Spontaneous self-assembly of pathogenic huntingtin exon 1 protein into amyloid structures

PolyQ (polyglutamine) diseases such as HD (Huntington's disease) or SCA1 (spinocerebellar ataxia type 1) are neurodegenerative disorders caused by abnormally elongated polyQ tracts in human proteins. PolyQ expansions promote misfolding and aggregation of disease-causing proteins, leading to the appearance of nuclear and cytoplasmic inclusion bodies in patient neurons. Several lines of experimental evidence indicate that this process is critical for disease pathogenesis. However, the molecular mechanisms underlying spontaneous polyQ-containing aggregate formation and the perturbation of neuronal processes are still largely unclear. The present chapter reviews the current literature regarding misfolding and aggregation of polyQ-containing disease proteins. We specifically focus on studies that have investigated the amyloidogenesis of polyQ-containing HTTex1 (huntingtin exon 1) fragments. These protein fragments are disease-relevant and play a critical role in HD pathogenesis. We outline potential mechanisms behind mutant HTTex1 aggregation and toxicity, as well as proteins and small molecules that can modify HTTex1 amyloidogenesis in vitro and in vivo. The potential implications of such studies for the development of novel therapeutic strategies are discussed

Evolution and function of CAG/polyglutamine repeats in protein-protein interaction networks

Expanded runs of consecutive trinucleotide CAG repeats encoding polyglutamine (polyQ) stretches are observed in the genes of a large number of patients with different genetic diseases such as Huntington's and several Ataxias. Protein aggregation, which is a key feature of most of these diseases, is thought to be triggered by these expanded polyQ sequences in disease-related proteins. However, polyQ tracts are a normal feature of many human proteins, suggesting that they have an important cellular function. To clarify the potential function of polyQ repeats in biological systems, we systematically analyzed available information stored in sequence and protein interaction databases. By integrating genomic, phylogenetic, protein interaction network and functional information, we obtained evidence that polyQ tracts in proteins stabilize protein interactions. This happens most likely through structural changes whereby the polyQ sequence extends a neighboring coiled-coil region to facilitate its interaction with a coiled-coil region in another protein. Alteration of this important biological function due to polyQ expansion results in gain of abnormal interactions, leading to pathological effects like protein aggregation. Our analyses suggest that research on polyQ proteins should shift focus from expanded polyQ proteins into the characterization of the influence of the wild-type polyQ on protein interactions

The pathobiology of perturbed mutant huntingtin protein-protein interactions in Huntington's disease

Mutations are at the root of many human diseases. Still, we largely do not exactly understand how they trigger pathogenesis. One, more recent, hypothesis has been that they comprehensively perturb protein-protein interaction (PPI) networks and significantly alter key biological processes. Under this premise, many rare genetic disorders with Mendelian inheritance, like e.g. Huntington's disease and several spinocerebellar ataxias, are likely to be caused by complex genotype-phenotype relationships involving abnormal PPIs. These altered PPI networks and their effects on cellular pathways are poorly understood at the molecular level. In this review, we focus on PPIs that are perturbed by the expanded pathogenic polyglutamine tract in huntingtin (HTT), the protein which, in its mutated form, leads to the autosomal dominant, neurodegenerative Huntington's disease. One aspect of perturbed mutant HTT interactions is the formation of abnormal protein species such as fibrils or large neuronal inclusions due to homotypic and heterotypic aberrant molecular interactions. This review focuses on abnormal PPIs that are associated with the assembly of mutant HTT aggregates in cells and their potential relevance in disease. Furthermore, the mechanisms and pathobiological processes that may contribute to phenotype development, neuronal dysfunction and toxicity in HD brains are also discussed

Ku70 alleviates neurodegeneration in drosophila models of Huntington's disease

DNA damage accumulates in genome DNA during the long life of neurons, thus DNA damage repair is indispensable to keep normal functions of neurons. We previously reported that Ku70, a critical molecule for DNA double strand break (DSB) repair, is involved in the pathology of Huntington's disease (HD). Mutant huntingtin (Htt) impaired Ku70 function via direct interaction, and Ku70 supplementation recovered phenotypes of a mouse HD model. In this study, we generate multiple Drosophila HD models that express mutant huntingtin (Htt) in eye or motor neuron by different drivers and show various phenotypes. In such fly models, Ku70 co-expression recovers lifespan, locomotive activity and eye degeneration. In contrast, Ku70 reduction by heterozygous null mutation or siRNA-mediated knock down accelerates lifespan shortening and locomotion disability. These results collectively support that Ku70 is a critical mediator of the HD pathology and a candidate therapeutic target in HD

Flexible web-based integration of distributed large-scale human protein interaction maps

Protein-protein interactions constitute the backbone of many molecular processes. This has motivated the recent construction of several large-scale human protein-protein interaction maps [1-10]. Although these maps clearly offer a wealth of information, their use is challenging: complexity, rapid growth, and fragmentation of interaction data hamper their usability. To overcome these hurdles, we have developed a publicly accessible database termed UniHI (Unified Human Interactome) for integration of human protein-protein interaction data. This database is designed to provide biomedical researchers a common platform for exploring previously disconnected human interaction maps. UniHI offers researchers flexible integrated tools for accessing comprehensive information about the human interactome. Several features included in the UniHI allow users to perform various types of network-oriented and functional analysis. At present, UniHI contains over 160,000 distinct interactions between 17,000 unique proteins from ten major interaction maps derived by both computational and experimental approaches [1-10]. Here we describe the details of the implementation and maintenance of UniHI and discuss the challenges that have to be addressed for a successful integration of interaction data

Myc inhibition impairs autophagosome formation

Autophagy, a major clearance route for most long-lived proteins and organelles, has long been implicated in cancer development. Myc is a proto-oncogene often found to be deregulated in many cancers, and thus presents as an attractive target for design of cancer therapy. Therefore, understanding the relationship between anti-Myc strategies and autophagy will be important for development of effective therapy. Here we show that Myc depletion inhibits autophagosome formation and impairs clearance of autophagy substrates. Myc suppression has an inhibitory effect on autophagy via reduction of JNK1 and Bcl2 phosphorylation. Additionally, the decrease in JNK1 phosphorylation observed with Myc knockdown is associated with a reduction in ROS production. Our data suggest that targeting Myc in cancer therapy might have the additional benefit of inhibiting autophagy in the case of therapy resistance associated with chemotherapy-induced autophagy

Epigallocatechin-3-gallate: a useful, effective and safe clinical approach for targeted prevention and individualised treatment of neurological diseases

Neurodegenerative disorders show an increasing prevalence in a number of highly developed countries. Often, these diseases require life-long treatment mostly with drugs which are costly and mostly accompanied by more or less serious side-effects. Their heterogeneous manifestation, severity and outcome pose the need for individualised treatment options. There is an intensive search for new strategies not only for treating but also for preventing these diseases. Green tea and green tea extracts seem to be such a promising and safe alternative. However, data regarding the beneficial effects and possible underlying mechanism, specifically in clinical trials, are rare and rather controversial or non-conclusive. This review outlines the existing evidence from preclinical studies (cell and tissue cultures and animal models) and clinical trials regarding preventive and therapeutic effects of epigallcatechin-3-gallate in neurodegenerative diseases and considers antioxidative vs. pro-oxidative properties of the tea catechin important for dosage recommendations

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 Alzheimer's 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 (A{beta}) fibrils in the brain. Recently, it was shown that extracellular A{beta} can enter brain cells, resulting in neurotoxicity. Methods: After internalization of A{beta}(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, A{beta}(42) aggregate amounts were significantly lower than in nonirradiated cells. Likewise, in EGCG-treated cells, A{beta}(42) aggregate amounts were significantly lower than in non-EGCG-treated cells. Except for the cells simultaneously laden with A{beta}(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 A{beta}(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 A{beta} 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

Aggregation of full length immunoglobulin light chains from AL amyloidosis patients is remodeled by epigallocatechin-3-gallate

Intervention into amyloid deposition with anti-amyloid agents like the polyphenol Epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and Multiple Myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL they form amyloid deposits in vivo. We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from urine of AL and MM patients. We quantified their thermodynamic stabilities, and monitored their aggregation under physiological conditions by ThT fluorescence, light scattering, SDS-stability and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ~50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-{beta} and {alpha}-synuclein

FEZ1 forms complexes with CRMP1 and DCC to regulate axon and dendrite development

Elaboration of neuronal processes is an early step in neuronal development. Guidance cues must work closely with intracellular trafficking pathways to direct expanding axons and dendrites to their target neurons during the formation of neuronal networks. However, how such coordination is achieved remains incompletely understood. Here, we characterize an interaction between FEZ1, an adapter involved in synaptic protein transport, and CRMP1, a protein that functions in growth cone guidance, at neuronal growth cones. We show that similar to CRMP1 loss-of-function mutants, FEZ1 deficiency in rat hippocampal neurons causes growth cone collapse and impairs axonal development. Strikingly, FEZ1-deficient neurons also exhibited a reduction in dendritic complexity stronger than that observed in CRMP1-deficient neurons, suggesting that the former could partake in additional developmental signaling pathways. Supporting this, FEZ1 colocalizes with VAMP2 in developing hippocampal neurons and forms a separate complex with Deleted in Colorectal Cancer and Syntaxin-1, components of the Netrin-1 signaling pathway that are also involved in regulating axon and dendrite development. Significantly, developing axons and dendrites of FEZ1-deficient neurons fail to respond to Netrin-1 or Netrin-1 and Sema3A treatment, respectively. Taken together, these findings highlight the importance of FEZ1 as a common effector to integrate guidance signaling pathways with intracellular trafficking to mediate axo-dendrite development during neuronal network formation. SIGNIFICANCE STATEMENT: Guidance cue-dependent elaboration of axons and dendrites towards their target neurons is a critical step in the formation of neuronal circuits during brain development. The elongating neurites require a constant supply of biomolecules, but it remains unclear how guidance cues cooperate with intracellular transport. Here, we show that the kinesin-1 adapter FEZ1 forms complexes with CRMP1 or DCC, which are downstream effectors of the Sema3A and Netrin-1 signaling pathway, respectively. FEZ1-deficient neurons not only exhibit abnormal axons and dendrites, they were also unresponsive to Sema3A- or Netrin-1-dependent regulation of axo-dendritic development. Our results highlight FEZ1 as a key convergence point where guidance cues and intracellular transport integrate to coordinate neuronal process development during neuronal network formation