Examining FKBP5 mRNA expression in human iPSC-derived neural cells

Abstract

In peripheral blood leukocytes, FKBP5 mRNA expression is upregulated following glucocorticoid receptor activation. The single nucleotide polymorphism rs1360780 in FKBP5 is associated with psychiatric illness and has functional molecular effects. However, examination of FKBP5 regulation has largely been limited to peripheral cells, which may not reflect regulation in neural cells. We used 27 human induced pluripotent stem cell lines (iPSCs) derived from 20 subjects to examine FKBP5 mRNA expression following GR activation. Following differentiation into forebrain-lineage neural cultures, cells were exposed to 1μM dexamethasone and mRNA expression of FKBP5 and NR3C1 analyzed. Results from the iPSC-derived neural cells were compared with those from 15 donor matched fibroblast lines. Following dexamethasone treatment, there was a 670% increase in FKBP5 expression in fibroblasts, mimicking findings in peripheral blood-derived cells, but only a 23% increase in iPSC-derived neural cultures. FKBP5 rs1360780 genotype did not affect the induction of FKBP5 mRNA in either fibroblasts or neural cells. These results suggest that iPSC-derived forebrain-lineage neurons may not be an optimal neural cell type in which to examine relationships between GR activation, FKBP5 expression, and genetic variation in human subjects. Further, FKBP5 induction following GR activation may differ between cell types derived from the same individual.

Alcohol-responsive genes identified in human iPSC-derived neural cultures

“…Following 12 weeks of neural maturation, iPSC-derived cultures contained dense Beta III-tubulin positive neurites ( Figure 1G), MAP2-positive neurons with pyramidal morphology (Figure 1H-I), and GFAP-positive astrocytes (Figure 1I). These findings are consistent with our prior work demonstrating this protocol efficiently generates mixed neural cell cultures with ~50% TBR1positive glutamate neurons that have the ability to generate trains of action potentials, display spontaneous synaptic activity indicative of synapse formation, and express functional glutamatergic and GABAergic ionotropic receptors, with no difference in the ability of iPSCs from control or AUD donors to generate neural cultures 24,25 .…”

“…Following differentiation and plating onto matrigel-coated glass coverslips, neural cells were cultured and matured for 12 weeks prior to experimentation. Our prior work has demonstrated that 8-12 weeks of growth under this protocol generates neural cultures with functional electrophysiological properties as evidenced by mature action potentials, spontaneous synaptic activity, and expression of ligand-gated ionotropic receptors 24,25 . Neural cell markers were examined in differentiated iPSC lines 12 weeks after plating via immunostaining, as we have described.…”

“…Neural cell markers were examined in differentiated iPSC lines 12 weeks after plating via immunostaining, as we have described. 25 Cells were fixed in 4% paraformaldehyde, permeabilized using 0.2 % triton X-100 (Sigma-Aldrich), and blocked in 5% donkey serum (Jackson ImmunoResearch). The following primary antibodies were diluted in 5% donkey serum and incubated for 24-48 hours at 4° Celsius: mouse anti-beta III-tubulin (1:500, MAB1637, Millipore), mouse anti-GFAP (1:500, MAB360, Millipore), and rabbit anti-MAP2 (1:500, AB5622, Millipore).…”

FKBP5 polymorphisms induce differential glucocorticoid responsiveness in primary CNS cells – First insights from novel humanized mice

“…In the human population, there is a wide variation in perception of stressful challenges, and variation in physiological expression of stress. Single nucleotide polymorphisms (SNPs) within the FKBP5 gene locus have been reported to impact stress responsivity, and risk or resilience to psychiatric disorders (Appel et al., 2011; Binder, 2009; Criado‐Marrero et al., 2019; Liebermann et al., 2017; Wilker et al., 2014). Differential expression levels of FKBP5 have preclinically been shown to impact on stress‐coping behavior and the SNP rs1360780 was reported to profoundly influence stress coping, suggesting FKBP5 genotypes as one source of human variation (Ising et al., 2008; Touma et al., 2011).…”

Uncovering True Cellular Phenotypes: Using Induced Pluripotent Stem Cell-Derived Neurons to Study Early Insults in Neurodevelopmental Disorders

“…A series of papers has focused on iPSC-derived neurons in relation to alcohol dependence, FKBP5 regulation, and glucocorticoid activation ( 95 – 97 ). These cells express voltage-gated inward and outward currents, fire APs in response to current injection, respond to puff application of glutamate and GABA, and display spontaneous synaptic activity.…”