NDP52 and OPTN S177D S473D: expression and purification
By onThis protocol describes how to express and purify human NDP52 and OPTN S177D S473D. The same procedure can be applied to purify wild type OPTN.
Evaluation of a novel method for spectral analysis of neurophysiology data
By onThis code implements a novel method for correcting a distortion effect that is present in the power spectra of neuronal spike trains.
pCAG-MBP-ATG9 (801-839)
By onPlasmid: To make monomeric ATG9 C-terminal tail for expression in mammalian cells.
Headplate surgery protocol for in vivo electrophysiological and optogenetic manipulation of basal ganglia neurons in awake head-fixed mice
By onStep by step protocol for headplate surgery for in vivo electrophysiological and optogenetic manipulation of basal ganglia neurons in awake mice.
Immunological detection of autophagy and mTORC1-related proteins
By onA general protocol for immunological detection by Western blotting MTOR, MTOR (pS2448), ULK1, ULK1 (pS757), p70S6K, p70S6K (pT389), SQSTM1, CALCOCO2, MAP1LC3B, GABARAP, TFEB, TFE3, PGRN, HSP90, and PCNA.
GFP Immunoprecipitation and Sample Preparation for Tandem Mass Tag (TMT) Mass Spectrometry Analysis
By onWe describe a method to identify potential interactors of any Green Fluorescent Protein (GFP) tagged protein expressed in mammalian cells by GFP immunoprecipitation coupled to Tandem Mass Tag (TMT) mass spectrometry analysis. As an example, we used a GFP-tagged phosphoRab interactor protein (RILPL1-GFP), and its non-binding mutant (RILPL1 -GFP, which cannot interact with phosphorylated Rab proteins) as a control.
Mitochondrial isolation protocol
By onThis protocol describes how to isolate crude mitochondrial fractions from HeLa cells.
α-synuclein promotes neuronal dysfunction and death by disrupting the binding of ankyrin to ß-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 ß-spectrin. Using males and females in a Drosophila model of α-synuclein-related disorders we demonstrate that ß-spectrin is critical for α-synuclein neurotoxicity. Further, the ankyrin binding domain of ß-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 leads to neuronal dysfunction and death.
Damaged mitochondria recruit the effector NEMO to activate NF-κB signaling
By onThe connections between molecular mechanisms like mitophagy and tissue-wide features like neuro-inflammation remain unclear. Here, the authors characterize a novel link between these two hallmarks of neurodegeneration.
ATP13A2 Regulates Cellular α-Synuclein Multimerization, Membrane Association, and Externalization
By onATP13A2 loss-of-function mutations are linked to Parkinson’s disease and alpha-synuclein pathology. The authors found that loss of ATP13A2 disrupts lysosomal membrane integrity and causes alpha-synuclein multimerization.
Three-step docking by WIPI2, ATG16L1 and ATG3 delivers LC3 to the phagophore
By onIn this manuscript, in a near-complete pathway from initial membrane recruitment to LC3 lipidation reaction, we show how a three-step targeting mechanism of the ATG12-ATG5-ATG16L1 machinery ensures a high level of regulatory control on autophagy.
pCAG-mcherry- WIPI2dR125E- cs-TEV -STREP
By onPlasmid for mammalian expression of human WIPI2d R125E with N-terminal mCherry and C-terminal Strep.
Constructs and generation of stable cell lines
By onProtocol used to generate stable Flp-In T-REx-HEK 293 cell lines expressing WT or mutant GCase (E326K or L444P) as a V5-FLAG-tagged protein using a tetracycline-inducible system.
pCAG-OSF-ATG13 (2-197)-F16D
By onPlasmid for expression of human ATG13 HORMA F16D mutant in mammalian cells.
Toward a standard model for autophagosome biogenesis
By onHere, we discuss two papers focusing on autophagosome biogenesis in mammals: Olivas et al. confirming ATG9A as an autophagosome component using biochemistry, while Broadbent et al. showing autophagy protein dynamics using particle tracking.
