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

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

Increased levels of CSF total but not oligomeric or phosphorylated forms of alpha-synuclein in patients diagnosed with probable Alzheimer's disease

Several studies reported an association between CSF alpha-synuclein (α-syn) and tau in Alzheimer's disease (AD), and demonstrated the significance of α-syn in improving the diagnostic sensitivity/specificity of classical AD CSF biomarkers. In the current study, we measured CSF levels of different α-syn species in a cohort of AD patients (n = 225) who showed a CSF profile typical of AD at baseline as well as in cognitively intact controls (n = 68). CSF total α-syn (t-α-syn) significantly increased in the AD group (p < 0.0001) compared to controls, while oligomeric- and phosphorylated-Ser129-α-syn did not change significantly. ROC analysis showed a sensitivity of 85% and a specificity of 84% (AUC = 0.88) in distinguishing AD from controls. T-α-syn levels correlated positively with tau species in AD group and negatively with baseline MMSE score. Our data support the added value of measurement of CSF α-syn species for further characterization of the CSF AD profile

CSF total and oligomeric α-Synuclein along with TNF-α as risk biomarkers for Parkinson\u27s disease: a study in LRRK2 mutation carriers

Background: Asymptomatic carriers of leucine-rich repeat kinase 2 (LRRK2) gene mutations constitute an idealpopulation for discovering prodromal biomarkers of Parkinson’s disease (PD). In this study, we aim to identify CSFcandidate 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, totaltau(tTau), phosphorylated threonine 181 tau (pTau), amyloid-beta 40 (Aβ-40), amyloid-beta-42 (Aβ-42) and 40inflammatory chemokines in symptomatic (n = 23) and asymptomatic (n = 51) LRRK2 mutation carriers, subjects witha clinical diagnosis of PD (n = 60) and age-matched healthy controls (n = 34). General linear models corrected forage and gender were performed to assess differences in CSF biomarkers between the groups. Markers that variedsignificantly between the groups were then analyzed using backward-elimination logistic regression analysis toidentify 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 bestdiscriminated asymptomatic LRRK2 mutation carriers from both symptomatic PD and healthy controls. Assessing thediscriminative 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 thedetection of PD at the prodromal stage. Our findings also highlight the dynamic interrelationships between CSFproteins and the importance of using a biomarkers’ panel approach for an accurate and timely diagnosis of PD.Keywords: Parkinson’s disease, LRRK2 mutation carriers, Alpha-synuclein oligomers, Biomarkers, Inflammatorymarker

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.Other Information Published in: Scientific Reports License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1038/s41598-020-65035-8</p

Lewy body-like alpha-synuclein inclusions trigger reactive microgliosis prior to nigral degeneration

Abstract Background Converging evidence suggests a role for microglia-mediated neuroinflammation in Parkinson’s disease (PD). Animal models of PD can serve as a platform to investigate the role of neuroinflammation in degeneration in PD. However, due to features of the previously available PD models, interpretations of the role of neuroinflammation as a contributor to or a consequence of neurodegeneration have remained elusive. In the present study, we investigated the temporal relationship of neuroinflammation in a model of synucleinopathy following intrastriatal injection of pre-formed alpha-synuclein fibrils (α-syn PFFS). Methods Male Fischer 344 rats (N = 114) received unilateral intrastriatal injections of α-syn PFFs, PBS, or rat serum albumin with cohorts euthanized at monthly intervals up to 6 months. Quantification of dopamine neurons, total neurons, phosphorylated α-syn (pS129) aggregates, major histocompatibility complex-II (MHC-II) antigen-presenting microglia, and ionized calcium-binding adaptor molecule-1 (Iba-1) immunoreactive microglial soma size was performed in the substantia nigra. In addition, the cortex and striatum were also examined for the presence of pS129 aggregates and MHC-II antigen-presenting microglia to compare the temporal patterns of pSyn accumulation and reactive microgliosis. Results Intrastriatal injection of α-syn PFFs to rats resulted in widespread accumulation of phosphorylated α-syn inclusions in several areas that innervate the striatum followed by significant loss (~ 35%) of substantia nigra pars compacta dopamine neurons within 5–6 months. The peak magnitudes of α-syn inclusion formation, MHC-II expression, and reactive microglial morphology were all observed in the SN 2 months following injection and 3 months prior to nigral dopamine neuron loss. Surprisingly, MHC-II immunoreactivity in α-syn PFF injected rats was relatively limited during the later interval of degeneration. Moreover, we observed a significant correlation between substantia nigra pSyn inclusion load and number of microglia expressing MHC-II. In addition, we observed a similar relationship between α-syn inclusion load and number of microglia expressing MHC-II in cortical regions, but not in the striatum. Conclusions Our results demonstrate that increases in microglia displaying a reactive morphology and MHC-II expression occur in the substantia nigra in close association with peak numbers of pSyn inclusions, months prior to nigral dopamine neuron degeneration, and suggest that reactive microglia may contribute to vulnerability of SNc neurons to degeneration. The rat α-syn PFF model provides an opportunity to examine the innate immune response to accumulation of pathological α-syn in the context of normal levels of endogenous α-syn and provides insight into the earliest neuroinflammatory events in PD

Correction to: Lewy body-like alpha-synuclein inclusions trigger reactive microgliosis prior to nigral degeneration

After publication of the original article [1] it was noted that the name of author, D. Luke Fisher, was erroneously typeset in both the PDF and online formats of the manuscript as Luke D. Fisher