Proteomic analysis of iNeurons lacking ER-phagy receptors
By onProteomic analysis of iNeurons and cells lacking ER-phagy receptors FAM134C, FAM134A, FAM134B, TEX264, and CCPG1 showed distinct protein profiles, highlighting their roles in ER-phagy pathways.
Nicotine-Mediated Rescue of α-Synuclein Toxicity Requires Synaptic Vesicle Glycoprotein 2 in Drosophila
By onBackground: Parkinson's disease (PD) is characterized by α-synuclein aggregation and loss of dopamine neurons. Risk of PD arises due to a combination of genetic and environmental factors, which may interact, termed gene-environment (G×E) interactions. An inverse association between smoking and the risk of PD is well established, and a previous genome-wide G×E interaction study identified genetic variation in the synaptic-vesicle glycoprotein 2C (SV2C) locus as an important mediator of the degree to which smoking is inversely associated with PD. Objective: We sought to determine the mechanism of the smoking-SV2C interaction in a Drosophila model of PD. Methods: Flies expressing human α-synuclein in all neurons develop the hallmarks of PD, including motor dysfunction, loss of dopaminergic (DA) neurons, and formation of α-synuclein inclusions. We assessed the effects of increasing doses of nicotine on these parameters of neurodegeneration, in the presence or absence of knockdown of two Drosophila orthologues of SV2, hereafter referred to as SV2L1 and SV2L2. Results: The α-synuclein-expressing flies treated with nicotine had improved locomotion, DA neuron counts, and α-synuclein aggregation. However, in α-synuclein-expressing flies in which SV2L1 and SV2L2 were knocked down, nicotine failed to rescue neurodegeneration. Conclusions: This work confirms a G×E interaction between nicotine and SV2, defines a role for this interaction in α-synuclein proteostasis, and suggests that future clinical trials on nicotine should consider genetic variation in SV2C. Furthermore, this provides proof of concept that our model can be used for the mechanistic study of G×E, paving the way for the investigation of additional G×E interactions or the identification of novel G×E. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Binding of Rab29 to the LRRK2 Armadillo domain by Microscale Thermophoresis
By onMicroscale thermophoresis is a powerful tool to measure the affinities of interactions between proteins. We present here our method for determining the binding of Rab29 GTPase to the LRRK2 N-terminal Armadillo domain.
Post-fibrillization nitration of alpha-synuclein abolishes its seeding activity and pathology formation in primary neurons and in vivo
By onIncreasing evidence points to post-translational modifications (PTMs) as key regulators of alpha-synuclein (α-Syn) function in health and disease. However, whether these PTMs occur before or after α-Syn pathology formation and their role in regulating α-Syn toxicity remain unclear. In this study, we demonstrate that post-fibrillization nitration of α-Syn fibrils induced their fragmentation, modified their surface and dynamic properties but not their structure, and nearly abolished their seeding activity in primary neurons and in vivo. Furthermore, we show that the dynamic and surface properties of the fibrils, rather than simply their length, are important determinants of α-Syn fibril seeding activity. Altogether, our work demonstrates that post-aggregation modifications of α-Syn may provide novel approaches to target a central process that contributes to pathology formation and disease progression. Finally, our results suggest that the pattern of PTMs on pathological aggregates, rather than simply their presence, could be a key determinant of their toxicity and neurodegeneration. This calls for reconsidering current approaches relying solely on quantifying and correlating the level of pathology to assess the efficacy of novel therapies, as not all α-Syn aggregates in the brain are pathogenic.
Primary data associated with doi: 10.1042/BCJ20220161 (Impact of 100 LRRK2 variants linked to Parkinson’s Disease on kinase activity and microtubule binding)
By onRaw immunoblotting data; tabular data for quantifications; numerical data for in vitro assays; immunofluorescence images; Prism files for statistical analysis.
Rat brain processing for histological analyses
By onProtocol for rat brain processing for histological analyses.
Indirect Proximity Ligation Assay (PLA) – Brightfield
By onThe authors describe the PLA protocol that is routinely used in the laboratory to detect nitrated alpha-synuclein and nitration of mitochondrial enzymes such as SOD2 and the mitochondrial complex 1 subunit NDUFB8.
Generation of ATG3 KO Hela cells stably expressing HaloTag-LC3B
By onThis protocol details generation of ATG3 KO HeLa cells stably expressing HaloTag-LC3B.
Single cell/nuclei RNAseq analysis
By onThis protocol describes the process for the single cell/nuclei RNA sequencing data of the manuscript "L1retrotransposons drive human neuronal transcriptome complexity and functional diversification " from fetal forebrain and adult prefrontal cortex tissue.
Iso-Seq mapping to L1HS/PA2 consensus sequence
By onThe protocol describes the steps to map HiFi reads to a consensus sequence and retrieve density plots.
Thawing, Passaging and Freezing of hPSCs on MEFs
By onThis collection contains protocols which describe the standard procedure of culturing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).
Subcloning of genotype-confirmed hPSCs clones
By onThis protocol describes a standard procedure for subcloning of genotype-confirmed human pluripotent stem cells (hPSCs).
Tet-off TauRD (P301L/V337M)
By onPlasmid: Tet-off TauRD (P301L/V337M). Associated with the following preprint: Saha et al., 2022 (published on biorxiv on February 19, 2022).
RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors
By onGain-of-function mutations in LRRK2 gene lead to by increasing phosphorylation of RAB GTPases. Over-phosphorylation of RAB3A disrupts synaptic vesicle transport in human neurons, altering synaptic protein distribution and likely contributing to PD.
CD8 Cell Density In Substantia Nigra And Cerebral Peduncle Image Analysi
By onQuPath is a bioimage analysis software designed for digital pathology and whole slide image analysis. This protocol describes how to measure CD8 density in the substantia nigra and cerebral peduncle using haematoxylin and DAB-stained brain sections.
The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines
By onThe GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in fibroblasts.
DOPA pheomelanin is increased in nigral neuromelanin of Parkinson’s disease
By onNeuromelanin in the substantia nigra may be a key factor contributing to dopaminergic neuron vulnerability in Parkinson’s disease. Here, the authors investigated the relative composition and specific roles of pheomelanin and eumelanin in PD.
