STUDY OF THE POTENTIAL NON-APOPTOTIC ROLE OF CASPASE-3 IN ASTROCYTE PROLIFERATION AND DIFFERENTIATION USING A NEUROSPHERE-DERIVED <EM>IN VITRO </EM>MODEL

Although caspase-3 is recognised as the pivotal mediator of apoptosis, its non-apoptotic functions are emerging. Since our previous studies identified a population of non-apoptotic cleaved caspase-3 positive (cC3⁺) cells, predominantly astrocytes, in postnatal rat spinal cord, we focused on investigating the potential role of cC3 in astrogliogenesis. We established an in vitro model derived from an intact neonatal rat spinal cord (postnatal day 7). Prepared neurospheres were cultivated in differentiation medium and analysed within 12 days (2-12DIV) to study the presence of cC3 in cell culture and its colocalisation with phenotypic and proliferation (Ki67) markers. In addition, to analyse the potential involvement of cC3 in the differentiation process, caspase inhibitors were applied to the medium. According to our results, high levels of cC3 were consistently maintained in both the control and inhibited groups throughout the experiment. Interestingly, cC3 colocalised with proliferating cells in both the control and treated groups. According to our data, the culture contains mostly GFAP⁺ astrocytes, which emerge early in differentiation (2DIV) and remain stable during the entire experimental period. Contrary to the control group, the percentage of nestin+ cells accumulated at the end of the experiment (12DIV) in the treated group, indicating potential delayed differentiation in the inhibited culture. Altogether, our results indicate potential involvement of cleaved caspase-3 in cell differentiation.
Acknowledgement: This work was funded by the EU NextGenerationEU through the Recovery and Resilience Plan for Slovakia under the project No. 09I03-03-V05-00008 and project APVV-23-0274.