Luminescent Conjugated Oligothiophenes (LCO) Staining
By onThe protocol describes Luminescent Conjugated Oligothiophenes staining on brain sections.
Whole-cell Patch-Clamp Recordings from Striatal Cholinergic Interneurons in ex vivo Mouse Brain Slices
By onProtocol for patch-clamp recording of mCherry-labelled striatal ChIs from mouse brain slices with added step for labelling astrocytes using SR101.
H9 ES AAVS1-NGN2 FAM134A-/-
By onSummary: H9 ES cells were modified using CRISPR/Cas9 to lack the ER-phagy receptor FAM134A. A NEUROG2 construct was introduced in the AAVS1 locus, creating iNeurons. The cells are derived from human embryonic stem cells at the blastocyst stage.
In vitro synthesis of PE2 (nCas9-MMLV RT fusion) polyA mRNA using T7 RNA polymerase
By onWe describe the preparation of synthetic capped and polyadenylated messenger RNA (mRNA) encoding a Cas9 nickase-reverse transcriptase fusion protein (PE2) for use in genome editing with a method called prime editing.
LRRK1 expression and purification
By onProtein purification protocol for full-length LRRK1 as done by Leschziner and Reck-Peterson Labs. Original protocol by Janice M. Reimer and Yu Xuan Lin.
Crystal structure of Wild-Type PPM1H phosphatase
By onCrystal structure of WT PPM1H phosphatase (as reported in 10.15252/embr.202152675) deposited in the Protein Data Bank with code 7L4J.
H9 ES AAVS1-NGN2 TEX264-/-
By onES cells modified using CRISPR/Cas9 lack ER-phagy receptor TEX264. They introduced a NEUROG2 construct in AAVS1 locus, derived from a female human embryonic stem cell at the blastocyst stage.
α-Synuclein Promotes Neuronal Dysfunction and Death by Disrupting the Binding of Ankyrin to β-Spectrin
By onα-Synuclein plays a key role in the pathogenesis of Parkinson's disease and related disorders, but critical interacting partners and molecular mechanisms mediating neurotoxicity are incompletely understood. We show that α-synuclein binds directly to β-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders, we demonstrate that β-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of β-spectrin is required for α-synuclein binding and neurotoxicity. A key plasma membrane target of ankyrin, Na+/K+ ATPase, is mislocalized when human α-synuclein is expressed in Drosophila Accordingly, membrane potential is depolarized in α-synuclein transgenic fly brains. We examine the same pathway in human neurons and find that Parkinson's disease patient-derived neurons with a triplication of the α-synuclein locus show disruption of the spectrin cytoskeleton, mislocalization of ankyrin and Na+/K+ ATPase, and membrane potential depolarization. Our findings define a specific molecular mechanism by which elevated levels of α-synuclein in Parkinson's disease and related α-synucleinopathies lead to neuronal dysfunction and death.SIGNIFICANCE STATEMENT The small synaptic vesicle associate protein α-synuclein plays a critical role in the pathogenesis of Parkinson's disease and related disorders, but the disease-relevant binding partners of α-synuclein and proximate pathways critical for neurotoxicity require further definition. We show that α-synuclein binds directly to β-spectrin, a key cytoskeletal protein required for localization of plasma membrane proteins and maintenance of neuronal viability. Binding of α-synuclein to β-spectrin alters the organization of the spectrin-ankyrin complex, which is critical for localization and function of integral membrane proteins, including Na+/K+ ATPase. These finding outline a previously undescribed mechanism of α-synuclein neurotoxicity and thus suggest potential new therapeutic approaches in Parkinson's disease and related disorders
Create Mask for Axonal Quantification Analysis with FIJI
By onThis protocol describes how to create a quantified mask from an image of fluorescence-tagged axonal projections using FIJI/ImageJ software.
Constitutive nuclear accumulation of endogenous alpha-synuclein in mice causes motor impairment and cortical dysfunction, independent of protein aggregation
By onThe authors created mice in which endogenous a-synuclein is localized to the nucleus. The mice show motor and GI deficits and motor cortex atrophy, suggesting that chronic nuclear a-synuclein can cause toxic phenotypes independent of its aggregation.
RNA and protein extraction from bulk dissections
By onProtocol for RNA and protein extraction from bulk dissections.
An open-source head-fixation and implant-protection system for mice
By onThis study presents a head-fixation and implant-protection system for mice including a headbar, headhat and head fixation station to facilitate experimental procedures in vivo. All 3D-printing files are open source and readily available and editable
Mouse Stereotaxic Surgery
By onThis protocol describes the steps for performing stereotaxic surgery in mice. It is applicable to intracranial injections (e.g. virus, drug) and placement of implants (e.g. optical fibers, electrode arrays) into targeted regions of mouse brains.
Validation of Genotyping Method for L444P Mice Ear-Clips.
By on|| Team Schapira || Authors Revi Shahar Golan, David ChauAbstractAim: the genotyping is used to identify if mice are heterozygote (hetero) or Wild-Type (WT), and the aim of the work is to validate the digestion method, and PCR program, the PCR primers, and the interpretation of the results. Associated with publication: doi: 10.1093/brain/awx221
Expression and purification MBP-ATG9 Constructs
By onExpression and purification from HEK cells of ATG13, ATG101 and FOLDON-ATG9A proteins.
Microsomal membrane isolation from cell culture
By onMicrosomal membrane isolation from cell culture
