Picomolar concentrations of oligomeric alpha-synuclein sensitizes TLR4 to play an initiating role in Parkinson’s disease pathogenesis

Funder: Alzheimer’s Research UK; doi: http://dx.doi.org/10.13039/501100002283Abstract: Despite the wealth of genomic and transcriptomic data in Parkinson’s disease (PD), the initial molecular events are unknown. Using LD score regression analysis, we show significant enrichment in PD heritability within regulatory sites for LPS-activated monocytes and that TLR4 expression is highest within human substantia nigra, the most affected brain region, suggesting a role for TLR4 inflammatory responses. We then performed extended incubation of cells with physiological concentrations of small alpha-synuclein oligomers observing the development of a TLR4-dependent sensitized inflammatory response with time, including TNF-α production. ROS and cell death in primary neuronal cultures were significantly reduced by TLR4 antagonists revealing that an indirect inflammatory mechanism involving cytokines produced by glial cells makes a major contribution to neuronal death. Prolonged exposure to low levels of alpha-synuclein oligomers sensitizes TLR4 responsiveness in astrocytes and microglial, explaining how they become pro-inflammatory, and may be an early causative event in PD

Nanobodies raised against monomeric alpha-synuclein inhibit fibril formation and destabilize toxic oligomeric species

BACKGROUND: The aggregation of the protein ɑ-synuclein (ɑS) underlies a range of increasingly common neurodegenerative disorders including Parkinson’s disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ɑS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ɑS aggregation in vitro in the presence of two ɑS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ɑS. RESULTS: We show that both nanobodies inhibit the formation of ɑS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers of ɑS, leading to a dramatic reduction in oligomer-induced cellular toxicity. CONCLUSIONS: The results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential

Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein

The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson’s disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule Fӧrster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.M.I. acknowledges Dr. Tayyeb-Hussain Scholarship. L.T. has been recipient of a grant PAT Post Doc Outgoing 2009 7th Framework Program Marie Curie COFUND actions. C.D.H. and C.E.B. acknowledge funding from Alzheimer’s Research UK. Augustus Newman Foundation is acknowledged

Nanobodies raised against monomeric ɑ-synuclein inhibit fibril formation and destabilize toxic oligomeric species

$\textbf{Background:}$ The aggregation of the protein ɑ-synuclein (ɑS) underlies a range of increasingly common neurodegenerative disorders including Parkinson’s disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ɑS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ɑS aggregation in vitro in the presence of two ɑS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ɑS. $\textbf{Results:}$ We show that both nanobodies inhibit the formation of ɑS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers of ɑS, leading to a dramatic reduction in oligomer-induced cellular toxicity. $\textbf{Conclusions:}$ The results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential.Parkinson’s UK (H-0903). EDG was supported by the Medical Research Council (MRC G1002272). DK was funded by ERC (669237) and the Royal Society

Self-Assembled Monolayer of Cr7Ni Molecular Nanomagnets by Sublimation

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Management of MDR-TB in HIV co-infected patients in Eastern Europe: Results from the TB:HIV study

Objectives Mortality among HIV patients with tuberculosis (TB) remains high in Eastern Europe (EE), but details of TB and HIV management remain scarce. Methods In this prospective study, we describe the TB treatment regimens of patients with multi-drug resistant (MDR) TB and use of antiretroviral therapy (ART). Results A total of 105 HIV-positive patients had MDR-TB (including 33 with extensive drug resistance) and 130 pan-susceptible TB. Adequate initial TB treatment was provided for 8% of patients with MDR-TB compared with 80% of those with pan-susceptible TB. By twelve months, an estimated 57.3% (95%CI 41.5\u201374.1) of MDR-TB patients had started adequate treatment. While 67% received ART, HIV-RNA suppression was demonstrated in only 23%. Conclusions Our results show that internationally recommended MDR-TB treatment regimens were infrequently used and that ART use and viral suppression was well below the target of 90%, reflecting the challenging patient population and the environment in which health care is provided. Urgent improvement of management of patients with TB/HIV in EE, in particular for those with MDR-TB, is needed and includes widespread access to rapid TB diagnostics, better access to and use of second-line TB drugs, timely ART initiation with viral load monitoring, and integration of TB/HIV care

Picomolar concentrations of oligomeric alpha-synuclein sensitizes TLR4 to play an initiating role in Parkinson’s disease pathogenesis

Funder: Alzheimer’s Research UK; doi: http://dx.doi.org/10.13039/501100002283Abstract: Despite the wealth of genomic and transcriptomic data in Parkinson’s disease (PD), the initial molecular events are unknown. Using LD score regression analysis, we show significant enrichment in PD heritability within regulatory sites for LPS-activated monocytes and that TLR4 expression is highest within human substantia nigra, the most affected brain region, suggesting a role for TLR4 inflammatory responses. We then performed extended incubation of cells with physiological concentrations of small alpha-synuclein oligomers observing the development of a TLR4-dependent sensitized inflammatory response with time, including TNF-α production. ROS and cell death in primary neuronal cultures were significantly reduced by TLR4 antagonists revealing that an indirect inflammatory mechanism involving cytokines produced by glial cells makes a major contribution to neuronal death. Prolonged exposure to low levels of alpha-synuclein oligomers sensitizes TLR4 responsiveness in astrocytes and microglial, explaining how they become pro-inflammatory, and may be an early causative event in PD