mHTT Seeding Activity: A Marker of Disease Progression and Neurotoxicity in Models of Huntington's Disease.

Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). Here, we report the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, we assessed the neurotoxicity of mHTT seeds in an inducible Drosophila model transgenic for HTTex1. We found a strong correlation between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.

The polyphenol EGCG directly targets intracellular amyloid-ß aggregates and promotes their lysosomal degradation.

The accumulation of amyloidogenic protein aggregates in neurons is a pathogenic hallmark of a large number of neurodegenerative diseases including Alzheimer's disease (AD). Small molecules targeting such structures and promoting their degradation may have therapeutic potential for the treatment of AD. Here, we searched for natural chemical compounds that decrease the abundance of stable, proteotoxic ß-sheet-rich amyloid-ß (Aß) aggregates in cells. We found that the polyphenol (-)-epigallocatechin gallate (EGCG) functions as a potent chemical aggregate degrader in SH-EP cells. We further demonstrate that a novel, fluorescently labeled EGCG derivative (EGC-dihydroxybenzoate (DHB)-Rhodamine) also shows cellular activity. It directly targets intracellular Aß42 aggregates and competes with EGCG for Aß42 aggregate binding in vitro. Mechanistic investigations indicated a lysosomal accumulation of Aß42 aggregates in SH-EP cells and showed that lysosomal cathepsin activity is critical for efficient EGCG-mediated aggregate clearance. In fact, EGCG treatment leads to an increased abundance of active cathepsin B isoforms and increased enzymatic activity in our SH-EP cell model. Our findings suggest that intracellular Aß42 aggregates are cleared through the endo-lysosomal system. We show that EGCG directly targets intracellular Aß42 aggregates and facilitates their lysosomal degradation. Small molecules, which bind to protein aggregates and increase their lysosomal degradation could have therapeutic potential for the treatment of amyloid diseases.

Allogeneic Liver Transplantation and Subsequent Syngeneic Hepatocyte Transplantation in a Rat Model: Proof of Concept for in vivo Tissue Engineering.

OBJECTIVES: Stable long-term functioning of liver cells after transplantation in humans is still not achieved successfully. A new approach for successful engraftment of liver cells may be the transplantation of syngeneic cells into an allogeneic liver graft. We therefore developed a new rat model for combined liver and liver cell transplantation (cLCTx) under stable immunosuppression. MATERIALS AND METHODS: After inducing a mitotic block, liver grafts from female donor rats (Dark Agouti) were transplanted into female recipients (Lewis). In male Lewis rats, liver cell proliferation was induced with subsequent cell isolation and transplantation into female recipients after organ transplantation. Y-chromosome detection of the transplanted male cells was performed by quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FisH) with localization of transplanted cells by immunohistochemistry. RESULTS: Immunohistochemistry demonstrated the engraftment of transplanted cells, as confirmed by FisH, showing repopulation of the liver graft with 15.6% male cells (± 1.8 SEM) at day 90. qPCR revealed 14.15% (± 5.09 SEM) male DNA at day 90. CONCLUSION: Engraftment of transplanted syngeneic cells after cLCTx was achieved for up to 90 days under immunosuppression. Immunohistochemistry indicated cell proliferation, and the FisH results were partly confirmed by qPCR. This new protocol in rats appears feasible for addressing long-term functioning and eventually the induction of operational tolerance in the future.

Sclerotiorin Stabilizes the Assembly of Nonfibrillar Abeta42 Oligomers with Low Toxicity, Seeding Activity, and Beta-sheet Content.

The self-assembly of the 42-residue amyloid-ß peptide, Aß42, into fibrillar aggregates is associated with neuronal dysfunction and toxicity in Alzheimer's disease (AD) patient brains, suggesting that small molecules acting on this process might interfere with pathogenesis. Here, we present experimental evidence that the small molecule sclerotiorin (SCL), a natural product belonging to the group of azaphilones, potently delays both seeded and nonseeded Aß42 polymerization in cell-free assays. Mechanistic biochemical studies revealed that the inhibitory effect of SCL on fibrillogenesis is caused by its ability to kinetically stabilize small Aß42 oligomers. These structures exhibit low ß-sheet content and do not possess seeding activity, indicating that SCL acts very early in the amyloid formation cascade before the assembly of seeding-competent, ß-sheet-rich fibrillar aggregates. Investigations with NMR WaterLOGSY experiments confirmed the association of Aß42 assemblies with SCL in solution. Furthermore, using ion mobility-mass spectrometry, we observed that SCL directly interacts with a small fraction of Aß42 monomers in the gas phase. In comparison to typical amyloid fibrils, small SCL-stabilized Aß42 assemblies are inefficiently taken up into mammalian cells and have low toxicity in cell-based assays. Overall, these mechanistic studies support a pathological role of stable, ß-sheet-rich Aß42 fibrils in AD, while structures with low ß-sheet content may be less relevant.

Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains.

Interactome maps are valuable resources to elucidate protein function and disease mechanisms. Here, we report on an interactome map that focuses on neurodegenerative disease (ND), connects ∼5,000 human proteins via ∼30,000 candidate interactions and is generated by systematic yeast two-hybrid interaction screening of ∼500 ND-related proteins and integration of literature interactions. This network reveals interconnectivity across diseases and links many known ND-causing proteins, such as α-synuclein, TDP-43, and ATXN1, to a host of proteins previously unrelated to NDs. It facilitates the identification of interacting proteins that significantly influence mutant TDP-43 and HTT toxicity in transgenic flies, as well as of ARF-GEP100 that controls misfolding and aggregation of multiple ND-causing proteins in experimental model systems. Furthermore, it enables the prediction of ND-specific subnetworks and the identification of proteins, such as ATXN1 and MKL1, that are abnormally aggregated in postmortem brains of Alzheimer's disease patients, suggesting widespread protein aggregation in NDs.

Self-assembly of Mutant Huntingtin Exon-1 Fragments into Large Complex Fibrillar Structures Involves Nucleated Branching.

Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.

DULIP: A Dual Luminescence-Based Co-Immunoprecipitation Assay for Interactome Mapping in Mammalian Cells.

Mapping of protein-protein interactions (PPIs) is critical for understanding protein function and complex biological processes. Here, we present DULIP, a dual luminescence-based co-immunoprecipitation assay, for systematic PPI mapping in mammalian cells. DULIP is a second-generation luminescence-based PPI screening method for the systematic and quantitative analysis of co-immunoprecipitations using two different luciferase tags. Benchmarking studies with positive and negative PPI reference sets revealed that DULIP allows the detection of interactions with high sensitivity and specificity. Furthermore, the analysis of a PPI reference set with known binding affinities demonstrated that both low- and high-affinity interactions can be detected with DULIP assays. Finally, using the well-characterized interaction between Syntaxin-1 and Munc18, we found that DULIP is capable of detecting the effects of point mutations on interaction strength. Taken together, our studies demonstrate that DULIP is a sensitive and reliable method of great utility for systematic interactome research. It can be applied for interaction screening and validation of PPIs in mammalian cells. Moreover, DULIP permits the specific analysis of mutation-dependent binding patterns.