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The differentiation of neural progenitors is a complex process that integrates different signals to drive transcriptional changes, which mediate metabolic, electrophysiological, and morphological cellular specializations. Understanding these adjustments is essential within the framework of stem cell and cancer research and therapy. Human neuroblastoma SH-SY5Y cells, widely used in neurobiology research, can be differentiated into neuronal-like cells through serum deprivation and retinoic acid (RA) supplementation. In our study, we observed that the differentiation process triggers the expression of Heat Shock Protein 70 (HSP70). Notably, inhibition of HSP70 expression by KNK437 causes a dramatic increase in cell death. While undifferentiated SH-SY5Y cells show a dose-dependent decrease in cell survival following exposure to hydrogen peroxide (H₂O₂), differentiated cells become resistant to H₂O₂-induced cell death. Interestingly, the differentiation process enhances the expression of SOD1 protein, and inhibition of HSP70 expression counteracts this effect and increases the susceptibility of differentiated cells to H₂O₂-induced cell death, suggesting that the cascade HSP70-SOD1 is involved in promoting survival against oxidative stress-dependent damage. Treatment of differentiated SH-SY5Y cells with Oxotremorine-M (Oxo), a muscarinic acetylcholine receptor agonist, enhances the expression of HSP70 and SOD1 and counteracts tert–Butyl hydroperoxide-induced cell death and reactive oxygen species (ROS) generation. It is worth noting that co-treatment with KNK437 reduces SOD1 expression and Oxo-induced protection against oxidative stress damage, suggesting the involvement of HSP70/SOD1 signaling in this beneficial effect. In conclusion, our findings demonstrate that manipulation of the HSP70 signal modulates SH-SY5Y differentiation and susceptibility to oxidative stress-dependent cell death and unravels novel mechanisms involved in Oxo neuroprotective functions. Altogether these data provide novel insights into the mechanisms underlying neuronal differentiation and preservation under stress conditions. Full article

A complex evaluation of agonist bias at G-protein coupled receptors at the level of G-protein classes and isoforms including non-preferential ones is essential for advanced agonist screening and drug development. Molecular crosstalk in downstream signaling and a lack of sufficiently sensitive and selective methods to study direct coupling with G-protein of interest complicates this analysis. We performed binding and functional analysis of 11 structurally different agonists on prepared fusion proteins of individual subtypes of muscarinic receptors and non-canonical promiscuous α-subunit of G₁₆ protein to study agonist bias. We have demonstrated that fusion of muscarinic receptors with Gα₁₆ limits access of other competitive Gα subunits to the receptor, and thus enables us to study activation of Gα₁₆ mediated pathway more specifically. Our data demonstrated agonist-specific activation of G₁₆ pathway among individual subtypes of muscarinic receptors and revealed signaling bias of oxotremorine towards Gα₁₆ pathway at the M₂ receptor and at the same time impaired Gα₁₆ signaling of iperoxo at M₅ receptors. Our data have shown that fusion proteins of muscarinic receptors with α-subunit of G-proteins can serve as a suitable tool for studying agonist bias, especially at non-preferential pathways. Full article