Rational design of structure-based vaccines targeting misfolded alpha-synuclein conformers of Parkinson's disease and related disorders.

Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are neurodegenerative disorders caused by the accumulation of misfolded alpha-synuclein protein. Developing effective vaccines against synucleinopathies is challenging due to the difficulty of stimulating an immune-specific response against alpha-synuclein without causing harmful autoimmune reactions, selectively targeting only pathological forms of alpha-synuclein. Previous attempts using linear peptides and epitopes without control of the antigen structure failed in clinical trials. The immune system was unable to distinguish between native alpha-synuclein and its amyloid form. The prion domain of the fungal HET-s protein was selected as a scaffold to introduce select epitopes from the surface of alpha-synuclein fibrils. Four vaccine candidates were generated by introducing specific amino acid substitutions onto the surface of the scaffold protein. The approach successfully mimicked the stacking of the parallel in-register beta-sheet structure seen in alpha-synuclein fibrils. All vaccine candidates induced substantial levels of IgG antibodies that recognized pathological alpha-synuclein fibrils derived from a synucleinopathy mouse model. Furthermore, the antisera recognized pathological alpha-synuclein aggregates in brain lysates from patients who died from DLB, MSA, or PD, but did not recognize linear alpha-synuclein peptides. Our approach, based on the rational design of vaccines using the structure of alpha-synuclein amyloid fibrils and strict control over the exposed antigen structure used for immunization, as well as the ability to mimic aggregated alpha-synuclein, provides a promising avenue toward developing effective vaccines against alpha-synuclein fibrils.

Treatment of Organophosphorus Poisoning with 6-Alkoxypyridin-3-ol Quinone Methide Precursors: Resurrection of Methylphosphonate-Aged Acetylcholinesterase.

Organophosphorus (OP) nerve agents inhibit acetylcholinesterase (AChE), creating a cholinergic crisis in which death can occur. The phosphylated serine residue spontaneously dealkylates to the OP-aged form, which current therapeutics cannot reverse. Soman's aging half-life is 4.2 min, so immediate recovery (resurrection) of OP-aged AChE is needed. In 2018, we showed pyridin-3-ol-based quinone methide precursors (QMPs) can resurrect OP-aged electric eel AChE in vitro, achieving 2% resurrection after 24 h of incubation (pH 7, 4 mM). We prepared 50 unique 6-alkoxypyridin-3-ol QMPs with 10 alkoxy groups and five amine leaving groups to improve AChE resurrection. These compounds are predicted in silico to cross the blood-brain barrier and treat AChE in the central nervous system. This library resurrected 7.9% activity of OP-aged recombinant human AChE after 24 h at 250 µM, a 4-fold increase from our 2018 report. The best QMP (1b), with a 6-methoxypyridin-3-ol core and a diethylamine leaving group, recovered 20.8% (1 mM), 34% (4 mM), and 42.5% (predicted maximum) of methylphosphonate-aged AChE activity over 24 h. Seven QMPs recovered activity from AChE aged with Soman and a VX degradation product (EA-2192). We hypothesize that QMPs form the quinone methide (QM) to realkylate the phosphylated serine residue as the first step of resurrection. We calculated thermodynamic energetics for QM formation, but there was no trend with the experimental biochemical data. Molecular docking studies revealed that QMP binding to OP-aged AChE is not the determining factor for the observed biochemical trends; thus, QM formation may be enzyme-mediated.

Vaccination with structurally adapted fungal protein fibrils induces immunity to Parkinson's disease.

The pathological misfolding and aggregation of soluble α-synuclein into toxic oligomers and insoluble amyloid fibrils causes Parkinson's disease, a progressive age-related neurodegenerative disease for which there is no cure. HET-s is a soluble fungal protein that can form assembled amyloid fibrils in its prion state. We engineered HET-s(218-298) to form four different fibrillar vaccine candidates, each displaying a specific conformational epitope present on the surface of α-synuclein fibrils. Vaccination with these four vaccine candidates prolonged the survival of immunized TgM83+/- mice challenged with α-synuclein fibrils by 8% when injected into the brain to model brain-first Parkinson's disease or by 21% and 22% when injected into the peritoneum or gut wall, respectively, to model body-first Parkinson's disease. Antibodies from fully immunized mice recognized α-synuclein fibrils and brain homogenates from patients with Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Conformation-specific vaccines that mimic epitopes present only on the surface of pathological fibrils but not on soluble monomers, hold great promise for protection against Parkinson's disease, related synucleinopathies and other amyloidogenic protein misfolding disorders.

Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm.

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.

Reliability and Variability of tDCS Induced Changes in the Lower Limb Motor Cortex.

BACKGROUND: Transcranial direct current stimulation (tDCS) is emerging as a promising adjuvant to enhance motor function. However, there has been increasing reservations about the reliability and variability of the neuromodulatory effects evoked by tDCS. OBJECTIVE/HYPOTHESIS: The main purpose of this study was to explore the test-retest reliability and inter-individual variability of tDCS of the lower limb M1 and the relationship between transcranial magnetic stimulation (TMS)-related measures and tDCS-induced changes. METHODS: Fifteen healthy participants received anodal tDCS of the lower limb M1 either when performing a lower limb motor task or when the limb was at rest. Each condition was tested twice. tDCS induced changes in corticomotor excitability of the tibialis anterior muscle were measured using TMS. A repeated measures ANOVA was performed to examine efficacy of tDCS between the two task conditions. Intraclass correlation coefficients (ICC) and variance component analyses were performed to examine reliability and variability respectively. RESULTS: A significant increase in in corticomotor excitability was noted for the tDCS-task condition at 140% active motor threshold (AMT) and when comparing recruitment curve slopes, but not at 120% and 130% AMT. Overall, ICC values between testing days for each stimulation condition ranged from 0.6-0.9. Higher ICCs were seen for higher TMS intensities (140% AMT) and recruitment curve slopes. Inter-individual variability contributed to 34% of the exhibited variance. CONCLUSIONS: Our data suggest that the TMS-related measure used to assess neuromodulation after tDCS has an effect on its perceived test-retest reliability and inter-individual variability. Importantly, we noticed that a high reliability and low variability does not necessarily indicate clinical efficacy of tDCS as some participants showed little to no modulation of corticomotor excitability consistently.

Timing-dependent priming effects of tDCS on ankle motor skill learning.

Transcranial direct current stimulation (tDCS) has gained increasing interest in neurorehabilitation with its ability to modulate cortical excitability, and thereby influence neural plasticity and functional recovery. While the beneficial effects of tDCS on motor learning and function have been recognized, there is no clear consensus regarding the timing of the tDCS priming protocol in relation to the intervention especially with respect to lower limb motor learning. Depending on the time of priming in relation to the training task, the neural mechanisms of priming (gating vs. homeostatic plasticity) are different and thereby subsequently affect motor learning. Hence, the aim of this study was to examine the interaction of tDCS with subsequent vs. concurrent motor learning using an ankle visuomotor skill learning paradigm. Twelve healthy participants were tested under three stimulation conditions: (1) anodal tDCS prior to the motor task (tDCS-before), (2) anodal tDCS during the motor task (tDCS-during) and (3) sham tDCS during the motor task (tDCS-sham). Results revealed that tDCS application during practice of a skilled motor task increased motor performance compared to tDCS applied prior to motor practice. Both tDCS groups demonstrated enhanced motor learning when tested 24 hours after practice. We conclude that the priming effects of tDCS are timing dependent, and maybe a critical regulatory feature in determining outcomes of priming with tDCS.