Biology subjects

Fedele, L.

Publications and source records attributed to Fedele, L..

7 recordsLinked to original sources

bioRxiv2026

Pathways regulating the excitability of sympathetic neurons derived from human induced pluripotent stem cells

Postganglionic sympathetic neurons not only regulate target organs but also their own intrinsic activity and can be further modulated by parasympathetic neurons. This crosstalk and automodulatory signalling have been implicated in cardiovascular disorders, with the majority of earlier work employing rodent models. Here, we have used human sympathetic neurons derived from induced pluripotent stem cells (hiPSCs), as a scalable human in vitro system with the aim to investigate these pathways. Efficient differentiation of hiPSCs into sympathetic neurons was confirmed using molecular characterisation for the expression of PHOX2B, DBH, TH, PRPH. We employed Ca2+ imaging and whole-cell patch-clamp electrophysiology, to examine the neuronal functional properties and found that hiPSC-derived sympathetic neurons recapitulate key physiological features of the rodent native counterparts. Most cells responded to nicotine and expressed functional 2 adrenergic and muscarinic receptors, involved in sympathetic autoregulation and parasympathetic crosstalk. We further demonstrated that 2 adrenergic and muscarinic receptors inhibit membrane excitability (increased rheobase, hyperpolarisation, reduced input resistance) and that both types of receptors converge on inwardly rectifying K+ channels (GIRK) as effectors. The GIRK blocker Tertiapin-Q significantly reduced the 2 adrenergic and muscarinic responses, while the activator ML297 mimicked their action. Analysis of mouse stellate scRNA-seq confirmed that the receptors and GIRK subtypes studied here are prominently expressed in native sympathetic neurons. Overall, our data show that GIRK channels play an important role in the regulation of sympathetic neurons excitability and that hiPSC-derived neurons provide an attractive in vitro tool for drug discovery to study sympathetic autoregulation and parasympathetic-sympathetic crosstalk. KEY POINTSO_LIhiPSC-sympathetic neurons recapitulate key cellular pathways of native counterparts C_LIO_LIThey express relevant receptors and ion channels involved in the inhibitory autoregulatory feedback and parasympathetic-sympathetic crosstalk C_LIO_LIUsing a combination of RT-qPCR and functional recordings we identified inwardly rectifying K+ channels as downstream effectors of both 2 adrenergic and M2 muscarinic receptors. We cross-validated our findings with a mouse transcriptomic dataset from thoracic sympathetic ganglia. C_LIO_LIOverall, our data suggest that hiPSC-sympathetic neurons can be employed as a human in vitro platform to study cellular pathways and for drug discovery purposes. C_LI O_FIG O_LINKSMALLFIG WIDTH=119 HEIGHT=200 SRC="FIGDIR/small/727423v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@14e37ddorg.highwire.dtl.DTLVardef@35c534org.highwire.dtl.DTLVardef@260837org.highwire.dtl.DTLVardef@e57af6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic abstract.C_FLOATNO Summary of proposed modulation of sympathetic neuron activity via 2ARs and muscarinic receptors via GIRK channels Functional recordings showed that muscarinic receptors and 2 adrenergic receptors reduce neuronal excitability, this effect was mimicked by activation of inwardly rectifying K+ channels (GIRK). Blockade of GIRK abolished the effects of both types of receptors, demonstrating that they converge on GIRK as a common effector. The muscarinic response was blocked by pertussis toxin, indicating an involvement of Gi/o downstream of the receptor, consistent with the high expression of CHRM2 by RT-qPCR. C_FIG

neuroscience↗

bioRxiv2025

Generation of parasympathetic neurons from hiPSC that reproduce the electrophysiological properties of native neurons and modulate the activity of hiPSC-atrial cardiomyocytes

Peripheral parasympathetic ganglia lie adjacent to their target organs; their dysfunction contributes to diseases like atrial fibrillation. Due to their challenging accessibility for primary culture, we provide a step-by-step protocol to generate parasympathetic neurons from human induced pluripotent stem cells (hiPSC). They reproduce numerous native physiological features: cholinergic and autonomic markers, functional nicotinic receptors and electrophysiological properties. We also describe a co-culture system with hiPSC-atrial cardiomyocytes allowing neuromodulation of cardiomyocyte activity and offering a platform for drug discovery and disease modelling. HighlightsO_LISteps to generate parasympathetic neurons from human induced pluripotent stem cells C_LIO_LIGuidance of the functional assessment of the neurons (whole-cell patch clamp and Ca2+ imaging) C_LIO_LIInstructions on how to co-culture these neurons with hiPSC-derived atrial cardiomyocytes C_LIO_LIGuidance of the functional assessment of the neuronal modulation of cardiomyocyte activity C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/684157v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1e77cfborg.highwire.dtl.DTLVardef@be6cbforg.highwire.dtl.DTLVardef@1d58aa6org.highwire.dtl.DTLVardef@81ba0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

A role for fibroblast and mural cell subsets in models of neuropathic pain.

Neuropathic pain is a particularly intractable type of chronic pain that can result from physical nerve damage due to surgery or entrapment. Here, we present data which suggest that a particular subclass of fibroblast and mural cells may be implicated in the sensory neuron dysfunction that is characteristic of this pain state. In a mouse model of traumatic painful neuropathy, we used cell sorting, nerve tissue clearing and RNA sequencing to study mesenchymal lineage cells. With cell sorting (n = 4 mouse nerves) and tissue clearing (n = 5), we show that fibroblasts and mural cells positive for the platelet-derived growth factor receptor beta (Pdgfrb) gene are increased in number for at least two months post-nerve damage. Moreover, single cell RNA sequencing data (n = 4) from our own lab and those of three other laboratories reveal that Pdgfrb+ cells express high levels of known and putative pro-algesic mediators. Bulk sequencing of sorted Pdgfrb+ fibroblasts (n = 10) and Pdgfrb+/Cd146+ mural cells (n = 11) further indicate that many of these mediators are upregulated in neuropathy. We go on to demonstrate that a human nerve pericyte line releases a selection of these pro-algesic mediators at protein level. Moreover, conditioned media from stimulated human pericytes induces intra-cellular changes in human induced pluripotent stem cell derived sensory neurons (n = 5 independent differentiations); these changes (phosphorylation of the transcription factor signal transducer and activator of transcription 3, STAT3) have been previously linked to sensory neuron activation. In summary, our data indicate that mesenchymal cell abnormalities should be considered when developing novel strategies to tackle neuropathic pain. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/627455v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@186492corg.highwire.dtl.DTLVardef@12deb90org.highwire.dtl.DTLVardef@1c43b8aorg.highwire.dtl.DTLVardef@211952_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Blockade of rheumatoid arthritis synovial fluid-induced sensory neuron activation by JAK inhibitors

ObjectiveClinical studies suggest that compared to anti-TNF treatment, JAK inhibitors (JAKi) are superior in reducing pain in rheumatoid arthritis (RA). The underlying mechanisms for this observation are still unknown. Sensory neurons transmit noxious signals from inflamed joints to the central nervous system, where a pain percept is generated. We investigated whether JAKi exert direct effects on sensory neurons. MethodsIn-house and public RNA sequencing datasets of sensory neurons were analysed for relevant JAK/STAT and cytokine-receptor gene expression. Human induced pluripotent stem cell (IPSC)-derived sensory neurons were stimulated with serum and synovial fluid (SF) from individuals with RA, or with selected cytokines that were found in RA SF by Luminex. Phosphorylation of STAT3 (pSTAT3) was assessed by Western blot. Sensory neuron activation was examined by recording neuronal firing using multi-electrode array and measuring expression levels of pain-relevant genes with STAT3-binding sites. ResultsCell-free RA synovial fluid induced pSTAT3 in IPSC-derived sensory neurons, an effect which was completely blocked by the JAKi tofacitinib. Compared to paired serum, RA SF was enriched for the JAK/STAT cytokines IL-6, IL-11, LIF, IFN-alpha and IFN-beta, with their requisite receptors present on sensory neurons. Stimulation of IPSC- derived sensory neurons with these recombinant cytokines recapitulated pSTAT3 induction in these cells. Furthermore, IL-6+sIL-6R or LIF upregulated expression of pain-relevant genes which was blocked by tofacitinib. Finally, we provided evidence that LIF can induce neuronal sensitisation. ConclusionOur data indicate that JAKi can act directly on sensory neurons, providing a potential mechanistic explanation for their suggested superior analgesic properties.

immunology↗

bioRxiv2024

Modelling inflammation-induced peripheral sensitization in a dish - more complex than expected?

Peripheral sensitization of nociceptors is believed to be a key driver of chronic pain states. Here, we sought to study the effects of a modified version of inflammatory soup on the excitability of human stem-cell derived sensory neurons. For this, we used a pre-existing and a novel stem cell line, modified to stably express the calcium sensor GCamP6f. Upon treatment with inflammatory soup, we observed no changes in neuronal transcription or functional responses upon calcium imaging, and only a very minor increase in resting membrane potential via whole cell patch clamping. Similarly small changes were observed when treating mouse primary sensory neurons with inflammatory soup. A semi-systematic re-examination of past literature further indicated that observed effects of inflammatory mediators on dissociated sensory neuron cultures are generally very small. We conclude that modelling inflammation-induced peripheral sensitization in vitro is non-trivial and will require careful selection of mediators and/or more complex, longitudinal multi-cellular setups. Especially in the latter, our novel GCamP6f induced-pluripotent stem cell line may be of value.

neuroscience↗

bioRxiv2021

The nuts and bolts of SARS-CoV-2 Spike Receptor Binding Domain heterologous expression

COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful "tool" to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.

biochemistry↗

bioRxiv2021

Mice lacking the cAMP effector protein POPDC1 show enhanced hippocampal synaptic plasticity

Extensive research has uncovered diverse forms of synaptic plasticity and a wide array of molecular signaling mechanisms that act as positive or negative regulators. Specifically, cAMP-dependent signaling pathways have been crucially implicated in long-lasting synaptic plasticity. In this study, we examine the role of POPDC1 (or BVES), a cAMP effector protein expressed in brain, in modulating hippocampal synaptic plasticity. Unlike other cAMP effectors, such as PKA and EPAC, POPDC1 is membrane-bound and the sequence of the cAMP-binding cassette differs from canonical cAMP-binding domains. These properties suggest that POPDC1 may have a unique role in cAMP-mediated signaling underlying synaptic plasticity. Our results show that POPDC1 is enriched in hippocampal synaptoneurosomes. Acute hippocampal slices from Popdc1 knockout (KO) mice exhibit enhanced long-term potentiation (LTP) induced by a variety of stimulation paradigms, particularly in response to weak stimulation paradigms that in slices from wildtype mice induce only transient LTP. Furthermore, Popdc1 KO mice did not display any further enhancement in forskolin-induced LTP observed following inhibition of phosphodiesterases (PDEs), suggesting a possible modulation of cAMP-PDE signaling by POPDC1. Taken together, these data reveal POPDC1 as a novel player in the regulation of hippocampal synaptic plasticity and as a potential target for cognitive enhancement strategies.

neuroscience↗