Preparing MEF-cultured hPSCs for nucleofection
By onThis protocol describes the procedure or preparing MEF-cultured human pluripotent stem cells (hPSCs) for the delivery of plasmids, mRNA, or ribonucleoprotein (RNP) using nucleofection.
The remote assessment of parkinsonism supporting the ongoing development of interventions in Gaucher disease
By onMutations in GBA which are causative of Gaucher disease in their biallelic form, are the most common genetic risk factor for Parkinson's disease (PD). The diagnosis of PD relies upon clinically defined motor features which appear after irreversible neurodegeneration. Prodromal symptoms of PD may provide a means to predict latent pathology, years before the onset of motor features. Previous work has reported prodromal features of PD in GBA mutation carriers, however this has been insufficiently sensitive to identify those that will develop PD. The Remote Assessment of Parkinsonism Supporting Ongoing Development of Interventions in Gaucher Disease (RAPSODI GD) study assesses a large cohort of GBA mutation carriers, to aid development of procedures for earlier diagnosis of PD.
LRRK2-G2019S Synergizes with Ageing and Low-Grade Inflammation to Promote Gut and Peripheral Immune Cell Activation that Precede Nigrostriatal Degeneration
By onBackground Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent cause of familial Parkinson’s disease (PD). The incomplete penetrance of LRRK2 mutations suggest that additional hits are required for disease onset. We hypothesized that chronic low-grade inflammation interacts with LRRK2 G2019S, the most frequent PD-associated mutation, to activate peripheral and central immune reactions and drive age-dependent neurodegeneration. Methods and Results We exposed wild-type and LRRK2 G2019S mice to a low chronic dose of lipopolysaccharide, and we performed a longitudinal analysis of central and peripheral immune reactions and neurodegeneration. Low-dose inflammation triggered nigrostriatal degeneration, macrophage/monocyte brain infiltration, and astro-/microgliosis. LRRK2 G2019S mice showed an early dysregulation of peripheral cytokines, increased CD4+ T-cell infiltration and α-synuclein aggregation in the colon. Interestingly, peripheral immune activation and colonic α-synuclein aggregation precede astro-/microgliosis and neurodegeneration. Conclusions Our study suggests an early role of the peripheral immune system and the gut in LRRK2 PD and provides a novel model to study early therapeutic immune targets and biomarkers.
Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy
By onSelective autophagy is essential for maintaining cellular homeostasis. Using in vitro reconstitution, the authors explored the details of mitophagy initiation from autophagy receptor engagement through LC3 lipidation.
Microscopy-based bead protein-protein interaction assay
By onAn assay to study protein-protein interaction
Dopaminergic axons track somatic signaling in behaving mice
By onHere, authors used genetic strategies to isolate key dopaminergic neuron subtypes and monitor their axonal and somatic signaling patterns in behaving mice.
Live imaging to investigate mitophagy kinetics and NEMO recruitment in HeLa-M cells
By onProtocol for live imaging to investigate mitophagy kinetics and NEMO recruitment in HeLa-M cells.
Gene editing of YIPF4 in hESCs V3
By onThis protocol describes the creation of YIPF4 knockout cell lines in H9 hESC cells using CRISPR-Cas9. Associated with preprint: https://doi.org/10.1101/2022.12.06.519342
Lentivirus production
By onThis protocol is associated with the following preprint, published on February 19, 2022: “The AAA+ chaperone VCP disaggregates Tau fibrils and generates aggregate seeds.”
Striatal dopamine measurement through HPLC
By onProtocol for striatal dopamine measurement in mouse brain
Primary data associated with the manuscript “Whole proteome copy number dataset in primary mouse cortical neurons” (doi: 10.1016/j.dib.2023.109336)
By onRaw data reported in Supplementary table in "Whole Proteome Copy Number Dataset in Primary Mouse Cortical Neurons."
Stereotaxic Injections
By onStereotaxic injections for Conserved and cell type-specific transcriptional responses to IFN-γ in the ventral midbrain.
CRISPResso analysis
By onThis protocol describes the process of using CRISPResso to analyze Next Generation Sequencing (NGS) data from CRISPR-targeted, genome-edited samples.
Primary data associated with the manuscript “CURTAIN – A Unique Web-based tool for exploration and sharing of MS-based proteomics data” (doi: https://doi.org/10.1101/2023.07.25.550405)
By onRaw immunoblotting data and their quantitation and graphs obtained using GraphPad Prism; PCR and sequencing data confirming that PPM1H-BromoTag clone E5 (used for all experiments in the manuscript) is a homozygous clone.
Raw tomogram of a TauRD-YFP aggregate in a TauRD-YFP expressing HEK293T cells and primary mouse neurons.
By onRaw tomogram of a TauRD-YFP aggregate in a TauRD-YFP expressing Hek293T cells. Accession numbers: HEK293T: https://www.ebi.ac.uk/emdb/EMD-13739 Primary mouse neurons: https://www.ebi.ac.uk/emdb/EMD-13740
Immunofluorescence of macrophages and microglia
By onThis protocol describes the fixation and staining of cultured cells in paraformaldehyde solution.
Disruption of lysosomal proteolysis in astrocytes facilitates midbrain proteostasis failure in an early-onset PD model
By onAccumulation of advanced glycation end products (AGEs) on biopolymers accompany cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early on-set Parkinson’s Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, α-synuclein (α-syn) phosphorylation to increase, and proteins to aggregate. These processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes’ capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in PD.
Glucose Tolerance Test
By onThe glucose tolerance test is performed to determine how quickly the glucose is cleared from the blood. It is used to test for diabetes or insulin resistance.
