Structural insights into the unique recognition module between α-synuclein peptide and nanobody.

Nanobodies are single-domain fragments of antibodies with comparable specificity and affinity to antibodies. They are emerging as versatile tools in biology due to their relatively small size. Here, we report the crystal structure of a specific nanobody Nbα-syn01, bound to a 14 amino acid long peptide of α-synuclein (αSyn), a 140-residue protein whose aggregation is associated with Parkinson's disease. The complex structure exhibits a unique binding pattern where the αSyn peptide replaces the N-terminal region of nanobody. Recognition is mediated principally by extended main chain interaction of the αSyn peptide and specificity of the interaction lies in the central 48-52 region of αSyn peptide. Structure-guided truncation of Nbα-syn01 shows tighter binding to αSyn peptide and improved inhibition of α-synuclein aggregation. The structure of the truncated complex was subsequently determined and was indistinguishable to full length complex as the full-length form had no visible electron density for the N-terminal end. These findings reveal the molecular basis for a previously unobserved binding mode for nanobody recognition of α-synuclein, providing an explanation for the enhanced binding, and potential for an alternate framework for structure-based protein engineering of nanobodies to develop better diagnostic and therapeutic tools.

Screening for Novel Inhibitors of Amyloid Beta Aggregation and Toxicity as Potential Drugs for Alzheimer's Disease.

AD is the most common neurodegenerative disorder characterized by progressive memory impairment and cognitive deficits. The pathology of AD is still unclear; however, several studies have shown that the aggregation of the Aß peptide in the CNS is an exclusively pathological process involved in AD. Currently, there is no proven medication to cure or prevent the disease progression. Nevertheless, various therapeutic approaches for AD show only relief of symptoms and mostly work on cognitive recovery. However, one of the promising approaches for therapeutic intervention is to use inhibitors for blocking the Aß peptide aggregation process. Recently, herbal phenolic compounds have been shown to have a therapeutic property for treatment of AD due to their multifaceted action. In this study, we investigated the effectiveness of SA, Gn Rb1, and DMyr on inhibiting the aggregation and toxicity of Aß40 and Aß42 using different biochemical and cell-based assays. Our results showed that SA and DMyr inhibit Aß40 and Aß42 fibrillation, seeded aggregation, and toxicity. Gn Rb1 did not have any effect on the aggregation or toxicity induced by Aß40 and Aß42. Moreover, SA and DMyr were able to disaggregate the preformed fibrils. Overall, these compounds may be used alone or synergistically and could be considered as a lead for designing new compounds that could be used as effective treatment of AD and related disorders.

Auto-immunoproteomics analysis of COVID-19 ICU patients revealed increased levels of autoantibodies related to the male reproductive system.

Background: Coronavirus disease (COVID-19) manifests many clinical symptoms, including an exacerbated immune response and cytokine storm. Autoantibodies in COVID-19 may have severe prodromal effects that are poorly understood. The interaction between these autoantibodies and self-antigens can result in systemic inflammation and organ dysfunction. However, the role of autoantibodies in COVID-19 complications has yet to be fully understood. Methods: The current investigation screened two independent cohorts of 97 COVID-19 patients [discovery (Disc) cohort from Qatar (case = 49 vs. control = 48) and replication (Rep) cohort from New York (case = 48 vs. control = 28)] utilizing high-throughput KoRectly Expressed (KREX) Immunome protein-array technology. Total IgG autoantibody responses were evaluated against 1,318 correctly folded and full-length human proteins. Samples were randomly applied on the precoated microarray slides for 2 h. Cy3-labeled secondary antibodies were used to detect IgG autoantibody response. Slides were scanned at a fixed gain setting using the Agilent fluorescence microarray scanner, generating a 16-bit TIFF file. Group comparisons were performed using a linear model and Fisher's exact test. Differentially expressed proteins were used for KEGG and WIKIpathway annotation to determine pathways in which the proteins of interest were significantly over-represented. Results and conclusion: Autoantibody responses to 57 proteins were significantly altered in the COVID-19 Disc cohort compared to healthy controls (p ≤ 0.05). The Rep cohort had altered autoantibody responses against 26 proteins compared to non-COVID-19 ICU patients who served as controls. Both cohorts showed substantial similarities (r 2 = 0.73) and exhibited higher autoantibody responses to numerous transcription factors, immunomodulatory proteins, and human disease markers. Analysis of the combined cohorts revealed elevated autoantibody responses against SPANXN4, STK25, ATF4, PRKD2, and CHMP3 proteins in COVID-19 patients. The sequences for SPANXN4 and STK25 were cross-validated using sequence alignment tools. ELISA and Western blot further verified the autoantigen-autoantibody response of SPANXN4. SPANXN4 is essential for spermiogenesis and male fertility, which may predict a potential role for this protein in COVID-19-associated male reproductive tract complications, and warrants further research.

Uncovering a neurological protein signature for severe COVID-19.

Coronavirus disease of 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has sparked a global pandemic with severe complications and high morbidity rate. Neurological symptoms in COVID-19 patients, and neurological sequelae post COVID-19 recovery have been extensively reported. Yet, neurological molecular signature and signaling pathways that are affected in the central nervous system (CNS) of COVID-19 severe patients remain still unknown and need to be identified. Plasma samples from 49 severe COVID-19 patients, 50 mild COVID-19 patients, and 40 healthy controls were subjected to Olink proteomics analysis of 184 CNS-enriched proteins. By using a multi-approach bioinformatics analysis, we identified a 34-neurological protein signature for COVID-19 severity and unveiled dysregulated neurological pathways in severe cases. Here, we identified a new neurological protein signature for severe COVID-19 that was validated in different independent cohorts using blood and postmortem brain samples and shown to correlate with neurological diseases and pharmacological drugs. This protein signature could potentially aid the development of prognostic and diagnostic tools for neurological complications in post-COVID-19 convalescent patients with long term neurological sequelae.

Production of α-Synuclein Fibrillar-Specific scFv from Inclusion Bodies.

Recombinant antibody fragments such as Fab, scFvs, and diabodies against α-syn have become a viable alternative to the conventional full-length antibodies in immunotherapeutic approaches due to their benefits which include smaller size, higher stability, specificity, and affinity. However, the majority of recombinant antibody fragments typically express as inclusion bodies (IBs) in E. coli, which makes their purification incredibly difficult. Here, we describe a method involving a mild solubilizing protocol followed by slow on-column refolding to purify active single-chain variable fragment (scFv-pF) antibody that can recognize the pathogenic α-syn fibrils.

Inhibition of α-Synuclein Seeding-Dependent Aggregation by ssDNA Aptamers Specific to C-Terminally Truncated α-Synuclein Fibrils.

Neuropathologically, Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of insoluble aggregates of α-synuclein (α-syn) in the Lewy bodies (LBs). In addition to full-length α-syn fibrils, C-terminally truncated α-syn is also abundant in the LBs that acts as seeds and facilitates the aggregation of the full-length α-syn in vitro and in vivo and induces toxicity. Hence, identifying molecules that can inhibit the seeding activity of these truncated forms is of great importance. Here, we report the first in vitro selection of aptamers targeting the fibrillar forms of different C-terminally truncated α-syn using systematic evolution by an exponential enrichment method followed by quantitative high-throughput DNA sequencing. We identify a panel of aptamers that bound with high specificity to different truncated forms of α-syn fibrils with no cross-reactivity toward other amyloid fibrils. Interestingly, two of the aptamers (named Apt11 and Apt15) show higher affinity to most C-terminally truncated forms of α-syn fibrils with an evident inhibition of α-syn-seeded aggregation in vitro by Apt11. This inhibition is further confirmed by circular dichroism, Congo red binding assay, and electronic microscopy. Moreover, Apt11 is also found to reduce the insoluble phosphorylated form of α-syn at Ser-129 (pS129-α-syn) in the cell model and also can inhibit α-syn aggregation using RT-QuIC reactions seeded with brain homogenates extracted from patients affected by PD. The aptamers discovered in this study represent potential useful tools for research and diagnostics or therapy toward PD and DLB.

α-Synuclein phosphorylation at serine 129 occurs after initial protein deposition and inhibits seeded fibril formation and toxicity.

α-Synuclein (α-syn) phosphorylation at serine 129 (pS129­α-syn) is substantially increased in Lewy body disease, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129­α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129­α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129­α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129­α-syn (WT­α-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129­α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129­α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129­α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129­α-syn as a measure of efficacy in clinical trials.

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.

Natural Alkaloid Compounds as Inhibitors for Alpha-Synuclein Seeded Fibril Formation and Toxicity.

The accumulation and aggregation of α-synuclein (α-syn) is the main pathologic event in Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. α-Syn-seeded fibril formation and its induced toxicity occupy a major role in PD pathogenesis. Thus, assessing compounds that inhibit this seeding process is considered a key towards the therapeutics of synucleinopathies. Using biophysical and biochemical techniques and seeding-dependent cell viability assays, we screened a total of nine natural compounds of alkaloid origin extracted from Chinese medicinal herbs. Of these compounds, synephrine, trigonelline, cytisine, harmine, koumine, peimisine, and hupehenine exhibited in vitro inhibition of α-syn-seeded fibril formation. Furthermore, using cell viability assays, six of these compounds inhibited α-syn-seeding-dependent toxicity. These six potent inhibitors of amyloid fibril formation and toxicity caused by the seeding process represent a promising therapeutic strategy for the treatment of PD and other synucleinopathies.

RT-QuIC Using C-Terminally Truncated α-Synuclein Forms Detects Differences in Seeding Propensity of Different Brain Regions from Synucleinopathies.

Aggregated α-synuclein (αSyn) protein is a core pathological feature of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Both PD and DLB demonstrate the presence of diverse intracellular α-synuclein (αSyn) species, including C-terminally truncated αSyn (C-αSyn), although it is unknown how C-αSyn species contribute to disease progression. Using recombinant C-αSyn and PD and DLB brain lysates as seeds in the real-time quaking-induced conversion (RT-QuIC) assay, we explored how C-αSyn may be involved in disease stratification. Comparing the seeding activity of aqueous-soluble fractions to detergent-soluble fractions, and using αSyn 1-130 as substrate for the RT-QuIC assay, the temporal cortex seeds differentiated PD and DLB from healthy controls. In contrast to the temporal cortex, where PD and DLB could not be distinguished, αSyn 1-130 seeded by the detergent-soluble fractions from the PD frontal cortex demonstrated greater seeding efficiency compared to the DLB frontal cortex. Moreover, proteinase K-resistant (PKres) fragments from the RT-QuIC end products using C-αSyn 1-130 or C-αSyn 1-115 were more obvious in the frontal cortex compared to the temporal cortex. Morphological examinations of RT-QuIC end products showed differences in the size of the fibrils between C-αSyn 1-130 and C-αSyn 1-115, in agreement with the RT-QuIC results. These data show that C-αSyn species can distinguish PD from DLB and suggest diversity in αSyn species across these synucleinopathies, which could play a role in disease progression.

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.

Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies.

Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E. coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.

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.

CSF total and oligomeric α-Synuclein along with TNF-α as risk biomarkers for Parkinson's disease: a study in LRRK2 mutation carriers.

BACKGROUND: Asymptomatic carriers of leucine-rich repeat kinase 2 (LRRK2) gene mutations constitute an ideal population for discovering prodromal biomarkers of Parkinson's disease (PD). In this study, we aim to identify CSF candidate risk biomarkers of PD in individuals with LRRK2 mutation carriers. METHODS: We measured the levels of CSF total- (t-), oligomeric (o-) and phosphorylated S129 (pS129-) α-syn, total-tau (tTau), phosphorylated threonine 181 tau (pTau), amyloid-beta 40 (Aß-40), amyloid-beta-42 (Aß-42) and 40 inflammatory chemokines in symptomatic (n = 23) and asymptomatic (n = 51) LRRK2 mutation carriers, subjects with a clinical diagnosis of PD (n = 60) and age-matched healthy controls (n = 34). General linear models corrected for age and gender were performed to assess differences in CSF biomarkers between the groups. Markers that varied significantly between the groups were then analyzed using backward-elimination logistic regression analysis to identify an ideal biomarkers panel of prodromal PD. RESULTS: Discriminant function analysis revealed low levels of CSF t-α-syn, high levels of CSF o-α-syn and TNF-α best discriminated asymptomatic LRRK2 mutation carriers from both symptomatic PD and healthy controls. Assessing the discriminative power using receiver operating curve analysis, an area under the curve > 0.80 was generated. CONCLUSIONS: The current study suggests that CSF t-, o-α-syn and TNF-α are candidate risk biomarkers for the detection of PD at the prodromal stage. Our findings also highlight the dynamic interrelationships between CSF proteins and the importance of using a biomarkers' panel approach for an accurate and timely diagnosis of PD.

Bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates.

In Parkinson's disease, synucleinopathy is hypothesized to spread from the enteric nervous system, via the vagus nerve, to the CNS. Here, we compare, in baboon monkeys, the pathological consequences of either intrastriatal or enteric injection of α-synuclein-containing Lewy body extracts from patients with Parkinson's disease. This study shows that patient-derived α-synuclein aggregates are able to induce nigrostriatal lesions and enteric nervous system pathology after either enteric or striatal injection in a non-human primate model. This finding suggests that the progression of α-synuclein pathology might be either caudo-rostral or rostro-caudal, varying between patients and disease subtypes. In addition, we report that α-synuclein pathological lesions were not found in the vagal nerve in our experimental setting. This study does not support the hypothesis of a transmission of α-synuclein pathology through the vagus nerve and the dorsal motor nucleus of the vagus. Instead, our results suggest a possible systemic mechanism in which the general circulation would act as a route for long-distance bidirectional transmission of endogenous α-synuclein between the enteric and the central nervous systems. Taken together, our study provides invaluable primate data exploring the role of the gut-brain axis in the initiation and propagation of Parkinson's disease pathology and should open the door to the development and testing of new therapeutic approaches aimed at interfering with the development of sporadic Parkinson's disease.

Investigating the presence of doubly phosphorylated α-synuclein at tyrosine 125 and serine 129 in idiopathic Lewy body diseases.

Aggregation of the protein α-synuclein (α-syn) into insoluble intracellular assemblies termed Lewy bodies (LBs) is thought to be a critical pathogenic event in LB diseases such as Parkinson's disease and dementia with LBs. In LB diseases, the majority of α-syn is phosphorylated at serine 129 (pS129), suggesting that this is an important disease-related post-translational modification (PTM). However, PTMs do not typically occur in isolation and phosphorylation at the proximal tyrosine 125 (pY125) residue has received considerable attention and has been inconsistently reported to be present in LBs. Furthermore, the proximity of Y125 to S129 means that some pS129 antibodies may have epitopes that include Y125, in which case phosphorylation of Y125 will impede recognition of α-syn. This would potentially lead to underestimating LB pathology burdens if pY125 occurs alongside pS129. To address the apparent controversy in the literature regarding the detection of pY125, we investigated its presence in the LB pathology. We generated pS129 antibodies whose epitope includes or does not include Y125 and compared the extent of α-syn pathology recognized in mouse models of α-synucleinopathies, human brain tissue lysates and fixed post-mortem brain tissues. Our study demonstrated no difference in α-syn pathology recognized between pS129 antibodies, irrespective of whether Y125 was part of the epitope or not. Furthermore, evaluation with pY125 antibodies whose epitope does not include S129 demonstrated no labeling of LB pathology. This study reconciles disparate results in the literature and demonstrates pY125 is not a key component of LB pathology in murine models or human tissues in idiopathic LB diseases.

Generation of monoclonal antibodies against phosphorylated α-Synuclein at serine 129: Research tools for synucleinopathies.

The majority of α-synuclein (α-syn) within Lewy bodies (LBs) has been reported to be phosphorylated at serine 129 (pS129-α-syn), suggesting a central role for phosphorylation in the pathogenesis of Parkinson's disease (PD) and related synucleinopathies. Various studies have investigated the effect of α-syn phosphorylation but have failed to reach a consensus as to whether this modification accelerates or inhibits α-syn aggregation. Nevertheless, pS129-α-syn is a reliable marker of α-syn aggregates and is widely evaluated in biomarkers and post-mortem studies. While several antibodies specific for pS129-α-syn exist, their reactivity with non-specific antigens appears to be a common challenge. To gain valuable insights into the role of α-syn phosphorylation in disease pathogenesis, antibodies that are highly specific to pS129-α-syn are necessary. In this study, we describe the generation of three mouse monoclonal antibodies (mAbs; 5B9, 6H5 and 9G1) using hybridoma technology. These were thoroughly characterized and validated in combination with our previously generated mAb (PS129), and the commercial ab51253 (Abcam). We demonstrated that our mAbs are highly specific for pS129-α-syn and do not cross react with wild-type α-syn. Results from staining of post-mortem human brain tissue showed that our mAbs detect pS129-α-syn pathology in patients with synucleinopathies. This study highlights three new antibodies as excellent and highly specific research tools to explore the role of pS129-α-syn inclusions in synucleinopathies.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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 ß-sheet content after subjecting them to proteinase K digestion. CONCLUSIONS: 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.