combination with microwave-plasma assisted atomic emission spectroscopy (MP-AES). The cytotoxic drug mitoxantrone was adsorbed on the protein shell and we showed that even in complex media it is slowly released with a close to zero order kinetics. We also describe an in vitro proof-of-concept assay in which we clearly showed that local enrichment of this SPION-drug conjugate with a magnet allows site-specific therapeutic effects.

Identification of Human Proteins That Modify Misfolding and Proteotoxicity of Pathogenic Ataxin-1

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.