Cortical neurons derived from human pluripotent stem cells lacking FMRP display altered spontaneous firing patterns

Abstract

Background: Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP). Methods: Electrophysiological whole-cell voltage-and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced. Results: Neurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca 2+activated K + currents and the persistent Na + current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na + channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines. Conclusions: Pharmacological manipulations can alter the action potential burst profiles in both control and FMRPnull human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered.

Impaired Functional Connectivity Underlies Fragile X Syndrome

“…However, differentiation of FX-PSCs led to poor neuronal maturation and decreased spike frequencies relative to control neurons [16,17]. Nevertheless, other studies demonstrated inconsistent results regarding the functional differences between FX-PSCs and control neurons [18][19][20]. In the current study, hESC isogenic subclones were generated, one with a full FX mutation and one that is free of the mutation (WT) but shares the same genetic background, enabling us to overcome variability between lines.…”

FMRP sustains presynaptic function via control of activity-dependent bulk endocytosis

“…It is likely that the observed reduction in ADBE reflects a compensatory or homeostatic mechanism to counteract the increased excitability in FXS and Fmr1 KO models (Booker et al, 2019; Das Sharma et al, 2020; Zhang et al, 2014). BK channel openers decrease hyperexcitability in Fmr1 KO models both in vitro and in vivo by increasing hyperpolarization, resulting in decreased calcium influx in nerve terminals.…”

“…A homeostatic reduction in ADBE in response to increased hyperexcitability would not be a homogenous adaptation across the brain, since neurons that display high firing rates would be disproportionately impacted. Therefore, even if reduced ADBE is not a causal mechanism in FXS, it may still be a valuable therapeutic intervention point to correct hyperexcitability in specific circuits (Booker et al, 2019; Das Sharma et al, 2020). Future studies are now required to determine whether modulation of ADBE can sculpt circuit activity in FXS (and other autism models that display hyperexcitability - such as SynGAP haploinsufficiency disorder (Gamache et al, 2020)).…”

“…Our finding that BK channel inhibition had no effect on ADBE in WT hippocampal cultures suggests that the inability of FMRP R138Q to restore ADBE in KO neurons is due to loss of BK channel (or other) interactions, rather than channel function itself. It is likely that the observed reduction in ADBE reflects a compensatory or homeostatic mechanism to counteract the increased excitability in FXS and Fmr1 KO models (Booker et al, 2019;Das Sharma et al, 2020;Zhang et al, 2014). BK channel openers decrease hyperexcitability in Fmr1 KO models both in vitro and in vivo by increasing hyperpolarization, resulting in decreased calcium influx in nerve terminals.…”

“…Hyperexcitability of neuronal circuits is a key feature of fragile X syndrome (FXS) (Booker et al, 2019; Das Sharma et al, 2020), one of the most common monogenic causes of intellectual disability (ID) and autism spectrum disorder (Mefford et al, 2012). Most FXS cases are caused by a CGG trinucleotide expansion in the 5’ untranslated region of the FMR1 gene which encodes FMRP, leading to hypermethylation of the promoter and silencing of the FMR1 gene.…”