ASAP is committed to accelerating the pace of discovery and informing a path to a cure for Parkinson’s disease through collaboration, research-enabling resources, and data sharing. We’ve created this catalog to showcase the research outputs and tools developed by ASAP-funded programs.
Published: PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074, and Thr1075) within the CORB GTPase domain
Published: This study describes how PKC isoforms are able to phosphorylate LRRK1 at three sites in a key regulatory domain of the protein (GTPase domain) inducing LRRK1’s kinase activity. Interestingly, this is not seen with the PD-associated LRRK2, suggesting that PKC isoforms do not regulate LRRK2. View original preprint.
Global ubiquitylation analysis of mitochondria in primary neurons identifies physiological Parkin targets following activation of PINK1
Published: Mutations in PINK1 and Parkin are implicated in PD via abherrant mitophagy. The authors identified ubiquitylated substrates of endogenous Parkin in mouse neurons by proteomic analysis. They identified and validated 22 protein targets of Parkin that are conserved in human neurons providing a functional Parkin landscape in neuronal cells. View original preprint.
Global ubiquitylation analysis of mitochondria in primary neurons identifies endogenous Parkin targets following activation of PINK1
Published: Loss-of-function mutations in Parkin cause disruption of mitophagy and are associated with PD. Yet, much of the biology surrounding Parkin function has taken place in artificial cell systems. The authors used human neurons to identify and validate 22 protein targets of Parkin, providing a functional Parkin landscape in neuronal cells.
PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074, and Thr1075) within the CORB GTPase domain
Leucine-rich-repeat-kinase 1 (LRRK1) and its homologue LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause PD. Here, the authors study the mechanism controlling LRRK1 activity and reveal a novel unexpected activation mechanism. View preprint.
Impact of 100 LRRK2 variants linked to Parkinson’s Disease on kinase activity and microtubule binding
Published: LRRK2 is a promising candidate for PD therapeutics via reduction of its kinase activity. The authors investigated 98 LRRK2 variants and their effects on function. They found 22 variants that robustly stimulated LRRK2 kinase activity and 12 variants that suppressed microtubule association in the presence of Type 1 kinase inhibitors. View original preprint.
PTEN-induced kinase 1 (PINK1) and Parkin: Unlocking a mitochondrial quality control pathway linked to Parkinson’s disease
Review: This review focuses on understanding the PINK1/Parkin-mediated mitochondrial quality control pathway through the lens of abherrant immune activation as a driver of dopaminerigic neuron loss following the loss of PINK and Parkin.
Creating pooled CRISPR-Cas9 knock-outs in NIH-3T3 cells
To validate a genome wide CRISPR screen, the authors select the top hits and create lentiviruses to validate the hits. Rather than screening each virus from single cell clones, the authors analyze the infected cells as pools according to this protocol.
CRISPR-Cas9 screen in NIH-3T3 cells to identify modulators of LRRK2 function
This protocol describes a pooled, CRISPR Cas9 screen to identify modulators of LRRK2 activity. The CRISPR-Cas9 based screen is carried out in mouse cells using a ready-to-use pooled guide RNA (gRNA) mouse library consisting of 78,637 gRNAs targeting 19,674 genes and an extra 1,000 control gRNAs.
Untargeted lipidomics analysis for Golgi immunopurification (Golgi-IP)
This protocol provides details for analyzing GolgiIP lipidomics samples using liquid chromatography mass spectrometry (LC-MS) for nonpolar lipid profiling.
Golgi immunopurification (Golgi-IP) for subcellular metabolite profiling
This protocol provides details for preparing Golgi-IP metabolomics samples.
Golgi immunopurification (Golgi-IP) for subcellular lipid profiling
This protocol provides details for preparing Golgi-IP lipidomics samples.
Untargeted metabolomics analysis for Golgi immunopurification (Golgi-IP)
This protocol provides details for analyzing GolgiIP metabolomics samples using liquid chromatography mass spectrometry (LC-MS) for polar metabolite profiling.
Primary data associated with the manuscript “Golgi-IP, a novel tool for multimodal analysis of Golgi molecular content” (doi.org/10.1101/2022.11.22.517583)
Raw data files used for the manuscript “Golgi-IP, a novel tool for multimodal analysis of Golgi molecular content” (doi.org/10.1101/2022.11.22.517583).