Inhibition of alpha-synuclein seeded fibril formation and toxicity by herbal medicinal extracts.

Recent studies indicated that seeded fibril formation and toxicity of α-synuclein (α-syn) play a main role in the pathogenesis of certain diseases including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. Therefore, examination of compounds that abolish the process of seeding is considered a key step towards therapy of several synucleinopathies. Using biophysical, biochemical and cell-culture-based assays, assessment of eleven compounds, extracted from Chinese medicinal herbs, was performed in this study for their effect on α-syn fibril formation and toxicity caused by the seeding process. Salvianolic acid B and dihydromyricetin were the two compounds that strongly inhibited the fibril growth and neurotoxicity of α-syn. In an in-vitro cell model, these compounds decreased the insoluble phosphorylated α-syn and aggregation. Also, in primary neuronal cells, these compounds showed a reduction in α-syn aggregates. Both compounds inhibited the seeded fibril growth with dihydromyricetin having the ability to disaggregate preformed α-syn fibrils. In order to investigate the inhibitory mechanisms of these two compounds towards fibril formation, we demonstrated that salvianolic acid B binds predominantly to monomers, while dihydromyricetin binds to oligomeric species and to a lower extent to monomers. Remarkably, these two compounds stabilized the soluble non-toxic oligomers lacking β-sheet content after subjecting them to proteinase K digestion. Eleven compounds were tested but only two showed inhibition of α-syn aggregation, seeded fibril formation and toxicity in vitro. These findings highlight an essential beginning for development of new molecules in the field of synucleinopathies treatment.The work conducted by Dr. El-Agnaf laboratory was supported by Qatar Biomedical Research Institute under the Start-up Fund SF 2017–007. Funding for this work was provided in part by NIH/NIA grant R37AG019391 to D.E. This study was made possible by NPRP grant 4–1371–1-223 from the Qatar National Research Fund (a member of Qatar Foundation). The funding bodies provided financial support for this study; they had no role in the study design, performance, data collection and analysis, decision to publish and preparation/writing of the manuscript

Fibrillar form of α-synuclein-specific scFv antibody inhibits α-synuclein seeds induced aggregation and toxicity

Synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are characterized by pathological accumulation of α-synuclein (α-syn). Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, antibody-based immunotherapy holds much promise. However, the large size of antibodies and corresponding difficulty in crossing the blood-brain barrier has limited development in this area. To overcome this issue, we engineered single-chain variable fragments (scFvs) against fibrillar α-syn, a putative disease-relevant form of α-syn. The purified scFvs showed specific activity towards α-syn fibrils and oligomers in comparison to monomers and recognized intracellular inclusions in human post-mortem brain tissue of Lewy body disease cases, but not aged controls. In vitro studies indicated scFvs inhibit the seeding of α-syn aggregation in a time-dependent manner, decreased α-syn seed-induced toxicity in a cell model of PD, and reduced the production of insoluble α-syn phosphorylated at Ser-129 (pS129-α-syn). These results suggest that our α-syn fibril-specific scFvs recognize α-syn pathology and can inhibit the aggregation of α-syn in vitro and prevent seeding-dependent toxicity. Therefore, the scFvs described here have considerable potential to be utilized towards immunotherapy in synucleinopathies and may also have applications in ante-mortem imaging modalities.Dr. El-Agnaf’s laboratory was funded by Qatar Biomedical Research Institute under the Start-up Fund SF 2017– 007. The Newcastle Brain Tissue Resource is funded in part by a grant from the UK Medical Research Council, by NIHR Newcastle Biomedical Research Centre awarded to the Newcastle upon Tyne NHS Foundation Trust and Newcastle University, and by a grant from the Alzheimer’s Society and Alzheimer’s Research UK as part of the Brains for Dementia Research Project

Novel engineered nanobodies specific for N-terminal region of alpha-synuclein recognize Lewy-body pathology and inhibit in-vitro seeded aggregation and toxicity.

Nanobodies (Nbs), the single-domain antigen-binding fragments of dromedary heavy-chain antibodies (HCAb), are excellent candidates as therapeutic and diagnostic tools in synucleinopathies because of their small size, solubility and stability. Here, we constructed an immune nanobody library specific to the monomeric form of alpha-synuclein (α-syn). Phage display screening of the library allowed the identification of a nanobody, Nbα-syn01, specific for α-syn. Unlike previously developed nanobodies, Nbα-syn01 recognized the N-terminal region which is critical for in vitro and in vivo aggregation and contains many point mutations involved in early PD cases. The affinity of the monovalent Nbα-syn01 and the engineered bivalent format BivNbα-syn01 measured by isothermal titration calorimetry revealed unexpected results where Nbα-syn01 and its bivalent format recognized preferentially α-syn fibrils compared to the monomeric form. Nbα-syn01 and BivNbα-syn01 were also able to inhibit α-syn-seeded aggregation in vitro and reduced α-syn-seeded aggregation and toxicity in cells showing their potential to reduce α-syn pathology. Moreover, both nanobody formats were able to recognize Lewy-body pathology in human post-mortem brain tissue from PD and DLB cases. Additionally, we present evidence through structural docking that Nbα-syn01 binds the N-terminal region of the α-syn aggregated form. Overall, these results highlight the potential of Nbα-syn01 and BivNbα-syn01 in developing into a diagnostic or a therapeutic tool for PD and related disorders

α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer

Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes

Age, Disease Severity and Ethnicity Influence Humoral Responses in a Multi-Ethnic COVID-19 Cohort

The COVID-19 pandemic has affected all individuals across the globe in some way. Despite large numbers of reported seroprevalence studies, there remains a limited understanding of how the magnitude and epitope utilization of the humoral immune response to SARS-CoV-2 viral anti-gens varies within populations following natural infection. Here, we designed a quantitative, multi-epitope protein microarray comprising various nucleocapsid protein structural motifs, including two structural domains and three intrinsically disordered regions. Quantitative data from the microarray provided complete differentiation between cases and pre-pandemic controls (100% sensitivity and specificity) in a case-control cohort (n = 100). We then assessed the influence of disease severity, age, and ethnicity on the strength and breadth of the humoral response in a multi-ethnic cohort (n = 138). As expected, patients with severe disease showed significantly higher antibody titers and interestingly also had significantly broader epitope coverage. A significant increase in antibody titer and epitope coverage was observed with increasing age, in both mild and severe disease, which is promising for vaccine efficacy in older individuals. Additionally, we observed significant differences in the breadth and strength of the humoral immune response in relation to ethnicity, which may reflect differences in genetic and lifestyle factors. Furthermore, our data enabled localization of the immuno-dominant epitope to the C-terminal structural domain of the viral nucleocapsid protein in two independent cohorts. Overall, we have designed, validated, and tested an advanced serological assay that enables accurate quantitation of the humoral response post natural infection and that has revealed unexpected differences in the magnitude and epitope utilization within a population

Gene-Based Therapeutics for Parkinson’s Disease

Parkinson’s disease (PD) is a complex multifactorial disorder that is not yet fully surmised, and it is only when such a disease is tackled on multiple levels simultaneously that we should expect to see fruitful results. Gene therapy is a modern medical practice that theoretically and, so far, practically, has demonstrated its capability in joining the battle against PD and other complex disorders on most if not all fronts. This review discusses how gene therapy can efficiently replace current forms of therapy such as drugs, personalized medicine or invasive surgery. Furthermore, we discuss the importance of enhancing delivery techniques to increase the level of transduction and control of gene expression or tissue specificity. Importantly, the results of current trials establish the safety, efficacy and applicability of gene therapy for PD. Gene therapy’s variety of potential in interfering with PD’s pathology by improving basal ganglial circuitry, enhancing dopamine synthesis, delivering neuroprotection or preventing neurodegeneration may one day achieve symptomatic benefit, disease modification and eradication

Soluble oligomers for the diagnosis of neurodegenerative diseases.

The formation of extracellular fibrous deposits of amyloid or intracellular inclusion bodies that contain abnormal protein fibrils is a common pathological feature of many neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies, multiple system atrophy, Huntington's disease, the transmissible prion dementias, and, possibly, motor-neuron disease.1 The early diagnosis of many of these diseases is problematic because of the long latency between the onset of damage to neurons in the brain and the appearance of clinical symptoms. For example, by the time PD is diagnosed, most of the dopaminergic neurons in the substantia nigra have been lost. There is no simple biochemical laboratory test that can confirm the diagnosis of any neurodegenerative disease associated with protein deposits. Genetic, transgenic, and biochemical studies suggest that accumulation of protein aggregates in the brain has a seminal role in the pathogenesis of these diseases. How monomeric proteins are converted to highly polymeric deposits is unclear, but much useful information has been obtained from the study of synthetic peptides and purified recombinant proteins. These studies suggest that amyloid proteins first coalesce to form small soluble oligomers. Further aggregation of these small oligomers produces high molecular weight assemblies—including so-called protofibrils and Aβ-derived diffusible ligands (ADDLs)—that eventually exceed solubility limits until they are deposited as amyloid fibrils. Our studies, and those of others, support the idea that soluble oligomers, rather than mature amyloid fibrils, are actually the pathogenetic species that cause neurodegeneration and neuronal death.2, 3, 4 and 5 Full-size image (24K) Soluble oligomers of synthetic Aβ peptide: pseudospheres (about 5 nm in diameter) and short curvilinear fibrils (up to 200 nm in length). Courtesy of Dominic Walsh View Within Article Recently, this hypothesis has been supported by Kayed and co-workers6 who have developed an antibody (anti-oligo) that recognises only soluble oligomeric forms of Aβ associated with AD.6 Temporal analysis of Aβ aggregation by electron microsocopy and dot blots with anti-oligo showed that the appearance of ADDLs7 and protofibrils8 and 9 was coincident with anti-oligo immuno-reactivity, which indicates that ADDLs and protofibrils share a common structural epitope. Surpisingly, anti-oligo also detects soluble oligomeric aggregates of -synuclein, islet amyloid polypeptide, polyglutamine, lysozyme, insulin, and prion protein 106–126. As with Aβ, anti-oligo did not detect the monomeric or fibrillar versions of these proteins. Thus, anti-oligo recognises a common structural epitope independent of primary protein sequence. Importantly, antioligo prevented oligomer-mediated toxicity of all the proteins tested, whereas other antibodies that were not specific for oligomers had no effect. By use of immunohistochemistry, the authors also examined the presence and distribution of soluble Aβ oligomers in the brains of patients with AD and in normal age-matched controls. Anti-oligo recognised Aβ oligomers that could not be detected by thioflavin-S in the brains of patients with AD. Furthermore, these oligomers were not detected in brain samples from normal individuals. These results support the idea that soluble oligomers of Aβ may be pathogenetic, and could also have important diagnostic implications, provided that oligomers can be detected in CSF or plasma. Support for this idea has come from recent studies where A oligomers were detected in CSF from AD patients10 and prion protein aggregates were detected in CSF from patients with Creutzfeldt-Jakob disease,11 but oligomers were not detected in control samples. Extensive clinical studies will be required to assess the validity of such procedures as potential diagnostic tests and to determine if there is any correlation between the detection of amyloid oligomers and the severity and/or stage of the disease. If oligomerisation occurs before most neurons are lost, then detection of oligomers may facilitate early diagnosis and treatment of various neurodegenerative diseases. Assays for detection of oligomers could also be used for high-throughput screening to identify small molecules that specifically bind to, and disrupt, the oligomer specific conformation. Moreover, oligomer specific assays may also be useful for the detection of proteopathies that have yet to be identified