Common variant genome-wide association studies (GWASs) have, to date, identified >24 risk loci for Parkinson’s disease (PD). To discover additional loci, we carried out a GWAS comparing 6,476 PD cases with 302,042 controls, followed by a meta-analysis with a recent study of over 13,000 PD cases and 95,000 controls at 9,830 overlapping variants. We then tested 35 loci (P < 1 × 10−6) in a replication cohort of 5,851 cases and 5,866 controls. We identified 17 novel risk loci (P < 5 × 10−8) in a joint analysis of 26,035 cases and 403,190 controls. We used a neurocentric strategy to assign candidate risk genes to the loci. We identified protein-altering or cis–expression quantitative trait locus (cis-eQTL) variants in linkage disequilibrium with the index variant in 29 of the 41 PD loci. These results indicate a key role for autophagy and lysosomal biology in PD risk, and suggest potential new drug targets for PD.

Botulinum neurotoxins (BoNTs) act within the synaptic terminal to block neurotransmitter release. The toxin enters the neuron by binding to neuronal membrane receptor(s), being taken up into an endosome-like compartment, and penetrating the endosome membrane via a pH-dependent translocation process. Once within the synaptic cytoplasm, BoNT serotypes A and E cleave separate sites on the C-terminus of the neuronal protein SNAP-25, one of the SNARE proteins required for synaptic vesicle fusion. In this study, we measured the effect of brief toxin exposure on SNAP-25 proteolysis in neuronal cell cultures as an indicator of toxin translocation. The results indicate that (1) uptake of both BoNT-A and -E is enhanced with synaptic activity induced by K+ depolarization in the presence of Ca2+ and (2) translocation of BoNT-A from the acidic endosomal compartment is slow relative to that of BoNT-E. Polyclonal antisera against each toxin protect cells when applied with the toxin during stimulation but has no effect when added immediately after toxin exposure, indicating that toxin endocytosis occurs with synaptic activity. Both serotypes cleave SNAP-25 at concentrations between 50 pM and 4 nM. IC50 values for SNAP-25 cleavage are approximately 0.5 nM for both serotypes. Inhibition of the pH-dependent translocation process by pretreating cultures with concanamycin A (Con A) prevents cleavage of SNAP-25 with IC50 values of approximately 25 nM. Addition of Con A at times up to 15 min after toxin exposure abrogated BoNT-A action; however, addition of Con A after 40 min was no longer protective. In contrast, Con A inhibited, but did not prevent, translocation of BoNT-E even when added immediately after toxin exposure, indicating that pH-dependent translocation of BoNT-E is rapid relative to that of BoNT-A. This study demonstrates that uptake of both BoNT-A and -E is enhanced with synaptic activity and that translocation of the toxin catalytic moiety into the cytosol occurs at different rates for these two serotypes.

The crystal structure of LRH-1 ligand binding domain bound to our previously reported agonist 3-(E-oct-4-en-4-yl)-1-phenylamino-2-phenyl-cis-bicyclo[3.3.0]oct-2-ene 5 is described. Two new classes of agonists in which the bridgehead anilino group from our first series was replaced with an alkoxy or 1-ethenyl group were designed, synthesized, and tested for activity in a peptide recruitment assay. Both new classes gave very active compounds, particularly against SF-1. Structure-activity studies led to excellent dual-LRH-1/SF-1 agonists (e.g., RJW100) as well as compounds selective for LRH-1 (RJW101) and SF-1 (RJW102 and RJW103). The series based on 1-ethenyl substitution was acid stable, overcoming a significant drawback of our original bridgehead anilino-substituted series. Initial studies on the regulation of gene expression in human cell lines showed excellent, reproducible activity at endogenous target genes.

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