pCAG-MBP-Foldon-ATG9 (692-839)
By onPlasmid for expression of ATG9 C-terminal tail trimer in mammalian cells.
LC3 Lipidation assay for ATG3 Mutants
By onProtocol describing the procedure to perform LC3 lipidation reaction assay on liposomes (SUVs, Small Unilamellar Vesicles).
Synaptotagmin-1-dependent phasic axonal dopamine release is dispensable for basic motor behaviors in mice
By onIn Parkinson’s disease (PD), motor dysfunctions only become apparent after extensive loss of DA innervation. This resilience has been hypothesized to be due to the ability of many motor behaviors to be sustained through a diffuse basal tone of DA; but experimental evidence for this is limited. Here we show that conditional deletion of the calcium sensor synaptotagmin-1 (Syt1) in DA neurons (Syt1 cKODA mice) abrogates most activity-dependent axonal DA release in the striatum and mesencephalon, leaving somatodendritic (STD) DA release intact. Strikingly, Syt1 cKODA mice showed intact performance in multiple unconditioned DA-dependent motor tasks and even in a task evaluating conditioned motivation for food. Considering that basal extracellular DA levels in the striatum were unchanged, our findings suggest that activity-dependent DA release is dispensable for such tasks and that they can be sustained by a basal tone of extracellular DA. Taken together, our findings reveal the striking resilience of DA-dependent motor functions in the context of a near-abolition of phasic DA release, shedding new light on why extensive loss of DA innervation is required to reveal motor dysfunctions in PD.
Design and preparation of synthetic reference peptides for APP/Aβ TOMAHAQ proteomics, version 2
By onThis protocol describes the design and preparation of synthetic reference peptides for APP/Aβ TOMAHAQ proteomics.
Thawing of feeder-free hPSCs
By onThis protocol describes the procedure of thawing feeder-free human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex.
The chaperone Clusterin in neurodegeneration−friend or foe?
By onThe authors review the diverse functions of Clusterin in the pathogenesis of neurodegenerative diseases, focusing on evidence that Clusterin may act either as a suppressor or enhancer of pathology.
H9 ES AAVS1-NGN2 FAM134C/A/B-/-; PiggyBac-Keima-RAMP4
By onES cells were modified to produce iNeurons lacking FAM134C, A, B receptors and expressing Keima-RAMP4 ER-phagy flux reporter. CRISPR/Cas9 was used to introduce NEUROG2 construct and knockout RETREG1, 2, 3 genes.
H9 ES AAVS1-NGN2 TEX264-/-; PiggyBac-Keima-RAMP4
By onES cells were modified using CRISPR/Cas9 to lack TEX264 and express Keima-RAMP4 ER-phagy flux reporter. NEUROG2 construct was introduced in AAVS1 locus. Cells are human embryonic stem cells with a fluorescent protein marker.
Assay for PhosphoRab activation of LRRK2 Kinase
By onThis protocol includes a method to produce phosphoRab8A protein and remove as much contaminating MST3 as possible, to enable use of the phosphoRab to test subsequent activation of LRRK2 kinase.
Interaction of an α-synuclein epitope with HLA-DRB1*15:01 triggers enteric features in mice reminiscent of prodromal Parkinson’s disease
By onInteraction of α-syn32-46 and HLA-DRB1*15:0 is critical for gut inflammation and CD4+ T cell-mediated loss of enteric neurons in humanized mice, suggesting potential mechanisms of prodromal enteric PD.
Organelle isolation from Mouse Embryonic Fibroblasts (MEFs) stably expressing organelle tags for subsequent immunoblotting or proteomic analysis
By onWe describe here a method to perform rapid isolation of intact organelles (including lysosomes and Golgi) from mouse embryonic fibroblasts stably expressing an organelle tag (TMEM192-3xHA, or LysoTag, and TMEM115-3xHA, or GolgiTag). First, cells are broken using a ball-bearing cell breaker, leading to plasma membrane rupture, while lysosomes and Golgi remain intact. Then, the cell homogenate is incubated with anti-HA magnetic beads to allow for immunopurification of HA-tagged lysosomes or Golgi in less than 15 minutes. The organelles purified using this method are highly enriched, intact, contaminant-free and, depending on solubilisation buffer, can be used for various downstream applications, including immunoblotting analysis and mass spectrometry proteomic analysis (as described here), but also metabolomic or lipidomic analysis. This protocol can be adapted to isolate organelles from commonly cultured cells, such as HEK293 and A549 cells, that express an organelle tag.
Images of adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain
By onDataset of images supporting the publication: Chuapoco, M.R., Flytzanis, N.C., Goeden, N. et al. Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain. Nat. Nanotechnol. (2023). https://doi.org/10.1038/s41565-023-01419-x Publication Abstract: Crossing the blood-brain-barrier in non-human primates (NHPs) is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust gene delivery to the brain through non-invasive, intravenous delivery. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here, we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and new-born macaques which has improved delivery efficiency in the brains of multiple NHP species: marmoset, rhesus macaque, and green monkey. CAP-Mac is neuron-biased in infant Old-World primates, exhibits broad tropism in adult rhesus macaques, and is vasculature-biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labeling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of NHPs.
LRRK2 suppresses lysosome degradative activity in macrophages and microglia through MiT-TFE transcription factor inhibition
By onCells maintain optimal levels of lysosome degradative activity to protect against pathogens, clear waste, and generate nutrients. Here, we show that LRRK2, a protein that is tightly linked to Parkinson’s disease, negatively regulates lysosome degradative activity in macrophages and microglia via a transcriptional mechanism. Depletion of LRRK2 and inhibition of LRRK2 kinase activity enhanced lysosomal proteolytic activity and increased the expression of multiple lysosomal hydrolases. Conversely, the kinase hyperactive LRRK2 G2019S Parkinson’s disease mutant suppressed lysosomal degradative activity and gene expression. We identified MiT-TFE transcription factors (TFE3, TFEB, and MITF) as mediators of LRRK2-dependent control of lysosomal gene expression. LRRK2 negatively regulated the abundance and nuclear localization of these transcription factors and their depletion prevented LRRK2-dependent changes in lysosome protein levels. These observations define a role for LRRK2 in controlling lysosome degradative activity and support a model wherein LRRK2 hyperactivity may increase Parkinson’s disease risk by suppressing lysosome degradative activity.
Manual isolation of nuclei from human brain using CellRaft device and single nucleus Whole Genome Amplification
By onProtocol for manual nuclear isolation from human brain tissue using Cell Raft device for single cell Whole Genome Amplification
Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy
By onGUV quantification
In vitro GCase activity assay (total cell lysate)
By onGlucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide (GlcCer), a membrane glyco-sphingolipid, to ceramide and glucose. This assay detects GBA activity by using a fluorogenic substrate that reacts with cell lysates previously treated with or without CBE (GBA1 inhibitor). This protocol is part of a Collection of protocols (dx.doi.org/10.17504/protocols.io.8epv593dng1b/v1) for the paper "Glucocerebrosidase, a Parkinson´s disease-associated protein, is imported into mitochondria and regulates complex I assembly and function" (https://doi.org/10.21203/rs.3.rs-1521848/v1)
ENS Quantification
By onLabelled enteric neurons in living mice with recombinant adeno-associated viruses (rAAVs) expressing fluorescent proteins, and used tissue clearing techniques to enhance visualization of intact GI tissue.
