Human pluripotent stem cell-derived neurons as an in vitro model for studying neural stress and CDK5 signaling

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

Vienna

Simposio; SY-STEM 3rd Symposium on Stem Cell Research; 2021

IMBA

Human pluripotent stem cells (hPSCs), which include embryonic and induced pluripotent stem cells (hESCs and hiPSCs, respectively), can differentiate into a wide range of specialized cells, including neurons. Moreover, hPSCs-derived neurons are proposed as a model for studying neurodegeneration. Neurodegeneration is a complex multifactorial process that causes progressive loss of structure and function of neurons. CDK5/p35 complex is involved in neuronal homeostasis and devel- opment. However, under pathological conditions p35 can be cleaved by calpain into p25. The resulting complex CDK5/ p25 engenders aberrant activity and phosphorylates a number of substrates abnormally. Although CDK5 signaling has been intensively studied in animal models, currently there are no-suitable in vitro models for studying its participation in human neuronal homeostasis 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 hPSCs. For this purpose, first we derived neural stem cells (NSC) from hESCs (H9 line) and hiPSCs (FN2.1 line), which were further differentiated into neurons using a 2D-based protocol. NSC and neuron-like phenotype were validated by electrophysiology and expression of lineage specific markers (Sox-1, Sox-2, Pax-6 and Nestin for NSC; MAP5, MAP2 and Tuj-1 for neurons) by immunofluorescence microscopy and RT-qPCR. Then, CDK5 and p35 mRNA and protein expression levels were analyzed in hPSCs, NSC and neurons by RT-qPCR and western blot. Interestingly, we observed that while CDK5 is ubiquitously expressed, p35 mRNA and protein are mainly expressed in neurons. We next evaluated how different types of stress (rotenone, glutamate and calcium ionophore A23187) affected hPSCs-derived neurons viability. We determined the percentage of cell viability after a 24 hours treatment with increasing concentrations of rotenone and glutamate and a 2 hours treatment with A23187 using a XTT vital dye assay. Cell viability fell down significantly in the case of rotenone and A23187 treatments in a concentration dependent manner, which was not accompanied by an increase in cell death. However, only a slight effect was observed with glutamate treat- ment. Moreover, an increase in mitochondrial membrane potential (measured using Mitoprobe JC-1 Assay and morphologic changes (axonal spheroid appearance) were found in derived neurons upon rotenone (1uM for 24h) and A23187 (2uM for 2h) stressful stimuli. Further, rotenone and A23187 treatments induced p35 cleavage to p25, which was mediated by calpains as proteolysis was inhibited with a calpain inhibitor (ALLN, 50uM). In conclusion, hPSCs derived neurons emerge as a potential in vitro human model for studying the relevance of CDK5 signaling in neural stress as they responded to stressful stimuli inducing calpain-mediated cleavage of p35 to p25.