Python script for Golgi cytoscape analysis
By onCustom Python script used for the cytoscape network analysis reported in doi.org/10.1101/2022.11.22.517583 (Golgi-IP, a novel tool for multimodal analysis of Golgi molecular content).
BPK1520-LRRK2-G2019S-ng
By onPlasmid: This resource can be used to introduce LRRK2-G2019S mutation using prime editing, in PE3 approach.
POLCAM: Instant molecular orientation microscopy for the life sciences
By onPOLCAM is a simplified single-molecule orientation localization microscopy method, using a polarization camera, enabling fast easy implementation on fluorescence microscopes. Here, we apply it to alpha-synuclein fibrils.
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.
LC3 Lipidation assay for ATG3 Mutants
By onProtocol describing the procedure to perform LC3 lipidation reaction assay on liposomes (SUVs, Small Unilamellar Vesicles).
pCAG-MBP-Foldon-ATG9 (692-839)
By onPlasmid for expression of ATG9 C-terminal tail trimer in mammalian cells.
H9 ES AAVS1-NGN2 FAM134C/A/B-/-;TEX264-/-; CCPG1-/-; PiggyBac-Keima-REEP5
By onES cells were modified to produce iNeurons lacking ER-phagy receptor genes and expressing Keima-REEP5 reporter. CRISPR/Cas9 was used to introduce NEUROG2 construct in AAVS1 locus, and various genes were knocked out.
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.
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.
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.
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.
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.
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
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.
