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.
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.
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
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.
GBA Variants and Parkinson Disease: Mechanisms and Treatments
By onThis review discusses the pathways associated with GBA-PD and highlights potential treatments which may act to target the lysosomal enzyme glucocerebrosidase and prevent neurodegeneration.
Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes
By onThe authors plate, culture, and transfect human iPSC-derived excitatory glutamatergic neurons for the purpose of observing transport of axonal cargoes under spinning disk confocal microscopy.
Freezing of feeder-free hPSCs
By onThis protocol describes the process of freezing feeder-free human pluripotent stem cells (hPSCs) using Accutase or ReLeSR
A fluorescence-based in vitro scrambling assay for yeast MCP1 and human XK
By onVPS13 proteins are proposed to function at contact sites between organelles as bridges for lipids to move directionally and in bulk between organellar membranes. VPS13s are found to interact with integral membrane proteins, like MCP1 in yeast or XK in humans. We showed that MCP1 and XK scramble phospholipids in vitro. Here I describe the detailed procedure of purification, reconstitution, and scrambling assay for both MCP1 and XK.
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.
Induction of non-selective bulk autophagy
By onThis protocol describes how to induce bulk (non-selective) autophagy in HeLa cells through nutrient starvation.
Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy
By onGUV quantification
Reduced Thalamic Excitation to Motor Cortical Pyramidal Tract Neurons in Parkinsonism
By onThe current data suggests that cell subtype- and synapse-specific adaptations in M1 contribute to altered cortical outputs in parkinsonism and are important aspects of PD pat
Glucocerebrosidase is imported into mitochondria and preserves complex I integrity and energy metabolism
By onMutations in GBA1, the gene encoding the lysosomal enzyme β-glucocerebrosidase (GCase), which cause Gaucher’s disease, are the most frequent genetic risk factor for Parkinson’s disease (PD). Here, we employ 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. We demonstrate that GCase can be imported from the cytosol into the mitochondria via recognition of internal mitochondrial targeting sequence-like signals. In mitochondria, GCase promotes the maintenance of mitochondrial complex I (CI) integrity and function. Furthermore, GCase interacts with the mitochondrial quality control proteins HSP60 and LONP1. Disease-associated mutations impair CI stability and function and enhance the interaction with the mitochondrial quality control machinery. These findings reveal a mitochondrial role of GCase and suggest that defective CI activity and energy metabolism may drive the pathogenesis of GCase-linked neurodegeneration.
