α-Synuclein expression in response to bacterial ligands and metabolites in gut enteroendocrine cells – an in vitro proof of concept study
By onCaudo-rostral migration of pathological forms of α-synuclein from the gut to the brain is proposed as an early feature in Parkinson disease pathogenesis, but the underlying mechanisms remain unknown. Intestinal epithelial enteroendocrine cells sense and respond to numerous luminal signals, including bacterial factors, and transmit this information to the brain via the enteric nervous system and vagus nerve. There is evidence that gut bacteria composition and their metabolites change in Parkinson disease patients and these alterations can trigger α-synuclein pathology in animal models of the disorder. Here we investigated the effect of toll-like receptor and free fatty acid receptor agonists on the intracellular level of α-synuclein and its release using mouse secretin tumour cell line 1 enteroendocrine cells. Secretin tumour cell line 1 enteroendocrine cells were treated for 24 or 48 hours with toll-like receptor agonists (toll-like receptor 4 selective lipopolysaccharide; toll-like receptor 2 selective Pam3CysSerLys4) and the free fatty acid receptor 2/3 agonists butyrate, propionate and acetate. The effect of selective receptor antagonists on the agonists effects after 24 hours was also investigated. The level of α-synuclein protein was measured in cell lysates and cell culture media by western blot and enzyme-linked immunosorbent assay. The level of α-synuclein and tumour necrosis factor messenger RNA was measured by quantitative reverse transcription real time polymerase chain reaction. Stimulation of secretin tumour cell line 1 enteroendocrine cells for 24 and 48 hours with toll-like receptor and free fatty acid receptor agonists significantly increased the amount of intracellular α-synuclein and the release of α-synuclein from the cells into the culture medium. Both effects were significantly reduced by antagonists selective for each receptor. toll-like receptor and free fatty acid receptor agonists also significantly increased tumour necrosis factor transcription and this was effectively inhibited by corresponding antagonists. Elevated intracellular α-synuclein increases the likelihood of aggregation and conversion to toxic forms. Factors derived from bacteria induce α-synuclein accumulation in secretin tumour cell line 1 enteroendocrine cells. Here we provide support for a mechanism by which exposure of enteroendocrine cells to specific bacterial factors found in Parkinson disease gut dysbiosis might facilitate accumulation of α-synuclein pathology in the gut.
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.
SINTBAD-GFP: expression and purification
By onThis protocol describes how to express and purify human SINTBAD tagged C-terminally with eGFP.
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.
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.
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.
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.
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.
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.
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.
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.
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-mcherry- WIPI2dR125E- cs-TEV -STREP
By onPlasmid for mammalian expression of human WIPI2d R125E with N-terminal mCherry and C-terminal Strep.
