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Resolving fundamental molecular and functional processes underlying human synaptic development is crucial for understanding normal brain function as well as dysfunction in disease. Based upon increasing evidence of species-divergent features of brain cell types, coupled with emerging studies of complex human disease genetics, we developed the first automated and quantitative high-content synaptic phenotyping platform using human neurons and astrocytes. To establish the robustness of our platform, we screened the effects of 376 small molecules on presynaptic density, neurite outgrowth, and cell viability, validating six small molecules that specifically enhanced human presynaptic density in vitro. Astrocytes were essential for mediating the effects of all six small molecules, underscoring the relevance of non-cell-autonomous factors in synapse assembly and their importance in synaptic screening applications. Bromodomain and extraterminal (BET) inhibitors emerged as the most prominent hit class and global transcriptional analyses using multiple BET inhibitors confirmed upregulation of synaptic gene expression. Through these analyses, we demonstrate the robustness of our automated screening platform for identifying potent synaptic modulators, which can be further leveraged for scaled analyses of human synaptic mechanisms and drug discovery efforts.

Astrocytes play essential roles in normal brain function, with dysfunction implicated in diverse developmental and degenerative disease processes. Emerging evidence of profound species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy, incompletely characterized, or require intermediate purification steps, limiting their utility for multi-cell line, adequately powered functional studies. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs) based upon transient Neurogenin 2 (NGN2) induction of neural progenitor-like cells followed by maturation in astrocyte media, which demonstrate remarkable homogeneity within the population and across 11 independent cell lines in the absence of additional purification steps. These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal maturation in vitro. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, most closely resembling human fetal astrocytes, and highly similar to hPSC-derived astrocytes generated using more complex approaches. Finally, the hiAs capture key molecular hallmarks in a trisomy 21 disease model. Thus, hiAs provide a valuable and practical resource well-suited for study of basic human astrocyte function and dysfunction in disease.

SYNGAP1haploinsufficiency in humans causes intellectual disability (ID). SYNGAP1 is highly expressed in cortical excitatory neurons and, reducing its expression in mice accelerates the maturation of excitatory synapses during sensitive developmental periods, restricts the critical period window for plasticity, and impairs cognition. However, its specific role in interneurons remains largely undetermined. In this study, we investigated the effects of conditionalSyngap1disruption in medial ganglionic eminence (MGE)-derived interneurons on hippocampal interneuron firing properties and excitatory synaptic inputs, as well as on pyramidal cell synaptic inhibition and synaptic integration. We show that conditionalSyngap1disruption in MGE-derived interneurons results in cell-specific impairment of firing properties of hippocampal Nkx2.1 fast-spiking interneurons, with enhancement of their AMPA receptor (AMPAR)-mediated excitatory synaptic inputs but compromised short-term plasticity. In contrast, regular-spiking Nkx2.1 interneurons are largely unaffected. These changes are associated with impaired pyramidal cell synaptic inhibition and enhanced summation of excitatory responses. Unexpectedly, we found that theSyngap1floxallele used in this study contains inverted loxP sites and that its targeted recombination in MGE-derived interneurons induces some cell loss during embryonic development and the reversible inversion of the sequence flanked by the loxP sites in postmitotic cells. Together, these results suggest thatSyngap1plays a role in cell-specific regulation of hippocampal interneuron function and inhibition of pyramidal cells in mice. However, because of our finding that theSyngap1floxallele used in this study contains inverted loxP sites, it will be important to further investigate interneuron function using a differentSyngap1conditional allele.

Resolving fundamental molecular and functional processes underlying human synaptic development is crucial for understanding normal brain function as well as dysfunction in disease. Based upon increasing evidence of species divergent features of brain cell types, coupled with emerging studies of complex human disease genetics, we developed the first automated and quantitative high-content synaptic phenotyping platform using human neurons and astrocytes. To establish the robustness of our platform, we screened the effects of 376 small molecules on presynaptic density, neurite outgrowth and cell viability, validating six small molecules which specifically enhanced human presynaptic density in vitro. Astrocytes were essential for mediating the effects of all six small molecules, underscoring the relevance of non-cell autonomous factors in synapse assembly and their importance in synaptic screening applications. Bromodomain and extraterminal (BET) inhibitors emerged as the most prominent hit class and global transcriptional analyses using multiple BET inhibitors confirmed upregulation of synaptic gene expression programs. Through these analyses, we demonstrate the robustness of our automated screening platform for identifying potent synaptic modulators, which can be further leveraged for scaled analyses of human synaptic mechanisms and drug discovery efforts.

Down syndrome (DS), driven by an extra copy of chromosome 21 (HSA21), and fragile X syndrome (FXS), driven by loss of the RNA-binding protein FMRP, are two common genetic causes of intellectual disability and autism. Based upon the number of DS-implicated transcripts bound by FMRP, we hypothesize that DS and FXS may share underlying mechanisms. Comparing DS and FXS human pluripotent stem cell (hPSC) and glutamatergic neuron models, we identify increased protein expression of select targets and overlapping transcriptional perturbations. Moreover, acute upregulation of endogenous FMRP in DS patient cells using CRISPRa is sufficient to significantly reduce expression levels of candidate proteins and reverse 40% of global transcriptional perturbations. These results pinpoint specific molecular perturbations shared between DS and FXS that can be leveraged as a strategy for target prioritization; they also provide evidence for the functional relevance of previous associations between FMRP targets and disease-implicated genes.

The Mechanistic Target Of Rapamycin Complex 1 (mTORC1) pathway controls several aspects of neuronal development. Mutations in regulators of mTORC1, such as Tsc1 and Tsc2, lead to neurodevelopmental disorders associated with autism, intellectual disabilities and epilepsy. The correct development of inhibitory interneurons is crucial for functional circuits. In particular, the axonal arborisation and synapse density of parvalbumin (PV)-positive GABAergic interneurons change in the postnatal brain. How and whether mTORC1 signaling affects PV cell development is unknown. Here, we show that Tsc1 haploinsufficiency causes a premature increase in terminal axonal branching and bouton density formed by mutant PV cells, followed by a loss of perisomatic innervation in adult mice. PV cell-restricted Tsc1 haploinsufficient and knockout mice show deficits in social behavior. Finally, we identify a sensitive period during the third postnatal week during which treatment with the mTOR inhibitor Rapamycin rescues deficits in both PV cell innervation and social behavior in adult conditional haploinsufficient mice. Our findings reveal a role of mTORC1 signaling in the regulation of the developmental time course and maintenance of cortical PV cell connectivity and support a mechanistic basis for the targeted rescue of autism-related behaviors in disorders associated with deregulated mTORC1 signaling., Regulators of mTORC1, such as Tsc1, are involved in neurodevelopmental disorders associated with autism, intellectual disabilities and epilepsy. The authors identify a role of mTORC1 signaling in the regulation and maintenance of cortical parvalbumin cell connectivity and social behaviour.

Mutations in regulators of the Mechanistic Target Of Rapamycin Complex 1 (mTORC1), such as Tsc1/2, lead to neurodevelopmental disorders associated with autism, intellectual disabilities and epilepsy. Whereas the effects of mTORC1 signaling dysfunction within diverse cell types are likely critical for the onset of the diverse neurological symptoms associated with mutations in mTORC1 regulators, they are not well understood. In particular, the effects of mTORC1 dys-regulation in specific types of inhibitory interneurons are unclear.Here, we showed that Tsc1 haploinsufficiency in parvalbumin (PV)-positive GABAergic interneurons either in cortical organotypic cultures or in vivo caused a premature increase in their perisomatic innervations, followed by a striking loss in adult mice. This effects were accompanied by alterations of AMPK-dependent autophagy in pre-adolescent but not adult mice. PV cell-restricted Tsc1 mutant mice showed deficits in social behavior. Treatment with the mTOR inhibitor Rapamycin restricted to the third postnatal week was sufficient to permanently rescue deficits in both PV cell innervation and social behavior in adult conditional haploinsufficient mice. All together, these findings identify a novel role of Tsc1-mTORC1 signaling in the regulation of the developmental time course and maintenance of cortical PV cell connectivity and provide a mechanistic basis for the targeted rescue of autism-related behaviors in disorders associated with deregulated mTORC1 signaling.

Preterm birth (PTB) is commonly accompanied by in utero fetal inflammation, and existing tocolytic drugs do not target fetal inflammatory injury. Of the candidate proinflammatory mediators, IL-1 appears central and is sufficient to trigger fetal loss. Therefore, we elucidated the effects of antenatal IL-1 exposure on postnatal development and investigated two IL-1 receptor antagonists, the competitive inhibitor anakinra (Kineret) and a potent noncompetitive inhibitor 101.10, for efficacy in blocking IL-1 actions. Antenatal exposure to IL-1β induced Tnfa, Il6, Ccl2, Pghs2, and Mpges1 expression in placenta and fetal membranes, and it elevated amniotic fluid IL-1β, IL-6, IL-8, and PGF2α, resulting in PTB and marked neonatal mortality. Surviving neonates had increased Il1b, Il6, Il8, Il10, Pghs2, Tnfa, and Crp expression in WBCs, elevated plasma levels of IL-1β, IL-6, and IL-8, increased IL-1β, IL-6, and IL-8 in fetal lung, intestine, and brain, and morphological abnormalities: e.g., disrupted lung alveolarization, atrophy of intestinal villus and colon-resident lymphoid follicle, and degeneration and atrophy of brain microvasculature with visual evoked potential anomalies. Late gestation treatment with 101.10 abolished these adverse outcomes, whereas Kineret exerted only modest effects and no benefit for gestation length, neonatal mortality, or placental inflammation. In a LPS-induced model of infection-associated PTB, 101.10 prevented PTB, neonatal mortality, and fetal brain inflammation. There was no substantive deviation in postnatal growth trajectory or adult body morphometry after antenatal 101.10 treatment. The results implicate IL-1 as an important driver of neonatal morbidity in PTB and identify 101.10 as a safe and effective candidate therapeutic.

Haploinsufficiency of the SYNGAP1 gene, which codes for a Ras GTPase-activating protein, impairs cognition both in humans and in mice. Decrease of Syngap1 in mice has been previously shown to cause cognitive deficits at least in part by inducing alterations in glutamatergic neurotransmission and premature maturation of excitatory connections. Whether Syngap1 plays a role in the development of cortical GABAergic connectivity and function remains unclear. Here, we show that Syngap1 haploinsufficiency significantly reduces the formation of perisomatic innervations by parvalbumin-positive basket cells, a major population of GABAergic neurons, in a cell-autonomous manner. We further show that Syngap1 haploinsufficiency in GABAergic cells derived from the medial ganglionic eminence impairs their connectivity, reduces inhibitory synaptic activity and cortical gamma oscillation power, and causes cognitive deficits. Our results indicate that Syngap1 plays a critical role in GABAergic circuit function and further suggest that Syngap1 haploinsufficiency in GABAergic circuits may contribute to cognitive deficits., Glutamatergic signalling regulation by Syngap1 has been linked to intellectual disabilities. Here, the authors find Syngap1 also regulates cortical GABAergic synaptic signalling development and that this reduced inhibitory signalling contributes to cognitive deficits in a mouse model.

De novo mutations in SYNGAP1, which codes for a RAS/RAP GTP-activating protein, cause nonsyndromic intellectual disability (NSID). All disease-causing point mutations identified until now in SYNGAP1 are truncating, raising the possibility of an association between this type of mutations and NSID. Here, we report the identification of the first pathogenic missense mutations (c.1084T>C [p.W362R], c.1685C>T [p.P562L]) and three novel truncating mutations (c.283dupC [p.H95PfsX5], c.2212_2213del [p.S738X], and (c.2184del [p.N729TfsX31]) in SYNGAP1 in patients with NSID. A subset of these patients also showed ataxia, autism, and a specific form of generalized epilepsy that can be refractory to treatment. All of these mutations occurred de novo, except c.283dupC, which was inherited from a father who is a mosaic. Biolistic transfection of wild-type SYNGAP1 in pyramidal cells from cortical organotypic cultures significantly reduced activity-dependent phosphorylated extracellular signal-regulated kinase (pERK) levels. In contrast, constructs expressing p.W362R, p.P562L, or the previously described p.R579X had no significant effect on pERK levels. These experiments suggest that the de novo missense mutations, p.R579X, and possibly all the other truncating mutations in SYNGAP1 result in a loss of its function. Moreover, our study confirms the involvement of SYNGAP1 in autism while providing novel insight into the epileptic manifestations associated with its disruption.