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Fragile X mental retardation 1 (FMR1) encodes the RNA binding protein FMRP. Loss of FMRP drives Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and a leading monogenic cause of autism. While cortical hyperexcitability is a hallmark of FXS, the reported phenotypes and underlying mechanisms, including alterations in synaptic transmission and ion channel properties, are heterogeneous and at times contradictory. Here, we report the generation of new isogenic FMR1y/+ and FMR1y/- human pluripotent stem cell (hPSC) lines using CRISPR-Cas9 to facilitate the study of how complete FMRP loss, independent of genetic background, drives molecular and cellular alterations relevant for FXS. After differentiating these stem cell tools into excitatory neurons, we systematically assessed the impact of FMRP loss on intrinsic membrane and synaptic properties over time. Using whole-cell patch clamp analyses, we found that FMR1y/- neurons overall showed an increased intrinsic membrane excitability compared to age-matched FMR1y/+ controls, with no discernable alternations in synaptic transmission. Surprisingly, longitudinal analyses of cell intrinsic defects revealed that a majority of significant changes emerged early following in vitro differentiation and some were not stable over time. Collectively, this study provides a new isogenic hPSC model which can be further leveraged by the scientific community to investigate basic mechanisms of FMR1 gene function relevant for FXS. Moreover, our results suggest that precocious changes in the intrinsic membrane properties during early developmental could be a critical cellular pathology ultimately contributing to cortical hyperexcitability in FXS.

The four voltage-gated sodium channels SCN1/2/3/8A have been associated with heterogeneous types of developmental disorders, each presenting with disease specific temporal and cell type specific gene expression. Using single-cell RNA sequencing transcriptomic data from humans and mice, we observe that SCN1A is predominantly expressed in inhibitory neurons. In contrast, SCN2/3/8A are profoundly expressed in excitatory neurons with SCN2/3A starting prenatally, followed by SCN1/8A neonatally. In contrast to previous observations from low resolution RNA screens, we observe that all four genes are expressed in both excitatory and inhibitory neurons, however, exhibit differential expression strength. These findings provide molecular evidence, at single-cell resolution, to support the hypothesis that the excitatory/inhibitory (E/I) neuronal expression ratios of sodium channels are important regulatory mechanisms underlying brain homeostasis and neurological diseases. Modulating the E/I expression balance within cell types of sodium channels could serve as a potential strategy to develop targeted treatment for NaV-associated neuronal developmental disorders.

© 2020 Elsevier Inc. Optogenetics is among the most widely employed techniques to manipulate neuronal activity. However, a major drawback is the need for invasive implantation of optical fibers. To develop a minimally invasive optogenetic method that overcomes this challenge, we engineered a new step-function opsin with ultra-high light sensitivity (SOUL). We show that SOUL can activate neurons located in deep mouse brain regions via transcranial optical stimulation and elicit behavioral changes in SOUL knock-in mice. Moreover, SOUL can be used to modulate neuronal spiking and induce oscillations reversibly in macaque cortex via optical stimulation from outside the dura. By enabling external light delivery, our new opsin offers a minimally invasive tool for manipulating neuronal activity in rodent and primate models with fewer limitations on the depth and size of target brain regions and may further facilitate the development of minimally invasive optogenetic tools for the treatment of neurological disorders.

Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and γ-aminobutyric acid type A (GABA(A)) receptors at GABAergic synapses in hippocampus and amygdala. Four collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all collybistin isoforms (CB2(SH3+), CB2(SH3-), CB3(SH3+), and CB3(SH3-)) target to and concentrate at GABAergic postsynapses. Moreover, in non-transfected neurons, collybistin concentrates at GABAergic synapses. Hippocampal neurons co-transfected with CB2(SH3-) and gephyrin developed very large postsynaptic gephyrin and GABA(A) receptor clusters (superclusters). This effect was accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents. Co-transfection with CB2(SH3+) and gephyrin induced the formation of many (supernumerary) non-synaptic clusters. Transfection with gephyrin alone did not affect cluster number or size, but gephyrin potentiated the clustering effect of CB2(SH3-) or CB2(SH3+). Co-transfection with CB2(SH3-) or CB2(SH3+) and gephyrin did not affect the density of presynaptic GABAergic terminals contacting the transfected cells, indicating that collybistin is not synaptogenic. Nevertheless, the synaptic superclusters induced by CB2(SH3-) and gephyrin were accompanied by enlarged presynaptic GABAergic terminals. The enhanced clustering of gephyrin and GABA(A) receptors induced by collybistin isoforms was not accompanied by enhanced clustering of neuroligin 2. Moreover, during the development of GABAergic synapses, the clustering of gephyrin and GABA(A) receptors preceded the clustering of neuroligin 2. We propose a model in which the SH3- isoforms play a major role in the postsynaptic accumulation of GABA(A) receptors and in GABAergic synaptic strength.

Cell adhesion molecules have been implicated as key organizers of synaptic structures, but there is still a need to determine how these molecules facilitate neurotransmitter receptor recruitment to developing synapses. Here, we identify erythrocyte protein band 4.1-like 3 (protein 4.1B) as an intracellular effector molecule of Synaptic Cell Adhesion Molecule 1 (SynCAM1) that is sufficient to recruit NMDA-type receptors (NMDARs) to SynCAM1 adhesion sites in COS7 cells. Protein 4.1B in conjunction with SynCAM1 also increased the frequency of NMDAR-mediated mEPSCs and area of presynaptic contact in an HEK293 cell/ neuron co-culture assay. Studies in cultured hippocampal neurons reveal that manipulation of protein 4.1B expression levels specifically affects NMDAR-mediated activity and localization. Finally, further experimentation in COS7 cells show that SynCAM1 may also interact with protein 4.1N to specifically effect AMPA type receptor (AMPAR) recruitment. Thus, SynCAM1 may recruit both AMPARs and NMDARs by independent mechanisms during synapse formation.

Neuroligins (NLGs) are postsynaptic cell adhesion molecules that are thought to function in synaptogenesis. To investigate the role of NLGs on synaptic transmission once the synapse is formed, we transfected neuroligin-2 (NLG-2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABA(A) (gamma-aminobutyric acid) receptor mediated miniature postsynaptic currents (mIPSCs). NLG-2 transfected cells had mIPSCs with faster decay than matching GFP expressing controls at young culture ages (days in vitro, DIV7-8). Down-regulation of NLG-2 by the isoform specific shRNA-NLG-2 resulted in an opposite effect. We and others have shown that the switch of alpha subunits of GABA(A)Rs from alpha2/3 to alpha1 underlies developmental speeding of the IPSC decay in various CNS regions, including the cerebellum. To assess whether the reduced decay time of mIPSCs by NLG-2 is due to the recruitment of more alpha1 containing GABA(A)Rs at the synapses, we examined the prolongation of current decay by the Zolpidem, which has been shown to preferentially enhance the activity of alpha1 subunit-containing GABA channel. The application of Zolpidem resulted in a significantly greater prolongation kinetics of synaptic currents in NLG-2 over-expressing cells than control cells, suggesting that NLG-2 over-expression accelerates synapse maturation by promoting incorporation of the alpha1 subunit-containing GABA(A)Rs at postsynaptic sites in immature cells. In addition, the effect of NLG-2 on the speeding of decay time course of synaptic currents was abolished when we used CGC cultures from alpha1-/- mice. Lastly, to exclude the possibility that the fast decay of mIPSCs induced by NLG-2 could be also due to the impacts of NLG-2 on the GABA transient in synaptic cleft, we measured the sensitivity of mIPSCs to the fast-off competitive antagonists TPMPA. We found that TPMPA similarly inhibits mIPSCs in control and NLG-2 over-expressing CGCs both at young age (DIV8) and old age (DIV14) of cultures. However, we confirm our previous finding of a greater inhibition of mIPSCs in young (DIV8) than more mature (DIV14) cultures. Together, our results suggest that NLG-2 does not alter uniquantal GABA release, and the fast decay of mIPSC induced by NLG-2 is due to the differential expression of postsynaptic GABA(A) receptor subtypes. Taken all together, we propose that NLG-2 plays important functional role in inhibitory synapse development and maturation.

The adaptor protein FE65 interacts with the beta-amyloid precursor protein (APP) via its C-terminal phosphotyrosine binding (PTB) domain and affects APP processing and Abeta production. Our previous data demonstrate that the apoE receptor ApoEr2 co-precipitated with APP and suggest that there are extracellular and intracellular interactions between these two transmembrane proteins. We hypothesized that FE65 acts as an intracellular link between ApoEr2 and APP. Co-immunoprecipitation experiments in COS7 cells demonstrated an interaction between ApoEr2 and FE65 that depended on the N-terminal PTB domain of FE65. Full-length FE65 increased co-immunoprecipitation of ApoEr2 and APP. Full-length FE65 also increased surface expression of ApoEr2, as determined by surface protein biotinylation and live cell surface staining. Constructs containing both the C- and N-terminal PTB domains of FE65 increased secreted APP, secreted ApoEr2, APP C-terminal fragment, and ApoEr2 C-terminal fragment, but constructs containing only single PTB domains did not affect APP or ApoEr2 processing. In addition, full-length FE65 decreased Abeta to a significantly greater extent than individual FE65 domains. These data suggest that FE65 can bind APP and ApoEr2 at the same time and affect the processing of each.

In our previous studies we showed that apoE treatment of neurons activated ERK 1/2 signaling, and activation was blocked by treatment with inhibitors of the low density lipoprotein receptor family, the N-methyl-d-aspartate (NMDA) receptor antagonist MK 801, and calcium channel blockers. We hypothesized an interaction between the low density lipoprotein receptor family members and the NMDA receptor. In the present study, we confirmed through co-immunoprecipitation experiments an interaction between the apoE receptor, ApoEr2, and NMDAR1 through their extracellular domains. We also found that the PDZ1 domain of PSD95, a postsynaptic scaffolding protein, interacted with the C terminus of ApoEr2 via an alternatively spliced, intracellular exon. This interaction between ApoEr2 and PSD95 in neurons was modulated by NMDA receptor activation and an ApoEr2 ligand. We also found that the PDZ2 domain of PSD95 interacted with the NR2A and NR2B subunits of NMDA receptors. Full-length PSD95 increased cell surface levels of ApoEr2 and its cleavage, resulting in increases in secreted ApoEr2 and C-terminal fragments of ApoEr2. These studies suggest that ApoEr2 can form a multiprotein complex with NMDA receptor subunits and PSD95.

We generated expression vectors for N-terminally green fluorescent protein -tagged NR2A and NR2B subunits (GFP-NR2A and GFP-NR2B). Both constructs expressed GFP and formed functional NMDA channels with similar properties to untagged controls when co-transfected with NR1 subunit partner in HEK293 cells. Primary cultured hippocampal neurons were transfected at five days in vitro with these vectors. Fifteen days after transfection, well-defined GFP clusters were observed for both GFP-NR2A and GFP-NR2B subunits being co-localized with endogenous NR1 subunit. Whole-cell recordings showed that the GFP-NR2A subunit determined the decay of NMDA-mediated miniature spontaneous excitatory postsynaptic currents (NMDA-mEPSCs) in transfected neurons. Live staining with anti-GFP antibody demonstrated the surface expression of GFP-NR2A and GFP-NR2B subunits that was partly co-localized a presynaptic marker. Localization of NMDA receptor clusters in dendrites was studied by co-transfection of CFP-actin and GFP-NR2 subunits followed by anti-GFP surface staining. Within one week after plating most surface NMDAR clusters were distributed on dendritic shafts. Later in development, a large portion of surface clusters for both GFP-NR2A and GFP-NR2B subunits were clearly localized at dendritic spines. Our report provides the basis for studies of NMDA receptor location together with dendritic dynamics in living neurons during synaptogenesis in vitro.