Krahn, A. I. | bioRxiv Science

bioRxiv2025

An automated workflow for quantifying the formation of synuclein aggregates in human dopaminergic neurons

Parkinsons disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (-syn) aggregates termed Lewy bodies. To model PD pathology in vitro, preformed fibrils of -syn (PFFs), which can be taken up by cells, provide a seed that drives misfolding and aggregation of endogenous -syn, with new aggregates amplifying this process. External application of PFFs to dopaminergic neurons (DNs) increases aggregate formation, marked by -syn phosphorylation at serine 129 (pS129-syn), a pathological PD marker. Building on this, we developed an automated synuclein seeding assay to quantify new -syn aggregates in iPSC-derived DNs. Using pS129-syn as a readout, we show that PFFs elicit a time- and dose-dependent increase in pS129-syn aggregates. Our high-throughput assay further revealed that aggregate formation depends on endogenous -syn levels. Treatment with PFFs produced a greater increase in pS129-syn aggregates in iPSC DNs derived from a PD patient with a triplication in the SNCA gene, which encodes the -syn protein and which elevates total -syn levels, relative to DNs from an isogenic iPSC line from the same individual, in which the SNCA gene mutation had been corrected by CRISPR/Cas9. In contrast, no pS129-syn signal was detected in neurons in which all copies of the SNCA gene had been knocked out (KO). This high-content imaging assay for synuclein seeding offers a platform for assessing compounds and therapeutics that may impede -syn aggregate formation.

neuroscience↗

bioRxiv2020

Defining the Neural Kinome: Strategies and Opportunities for Small Molecule Drug Discovery to Target Neurodegenerative Diseases

Kinases are highly tractable drug targets that have reached unparalleled success in fields such as cancer but whose potential has not yet been realized in neuroscience. There are currently 55 approved small molecule kinase-targeting drugs, 48 of which have an anti-cancer indication. The intrinsic complexity linked to central nervous system (CNS) drug development and a lack of validated targets has hindered progress in developing kinase inhibitors for CNS disorders when compared to other therapeutic areas such as oncology. Identification and/or characterization of new kinases as potential drug targets for neurodegenerative diseases will create opportunities for development of CNS drugs in the future. The track record of kinase inhibitors in other disease indications supports the idea that with the best targets identified small molecule kinase modulators will become impactful therapeutics for neurodegenerative diseases. KEY CONCEPTSChemical probe: a high-quality small molecule that is potent, selective, and cell-active that meets the following criteria: (1) in vitro biochemical IC50 < 50 nM, (2) [≥] 30-fold selectivity relative to other kinases in a large assay panel such as DiscoverX scanMAX, and (3) cellular activity or target engagement with an IC50 < 1 M Narrow spectrum: a selectivity threshold that can be defined as potently inhibiting [~]10% or less of all kinases screened Kinome: all human kinases Kinase chemogenomic set (KCGS): publicly-available curated physical library of narrow spectrum and potent kinase inhibitors for which the SGC-UNC has received permission to share the compounds; subsequent releases will increase kinome-wide coverage Illuminating the Druggable Genome (IDG) program: several interconnected projects currently funded by the National Institutes of Health to provide information on historically understudied members within protein families that have provided drug targets; the three main focus areas are kinases, G-protein coupled receptors, and ion channels IDG kinase: a kinase that was nominated as dark (understudied) by the National Institutes of Health IDG program (curated list found here: https://druggablegenome.net/IDGProteinList); IDG consortium members generate data and resources to aid in the illumination of the function of these kinases DK tool: a narrow spectrum inhibitor that exhibits a defined selectivity score (S10(1 M) < 0.05) and cellular target engagement with an IC50 < 1 M; S10(1 M) is a measure of selectivity equal to the percentage of screened kinases biochemically inhibited by >90% at 1 M

neuroscience↗