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.
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.
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.
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.
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.
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.
Freezing of feeder-free hPSCs
By onThis protocol describes the process of freezing feeder-free human pluripotent stem cells (hPSCs) using Accutase or ReLeSR
