RESUMO
We investigated the impact of the human-specific gene CHRFAM7A on the function of α7 nicotinic acetylcholine receptors (α7 nAChRs) in two different types of neurons: human-induced pluripotent stem cell (hiPSC)-derived cortical neurons, and superior cervical ganglion (SCG) neurons, taken from transgenic mice expressing CHRFAM7A. dupα7, the gene product of CHRFAM7A, which lacks a major part of the extracellular N-terminal ligand-binding domain, co-assembles with α7, the gene product of CHRNA7. We assessed the receptor function in hiPSC-derived cortical and SCG neurons with Fura-2 calcium imaging and three different α7-specific ligands: PNU282987, choline, and 4BP-TQS. Given the short-lived open state of α7 receptors, we combined the two orthosteric agonists PNU282987 and choline with the type-2 positive allosteric modulator (PAM II) PNU120596. In line with different cellular models used previously, we demonstrate that CHRFAM7A has a major impact on nicotinic α7 nAChRs by reducing calcium transients in response to all three agonists.
Assuntos
Células-Tronco Pluripotentes Induzidas , Camundongos Transgênicos , Neurônios , Receptor Nicotínico de Acetilcolina alfa7 , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genética , Animais , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Camundongos , Colina/farmacologia , Colina/metabolismo , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/metabolismo , Compostos Bicíclicos com Pontes/farmacologia , Agonistas Nicotínicos/farmacologia , Benzamidas/farmacologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Córtex Cerebral/efeitos dos fármacos , Cálcio/metabolismo , Isoxazóis , Compostos de FenilureiaRESUMO
Rett Syndrome (RTT) is a severe neurodevelopmental disorder, afflicting 1 in 10,000 female births. It is caused by mutations in the X-linked methyl-CpG-binding protein gene (MECP2), which encodes for the global transcriptional regulator methyl CpG binding protein 2 (MeCP2). As human brain samples of RTT patients are scarce and cannot be used for downstream studies, there is a pressing need for in vitro modeling of pathological neuronal changes. In this study, we use a direct reprogramming method for the generation of neuronal cells from MeCP2-deficient and wild-type human dermal fibroblasts using two episomal plasmids encoding the transcription factors SOX2 and PAX6. We demonstrated that the obtained neurons exhibit a typical neuronal morphology and express the appropriate marker proteins. RNA-sequencing confirmed neuronal identity of the obtained MeCP2-deficient and wild-type neurons. Furthermore, these MeCP2-deficient neurons reflect the pathophysiology of RTT in vitro, with diminished dendritic arborization and hyperacetylation of histone H3 and H4. Treatment with MeCP2, tethered to the cell penetrating peptide TAT, ameliorated hyperacetylation of H4K16 in MeCP2-deficient neurons, which strengthens the RTT relevance of this cell model. We generated a neuronal model based on direct reprogramming derived from patient fibroblasts, providing a powerful tool to study disease mechanisms and investigating novel treatment options for RTT.