title: Screening assay for small-molecule inhibitors of synaptojanin 1, a synaptic phosphoinositide phosphatase.
abstract: Elevation of amyloid β-peptide (Aβ) is critically associated with Alzheimer disease (AD) pathogenesis. Aβ-induced synaptic abnormalities, including altered receptor trafficking and synapse loss, have been linked to cognitive deficits in AD. Recent work implicates a lipid critical for neuronal function, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], in Aβ-induced synaptic and behavioral impairments. Synaptojanin 1 (Synj1), a lipid phosphatase mediating the breakdown of PI(4,5)P2, has been shown to play a role in synaptic vesicle recycling and receptor trafficking in neurons. Heterozygous deletion of Synj1 protected neurons from Aβ-induced synaptic loss and restored learning and memory in a mouse model of AD. Thus, inhibition of Synj1 may ameliorate Aβ-associated impairments, suggesting Synj1 as a potential therapeutic target. To this end, we developed a screening assay for Synj1 based on detection of inorganic phosphate liberation from a water-soluble, short-chain PI(4,5)P2. The assay displayed saturable kinetics and detected Synj1's substrate preference for PI(4,5)P2 over PI(3,4,5)P3. The assay will enable identification of novel Synj1 inhibitors that have potential utility as chemical probes to dissect the cellular role of Synj1 as well as potential to prevent or reverse AD-associated synaptic abnormalities.
authors.affiliation: 1Department of Pathology and Cell Biology, and the Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University Medical Center, New York, NY, USA.
authors.forename: Laura Beth J
authors.initials: LB
authors.lastname: McIntire
authors.forename: Kyu-In
authors.initials: KI
authors.lastname: Lee
authors.forename: Belle
authors.initials: B
authors.lastname: Chang-Ileto
authors.forename: Gilbert
authors.initials: G
authors.lastname: Di Paolo
authors.forename: Tae-Wan
authors.initials: TW
authors.lastname: Kim
mesh_terms: D002460:Cell Line; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D057166:High-Throughput Screening Assays; D006801:Humans; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D011993:Recombinant Fusion Proteins; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D049452:Green Fluorescent Proteins; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases
keywords: Alzheimer disease; drug discovery; lipid phosphatase; phosphatidylinositol-4,5-bisphosphate; small-molecule screening; src homology 2 domain-containing inositol-5-phosphatase 2 (SHIP2); synaptojanin 1