ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA-dependent STING signaling
By onMutations in VPS13C cause early onset, autosomal recessive Parkinson’s Disease (PD). We have established that VPS13C encodes a lipid transfer protein localized to contact sites between the endoplasmic reticulum (ER) and late endosomes/lysosomes. In the current study, we demonstrate that depleting VPS13C in HeLa cells causes an accumulation of lysosomes with an altered lipid profile, including an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS/STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation and place VPS13C in pathways relevant to PD pathogenesis.
NHS-ester-protein-labeling
By onProtocol for labeling a purified protein with an NHS ester fluorescent dye.
Ex Vivo Electrophysiology
By onThis protocol describes preparation of brain slices, setup of electrophysiology rig, and solutions for collecting whole cell and cell attached recordings.
Pole Test – mice
By onThis test assesses motor coordination in mice by assessing a mouse's ability to turn and descend on a vertical pole.
Chemical degradation and determination of pheomelanin and eumelanin markers
By onThis is protocol involving chemical oxidation and reduction methods followed by high performance liquid chromatography (HPLC) detection of markers for pheomelanin and eumelanin.
Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK
By onMuch of the biology surrounding Parkin function has taken place in artificial cell systems. The authors used human neurons to identify and validate 22 protein targets of Parkin, providing a functional Parkin landscape in neuronal cells.
Preparation of Acute Brain Slices
By onThis protocol describes the steps for preparing acute brain slices.
Neuromelanin-positive Neuron Density in Substantia Nigra Image Analysis
By onThe protocol covers the steps to measure neuromelanin-positive neuron density in substantia nigra using image analysis tools, including NZConnect (Hamamatsu), a web-based whole-slide image (WSI) viewer, Cellpose, and QuPath.
Three-step docking by WIPI2, ATG16L1 and ATG3 delivers LC3 to the phagophore: Molecular dynamics simulation data
By onAtomistic molecular dynamics simulation data set accompanying manuscript "Three-step docking by WIPI2, ATG16L1 and ATG3 delivers LC3 to the phagophore".
ATG3 construct cloning
By onProtocol describing the cloning of ATG3 construct for production of recombinant (GFP tagged) ATG3 variants.
Analysis of glycosphingolipids from human cerebrospinal fluid
By onThe method uses the fluorescent compound anthranilic acid (2-AA) to label oligosaccharides prior to analysis using normal-phase high-performance liquid chromatography. The labeling procedure is rapid, selective, and easy to perform.
Plasmid Construction and Gibson cloning
By onThis protocol describes plasmid construction using Gibson cloning.
GFP-YIPF3 Immunoprecipitation
By onThe protocol outlines the immunoprecipitation procedure for GFP-YIPF3 protein.
