Neurons derived from human pluripotent stem cells as a model for studying neural stress and CDK5 signaling

MUCCI, SOFÍA; RODRÍGUEZ VARELA, MARÍA SOLEDAD; ISAJA, LUCIANA; SEVLEVER, GUSTAVO EMILIO; SCASSA, MARÍA ELIDA; ROMORINI, LEONARDO

Buenos Aires

Congreso; Reunión de Sociedades Biociencias 2020, SAIC.SAI.SAFIS; 2020

SAIC SAI SAFIS

Neurodegeneration is a complex multifactorial process that caus- es progressive loss of structure or function of neurons. CDK5/p35 complex is involved in neuronal homeostasis. However, stressful stimuli induce calpain-mediated cleavage of p35 to p25. p25 in turns forms a more stable CDK5/p25 hyperactive complex that participate in neurodegenerative mechanisms. Although CDK5 signaling has been intensively study in animal models, there are currently no good in vitro models for studying its participation in human neuronal ho- meostasis and neurodegenerative processes. In this work we aimed to generate an in vitro human model for studying CDK5 signaling and neural stress based on the neuronal differentiation of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs, respectively). First, hESCs (H9 line) and hiPSCs (FN2.1 line) were derived to neural stem cells (NSC), which were then differentiated into generic neurons using 2D protocol. Phenotypes were validat- ed by immunofluorescence of lineage specific markers (Sox-1 and Sox-2 for NSC, MAP2 and Tuj-1 for neurons). Next, neurons were subjected to stressful stimuli (rotenone 1μM for 24h and calcium ionophore A23187 2μM for 2h) and an increase in mitochondrial membrane potential was observed (measured using Mitoprobe JC-1 Assay). Moreover, morphologic changes (axonal spheroids appear- ance) were found in derived-neurons upon stressful stimuli. Final- ly, we found that p35 is highly expressed (quantified by RT-qPCR and western blot) in derived-neurons and that rotenone and A23187 treatments induced its cleavage to p25 (analyzed by western blot). Further, p35 cleavage was mediated by calpains as proteolysis was inhibited with a calpain inhibitor (ALLN, 50μM). In conclusion, neu- rons-derived from hESCs and hiPSCs are potentially a good in vitro human model for studying the relevance of CDK5 signaling in neural stress as they responded to stressful stimuli inducing calpain-medi- ated cleavage of p35 to p25.