Pharmacological modulation of developmental and synaptic phenotypes in human SHANK3 deficient stem cell-derived neuronal models.

Phelan-McDermid syndrome (PMDS) arises from mutations in the terminal region of chromosome 22q13, impacting the SHANK3 gene. The resulting deficiency of the postsynaptic density scaffolding protein SHANK3 is associated with autism spectrum disorder (ASD). We examined 12 different PMDS patient and CRISPR-engineered stem cell-derived neuronal models and controls and found that reduced expression of SHANK3 leads to neuronal hyperdifferentiation, increased synapse formation, and decreased neuronal activity. We performed automated imaging-based screening of 7,120 target-annotated small molecules and identified three compounds that rescued SHANK3-dependent neuronal hyperdifferentiation. One compound, Benproperine, rescued the decreased colocalization of Actin Related Protein 2/3 Complex Subunit 2 (ARPC2) with ß-actin and rescued increased synapse formation in SHANK3 deficient neurons when administered early during differentiation. Neuronal activity was only mildly affected, highlighting Benproperine's effects as a neurodevelopmental modulator. This study demonstrates that small molecular compounds that reverse developmental phenotypes can be identified in human neuronal PMDS models.

UBAP2L-dependent coupling of PLK1 localization and stability during mitosis.

PLK1 is an important regulator of mitosis whose protein levels and activity fluctuate during the cell cycle. PLK1 dynamically localizes to various mitotic structures to regulate chromosome segregation. However, the signaling pathways linking localized PLK1 activity to its protein stability remain elusive. Here, we identify the Ubiquitin-Binding Protein 2-Like (UBAP2L) that controls both the localization and the protein stability of PLK1. We demonstrate that UBAP2L is a spindle-associated protein whose depletion leads to severe mitotic defects. UBAP2L-depleted cells are characterized by increased PLK1 protein levels and abnormal PLK1 accumulation in several mitotic structures such as kinetochores, centrosomes and mitotic spindle. UBAP2L-deficient cells exit mitosis and enter the next interphase in the presence of aberrant PLK1 kinase activity. The C-terminal domain of UBAP2L mediates its function on PLK1 independently of its role in stress response signaling. Importantly, the mitotic defects of UBAP2L-depleted cells are largely rescued by chemical inhibition of PLK1. Overall, our data suggest that UBAP2L is required to fine-tune the ubiquitin-mediated PLK1 turnover during mitosis as a means to maintain genome fidelity.

High-content phenotyping of Parkinson's disease patient stem cell-derived midbrain dopaminergic neurons using machine learning classification.

Combining multiple Parkinson's disease (PD) relevant cellular phenotypes might increase the accuracy of midbrain dopaminergic neuron (mDAN) in vitro models. We differentiated patient-derived induced pluripotent stem cells (iPSCs) with a LRRK2 G2019S mutation, isogenic control, and genetically unrelated iPSCs into mDANs. Using automated fluorescence microscopy in 384-well-plate format, we identified elevated levels of α-synuclein (αSyn) and serine 129 phosphorylation, reduced dendritic complexity, and mitochondrial dysfunction. Next, we measured additional image-based phenotypes and used machine learning (ML) to accurately classify mDANs according to their genotype. Additionally, we show that chemical compound treatments, targeting LRRK2 kinase activity or αSyn levels, are detectable when using ML classification based on multiple image-based phenotypes. We validated our approach using a second isogenic patient-derived SNCA gene triplication mDAN model which overexpresses αSyn. This phenotyping and classification strategy improves the practical exploitability of mDANs for disease modeling and the identification of novel LRRK2-associated drug targets.