Sphingolipid changes in Parkinson L444P GBAmutation fibroblasts promote α-synuclein aggregation
By onIntraneuronal accumulation of aggregated α-synuclein is a pathological hallmark of Parkinson’s disease. Therefore, mechanisms capable of promoting α-synuclein deposition bear important pathogenetic implications. Mutations of the glucocerebrosidase 1 (GBA) gene represent a prevalent Parkinson’s disease risk factor. They are associated with loss of activity of a key enzyme involved in lipid metabolism, glucocerebrosidase, supporting a mechanistic relationship between abnormal α-synuclein–lipid interactions and the development of Parkinson pathology. In this study, the lipid membrane composition of fibroblasts isolated from control subjects, patients with idiopathic Parkinson’s disease and Parkinson's disease patients carrying the L444P GBA mutation (PD-GBA) was assayed using shotgun lipidomics. The lipid profile of PD-GBA fibroblasts differed significantly from that of control and idiopathic Parkinson’s disease cells. It was characterized by an overall increase in sphingolipid levels. It also featured a significant increase in the proportion of ceramide, sphingomyelin and hexosylceramide molecules with shorter chain length and a decrease in the percentage of longer-chain sphingolipids. The extent of this shift was correlated to the degree of reduction of fibroblast glucocerebrosidase activity. Lipid extracts from control and PD-GBA fibroblasts were added to recombinant α-synuclein solutions. The kinetics of α-synuclein aggregation were significantly accelerated after addition of PD-GBA extracts as compared to control samples. Amyloid fibrils collected at the end of these incubations contained lipids, indicating α-synuclein–lipid co-assembly. Lipids extracted from α-synuclein fibrils were also analysed by shotgun lipidomics. Data revealed that the lipid content of these fibrils was significantly enriched by shorter-chain sphingolipids. In a final set of experiments, control and PD-GBA fibroblasts were incubated in the presence of the small molecule chaperone ambroxol. This treatment restored glucocerebrosidase activity and sphingolipid levels and composition of PD-GBA cells. It also reversed the pro-aggregation effect that lipid extracts from PD-GBA fibroblasts had on α-synuclein. Taken together, the findings of this study indicate that the L444P GBA mutation and consequent enzymatic loss are associated with a distinctly altered membrane lipid profile that provides a biological fingerprint of this mutation in Parkinson fibroblasts. This altered lipid profile could also be an indicator of increased risk for α-synuclein aggregate pathology.
Feed-forward metabotropic signaling by Cav1 Ca2+ channels supports pacemaking in pedunculopontine cholinergic neurons
By onCholinergic neurons (CNs) in the pedunculopontine nucleus (PPN) are lost in the course of Parkinson’s disease. PPN CNs have a distinctive physiological phenotype that shares only some of the features of other selectively vulnerable neurons in PD.
Fixation and imaging of HeLa cells after mitochondrial depolarization
By onA protocol for the fixation and imaging of HeLa cells after mitochondrial depolarization
PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the CORB GTPase domain
By onLeucine-rich-repeat-kinase 1 (LRRK1) and its homologue LRRK2 are multidomain kinases. Here, the authors study the mechanism controlling LRRK1 activity and reveal a novel unexpected activation mechanism.
Subtomogram averaging and classification
By onThis protocol describes the procedure of subtomogram averaging and 3D classification of VPS13C rod-shaped densities inside cryotomograms of mammalian cells. Sub-tomogram averaging package i3 was used for 3D alignment and classification.
Proximity biotinylation of ATG8 proteins and selective autophagy receptors
By onAutophagy forms autophagosomes around cellular cargo for degradation in lysosomes. Proximity biotinylation with APEX2 fused to ATG8 proteins identifies proteins near autophagic cargo receptors during selective autophagy activation by nutrient stress
pCAG-mcherry- WIPI2dK88E-cs- TEV -STREP
By onPlasmid for mammalian expression of human WIPI2d K88E with N-terminal mCherry and C-terminal Strep.
Raw tomogram of GFP-a-synuclein inclusion in primary mouse neuron expressing GFP-a-synuclein, seeded with MSA aggregates
By onRaw data associated with DOI: 10.1038/s41467-021-22108-0
Visualisation and quantification of dendritic spines in cultured human Medium Spiny Neurons (MSNs)
By onThis protocol describes the visualisation of dendritic spines of human neurons cultured on coverslips in vitro and subsequent quantification using the software Imaris.
Preparing of genomic DNA from in vitro cultured cells
By onThis protocol describes a standard procedure for preparing crude cell lysate which can be further analyzed by PCR.
Regular maintenance of human pluripotent stem cells
By onThis protocol describes the regular maintenance and passaging human iPSCs.
Adapting hPSCs cultured on MEFs to feeder-free system
By onThis protocol describes the procedure of adapting human pluripotent stem cells (hPSCs) to feeder-free culturing conditions using mTeSR-plus or StemFlex
AAV.CAP-Mac analysis code
By onFirst release of AAV.CAP-Mac analysis code and files to generate figures.
Spatial transcriptomics reveals molecular dysfunction associated with Lewy pathology
By onThe results identify neurons vulnerable to Lewy pathology in the PD cortex and identify a conserved signature of molecular dysfunction in both mice and humans.
