While advances in omics profiling rapidly expand the catalog of genes associated with brain activity in health and disease, functional annotation lags far behind. Here, we establish a high-throughput functional genomics platform that couples the calcium-integrating sensor CaMPARI2 with CRISPRi screening in human iPSC-derived neurons. By converting cumulative neuronal activity into a stable, flow cytometry-readable signal, this approach enables systematic interrogation through pooled screening. Using a focused library of memory-associated genes, we recover known regulators and identify TMEM50A, a previously uncharacterized protein, as an essential regulator of neuronal activity. TMEM50A forms a complex with LEPROTL1 and associates with ESCRT-III machinery on multivesicular bodies (MVBs). TMEM50A loss impairs MVBs function, remodels the neuronal surface proteome, reduces synapse density, and alters behavior in mice. This platform enables systematic discovery of neuronal activity regulators and reveals a critical role for TMEM50A-dependent MVB function in maintaining synaptic integrity and behavior.
Understanding the complex interplay between gene expression and neuronal activity is crucial for unraveling the molecular mechanisms underlying cognitive function and neurological disorders. Here, we developed pooled screens using CRISPR interference (CRISPRi) and the fluorescent calcium integrator CaMPARI2 to evaluate genetic modifiers of neuronal depolarization. Using this screening method, we evaluated 1343 genes for their effect on depolarization in a human iPSC-derived neuron model, revealing potential links to neurodegenerative and neurodevelopmental disorders. These genes include known regulators of neuronal excitability, such as TARPs and ion channels, as well as genes associated with autism spectrum disorder and Alzheimer’s disease not previously described to affect neuronal depolarization. This CRISPRi-based screening platform offers a versatile tool to uncover molecular mechanisms controlling neuronal function in health and disease. We currently lack scalable approaches to systematically reveal the genetic underpinnings of neuronal function in health and disease. Here, the authors develop a platform to uncover genes affecting neuronal excitability in scalable CRISPR screens.
Steven C. Boggess, Vaidehi Gandhi, Ming-Chi Tsai et al.· Nature Communications· 0 citations