Biology subjects

Leuenberger, J.

Publications and source records attributed to Leuenberger, J..

3 recordsLinked to original sources

bioRxiv2025

Advancing Human iPSC-Derived Motor Neuron Models Using Glutamatergic Modulators for Synaptic Function Studies

Understanding human synapse structure and function at nanoscale resolution requires experimental systems that support both high-quality imaging and robust synaptogenesis. While brain tissue and primary neuronal cultures have been widely used, human induced pluripotent stem cells (iPSCs) offer key advantages: they enable patient-specific modeling and provide a renewable source of human neurons without relying on animal models. However, reproducible synapse formation in iPSC-derived neurons, particularly motor neurons (MNs), remains challenging due to variable differentiation efficiencies and low synaptic density. Here, we optimized MN differentiation to enhance synaptogenesis and enable high-resolution structural analysis using cryo-electron tomography (cryo-ET). Within 28 days, human iPSC-derived MNs developed into morphologically and functionally mature neuronal networks, characterized by phase-bright somata, elongated axons, and dense axodendritic branching. To further promote synaptic connectivity, we applied the glutamatergic modulators CX516 and CDPPB during differentiation. Their effects were validated by immunolabeling of synaptic markers, electrophysiology, calcium imaging, live-cell synaptic vesicle recycling assays, and ultrastructural imaging, including cryo-ET. This combined approach of optimized differentiation and targeted neuromodulation yielded reproducible MN networks with enhanced synaptic density and function, providing a robust in vitro platform for investigating human MN physiology, synaptic mechanisms, and disease-relevant synaptopathies.

neuroscience↗

bioRxiv2025

Differentiated SH-SY5Y Cells Exhibit Neuronal Features but Lack Synaptic Maturity

A vital question in neuroscience is if and how efficiently cellular models may be differentiated into functional neuronal cells in culture. Despite the frequent use of the human neuroblastoma cell line SH-SY5Y, differentiation protocols vary extensively, with the most common being differentiation via the addition of retinoic acid and brain-derived neurotrophic factor. However, due to the lack of a reliable evaluation method, their adequacy as synaptic models remains unclear. Here, we investigate whether SH-SY5Y cells constitute a functional model for synaptic studies by phenotypically and ultra structurally analyzing synaptogenesis in SH-SY5Y cells subjected to different differentiation protocols. Electron microscopy (EM) techniques, including conventional EM, cryo-EM, and cryo-electron tomography, were systematically applied to characterize synaptogenesis in SH-SY5Y cells. Further characterization was performed through immunostaining and functional assays, such as live exocytosis assays and whole-cell patch-clamp electrophysiology. Despite exhibiting some presynaptic-like features, differentiated SH-SY5Y cells do not form morphologically or functionally complete synapses under the conditions tested. Immunostaining results were consistent with previous findings, showing synaptic markers. However, functional investigations did not detect synaptic activity. High-throughput EM analyses revealed an absence of synaptic structures in these cells. Additionally, an alternative differentiation approach incorporating additional neurotrophic factors promoted the formation of pre-synaptic-like compartments containing synapse-like synaptic vesicles (SVLVs). Though these SVLVs exhibited pleomorphic size distributions, differing from typical synaptic vesicles, and lacked connectors. These findings emphasize the need for cautious interpretation of results derived from SH-SY5Y cells when studying molecular synaptic architecture or neurodegenerative diseases.

neuroscience↗

bioRxiv2019

Y-chromosome haplotypes of varying differentiation to the X are not associated with male fitness in common frogs

The canonical model of sex-chromosome evolution assigns a key role to sexually antagonistic (SA) genes on the arrest of recombination and ensuing degeneration of Y chromosomes. This assumption cannot be tested in organisms with highly differentiated sex chromosomes, such as mammals or birds, owing to the lack of polymorphism. Fixation of SA alleles, furthermore, might be the consequence rather than the cause of recombination arrest. Here we focus on a population of common frogs (Rana temporaria) where XY males with genetically differentiated Y chromosomes (non-recombinant Y haplotypes) coexist with both XY{degrees} males with proto-Y chromosomes (only differentiated from X chromosomes in the immediate vicinity of the candidate sex-determining locus Dmrt1) and XX males with undifferentiated sex chromosomes (genetically identical to XX females). Our study shows no effect of sex-chromosome differentiation on male phenotype, mating success or fathering success. Our conclusions rejoin genomic studies that found no differences in gene expression between XY, XY{degrees} and XX males. Sexual dimorphism in common frogs seems to result from the differential expression of autosomal genes rather than sex-linked SA genes. Among-male variance in sex-chromosome differentiation is better explained by a polymorphism in the penetrance of alleles at the sex locus, resulting in variable levels of sex reversal (and thus of X-Y recombination in XY females), independent of sex-linked SA genes.\n\nImpact Summary\n\nHumans, like other mammals, present highly differentiated sex chromosomes, with a large, gene-rich X chromosome contrasting with a small, gene-poor Y chromosome. This differentiation results from a process that started approximately 160 Mya, when the Y first stopped recombining with the X. How and why this happened, however, remain controversial. According to the canonical model, the process was initiated by sexually antagonistic selection; namely, selection on the proto-Y chromosome for alleles that were beneficial to males but detrimental to females. The arrest of XY recombination then allowed such alleles to be only transmitted to sons, not to daughters. Although appealing and elegant, this model can no longer be tested in mammals, as it requires a sex-chromosome system at an incipient stage of evolution. Here we focus on a frog that displays within-population polymorphism is sex-chromosome differentiation, where XY males with differentiated chromosomes coexist with XX males lacking Y chromosomes. We find no effect of sex-chromosome differentiation on male phenotype or mating success, opposing expectations from the standard model. Sex linked genes do not seem to have a disproportionate effect on sexual dimorphism. From our results, sexually antagonistic genes show no association with sex-chromosome differentiation in frogs, which calls for alternative models of sex-chromosome evolution.

evolutionary biology↗