Generation of locus coeruleus norepinephrine neurons from human pluripotent stem cells.

Central norepinephrine (NE) neurons, located mainly in the locus coeruleus (LC), are implicated in diverse psychiatric and neurodegenerative diseases and are an emerging target for drug discovery. To facilitate their study, we developed a method to generate 40-60% human LC-NE neurons from human pluripotent stem cells. The approach depends on our identification of ACTIVIN A in regulating LC-NE transcription factors in dorsal rhombomere 1 (r1) progenitors. In vitro generated human LC-NE neurons display extensive axonal arborization; release and uptake NE; and exhibit pacemaker activity, calcium oscillation and chemoreceptor activity in response to CO2. Single-nucleus RNA sequencing (snRNA-seq) analysis at multiple timepoints confirmed NE cell identity and revealed the differentiation trajectory from hindbrain progenitors to NE neurons via an ASCL1-expressing precursor stage. LC-NE neurons engineered with an NE sensor reliably reported extracellular levels of NE. The availability of functional human LC-NE neurons enables investigation of their roles in psychiatric and neurodegenerative diseases and provides a tool for therapeutics development.

Species-specific FMRP regulation of RACK1 is critical for prenatal cortical development.

Fragile X messenger ribonucleoprotein 1 protein (FMRP) deficiency leads to fragile X syndrome (FXS), an autism spectrum disorder. The role of FMRP in prenatal human brain development remains unclear. Here, we show that FMRP is important for human and macaque prenatal brain development. Both FMRP-deficient neurons in human fetal cortical slices and FXS patient stem cell-derived neurons exhibit mitochondrial dysfunctions and hyperexcitability. Using multiomics analyses, we have identified both FMRP-bound mRNAs and FMRP-interacting proteins in human neurons and unveiled a previously unknown role of FMRP in regulating essential genes during human prenatal development. We demonstrate that FMRP interaction with CNOT1 maintains the levels of receptor for activated C kinase 1 (RACK1), a species-specific FMRP target. Genetic reduction of RACK1 leads to both mitochondrial dysfunctions and hyperexcitability, resembling FXS neurons. Finally, enhancing mitochondrial functions rescues deficits of FMRP-deficient cortical neurons during prenatal development, demonstrating targeting mitochondrial dysfunction as a potential treatment.

Multimodal analysis reveals genes driving neuronal maturation in the primate prefrontal cortex.

The dorsolateral prefrontal cortex (dlPFC) is an evolutionarily derived cortical region in primates critical for high-level cognitive functions and implicated in various neuropsychiatric disorders. The cells that compose the dlPFC, especially excitatory and inhibitory neurons, undergo extensive maturation throughout midfetal and late-fetal development, during which critical neurodevelopmental events, such as circuit assembly and electrophysiological maturation of neurons, occur. Despite the relevance of neuronal maturation in several neurodevelopmental and psychiatric disorders, the molecular mechanisms underlying this process remain largely unknown. Here, we performed an integrated Patch-seq and single-nucleus multiomic analysis of the rhesus macaque dlPFC to identify genes governing neuronal maturation from midfetal to late-fetal development. Our multimodal analysis identified gene pathways and regulatory networks important for the maturation of distinct neuronal populations, including upper-layer intratelencephalicprojecting neurons. We identified genes underlying the maturation of specific electrophysiological properties of these neurons. Furthermore, gene knockdown in organotypic slices revealed that RAPGEF4 regulates the maturation of resting membrane potential and inward sodium current. Using this strategy, we also found that the knockdown of CHD8, a high-confidence autism spectrum disorder risk gene, in human slices led to deficits in neuronal maturation, via the downstream downregulation of several key genes, including RAPGEF4. Our study revealed novel regulators of neuronal maturation during a critical period of prefrontal development in primates and implicated such regulators in molecular processes underlying autism.

Elevated levels of FMRP-target MAP1B impair human and mouse neuronal development and mouse social behaviors via autophagy pathway.

Fragile X messenger ribonucleoprotein 1 protein (FMRP) binds many mRNA targets in the brain. The contribution of these targets to fragile X syndrome (FXS) and related autism spectrum disorder (ASD) remains unclear. Here, we show that FMRP deficiency leads to elevated microtubule-associated protein 1B (MAP1B) in developing human and non-human primate cortical neurons. Targeted MAP1B gene activation in healthy human neurons or MAP1B gene triplication in ASD patient-derived neurons inhibit morphological and physiological maturation. Activation of Map1b in adult male mouse prefrontal cortex excitatory neurons impairs social behaviors. We show that elevated MAP1B sequesters components of autophagy and reduces autophagosome formation. Both MAP1B knockdown and autophagy activation rescue deficits of both ASD and FXS patients' neurons and FMRP-deficient neurons in ex vivo human brain tissue. Our study demonstrates conserved FMRP regulation of MAP1B in primate neurons and establishes a causal link between MAP1B elevation and deficits of FXS and ASD.

FXR1 regulation of parvalbumin interneurons in the prefrontal cortex is critical for schizophrenia-like behaviors.

Parvalbumin interneurons (PVIs) are affected in many psychiatric disorders including schizophrenia (SCZ), however the mechanism remains unclear. FXR1, a high confident risk gene for SCZ, is indispensable but its role in the brain is largely unknown. We show that deleting FXR1 from PVIs of medial prefrontal cortex (mPFC) leads to reduced PVI excitability, impaired mPFC gamma oscillation, and SCZ-like behaviors. PVI-specific translational profiling reveals that FXR1 regulates the expression of Cacna1h/Cav3.2 a T-type calcium channel implicated in autism and epilepsy. Inhibition of Cav3.2 in PVIs of mPFC phenocopies whereas elevation of Cav3.2 in PVIs of mPFC rescues behavioral deficits resulted from FXR1 deficiency. Stimulation of PVIs using a gamma oscillation-enhancing light flicker rescues behavioral abnormalities caused by FXR1 deficiency in PVIs. This work unveils the function of a newly identified SCZ risk gene in SCZ-relevant neurons and identifies a therapeutic target and a potential noninvasive treatment for psychiatric disorders.

RGS6 Mediates Effects of Voluntary Running on Adult Hippocampal Neurogenesis.

Voluntary running enhances adult hippocampal neurogenesis, with consequences for hippocampal-dependent learning ability and mood regulation. However, the underlying mechanism remains unclear. Here, we show that voluntary running induces unique and dynamic gene expression changes specifically within the adult-born hippocampal neurons, with significant impact on genes involved in neuronal maturation and human diseases. We identify the regulator of G protein signaling 6 (RGS6) as a key factor that mediates running impact on adult-born neurons. RGS6 overexpression mimics the positive effects of voluntary running on morphological and physiological maturation of adult new neurons and reduced sensitivity of adult-born neurons to the inhibitory effect of GABAB (γ-Aminobutyric acid B) receptor activation. Knocking down RGS6 abolishes running-enhanced neuronal maturation and hippocampal neurogenesis-dependent learning and anxiolytic effect. Our study provides a data resource showing genome-wide intrinsic molecular changes in adult-born hippocampal neurons that contribute to voluntary running-induced neurogenesis.

An Astrocytic Influence on Impaired Tonic Inhibition in Hippocampal CA1 Pyramidal Neurons in a Mouse Model of Rett Syndrome.

Rett syndrome (RTT) is a severe neurodevelopmental disease caused by mutations in the methyl-CpG binding protein 2 (MECP2) gene. Although altered interneuron development and function are clearly demonstrated in RTT mice, a particular mode of inhibition, tonic inhibition, has not been carefully examined. We report here that tonic inhibition is significantly reduced in pyramidal neurons in the CA1 region of the hippocampus in mice where Mecp2 is deleted either in all cells or specifically in astrocytes. Since no change is detected in the level of GABA receptors, such a reduction in tonic inhibition is likely a result of decreased ambient GABA level in the extracellular space. Consistent with this explanation, we observed increased expression of a GABA transporter, GABA transporter 3 (GAT3), in the hippocampus of the Mecp2 KO mice, as well as a corresponding increase of GAT3 current in hippocampal astrocytes. These phenotypes are relevant to RTT because pharmacological blockage of GAT3 can normalize tonic inhibition and intrinsic excitability in CA1 pyramidal neurons, and rescue the phenotype of increased network excitability in acute hippocampal slices from the Mecp2 KO mice. Finally, chronic administration of a GAT3 antagonist improved a composite symptom score and extended lifespan in the Mecp2 KO mice. Only male mice were used in this study. These results not only advance our understanding of RTT etiology by defining a new neuronal phenotype and revealing how it can be influenced by astrocytic alterations, but also reveal potential targets for intervention.SIGNIFICANCE STATEMENT Our study reports a novel phenotype of reduced tonic inhibition in hippocampal CA1 pyramidal neurons in the Rett syndrome mice, reveal a potential mechanism of increased GABA transporter expression/activity in the neighboring astrocytes, describe a disease-relevant consequence in hyperexcitability, and provide preliminary evidence that targeting this phenotype may slow down disease progression in Rett syndrome mice. These results help our understanding of the disease etiology and identify a new therapeutic target for treating Rett syndrome.

Mechanism and consequence of abnormal calcium homeostasis in Rett syndrome astrocytes.

Astrocytes play an important role in Rett syndrome (RTT) disease progression. Although the non-cell-autonomous effect of RTT astrocytes on neurons was documented, cell-autonomous phenotypes and mechanisms within RTT astrocytes are not well understood. We report that spontaneous calcium activity is abnormal in RTT astrocytes in vitro, in situ, and in vivo. Such abnormal calcium activity is mediated by calcium overload in the endoplasmic reticulum caused by abnormal store operated calcium entry, which is in part dependent on elevated expression of TRPC4. Furthermore, the abnormal calcium activity leads to excessive activation of extrasynaptic NMDA receptors (eNMDARs) on neighboring neurons and increased network excitability in Mecp2 knockout mice. Finally, both the abnormal astrocytic calcium activity and the excessive activation of eNMDARs are caused by Mecp2 deletion in astrocytes in vivo. Our findings provide evidence that abnormal calcium homeostasis is a key cell-autonomous phenotype in RTT astrocytes, and reveal its mechanism and consequence.

CREB Signaling Is Involved in Rett Syndrome Pathogenesis.

Rett syndrome (RTT) is a debilitating neurodevelopmental disorder caused by mutations in the MECP2 gene. To facilitate the study of cellular mechanisms in human cells, we established several human stem cell lines: human embryonic stem cell (hESC) line carrying the common T158M mutation (MECP2T158M/T158M ), hESC line expressing no MECP2 (MECP2-KO), congenic pair of wild-type and mutant RTT patient-specific induced pluripotent stem cell (iPSC) line carrying the V247fs mutation (V247fs-WT and V247fs-MT), and iPSC line in which the V247fs mutation was corrected by CRISPR/Cas9-based genome editing (V247fs-MT-correction). Detailed analyses of forebrain neurons differentiated from these human stem cell lines revealed genotype-dependent quantitative phenotypes in neurite growth, dendritic complexity, and mitochondrial function. At the molecular level, we found a significant reduction in the level of CREB and phosphorylated CREB in forebrain neurons differentiated from MECP2T158M/T158M , MECP2-KO, and V247fs-MT stem cell lines. Importantly, overexpression of CREB or pharmacological activation of CREB signaling in those forebrain neurons rescued the phenotypes in neurite growth, dendritic complexity, and mitochondrial function. Finally, pharmacological activation of CREB in the female Mecp2 heterozygous mice rescued several behavioral defects. Together, our study establishes a robust in vitro platform for consistent quantitative evaluation of genotype-dependent RTT phenotypes, reveals a previously unappreciated role of CREB signaling in RTT pathogenesis, and identifies a potential therapeutic target for RTT.SIGNIFICANCE STATEMENT Our study establishes a robust human stem cell-based platform for consistent quantitative evaluation of genotype-dependent Rett syndrome (RTT) phenotypes at the cellular level. By providing the first evidence that enhancing cAMP response element binding protein signaling can alleviate RTT phenotypes both in vitro and in vivo, we reveal a previously unappreciated role of cAMP response element binding protein signaling in RTT pathogenesis, and identify a potential therapeutic target for RTT.

Misregulation of Alternative Splicing in a Mouse Model of Rett Syndrome.

Mutations in the human MECP2 gene cause Rett syndrome (RTT), a severe neurodevelopmental disorder that predominantly affects girls. Despite decades of work, the molecular function of MeCP2 is not fully understood. Here we report a systematic identification of MeCP2-interacting proteins in the mouse brain. In addition to transcription regulators, we found that MeCP2 physically interacts with several modulators of RNA splicing, including LEDGF and DHX9. These interactions are disrupted by RTT causing mutations, suggesting that they may play a role in RTT pathogenesis. Consistent with the idea, deep RNA sequencing revealed misregulation of hundreds of splicing events in the cortex of Mecp2 knockout mice. To reveal the functional consequence of altered RNA splicing due to the loss of MeCP2, we focused on the regulation of the splicing of the flip/flop exon of Gria2 and other AMPAR genes. We found a significant splicing shift in the flip/flop exon toward the flop inclusion, leading to a faster decay in the AMPAR gated current and altered synaptic transmission. In summary, our study identified direct physical interaction between MeCP2 and splicing factors, a novel MeCP2 target gene, and established functional connection between a specific RNA splicing change and synaptic phenotypes in RTT mice. These results not only help our understanding of the molecular function of MeCP2, but also reveal potential drug targets for future therapies.

Mutant astrocytes differentiated from Rett syndrome patients-specific iPSCs have adverse effects on wild-type neurons.

The disease mechanism of Rett syndrome (RTT) is not well understood. Studies in RTT mouse models have suggested a non-cell-autonomous role for astrocytes in RTT pathogenesis. However, it is not clear whether this is also true for human RTT astrocytes. To establish an in vitro human RTT model, we previously generated isogenic induced pluripotent stem cell (iPSC) lines from several RTT patients carrying different disease-causing mutations. Here, we show that these RTT iPSC lines can be efficiently differentiated into astroglial progenitors and glial fibrillary acidic protein-expressing (GFAP(+)) astrocytes that maintain isogenic status, that mutant RTT astrocytes carrying three different RTT mutations and their conditioned media have adverse effects on the morphology and function of wild-type neurons and that the glial effect on neuronal morphology is independent of the intrinsic neuronal deficit in mutant neurons. Moreover, we show that both insulin-like growth factor 1 (IGF-1) and GPE (a peptide containing the first 3 amino acids of IGF-1) are able to partially rescue the neuronal deficits caused by mutant RTT astrocytes. Our findings confirm the critical glial contribution to RTT pathology, reveal potential cellular targets of IGF-1 therapy and further validate patient-specific iPSCs and their derivatives as valuable tools to study RTT disease mechanism.

Astrocytic Ca(2+) waves mediate activation of extrasynaptic NMDA receptors in hippocampal neurons to aggravate brain damage during ischemia.

Excitotoxicity plays a central role in the neuronal damage during ischemic stroke. Although growing evidence suggests that activation of extrasynaptic NMDA receptors initiates neuronal death, no direct evidence demonstrated their activation during ischemia. Using rat hippocampal slices, we detected oxygen-glucose deprivation (OGD) induced slow inward currents (SICs) mediated by extrasynaptic NMDA receptors in CA1 pyramidal neurons. Moreover, Ca(2+) chelator BAPTA dialysis into astrocytic network decreased the frequency of OGD induced SICs, indicating that the activation of extrasynaptic NMDA receptors depended on astrocytic Ca(2+) activity. To further demonstrate the importance of astrocytic Ca(2+) activity, we tested hippocampal slices from inositol triphosphate receptor type 2 (IP3R2) knock-out mice which abolished the astrocytic Ca(2+) activity. As expected, the frequency of OGD induced SICs was reduced. Using two-photon Ca(2+) imaging, we characterized the astrocytic Ca(2+) dynamics. By controlling Ca(2+) level in the individual astrocytes using targeted photolysis, we found that OGD facilitated the propagation of intercellular Ca(2+) waves, which were inhibited by gap junction blocker carbenoxolone (CBX). CBX also inhibited the Ca(2+) activity of the astrocytic network and decreased the SIC frequency during OGD. Functionally, the infarct volumes from brain ischemia were reduced in IP3R2 knock-out mice and in rat intracerebrally delivered with CBX. Our results demonstrate that enhanced Ca(2+) activity of the astrocytic network plays a key role on the activation of extrasynaptic NMDA receptors in hippocampal neurons, which enhances brain damage during ischemia.

Morphine protects against intracellular amyloid toxicity by inducing estradiol release and upregulation of Hsp70.

Certain experimental models support morphine can play a beneficial role against damage in the neuronal system. In this study, we find morphine as well as endomorphin-1 and endomorphin-2 can protect against intracellular amyloid ß (iAß) toxicity in human and rat primary neuronal cultures and in rat brains in vivo. Morphine reverses the electrophysiological changes induced by iAß, including current density, resting membrane potential and capacitance. Also morphine improves the spatial memory performance in rats infected by iAß packaged virus and in APP/PS1 mice in Morris water maze tests. Morphine protection is mediated through inducing estradiol release in hippocampal neurons measured by ELISA and liquid chromatography-mass spectrometry, possibly by increasing P450 cytochrome aromatase activity. Released estradiol induces upregulation of heat shock protein 70 (Hsp70). Hsp70 protects against intracellular amyloid toxicity by rescuing proteasomal activity which is impaired by iAß. This is the first time, to our knowledge, that induction of estradiol release in hippocampal neurons by morphine is reported. Our data may contribute to both Alzheimer's disease therapy and pain clinics where morphine is widely used.

Watching moving images specifically promotes development of medial area of secondary visual cortex in rat.

It is generally accepted that the cortex can be divided into numerous regions depending on the type of information each processes, and that specific input is effective in improving the development of related regions. In visual cortex, many subareas are distinguished on the basis of their adequate information. However, whether the development of a subarea can be specifically improved by its particular input is still largely unknown. Here, we show the specific effects of motion information on the development of the medial area of secondary visual cortex (V2M), a subarea associated with processing the movement component of visual information. Although watching a moving or a still image had similar effects in primary visual cortex, the moving image induced multistage development of V2M in dark-reared rats: both mRNA and protein levels of GluR2 were upregulated, the density and protein content of GluR2-positive synapses increased, and the spine density and the frequency of spontaneous excitatory postsynaptic currents (EPSCs) of pyramidal neurons in Layer 5 were elevated. Our results suggest that rats are able to identify motion information, distribute it to V2M, and then use this input to specifically improve the development of V2M.