Colon-Mercado, J. M. | bioRxiv Science

bioRxiv2025

One-step induction of human GABAergic neurons promotes presynaptic development & synapse maturation

Human induced pluripotent stem cells (iPSCs) present a powerful approach to study human brain physiology and disease, yet robust, pure GABAergic induction has remained difficult. Here we present improved, single-step, transposon-based GABAergic induction with Ascl1/Dlx2, which yields, unlike lentiviral approaches, exclusively GABAergic neurons and was validated across three independent iPSC lines. Co-seeding with Ngn2-induced excitatory neurons created stable networks of predefined excitation/inhibition ratios, with corresponding synapse ratios. Proteomic and electrophysiological characterization at different developmental time points showed that the single-step induced GABAergic neurons gain a proteomic profile that maps to different cortical interneuron subtypes and display typical GABAergic synaptic properties, producing large, synchronous and picrotoxin-sensitive currents. During early development, synaptic strength increased threefold, which was accompanied by an increase in expression of proteins exclusively enriched for presynaptic SYNGO terms. Synaptic strength continued to increase during late development but with only minor proteomic changes. Taken together, transposon-based GABAergic induction yields exclusively mature GABAergic neurons suitable for studying genes involved in synaptic maturation and to build excitation/inhibition networks for disease modelling.

neuroscience↗

bioRxiv2021

Myotubularin-related phosphatase 5 is a critical determinant of autophagy in neurons

Autophagy is a conserved, multi-step process of capturing proteolytic cargo in autophagosomes for lysosome degradation. The capacity to remove toxic proteins that accumulate in neurodegenerative disorders attests to the disease-modifying potential of the autophagy pathway. However, neurons respond only marginally to conventional methods for inducing autophagy, limiting efforts to develop therapeutic autophagy modulators for neurodegenerative diseases. The determinants underlying poor autophagy induction in neurons and the degree to which neurons and other cell types are differentially sensitive to autophagy stimuli are incompletely defined. Accordingly, we sampled nascent transcript synthesis and stabilities in fibroblasts, induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iNeurons), thereby uncovering a neuron-specific stability of transcripts encoding myotubularin-related phosphatase 5 (MTMR5). MTMR5 is an autophagy suppressor that acts with its binding partner, MTMR2, to dephosphorylate phosphoinositides critical for autophagy initiation and autophagosome maturation. We found that MTMR5 is necessary and sufficient to suppress autophagy in iNeurons and undifferentiated iPSCs. Using optical pulse labeling to visualize the turnover of endogenously-encoded proteins in live cells, we observed that knockdown of MTMR5 or MTMR2, but not MTMR9, significantly enhances neuronal degradation of TDP-43, an autophagy substrate implicated in several neurodegenerative diseases. Accordingly, our findings establish a regulatory mechanism of autophagy intrinsic to neurons and targetable for clearing disease-related proteins in a cell type-specific manner. In so doing, our results not only unravel novel aspects of neuronal biology and proteostasis, but also elucidate a strategy for modulating neuronal autophagy that could be of high therapeutic potential for multiple neurodegenerative diseases.

neuroscience↗