Astrocytes are essential for the formation and maintenance of neural networks. However, a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Despite recent advances in human pluripotent stem cell (hPSC) techniques, primary rodent astrocytes remain the gold standard in coculture with human neurons. We demonstrate that a combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) directs hPSC-derived neural precursor cells to a highly pure population of astroglia in 28 d. Using single-cell RNA sequencing, we confirm the astroglial identity of these cells and highlight profound transcriptional adaptations in cocultured hPSC-derived astrocytes and neurons, consistent with their further maturation. In coculture with human neurons, multielectrode array recordings revealed robust network activity of human neurons in a coculture with hPSC-derived or rat astrocytes [3.63 +/- 0.44 min(-1 )(hPSC-derived), 2.86 +/- 0.64 min(-1 )(rat); p = 0.19]. In comparison, we found increased spike frequency within network bursts of human neurons cocultured with hPSC-derived astrocytes [56.31 +/- 8.56 Hz (hPSC-derived), 24.77 +/- 4.04 Hz (rat); p < 0.01], and whole-cell patch-clamp recordings revealed an increase of postsynaptic currents [2.76 +/- 0.39 Hz (hPSC-derived), 1.07 +/- 0.14 Hz (rat); p < 0.001], consistent with a corresponding increase in synapse density [14.90 +/- 1.27/100 mu m(2 )(hPSC-derived), 8.39 +/- 0.63/100 mu m(2) (rat); p < 0.001]. Taken together, we show that hPSC-derived astrocytes compare favorably with rat astrocytes in supporting human neural network activity and maturation, providing a fully human platform for investigating astrocyte function and neuronal-glial interactions.

ABSTRACT Astrocytes are essential for the formation and maintenance of neural networks through metabolic support, facilitation of synaptic function, and optimization of electrophysiological activity. However, a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Here we present a novel method to efficiently differentiate human pluripotent stem cell (hPSC)-derived neural progenitors to functional astrocytes in 28 days using a culture medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). This approach yields highly pure populations of astrocytes expressing canonical astrocyte markers, which we confirmed by immunofluorescence, flow cytometry and RNA sequencing. Human PSC-derived astrocytes efficiently buffer glutamate and robustly support neural network activity. Co-cultures of hPSC-derived astrocytes and neurons on multi-electrode arrays generated robust network activity within 2 days and synchronous network bursts after 6 days. Whole cell patch-clamp recordings revealed an increased frequency of postsynaptic currents in human hPSC-derived neurons co-cultured with hPSC-derived versus primary rodent astrocytes, consistent with a corresponding increase in synapse density. Furthermore, hPSC-derived astrocytes retained their hominid morphology when transplanted into a mouse brain. In conclusion, we present a novel protocol to obtain functional astrocytes from human pluripotent stem cells, providing a platform for investigating human astrocyte function and neuronal-glial interactions.

Sparse populations of neurons in the dentate gyrus (DG) of the hippocampus are causally implicated in the encoding of contextual fear memories. However, engram-specific molecular mechanisms underlying memory consolidation remain largely unknown. Here we perform unbiased RNA sequencing of DG engram neurons 24 h after contextual fear conditioning to identify transcriptome changes specific to memory consolidation. DG engram neurons exhibit a highly distinct pattern of gene expression, in which CREB-dependent transcription features prominently ( P = 6.2 × 10 −13 ), including Atf3 ( P = 2.4 × 10 −41 ), Penk ( P = 1.3 × 10 −15 ), and Kcnq3 ( P = 3.1 × 10 −12 ). Moreover, we validate the functional relevance of the RNAseq findings by establishing the causal requirement of intact CREB function specifically within the DG engram during memory consolidation, and identify a novel group of CREB target genes involved in the encoding of long-term memory.

Noncoding RNAs have been widely recognized as essential mediators of gene regulation. However, in contrast to protein-coding genes, much less is known about the influence of noncoding RNAs on human diseases. Here we examined the association of genetic variants located in primary microRNA sequences and long noncoding RNAs (lncRNAs) with Alzheimer disease (AD) by leveraging data from the largest genome-wide association meta-analysis of late-onset AD. Variants annotated to 5 miRNAs and 10 lncRNAs (in seven distinct loci) exceeded the Bonferroni-corrected significance threshold (p < 1.02 × 10-6 ). Among these, a leading variant (rs2526377:A>G) at the 17q22 locus annotated to two noncoding RNAs (MIR142 and BZRAP1-AS) was significantly associated with a reduced risk of AD and fulfilled predefined criteria for being a functional variant. Our functional genomic analyses revealed that rs2526377 affects the promoter activity and decreases the expression of miR-142. Moreover, differential expression analysis by RNA-Seq in human iPSC-derived neural progenitor cells and the hippocampus of miR-142 knockout mice demonstrated multiple target genes of miR-142 in the brain that are likely to be involved in the inflammatory and neurodegenerative manifestations of AD. These include TGFBR1 and PICALM, of which their derepression in the brain due to reduced expression levels of miR-142-3p may reduce the risk of AD.

Memories are encoded within sparsely distributed neuronal ensembles. However, the defining cellular properties of neurons within a memory trace remain incompletely understood. Using a fluorescence-based Arc reporter, we were able to visually identify the distinct subset of lateral amygdala (LA) neurons activated during auditory fear conditioning. We found that Arc- expressing neurons have enhanced intrinsic excitability and are preferentially recruited into newly encoded memory traces. Furthermore, synaptic potentiation of thalamic inputs to the LA during fear conditioning is learning-specific, postsynaptically mediated and highly localized to Arc -expressing neurons. Taken together, our findings validate the immediate-early gene Arc as a molecular marker for the LA neuronal ensemble recruited during fear learning. Moreover, these results establish a model of fear memory formation in which intrinsic excitability determines neuronal selection, whereas learning-related encoding is governed by synaptic plasticity.

Angiotensin-induced vasodilation, involving type 2 receptor (AT(2)R)-induced generation of nitric oxide (NO; by endothelial NO synthase) and endothelium-derived hyperpolarizing factors, may be limited to women. To distinguish the contribution of female sex hormones and chromosomes to AT(2)R function and endothelium-derived hyperpolarizing factor-mediated vasodilation, we made use of the four-core genotype model, where the testis-determining Sry gene has been deleted (Y-) from the Y chromosome, allowing XY- mice to develop a female gonadal phenotype. Simultaneously, by incorporating the Sry gene onto an autosome, XY(-)Sry and XXSry transgenic mice develop into gonadal male mice. Four-core genotype mice underwent a sham or gonadectomy (GDX) operation, and after 8 weeks, iliac arteries were collected to assess vascular function. XY(-)Sry male mice responded more strongly to angiotensin than XX female mice, and the AT(2)R antagonist PD123319 revealed that this was because of a dilator AT(2)R-mediated effect occurring exclusively in XX female mice. The latter could not be demonstrated in XXSry male and XY- female mice nor in XX female mice after GDX, suggesting that it depends on both sex hormones and chromosomes. Indeed, treating C57bl/6 GDX male mice with estrogen could not restore angiotensin-mediated, AT(2)R-dependent relaxation. To block acetylcholine-induced relaxation of iliac arteries obtained from four-core genotype XX mice, both endothelial NO synthase and endothelium-derived hyperpolarizing factor inhibition were required, whereas in four-core genotype XY animals, endothelial NO synthase inhibition alone was sufficient. These findings were independent of gonadal sex and unaltered after GDX. In conclusion, AT(2)R-induced relaxation requires both estrogen and the XX chromosome sex complement, whereas only the latter is required for endothelium-derived hyperpolarizing factors.

Objective: Angiotensin II induces vasoconstriction via its type 1 receptors (AT1R), while type 2 (AT2) R are believed to be vasodilator. The latter is not a universal finding and may be limited to women. AT2R-induced vasodilation, if occurring, is mediated via nitric oxide (generated by endothelial NO synthase, eNOS) and/or endothelium-derived hyperpolarizing factors (EDHFs). Studies in eNOS knockout mice suggest that EDHF predominate in women. To distinguish the contribution of female sex hormones and chromosomes to AT2R function and EDHF-mediated vasodilation, we made use of the four core genotype (FCG) model, where the testis-determining Sry gene has been deleted (Y-) from the Y chromosome, allowing XY- mice to develop a female gonadal phenotype. Simultaneously, by incorporating the Sry gene onto an autosome, XY-Sry and XXSry transgenic mice develop into gonadal males. Design and method: FCG mice underwent a sham or gonadectomy (GDX) operation, and after 8 weeks, animals were sacrificed and iliac arteries were collected to assess vascular function. Vascular function was also studied in C57bl/6 males treated with estrogen after GDX. Results: XY-Sry males responded more strongly to Ang II than XX females, and the AT2R antagonist PD123319 revealed that this was due to a dilator AT2R-mediated effect occurring exclusively in XX females. The latter could not be demonstrated in XXSry males and XY- females, nor in XX females after GDX, suggesting that it depends on both sex hormones and chromosomes. Indeed, treating C57bl/6 GDX males with estrogen could not restore Ang II-mediated, AT2R-dependent relaxation. To block acetylcholine-induced relaxation of iliac arteries obtained from FCG XX mice, both eNOS - and EDHF inhibition were required, while in FCG XY animals eNOS inhibition alone was sufficient. These findings were independent of gonadal sex, and unaltered after GDX. Conclusions: AT2 receptor-induced relaxation requires both estrogen and the XX chromosome sex complement, while only the latter is required for EDHF. Estrogen treatment of male mice confirms that this approach is insufficient to re-introduce AT2 receptor-induced relaxation.

The α isoform of the calcium/calmodulin-dependent protein kinase II (αCaMKII) has been implicated extensively in molecular and cellular mechanisms underlying spatial and contextual learning in a wide variety of species. Germline deletion of Camk2a leads to severe deficits in spatial and contextual learning in mice. However, the temporal and region-specific requirements for αCaMKII have remained largely unexplored. Here, we generated conditional Camk2a mutants to examine the influence of spatially restricted and temporally controlled expression of αCaMKII. Forebrain-specific deletion of the Camk2a gene resulted in severe deficits in water maze and contextual fear learning, whereas mice with deletion restricted to the cerebellum learned normally. Furthermore, we found that temporally controlled deletion of the Camk2a gene in adult mice is as detrimental as germline deletion for learning and synaptic plasticity. Together, we confirm the requirement for αCaMKII in the forebrain, but not the cerebellum, in spatial and contextual learning. Moreover, we highlight the absolute requirement for intact αCaMKII expression at the time of learning.