Viral-mediated short-hairpin RNA knockdown
By Julia Julia Leonard onStereotaxic injection of viral vectors for gene knockdown experiments: The AAV vectors for knocking down the gene of interest were custom-made with Vector Biolabs using AAV-GFP-U6 vector for AAV1 packaging (Cat no, 7040, Vector Biolabs). They were then unilaterally injected into the striatum of mice as the protocol details.
Preparation of fresh paraformaldehyde for mouse perfusion and tissue fixation
By Julia Julia Leonard onPreparation of fresh paraformaldehyde for mouse perfusion and tissue fixation.
Quantification of extracellular 𝜶-synuclein assay
By Julia Julia Leonard onThis protocol uses a classical ELISA experimental design with sensitive electrochemical detection to provide a measure of 𝜶-synuclein concentration in the conditioned media of cultured iPSC-derived human cells.
Mechanism of human PINK1 activation at the TOM complex in a reconstituted system
By Julia Julia Leonard onPublished: The authors demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These molecular findings will aid in the development of small molecule activators of PINK1 as a therapeutic strategy for PD. View original preprint.
Tandem Mass Tag (TMT) label-based mass spectrometry analysis of RILPL1 interactors
By Julia Julia Leonard onPrimary mass spectrometry data associated with Fig.5 in the manuscript, "Parkinson’s VPS35 mutation induces LRRK2 mediated lysosomal association of RILPL1 and TMEM55B" (Association of RILPL1 with a lysosomal membrane integral protein, TMEM55B mediated by LRRK2 activity). Curtain link for the dataset: https://curtain.proteo.info/#/4f15d6c8-9192-4bb2-a2ff-da7aa542025c
Mass spectrometry analysis of proteome changes in VPS35-WT vs VPS35[D620N] MEFs and VPS35[D620N] MEFs vs LRRK2 inhibitor in whole cell lysates and lysosomes
By Julia Julia Leonard onPrimary mass spectrometry data associated with Fig.1 and Fig.2 in the manuscript "Parkinson’s VPS35 mutation induces LRRK2 mediated lysosomal association of RILPL1 and TMEM55B" (Phospho-Rab and RILPL1 enrichment at VPS35 mutant lysosome and Lysosomal recruitment of RILPL1 is dependent on LRRK2 kinase activity). Curtain links for the datasets: https://curtain.proteo.info/#/0e673d58-d8f2-4368-996b-0869d5513d46; https://curtain.proteo.info/#/0e673d58-d8f2-4368-996b-0869d5513d46; https://curtain.proteo.info/#/d864df78-e2a5-4a64-99fb-8c5d8b2e1ab8; https://curtain.proteo.info/#/062e0a64-1fd3-4cc1-833a-a5b007d95a3c.
Detection of accessible cholesterol in primary cilia using purified His-ALOD4-mNeon in 3T3 Fibroblasts
By Julia Julia Leonard onThe protocol described here is based upon previously established methods (Kinnebrew et al., 2019; Johnson and Radhakrishnan, 2021) and has also been used successfully to label cilia in RPE cells.
In vivo reduction of age-dependent neuromelanin accumulation mitigates features of Parkinson’s disease
By savannah onCollection of all datasets (Metabolomics, microscopy, and behavioral data) included in the article entitled "In vivo reduction of age-dependent neuromelanin accumulation mitigates features of Parkinson’s disease."
Unique functional responses differentially map onto genetic subtypes of dopamine neurons
By savannah onPublished: Genetic strategies to isolate dopaminergic subtypes lead to the establishment of a novel subtype of dopamine neurons within the mouse substantia nigra. The results show that the neural activity patterns of the genetically identified subtypes map to differing features of locomotion, such as acceleration, deceleration, and appetitive. View original preprint.
Astrocytic LRRK2 controls synaptic connectivity through ERM phosphorylation
By savannah onThis work reveals a causal link between the astrocytic and synaptic dysfunction induced by the PD-linked mutation, LRRK2 G2019S, providing a non-cell autonomous mechanism for rescuing aberrant cortical wiring
Testing AAVrg-hSyn-mCherry in the spinal cord of Rhesus Macaques
By Emma Sherrell onViral Vector: Two rhesus macaque animals were tested for AAVrg-hSyn-mCherry in the spinal cord. The authors observed abundant mCherry-positive neurons in expected targets (i.e., motor cortical regions known to project to the spinal cord).
Preparation of soluble and insoluble mitochondrial protein fractions for mass spectrometry analysis
By Emma Sherrell onPreparation of soluble and insoluble mitochondrial protein fractions from HeLa cells for mass spectrometry analysis.
Sex distribution of GBA1 variant carriers with dementia with Lewy Bodies and Parkinson’s disease
By savannah onSex distribution of GBA1 variant carriers with dementia with Lewy Bodies and Parkinson's disease.
Primary data associated with the manuscript 10.1073/pnas.2315171120 (“Localization of PPM1H phosphatase tunes Parkinson’s disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis”)
By Emma Sherrell onPrimary data associated with the manuscript 10.1073/pnas.2315171120 ("Localization of PPM1H phosphatase tunes Parkinson’s disease-linked LRRK2 kinase-mediated Rab GTPase phosphorylation and ciliogenesis").
Transfection by electroporation of GFP-LRRK2 and Immunofluorescent imaging of MEFs VPS35 (D620N) mutants stably expressing LysoTag
By Emma Sherrell onThe authors investigated the colocalization of GFP-LRRK2 with a lysosomal localized TMEM192-3xHA.
Generation of A549 PPM1H BromoTag CRISPR/CAS9 knock-in cell line
By Emma Sherrell onThis protocol details the generation of A549 PPM1H BromoTag CRISPR/CAS9 knock-in cell line.
Video tutorials for Curtain and Curtain-PTM
By Emma Sherrell onVideo tutorials to assist users of Curtain and Curtain-PTM proteomic data analysis web tools (described in https://www.biorxiv.org/content/10.1101/2023.07.25.550405v1).
Immunoblots
By Emma Sherrell onThe steps of this protocol are similar for different sources of protein such as cell lysate, cytosol, or membrane samples, and EV samples. However, some specific changes depend on the protein of interest, endogenous alpha-synuclein, or type of cells, hiPSC dopamine neurons.
shRNA knockdown
By Emma Sherrell onThis protocol describes a standard procedure to generate a stable cell line for a DNAJC5 knockdown using shRNA.
Differentiation of SH-SY5Y cells
By Emma Sherrell onThis protocol describes a standard procedure to differentiate SH-SY5Y cells into dopaminergic neurons using retinoic acid.