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

SOFIA MUCCI; LUCIANA ISAJA; MARIA SOLEDAD RODRIGUEZ VARELA; GUSTAVO E. SEVLEVER; MARÍA E. SCASSA; LEONARDO ROMORINI

Viena

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

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 or function of neurons. CDK5/p35 complex is involved in neuronal homeostasis and development. However, its function is deregulated in neurodegeneration by p35 cleavage into p25, which allows the aberrant phosphorylation of targets through the constitution of a more stable CDK5/p25 hyperactive complex. Although CDK5 signaling has been intensively studied in animal models, there are currently no-good 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, we first 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 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 although CDK5 was ubiquitous, p35 mRNA and protein were mainly expressed in neurons. We next evaluated how different stress stimuli (rotenone, glutamate and calcium ionophore A23187) affected hPSCs-derived neurons viability. We determined the percentage of cell viability after 24 hours treatment with increasing concentrations of rotenone and glutamate and 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 concomitant with an increase in cell death. However, only a little effect was observed with glutamate treatment. 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 (1μM for 24h) and A23187 (2μM 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, 50μM). In conclusion, neurons-derived from hPSCs 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-mediated cleavage of p35 to p25.