Functional assays for the assessment of the pathogenicity of variants of GOSR2, an ER-to-Golgi SNARE involved in progressive myoclonus epilepsies.

Progressive myoclonus epilepsies (PMEs) are inherited disorders characterized by myoclonus, generalized tonic-clonic seizures, and ataxia. One of the genes that is associated with PME is the ER-to-Golgi Qb-SNARE GOSR2, which forms a SNARE complex with syntaxin-5, Bet1 and Sec22b. Most PME patients are homo-zygous for a p.Gly144Trp mutation and develop similar clinical presentations. Recently, a patient who was compound heterozygous for p.Gly144Trp and a previously unseen p.Lys164del mutation was identified. Because this patient presented with a milder disease phenotype, we hypothesized that the p.Lys164del mutation may be less severe compared to p.Gly144Trp. To characterize the effect of the p.Gly144Trp and p.Lys164del mutations, both of which are present in the SNARE motif of GOSR2, we examined the corresponding mutations in the yeast ortholog Bos1. Yeasts expressing the orthologous mutants in Bos1 showed impaired growth, suggesting a partial loss of function, which was more severe for the Bos1 p.Gly176Trp mutation. Using anisotropy and gel filtration, we report that Bos1 p.Gly176Trp and p.Arg196del are capable of complex formation, but with partly reduced activity. Molecular dynamics (MD) simulations showed that the hydrophobic core, which triggers SNARE complex formation, is compromised due to the glycine-to-tryptophan substitution in both GOSR2 and Bos1. In contrast, the deletion of residue p.Lys164 (or p.Arg196del in Bos1) interferes with the formation of hydrogen bonds between GOSR2 and syntaxin-5. Despite these perturbations, all SNARE complexes stayed intact during longer simulations. Thus, our data suggest that the milder course of disease in compound heterozygous PME is due to less severe impairment of the SNARE function

Two C-terminal Sequence Variations Determine Differential Neurotoxicity Between Human and Mouse α-synuclein

BACKGROUND: α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson\u27s disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. METHODS: Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey\u27s multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn\u27s multiple comparisons test or a two-tailed Mann-Whitney test. RESULTS: Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. CONCLUSIONS: Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders

Raw sequencing data of GBA1 (all exons) and LRRK2 (Exon 31 and 41)

Sequencing of GBA1 (all exons) and LRRK2 (Exon 31 and 41) for all lines included in the manuscript: LRRK2 kinase activity regulates lysosomal glucocerebrosidase in Parkinson's disease pathogenesis. We do not observed unexpected point mutations in theses genes in non-mutant cell lines

"LRRK2 kinase activity regulates lysosomal glucocerebrosidase in Parkinson's disease pathogenesis" Sequencing Data

Sequencing of GBA1 (all exons) and LRRK2 (Exon 31 and 41) for all lines included in the manuscript: LRRK2 kinase activity regulates lysosomal glucocerebrosidase in Parkinson's disease pathogenesis. We do not observed unexpected point mutations in these genes in non-mutant cell lines

Sequencing of LRRK2 and GBA1

Sequencing of GBA1 (all exons) and LRRK2 (Exon 31 and 41) for all lines included in the manuscript: LRRK2 kinase activity regulates lysosomal glucocerebrosidase in Parkinson's disease pathogenesis. We do not observed unexpected point mutations in theses genes in non-mutant cell lines

"LRRK2 kinase activity regulates lysosomal glucocerebrosidase in Parkinson's disease pathogenesis" Sequencing Data

Sequencing of GBA1 (all exons) and LRRK2 (Exon 31 and 41) for all lines included in the manuscript: LRRK2 kinase activity regulates lysosomal glucocerebrosidase in Parkinson's disease pathogenesis. We do not observed unexpected point mutations in these genes in non-mutant cell lines

Inhibition of Membrane-Induced Alpha-Synuclein (aSyn) Aggregation: A Strategy to Interfere with aSyn Neurotoxicity in Parkinson's Disease (PD)

Branfman Family Foundation, Indiana CTSI Core Pilot Grant, Indiana CTSI Collaboration in Translational Research Grant, Richard F. Borch Research Enhancement Awar

Lysosomal integral membrane protein-2 as a phospholipid receptor revealed by biophysical and cellular studies

Lysosomal integral membrane protein-2 (LIMP-2) is a glucocerebrosidase receptor, which is linked to kidney failure and other diseases. Here the authors show that LIMP-2 is also a phospholipid receptor and present the lipid-bound structure of the LIMP-2 luminal domain dimer and discuss its lipid trafficking mechanism