iPSC model of CHRFAM7A effect on α7 nicotinic acetylcholine receptor function in the human context

The α7 nicotinic acetylcholine receptor (α7nAChR) has been a promising target for diseases affecting cognition and higher cortical functions; however, the effect observed in animal models failed to translate into human clinical trials identifying a translational gap. CHRFAM7A is a human-specific fusion gene with properties that enable incorporation into the α7nAChR and, being human specific, CHRFAM7A effect was not accounted for in preclinical studies. We hypothesized that CHRFAM7A may account for this translational gap and understanding its function may offer novel insights when exploring α7nAChR as a drug target. CHRFAM7A is present in different copy number variations (CNV) in the human genome with high frequency. To study the functional consequences of the presence of the CHRFAM7A, two induced pluripotent stem cell (iPSC) lines (0 copy and 1 copy direct) were developed. The 0 copy line was rescued with CHRFAM7A transfection to control for genetic heterogeneity. As readouts for genotype–phenotype correlation, α7nAChR synaptic transmission and amyloid beta 1–42 (Aβ1–42) uptake were tested. Synaptic transmission in the presence of CHRFAM7A demonstrated that PNU-modulated desensitization of α7nAChR currents increased as a function of CHRFAM7A dosage. CHRFAM7A mitigated the dose response of Aβ1–42 uptake suggesting a protective effect beyond physiological concentrations. Furthermore, in the presence of CHRFAM7A Aβ1–42 uptake activated neuronal interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) without activating the canonical inflammasome pathway. Lead optimization may identify more potent molecules when the screen has a model harboring CHRFAM7A. Incorporating pharmacogenetics into clinical trials may enhance signals in efficacy measures.

NIM with 2% B27 (Life Technologies) to form neurospheres. The cultures were fed every other day. On day 25, the neural progenitor cells, NPCs, were dissociated with Accutase (Stem Cell Technonlogies) and plated onto 6-well plates for characterization, expansion, and further differentiation to GABA interneurons and BFCN.

Quantitative Polymerase Chain Reaction (qPCR)
Total RNA was isolated from cell cultures at different stages of neuronal differentiation (D0 pluripotent stem cells, D25-neural progenitor cells, D40 -neurons) using Trizol (Invitrogen) according to manufacturer's protocol. cDNA was synthesized from 500ng of total RNA using ImProm-II reverse transcriptase (Promega) and oligo (DT) (Promega). The reaction was carried out at 42°C for one hour. For quantitative gene expression, standard RT-qPCR was performed using the primers (IDT) listed in Supplementary data, Table 1. qPCR was performed using the SYBR green master mix (Biotool) and run on Bio-Rad CFX Connect cycler (Bio-Rad).
Samples were assayed with 3 technical replicates, and data was analyzed using the ∆∆C T method and normalized to GAPDH expression. Data are presented as the average of the triplicates ± standard error of the mean.

Immunocytochemistry and Confocal Microscopy
Cells plated on glass coverslips or 8-well glass chambers (ThermoFisher) were fixed with 4% paraformaldehyde (Mallinckrodt Baker) for 15 minutes, permeabilized with 0.1% Triton X100 (Mallinckrodt Baker) for 10 minutes, and blocked with blocking buffer (5% BSA in PBS) for 1 hour at room temperature (RT). Cells were incubated overnight at 4 o C with primary antibodies (Supplementary data, Table 2). On the next day, cells were incubated for 1h at RT with secondary antibodies. Both primary and secondary antibodies were diluted in blocking buffer. Slides were mounted with a Prolong ® Gold antifade reagent with DAPI (Life Technologies), and confocal images were captured by using LSM510 Meta microscope (40 x objective). Images were acquired using ZEN black software.
Because of the high extracellular [K + ], the membrane potential V m was ~0 mV. In most cases (except in action current recordings), we routinely added 100 nM Tetrodotoxin (TTX) and 10 mM tetraethylammonium (TEA) to block Na v and K v currents.