Glucocerebrosidase, a Parkinson´s disease-associated protein, is imported into mitochondria and regulates complex I assembly and function
By savannah onMutations in the lysosomal enzyme β-glucocerebrosidase (GCase), which cause Gaucher's disease, are the most frequent genetic risk factor for Parkinson’s disease (PD). Here, the authors employed global proteomic and single-cell genomic approaches in stable cell lines as well as induced pluripotent stem cell (iPSC)-derived neurons and midbrain organoids to dissect the mechanisms underlying GCase-related neurodegeneration.
Cryosectioning mouse brain
By savannah onThis protocol details the cryosectioning of the mouse brain.
Plasmid-reprogramming of human iPSCs
By savannah onThis protocol details about plasmid-reprogramming of human fibroblasts.
Protein network analysis links the NSL complex to Parkinson’s disease and mitochondrial biology
By savannah onA bioinformatics approach was taken to investigate the proteome of the NSL complex, to unpick its relevance to PD progression. The authors’ data points to NSL complex members OGT and WDR5 as key drivers of this increased PD association. These findings strengthen a role for mitochondrial quality control in both familial and sporadic disease.
AAVS1-SA-neo-CAGGS-PE2-2A-GFP
By savannah onThis resource can be used to generate knock-in cell line faciliating prime editing.
A combined cell and gene therapy approach for homotopic reconstruction of midbrain dopamine pathways using human pluripotent stem cells
By savannah onThese results demonstrate the remarkable capacity for achieving functional and anatomically precise reconstruction of long-distance circuitry in the adult brain by matching appropriate growth-factor signaling to grafting of specific cell types.
A prebiotic diet modulates microglia response and motor deficits in α-synuclein overexpressing mice (Dataset)
By savannah onThese studies uncover a novel microglia-dependent interaction between diet and motor symptoms in mice, findings that may have implications for neuroinflammation and PD.
ASAP Blueprint for Collaborative Open Science
By savannah onThis Blueprint presents initial findings on how ASAP’s approach to open science has solidified and evolved over its first three years, data and metrics on progress, and CC-BY versions of assets that can be adopted and adapted by others. ASAP plans to update the Blueprint with new findings and assets on a regular basis.
Basal ganglia neurons in healthy and Parkinsonian primates generate recurring sequences of spikes
By savannah onThe authors conclude that basal ganglia neurons fire in recognizable sequences of ISIs, whose incidence is influenced by the induction of parkinsonism.
Brain repair by cell replacement via in situ neuronal reprogramming
By savannah onThis review focuses on key advances in generating new neurons through in situ neuronal reprogramming, which is tied to fundamental questions regarding adult neurogenesis, cell source, and mechanisms for neuronal reprogramming, as well as the ability of new neurons to integrate into the existing circuitry.
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 onHere the authors examined the impact of gene variants on mortality and cognitive impairment in PD.
HyDrop: droplet-based scATAC-seq and scRNA-seq using dissolvable hydrogel beads
By savannah onThe 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
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 onAt acid pH B–glucosidase hydrolyses the substrate 4–methylumbelliferyl–B–D–glucopyranoside to 4–methylumbelliferone and glucose. Adding alkaline buffer stops the enzyme reaction and causes 4–methylumbelliferone to fluoresce at a different wavelength from unhydrolyzed substrate, thereby permitting its measurement in the presence of a vast excess of unhydrolyzed substrate.
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.
