Structure-based inhibitors of amyloid beta core suggest a common interface with tau.

Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline

Contribution to drug discovery and development for tauopathies using yeast as a model

This work aimed to contribute to drug discovery and development (DDD) for tauopathies, while expanding our knowledge on this group of neurodegenerative disorders, including Alzheimer’s disease (AD). Using yeast, a recognized model for neurodegeneration studies, useful models were produced for the study of tau interaction with beta-amyloid (Aβ), both AD hallmark proteins. The characterization of these models suggests that these proteins co-localize and that Aβ1-42, which is toxic to yeast, is involved in tau40 phosphorylation (Ser396/404) via the GSK-3β yeast orthologue, whereas tau seems to facilitate Aβ1-42 oligomerization. The mapping of tau’s interactome in yeast, achieved with a tau toxicity enhancer screen using the yeast deletion collection, provided a novel framework, composed of 31 genes, to identify new mechanisms associated with tau pathology, as well as to identify new drug targets or biomarkers. This genomic screen also allowed to select the yeast strain mir1Δ-tau40 for development of a new GPSD2TM drug discovery screening system. A library of unique 138 marine bacteria extracts, obtained from the Mid-Atlantic Ridge hydrothermal vents, was screened with mir1Δ-tau40. Three extracts were identified as suppressors of tau toxicity and constitute good starting points for DDD programs. mir1Δ strain was sensitive to tau toxicity, relating tau pathology with mitochondrial function. SLC25A3, the human homologue of MIR1, codes for the mitochondrial phosphate carrier protein (PiC). Resorting to iRNA, SLC25A3 expression was silenced in human neuroglioma cells, as a first step towards the engineering of a neural model for replicating the results obtained in yeast. This model is essential to understand the mechanisms of tau toxicity at the mitochondrial level and to validate PiC as a relevant drug target. The set of DDD tools here presented will foster the development of innovative and efficacious therapies, urgently needed to cope with tau-related disorders of high human and social-economic impact

Intrinsically Disordered Proteins and Chronic Diseases

This book is an embodiment of a series of articles that were published as part of a Special Issue of Biomolecules. It is dedicated to exploring the role of intrinsically disordered proteins (IDPs) in various chronic diseases. The main goal of the articles is to describe recent progress in elucidating the mechanisms by which IDPs cause various human diseases, such as cancer, cardiovascular disease, amyloidosis, neurodegenerative diseases, diabetes, and genetic diseases, to name a few. Contributed by leading investigators in the field, this compendium serves as a valuable resource for researchers, clinicians as well as postdoctoral fellows and graduate student

Splicing factor SC35 promotes tau expression through stabilization of its mRNA

AbstractAltered alternative splicing and accumulation of brain microtubule-associated protein tau are found in several tauopathies and are believed to lead to these neurodegenerative diseases. We found that in addition to promoting tau exon 10 inclusion, splicing factor SC35 also promoted tau expression in HEK-293T cells. The activity of SC35 in promotion of tau expression was limited to exon 10 containing tau isoforms. SC35 did not affect tau transcription, but stabilized tau mRNA by binding to the SC35-like element of exon 10. These results provide novel insight into the regulation of tau expression and a molecular mechanism of tauopathies

Splicing factor SC35 promotes tau expression through stabilization of its mRNA

AbstractAltered alternative splicing and accumulation of brain microtubule-associated protein tau are found in several tauopathies and are believed to lead to these neurodegenerative diseases. We found that in addition to promoting tau exon 10 inclusion, splicing factor SC35 also promoted tau expression in HEK-293T cells. The activity of SC35 in promotion of tau expression was limited to exon 10 containing tau isoforms. SC35 did not affect tau transcription, but stabilized tau mRNA by binding to the SC35-like element of exon 10. These results provide novel insight into the regulation of tau expression and a molecular mechanism of tauopathies

Mutant HTT seeding activity: a marker of disease progression and neurotoxicity in models of Huntington’s disease

Self-propagating, amyloidogenic mutant huntingtin (HTT) aggregates may drive progression of Huntington’s disease (HD). Here, we report the development of a FRET-based HTT aggregate seeding (FRASE) biosensor assay that enables the quantification of mutant HTT 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 knock-in mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that HSA is high in fractions that contain small HTT fibrils but is low in fractions with large, insoluble HTT aggregates, indicating that small rather than large mutant HTT structures possess HSA. Finally, we assessed the neurotoxicity of mutant HTT seeds in an inducible Drosophila model transgenic for HTT. We found a strong correlation between HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mutant HTT structures with severe phenotypic consequences in vivo

Bispecific Tau Antibodies with Additional Binding to C1q or Alpha-Synuclein

BACKGROUND: Alzheimer’s disease (AD) and other tauopathies are neurodegenerative disorders characterized by cellular accumulation of aggregated tau protein. Tau pathology within these disorders is accompanied by chronic neuroinflammation, such as activation of the classical complement pathway by complement initiation factor C1q. Additionally, about half of the AD cases present with inclusions composed of aggregated alpha-synuclein called Lewy bodies. Lewy bodies in disorders such as Parkinson’s disease and Lewy body dementia also frequently occur together with tau pathology. OBJECTIVE: Immunotherapy is currently the most promising treatment strategy for tauopathies. However, the presence of multiple pathological processes within tauopathies makes it desirable to simultaneously target more than one disease pathway. METHODS: Herein, we have developed three bispecific antibodies based on published antibody binding region sequences. One bispecific antibody binds to tau plus alpha-synuclein and two bispecific antibodies bind to tau plus C1q. RESULTS: Affinity of the bispecific antibodies to their targets compared to their monospecific counterparts ranged from nearly identical to one order of magnitude lower. All bispecific antibodies retained binding to aggregated protein in patient-derived brain sections. The bispecific antibodies also retained their ability to inhibit aggregation of recombinant tau, regardless of whether the tau binding sites were in IgG or scFv format. Mono- and bispecific antibodies inhibited cellular seeding induced by AD-derived pathological tau with similar efficacy. Finally, both Tau-C1q bispecific antibodies completely inhibited the classical complement pathway. CONCLUSIONS: Bispecific antibodies that bind to multiple pathological targets may therefore present a promising approach to treat tauopathies and other neurodegenerative disorders

Functional details of human HtrA2 protease studied by NMR spectroscopy

Cells rely on an array of cellular machineries in the protein quality control system (PQC) to maintain the health of the collective proteome. The HtrA family of serine proteases are found in all kingdoms of life and function in the PQC by degrading damaged and aggregated proteins as well as by acting as molecular chaperones. The human mitochondrial serine protease HtrA2 functions as a pro-apoptotic agent in addition to its protease function in the mitochondrial PQC where it targets a diverse set of proteins including presenilins, α-synuclein and amyloid-β42. Dysfunction of HtrA2 is associated to multiple diseases including Parkinson’s disease, Alzheimer’s disease, ischemic cerebral small-vessel disease, and multiple types of cancer. As such, HtrA2 is an important target of study and the structural details of the HtrA2 functional cycle remain incomplete to date. In this thesis, I have focused on detailing the allosteric activation of HtrA2 as well as characterizing the interaction between HtrA2 and its natural substrates XIAP and -synuclein by using solution nuclear magnetic resonance (NMR) spectroscopy as my main method of choice. I showed that HtrA2 protease activity can be modulated by divalent cations binding to the HtrA2-PDZ domain, providing novel insight into the link between how metal dyshomeostasis can influence the PQC and subsequently lead to disease. I further detailed the allosteric activation pathway of HtrA2 and show for the first time that also the amino-terminal helix of HtrA2, harboring a motif critical for interaction with inhibitor of apoptosis (IAP) proteins, is affected by allosteric activation. My characterization of the interaction between HtrA2 and XIAP shows previously unreported weak interactions between XIAP and the HtrA2-PDZ domain and reveals that while mutations in the IAP-binding motif of HtrA2 drastically diminishes the interaction with XIAP, it does not alter the proteolytic efficiency of HtrA2 towards XIAP but hampers the ability to stimulate the HtrA2 proteolytic activity by use of divalent cations. Further, I show that HtrA2 can degrade monomeric α-synuclein and Tau isoforms Tau-39 and Tau-40. In conclusion, my results provide novel insights into the structural details of the HtrA2 functional cycle in atomical resolution and widens our understanding of the role of HtrA2 in apoptotic cell regulation and in the progression of neurological disease

Folding of Alzheimer's core PHF subunit revealed by monoclonal antibody 423

AbstractAt present, the conformation-dependent monoclonal antibodies (mAb) provide the only information on folding of tau in the core PHF. Monoclonal antibody MN423 recognizes all and only those Alzheimer's disease (AD) core paired helical filaments (PHFs) subunits, which terminate at Glu391. Using recombinant analogs of the core PHF subunit corresponding to tau residues τ297–391, we found that the C-terminal pentapeptide 387DHGAE391 represented only one component of the structure recognized by mAb 423. Therefore, deletion mutants of the core subunit were generated to identify assembled parts of this conformational structure. We localized two spatially close components in the region 306–325 (306VQIVYK311 and 321KCGSL325) contributing to formation of the structure identified by mAb 423. Thus, the spatial proximity of three subunit segments 306VQIVYK311, 321KCGSL325 and 387DHGAE391 represents constraints for intramolecular folding of the core PHF subunit. Since PHF represents a compelling drug target in AD, structural knowledge presented could contribute to structure-based drug design