Biology subjects

Suciu, I.

Publications and source records attributed to Suciu, I..

2 recordsLinked to original sources

bioRxiv2022

Generation of human nociceptor-enriched sensory neurons for the study of pain-related dysfunctions

In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically-useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly-enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9,000 cells were used to establish the "fraction of reactive cells" in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.

pharmacology and toxicology↗

bioRxiv2022

Specific attenuation of purinergic signaling during bortezomib-induced peripheral neuropathy

Human peripheral neuropathies are poorly-understood, and the availability of experimental models limits further research. The PeriTox test uses immature dorsal root ganglia (DRG)-like neurons, derived from induced pluripotent stem cells (iPSC), to assess cell death and neurite damage. Here, we explored the suitability of matured peripheral neuron cultures for detection of sub-cytotoxic endpoints, such as altered responses of pain-related P2X receptors. A 2-step differentiation protocol, involving transient expression of ectopic neurogenin-1 (NGN1), allowed for the generation of homogeneous cultures of sensory neurons. After > 38 days-of-differentiation, they showed a robust response (Ca2+-signalling) to the P2X3 ligand ,{beta}-methylene ATP. The clinical proteasome inhibitor bortezomib abolished the P2X3 signal at [≥] 5 nM, while 50-200 nM were required in the PeriTox test to identify neurite damage and cell death. A 24 h treatment with low nM concentrations of bortezomib led to moderate increases in resting cell intracellular [Ca2+], but signalling through transient receptor potential-V1 (TRPV1) receptors or depolarization-triggered Ca2+-influx remained unaffected. We interpret the specific attenuation of purinergic signalling as functional cell stress response. A reorganization of tubulin to dense structures around the cell somata confirmed a mild, non-cytotoxic stress triggered by low concentrations of bortezomib. The proteasome inhibitors carfilzomib, delanzomib, epoxomycin and MG-132 showed similar stress responses. Thus, the model presented here may be used for profiling of new proteasome inhibitors as to their side effect (neuropathy) potential, or for pharmacological studies on the attenuation of their neurotoxicity. P2X3 signalling proved useful as endpoint to assess potential neurotoxicants in peripheral neurons.

pharmacology and toxicology↗