Cell line construction and maintenance for Lyso-IP with or without genes linked with lysosomal storage disease
By savannah onAssociated with the following preprint (published September 30th 2021): Progranulin deficiency results in reduced bis(monoacylglycero)phosphate (BMP) levels and gangliosidosis. bioRxiv 2021.09.30.461806; doi: https://doi.org/10.1101/2021.09.30.461806
Endosomal and lysosomal immunoprecipitation for proteomics, lipidomics, and TEM
By savannah onHere, authors describe an approach for purification of early/sorting endosomes, providing a means by which to examine early aspects of the endolysosomal system (Endo-IP) and to combine this with lysosome purification using Lyso-IP. This allows one to examine the proteome, lipidome, as well as electron microscopy imaging of endosomes.
Genome-wide determinants of mortality and clinical progression in Parkinson’s disease
By savannah onPublished: The authors examined the impact of gene variants on mortality and cognitive impairment in PD. Only the non-Gaucher disease causing GBA PD risk variant E326K, of the known PD risk variants, was associated with progression in PD. View original preprint.
HyDrop: droplet-based scATAC-seq and scRNA-seq using dissolvable hydrogel beads. Article
By savannah onPublished: The data available in this repository can be used to replicate all the figures in the authors’ manuscript using their data analysis tutorial available here: https://github.com/aertslab/hydrop_data_analysis. View original preprint.
Immunofluorescence of RAB5 and FLAG-EEA1 puncta after Dynamin-1 and -2 inhibition with Dyngo4a
By savannah onHere the authors present an immunofluorescence protocol to assess the extent of colocalization between FLAG-EEA1 and RAB5 with and without the Dynamin-1 and -2 (DNM1/2) inhibitor Dyngo4a.
In vitro GCase activity assay (total cell lysate)
By savannah 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).
Lipidomic analysis of tissue culture cells, tissues, and purified organelles
By savannah onThe objective of this protocol is to provide directive on how to extract lipids from plasma, cells, tissue, and purified organelles for analysis by liquid chromatography (LC)-MS. This will typically yield quantitative data for more than 200 lipids, depending on the sample type analyzed, across a range of lipid classes: phospholipids, cardiolipins, sphingolipids, di- and triacyclglycerols, and cholesterylesters.
Neurite_FISH_Quant – Python code for quantification of FISH puncta in neurites
By savannah onThis repository contains Jupyter Notebooks with Python 3 code for quantification of FISH puncta within neuronal dendrites or axons. However, prior identification of FISH puncta in the images (and optional neurite segmentation) is required. We use ImageJ plugins for this purpose. The workflow assumes Z-stack, multi-channel fluorescence images, with one channel used for identification of neurites and other channels for FISH.
Passaging of hPSCs grown on MEFs
By savannah onThis protocol describes the standard procedure of using collagenase to passage human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).
PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074, and Thr1075) within the CORB GTPase domain
By savannah onLeucine-rich-repeat-kinase 1 (LRRK1) and its homologue LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause PD. Here, the authors study the mechanism controlling LRRK1 activity and reveal a novel unexpected activation mechanism. View preprint.
pET30a(+)-PE2
By savannah onThis resource can be used to express and purify PE2 protein for RNP delivery.
pU6-pegRNA-HEK3-CTTins-px330-scaffold
By savannah onThis resource can be used to introduce HEK3 CTT insertion using prime editing. It serves as a good positive control for prime editing.
pU6-pegRNA-SNCA-A30P
By savannah onThis resource can be used to introduce SNCA-A30P mutation using prime editing.
pU6-pegRNA-SNCA-A53T
By savannah onThis resource can be used to introduce SNCA-A53T mutation using prime editing.
pCAG-MBP-Foldon-ATG9 (801-839)
By savannah onTo make ATG9 C-terminal tail trimer for expression in mammalian cells.
LRRK2 RCKW protein purification
By savannah onProtein purification protocol for tag-less LRRK2RCKW as done by Leschziner and Reck-Peterson Labs. Same protocol can be used to purify LRRK1RCKW as well. Original protocol by David Snead. Modified by Yu Xuan Lin and Mariusz Matyszewski for publication.
x4 GBA plasmids
By savannah onThe below plasmids are deposited and available via Addgene: https://www.addgene.org/Anthony_Schapira/. These have been used in publication: 10.1093/hmg/ddac233 188580 WT GBA pcDNA3.1 GBA (Homo sapiens) 188581 E326K GBA pcDNA3.1 GBA (Homo sapiens) 188582 L444P GBA pcDNA3.1 GBA (Homo sapiens) 188583 N370S GBA pcDNA3.1 GBA (Homo sapiens)
The mass spectrometry proteomics data associated of TauRD-Y interactome from TauRD-Y and TauRD-Y* cells
By savannah onThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE84 partner repository with the dataset identifier PXD023400. Associated with the following preprint: Saha et al., 2022 (published on biorxiv on February 19, 2022). https://www.biorxiv.org/content/10.1101/2022.02.18.481043v1.full
The non-specific lethal complex regulates genes and pathways genetically linked to Parkinson’s disease
By savannah onTwo Parkinson's disease candidate genes, KAT8 and KANSL1, identified through genome-wide studies and a PINK1-mitophagy screen, encode part of the histone acetylating non-specific lethal complex. Here, the authors sought to identify whether the non-specific lethal complex has potential regulatory relationships with other genes associated with Parkinson's disease in human brain.
Thawing, passaging, and freezing of hPSCs on MEFs
By savannah onThis collection contains protocols which describe the standard procedure of culturing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).
