Determination of edits in CRISPR-edited cell lines by sequencing
By onThis protocol details the procedure of determination of edits in CRISPR-edited cell lines by sequencing.
Formalin Fixed Paraffin Embedded (FFPE) Tissue Preparation for Digital Pathology Quantification Using QuPath
By onQuPath is an open-source digital pathology analysis software. Here, it is used to quantify various misfolded proteins across anatomical regions.
Subcellular and regional localization of mRNA translation in midbrain dopamine neurons
By onLocal translation within excitatory and inhibitory neurons is known to be involved in neuronal development and synaptic plasticity. Despite the extensive dendritic and axonal arborizations of monoaminergic neurons, the subcellular localization of protein synthesis has not been well-characterized in these populations. Here, we investigated mRNA localization in midbrain dopaminergic (mDA) neurons, cells with enormous axonal and dendritic projections, both of which can release dopamine (DA). Using highly-sensitive sequencing and imaging approaches in mDA axons, we found no evidence for axonal mRNA localization or translation. In contrast, we found that mDA neuronal dendritic projections into the substantia nigra reticulata (SNr) contain ribosomes and mRNAs encoding the DA synthesis, release, and reuptake machinery. Surprisingly, we found dendritic localization of mRNAs encoding synaptic vesicular release proteins in mDA neurons. Our results are consistent with a role for local translation in the regulation of DA transmission from dendrites, but not striatal axons. Finally, we defined a molecular signature of sparse mDA neurons in the SNr, including enrichment of an ER calcium pump previously undescribed in mDA neurons. RNA-sequencing of brain tissue, RiboTagged dopamine neurons, and synaptosomes using PLATE-seq 3'-tag sequencing or Clontech SMARTer full-length RNA library preparation This dataset is a part of "Subcellular and regional localization of mRNA translation in midbrain dopamine neurons" (Hobson et. al, 2022).
Neuronal Presentation of Antigen and Its Possible Role in Parkinson’s Disease
By onPatients with Parkinson's disease and synucleinopathies show autoimmune features, with T cells recognizing alpha-synuclein. Studies explore T cell-mediated neuronal death in PD and other disorders.
Expansion microscopy with R1441C LRRK2 MEF cells: visualization of Myc-RILPL1 and TMEM55B
By onExpansion microscopy is a super-resolution imaging technique that increases the physical distance between fluorophores from fixed cells on coverslips. Here, the authors use expansion techniques to confocal image TMEM55B and RILPL1.
Harvesting and irradiation of mouse embryonic fibroblasts (MEFs) for hPSC cultures
By onThis protocol describes the process of harvesting and irradiating mouse embryonic fibroblasts (MEFs) to use as feeder cells for human pluripotent stem cell (hPSC) culture.
Brain slice physiology and optogenetics
By onThe protocol outlines standard external solutions for electrophysiology experiments.
Native gel lipid binding assay
By onThis protocol details how to perform a native gel lipid binding assay to evaluate the number of lipids bound per molecule of 3xFLAG-SHIP164Δ901-1099
Co-immunoprecipitation using GFP-trap
By onProtocol describing the procedure to perform co-immunoprecipitation experiments in HEK293 cells using GFP-trap beads
Sst+ GPi output neurons provide direct feedback to key nodes of the basal ganglia and drive behavioral flexibility
By onHere, the authors propose that GPi Sst+ neurons drive behavioral flexibility through a direct feedback signal to balance the activity of key nodes in the basal ganglia.
Single-cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, aging, and disease
By onThis study used two complementary approaches in a Parkinson's disease mouse model and revealed insights into dopaminergic neuron characteristics, aging markers, and potential disease genes like CASR.
Preparation and imaging of enriched Golgi from GolgiTAG-IP using Transmission Electron Microscopy
By onA protocol where Golgi, isolated from cells by GolgiTAG immunoprecipitation (IP) can be prepared and imaged using TEM. This protocol can also be used to image any organelles isolated using various organelle-IP protocols that are available.
pCMV-pOTC-3H-GFP
By onExpression of GFP fused with pOTC-3H, a leader peptide from mitochondrial matrix enzyme ornithine transcarbamylase and a thermostable three helix-bundle
pCAG- WIPI2dK128E- cs-TEV -STREP
By onPlasmid: Mammalian expression of human WIPI2d K128E with C-terminal Strep.
H9 ES AAVS1-NGN2 FAM134B-/-; PiggyBac-Keima-RAMP4
By onES cells modified to lack FAM134B and express Keima-RAMP4 ER-phagy reporter using CRISPR/Cas9. NEUROG2 construct added to AAVS1 locus. Transfected with SERP1 and mKeima. Derived from human embryonic stem cells at blastocyst stage.
pCAG-TSF-ATG13 (363-517)
By onPlasmid: Mammalian expression of TwinStrep-Flag tagged ATG13 (363-517).
Sample Collection and Measurement of Serum Neurofilament Light (NfL)
By onThis protocol details the steps for the preparation of serum from human blood samples and the measurement of NfL concentration using the NF-Light Advantage kit on the HD-X Analyzer (Quanterix, Billerica, MA).
Expression and purification GST-tagged ATG13:ATG101 constructs
By onExpression and purification of GST-tagged ATG13/ATG101 constructs.