Mental Illnesses-Associated Fxr1 and Its Negative Regulator Gsk3β Are Modulators of Anxiety and Glutamatergic Neurotransmission

Abstract

Genetic variants of the fragile X mental retardation syndrome-related protein 1 (FXR1) have been associated to mood regulation, schizophrenia, and bipolar disorders. Nonetheless, genetic association does not indicate a functional link of a given gene to neuronal activity and associated behaviors. In addition, interaction between multiple genes is often needed to sculpt complex traits such as behavior. Thus, modulation of neuronal functions by a given gene product, such as Fxr1, has to be thoroughly studied in the context of its interactions with other gene products. Glycogen synthase kinase-3 beta (GSK3β) is a shared target of several psychoactive drugs. In addition, interaction between functional polymorphisms of GSK3b and FXR1 has been implicated in mood regulation in healthy subjects and bipolar patients. However, the mechanistic underpinnings of this interaction remain unknown. We used somatic CRISPR/Cas9 mediated knockout and overexpression to investigate the impact of Fxr1 and its regulator Gsk3β on neuronal functions directly in the adult mouse brain. Suppression of Gsk3β or increase of Fxr1 expression in medial prefrontal cortex neurons leads to anxiolytic-like responses associated with a decrease in AMPA mediated excitatory postsynaptic currents. Furthermore, Fxr1 and Gsk3β modulate glutamatergic neurotransmission via regulation of AMPA receptor subunits GluA1 and GluA2 as well as vesicular glutamate transporter VGlut1. These results underscore a potential mechanism underlying the action of Fxr1 on neuronal activity and behaviors. Association between the Gsk3β-Fxr1 pathway and glutamatergic signaling also suggests how it may contribute to emotional regulation in response to mood stabilizers, or in illnesses like mood disorders and schizophrenia.

Fxr1 regulates sleep and synaptic homeostasis

“…4). The interaction between Fxr1 and Gsk3b have also been reported to affects anxiety-related behaviors in mice 25 and self-reported emotional stability in humans 18 .…”

“…2a-c). Then, the most efficient Fxr1 targeting gRNA and a previously characterized Gsk3b targeting gRNA 25 were used to make Crispr/Cas9 constructs to target Fxr1 and Gsk3β in neuronal cultures. All control plasmids contained scrambled gRNAs.…”

“…Thus, we examined whether Fxr1 and its negative regulator Gsk3β can block increase of mEPSCs and synaptic GluA1 during SD. To address this question AAV SYN GFP-Fxr1 (Fxr1over), AAV Gsk3sgRNA/GFP + AAV SpCas9 (Gsk3sKO) 25 , and AAV SYN GFP (Ctrl) were injected into the prefrontal cortex three weeks prior to SD ( Fig. 6a).…”

“…For primary neuronal culture transfection previously characterized Gsk3b gRNA oligonucleotide 25 Sequences of all constructs were verified. To test the activity of Fxr1 sgRNA by western blot (Fig.…”

“…Fxr2, and Fmrp have been shown to regulate synaptic expression of the AMPA receptor GluA1 subunit in different brain regions [24][25][26] . Fxr1 has been shown to regulate de novo synthesis of the GluA2 AMPA receptor subunit during long-lasting synaptic potentiation of hippocampal neurons 27 .…”