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Spatiotemporal regulation of the mechanistic target of rapamycin (mTOR) pathway is pivotal for establishment of brain architecture. Dysregulation of mTOR signaling is associated with a variety of neurodevelopmental disorders. Here, we demonstrate that the UBE4B-KLHL22 E3 ubiquitin ligase cascade regulates mTOR activity in neurodevelopment. In a mouse model with UBE4B conditionally deleted in the nervous system, animals display severe growth defects, spontaneous seizures and premature death. Loss of UBE4B in the brains of mutant mice results in depletion of neural precursor cells and impairment of neurogenesis. Mechanistically, UBE4B polyubiquitylates and degrades KLHL22, an E3 ligase previously shown to degrade the GATOR1 component DEPDC5. Deletion of UBE4B causes upregulation of KLHL22 and hyperactivation of mTOR, leading to defective proliferation and differentiation of neural precursor cells. Suppression of KLHL22 expression reverses the elevated activity of mTOR caused by acute local deletion of UBE4B. Prenatal treatment with the mTOR inhibitor rapamycin rescues neurogenesis defects in Ube4b mutant mice. Taken together, these findings demonstrate that UBE4B and KLHL22 are essential for maintenance and differentiation of the precursor pool through fine-tuning of mTOR activity.

The toxic effect of di(2-ethylhexyl) phthalate (DEHP) on prepubertal testes was examined in this study. We treated 3-week-old male mice with 4.8 mg/kg/day (milligram/kilogram/day) (no observed adverse effect level), 30 mg/kg/day (high exposure dose relative to humans), 100 mg/kg/day (level causing a reproductive system disorder), and 500 mg/kg/day (dose causing a multigenerational reproductive system disorder) of DEHP via gavage. Obvious abnormalities in the testicular organ coefficient, spermatogenic epithelium, and testosterone levels occurred in the 500 mg/kg DEHP group. Ribonucleic acid sequencing (RNA-seq) showed that differentially expressed genes (DEGs) in each group could enrich reproduction and reproductive process terms according to the gene ontology (GO) results, and coenrichment of metabolism pathway was observed by the Reactome pathway analysis. Through the analysis of common genes in the metabolism pathway, we discovered that DEHP exposure at 4.8 to 500 mg/kg or 100 mg/kg caused the same damages to the prepubertal testis. In general, we identified two key transcriptional biomarkers (fatty acid binding protein 3 (Fabp3) and carboxylesterase (Ces) 1d), which provided new insight into the gene regulatory mechanism associated with DEHP exposure and will contribute to the prediction and diagnosis of prepuberty testis injury caused by DEHP.

Human brain organoids can provide not only promising models for physiological and pathological neurogenesis but also potential therapies in neurological diseases. However, technical issues such as surgical lesions due to transplantation still limit their applications.Instead of applying mature organoids, we innovatively developed human brain organoids in vivo by injecting small premature organoids into corpus striatum of adult SCID mice. Two months after injection, single-cell transcriptome analysis was performed on 6131 GFP-labeled human cells from transplanted mouse brains.Eight subsets of cells (including neuronal cells expressing striatal markers) were identified in these in vivo developed organoids (IVD-organoids) by unbiased clustering. Compared with in vitro cultured human cortical organoids, we found that IVD-organoids developed more supporting cells including pericyte-like and choroid plexus cells, which are important for maintaining organoid homeostasis. Furthermore, IVD-organoids showed lower levels of cellular stress and apoptosis.Our study thus provides a novel method to generate human brain organoids, which is promising in various applications of disease models and therapies.

Focal cortical dysplasia (FCD) is one of the most common causes of drug-resistant epilepsy. Dysmorphic neurons are the major histopathological feature of type II FCD, but their role in seizure genesis in FCD is unclear. Here we performed whole-cell patch-clamp recording and morphological reconstruction of cortical principal neurons in postsurgical brain tissue from drug-resistant epilepsy patients. Quantitative analyses revealed distinct morphological and electrophysiological characteristics of the upper layer dysmorphic neurons in type II FCD, including an enlarged soma, aberrant dendritic arbors, increased current injection for rheobase action potential firing, and reduced action potential firing frequency. Intriguingly, the upper layer dysmorphic neurons received decreased glutamatergic and increased GABAergic synaptic inputs that were coupled with upregulation of the Na

Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor that accumulates in organisms in various ways and induces male reproductive system disorders. In this study, we established a testicular injury model by gavage with different concentrations of DEHP. The testes were then collected for RNA sequencing (RNA-seq), and the results were analyzed by bioinformatics and verified by experiments. Our research results show that different concentrations of DEHP interfere with testicular development differently. Weighted gene coexpression network analysis (WGCNA) generated sixteen modules and identified the turquoise module as key. Then, estrogen receptor 1 (ESR1), filamin A (Flna) and Furin were identified as hub genes. qPCR and immunohistochemistry results revealed that all three hub genes were upregulated. We detected the locations of these genes by immunohistochemistry. ESR1 was mainly located in Leydig cells; Flna immunostaining is observed in the Leydig and some germ cells and Furin staining was seen in almost all types of testicular cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed enrichment mainly in MAPK signaling pathways, p53 signaling pathways, HIF-1 signaling pathways, protein processing in the endoplasmic reticulum, apoptosis, the cell cycle, RNA degradation, etc. This is the first study using WGCNA to investigate the mechanism of DEHP-induced injury in the prepubertal testis, providing new research angles to further understand the mechanism of DEHP-induced injury in the prepubertal testis.

Flight, an active fear response to imminent threat, is dependent on the rapid risk assessment of sensory information processed by the cortex. The thalamic reticular nucleus (TRN) filters information between the cortex and the thalamus, but whether it participates in the regulation of flight behavior remains largely unknown. Here, we report that activation of parvalbumin-expressing neurons in the limbic TRN, but not those in the sensory TRN, mediates flight. Glutamatergic inputs from the cingulate cortex (Cg) selectively activate the limbic TRN, which in turn inhibits the intermediodorsal thalamic nucleus (IMD). Activation of this Cg→limbic TRN→IMD circuit results in inhibition of the IMD and produces flight behavior. Conversely, removal of inhibition onto the IMD results in more freezing and less flight, suggesting that the IMD may function as a pro-freeze center. Overall, these findings reveal a novel corticothalamic circuit through the TRN that controls the flight response.

Di-(2-ethylhexyl) phthalate (DEHP) is an extensively used plasticizer and has been shown to cause reproductive dysfunction in humans and model animals. However, the exact mechanisms of testicular injury induced by DEHP exposure have not been fully clarified. Using gas chromatography-mass spectrometry, we found that mono-2-ethylhexyl ester (MEHP, a major biometabolite of DEHP) and DEHP concentrations were elevated in mouse serum after DEHP exposure. Using RNA-seq, we found that ferroptosis and HIF-1 signaling pathways might be involved in testicular injury due to prepubertal DEHP exposure. Subsequent Western blotting, ferrous iron and MDA measurements, and immunofluorescence of testicular sections verified the RNA-seq findings. Consistently, based on the RNA-seq findings, we found that ferroptosis and HIF-1 signaling pathways might play crucial roles in Leydig and Sertoli cell injury due to MEHP exposure in vitro. Further experiments also confirmed ferroptosis in Leydig and Sertoli cells. Using Western blotting, cellular immunofluorescence and ChIP-qPCR, we found that MEHP exposure caused HIF-1α accumulation and stabilization, resulted in HIF-1α translocation into the nucleus, and induced HIF-1α/Hmox1 binding in Leydig and Sertoli cells. To clarify whether HIF-1α plays a pivotal role in MEHP-induced ferroptosis, we knocked out Hif-1α using the CRISPR/Cas9 technique. We found that Hif-1α knockout rescued MEHP-induced ferroptosis. In summary, our findings certified that prepubertal DEHP exposure led to ferroptosis in mouse testes via the HIF-1α/HO-1 signaling pathway.

Di-(2-ethylhexyl) phthalate (DEHP) is the most common phthalate that can affect the male reproductive system. DEHP exposure at the prepubertal stage could lead to the injury of immature testes, but the mechanism has not been fully clarified. In the present study, we elucidated the possible underlying mechanism of DEHP-induced prepubertal testicular injury through stereological analysis and transcriptome profiling. Compared with the control group, the DEHP-treated rats had lower body weight gain and decreased testicular weight and organ coefficient. Moreover, lower serum levels of testosterone and LH were observed in the DEHP group, in contrast to the increased FSH level. Additionally, the serum level of estradiol had no significant difference after DEHP exposure. Stereological analysis showed significant reduction in volumes of most testicular structures, especially in the seminiferous tubule and seminiferous epithelium, along with a vast decrease of spermatogenic cells and obvious structural damages with substantial pathological signs (germ cracks, cytoplasmic vacuolization, sloughing, multinucleated giant cell formation, chromatolysis desquamation and dissolution, pyknosis of nuclei) in the seminiferous tubule upon DEHP exposure at the prepubertal stage. Furthermore, transcriptome profiling identified 5548 differentially expressed genes (DEGs) upon DEHP exposure. Pathway enrichment analysis revealed several crucial signaling pathways related to retinol metabolism, oxidative phosphorylation, steroid hormone biosynthesis, and cell adhesion molecules (CAMs). In addition, seven DEGs selected from RNA-seq data were validated by quantitative real-time polymerase chain reaction (qRT-PCR), and the results showed the same trends as the RNA-seq results. In conclusion, the above findings provide basic morphological data and lay a foundation for systematic research on transcriptome profiling in prepubertal testicular injury induced by DEHP.

Summary Pain is a source of substantial discomfort. Abnormal activity in both the zona incerta (ZI) and posterior complex of the thalamus (Po) are implicated in neuropathic pain, but their exact roles remain unclear. In particular, the precise cell types and molecular mechanisms of the ZI-Po circuit that regulate nociception are largely uncharacterized. Here, we found that parvalbumin (PV)-positive neuronal projections from the ventral ZI (ZIv) to the Po (ZIv-Po) are critical for promoting nocifensive behaviors, whereas selectively inhibiting ZIv-Po activity reduces nocifensive withdrawal responses. Furthermore, cannabinoid type 1 receptors (CB1Rs) are expressed specifically at ZIv-Po axon terminals in this circuit, and cannabinoids attenuate nocifensive responses through presynaptic inhibition. Selective inhibition of the ZIv-Po circuit or administration of cannabinoids into the Po are sufficient to ameliorate pathological pain. These findings identify the critical role of the ZIv-Po circuit and its modulation by endocannabinoids in controlling nocifensive behaviors.

In the developing human neocortex, progenitor cells generate diverse cell types prenatally. Progenitor cells and newborn neurons respond to signaling cues, including neurotransmitters. While single-cell RNA sequencing has revealed cellular diversity, physiological heterogeneity has yet to be mapped onto these developing and diverse cell types. By combining measurements of intracellular Ca2+ elevations in response to neurotransmitter receptor agonists and RNA sequencing of the same single cells, we show that Ca2+ responses are cell-type-specific and change dynamically with lineage progression. Physiological response properties predict molecular cell identity and additionally reveal diversitynot captured by single-cell transcriptomics. We find that the serotonin receptor HTR2A selectively activates radial glia cells in the developing human, butnot mouse, neocortex, and inhibiting HTR2A receptorsin human radial glia disrupts the radial glial scaffold. We show highly specific neurotransmittersignaling during neurogenesis in the developing human neocortex and highlight evolutionarilydivergent mechanisms of physiological signaling.

Di(2-ethylhexyl) phthalate (DEHP) is a widely-used plasticizer and has long been recognized as an endocrine-disrupting chemical with male reproductive toxicities. DEHP exposure at the prepubertal stage may lead to extensive testicular injury. However, the underlying mechanisms remain to be elucidated. In the present study, we gavaged male C57BL/6 mice with different concentrations of DEHP (0, 250, and 500 mg/kg-bw·d) from postnatal day 22–35, and exposed TM3 Leydig cells with 0, 100, 200, 300, and 400 μM of MEHP (bioactive metabolite of DEHP) for 12–48 h. RNA sequencing was performed both in testicular tissue and TM3 cells. The results showed that DEHP disrupts testicular development and reduces serum testosterone levels in male prepubertal mice. Bioinformatic analysis and experimental verification have revealed that DEHP/MEHP induces cell cycle arrest in TM3 cells and increases apoptosis both in vivo and in vitro. Furthermore, the p53 signaling pathway was found to be activated upon DEHP/MEHP treatment. The inhibition of p53 by pifithrin-α significantly reduced MEHP-induced injuries in TM3 cells. Cumulatively, these findings revealed the involvement of the p53 signaling pathway in DEHP-induced prepubertal testicular injury by promoting cell apoptosis and inhibiting cell proliferation of Leydig cells.

Neuropathic pain and bladder dysfunction represent significant quality-of-life issues for many spinal cord injury patients. Loss of GABAergic tone in the injured spinal cord may contribute to the emergence of these symptoms. Previous studies have shown that transplantation of rodent inhibitory interneuron precursors from the medial ganglionic eminence (MGE) enhances GABAergic signaling in the brain and spinal cord. Here we look at whether transplanted MGE-like cells derived from human embryonic stem cells (hESC-MGEs) can mitigate the pathological effects of spinal cord injury. We find that 6 months after transplantation into injured mouse spinal cords, hESC-MGEs differentiate into GABAergic neuron subtypes and receive synaptic inputs, suggesting functional integration into host spinal cord. Moreover, the transplanted animals show improved bladder function and mitigation of pain-related symptoms. Our results therefore suggest that this approach may be a valuable strategy for ameliorating the adverse effects of spinal cord injury.

Heterosynaptic long-term depression (hLTD) at untetanized synapses accompanying the induction of long-term potentiation (LTP) spatially sharpens the activity-induced synaptic potentiation; however, the underlying mechanism remains unclear. We found that hLTD in the hippocampal CA1 region is caused by stimulation-induced ATP release from astrocytes that suppresses transmitter release from untetanized synaptic terminals via activation of P2Y receptors. Selective stimulation of astrocytes expressing channelrhodopsin-2, a light-gated cation channel permeable to Ca2+, resulted in LTD of synapses on neighboring neurons. This synaptic modification required Ca2+ elevation in astrocytes and activation of P2Y receptors, but not N-methyl-D-aspartate receptors. Furthermore, blocking P2Y receptors or buffering astrocyte intracellular Ca2+ at a low level prevented hLTD without affecting LTP induced by SC stimulation. Thus, astrocyte activation is both necessary and sufficient for mediating hLTD accompanying LTP induction, strongly supporting the notion that astrocytes actively participate in activity-dependent synaptic plasticity of neural circuits. (c) 2012 Wiley Periodicals, Inc.

Circuits for brain building The brain is made up of circuits. Chen and Kriegstein analyzed how one particular circuit functions differently in developing and mature mouse brains (see the Perspective by Spitzer). This circuit connects the brain's diencephalon to the cortex. Early in development, the circuit is excitatory and affects how the cortical neurons it touches build their dendrites and synapses. Later on, after the fundamentals of brain construction are in place, the circuit switches to inhibitory functions, helping the brain resist epileptiform activity. Science , this issue p. 554 ; see also p. 510

Non-neurogenic cell types, such as cortical astroglia and fibroblasts, can be directly converted into neurons by the overexpression of defined transcription factors. Normally, the cellular phenotype of such differentiated cells is remarkably stable and resists direct cell transdifferentiation. Here we show that theInk4a/Arf(also known asCdkn2a) locus is a developmental barrier to direct neuronal transdifferentiation induced by transcription factor overexpression. With serial passagein vitro, wild-type postnatal cortical astroglia become progressively resistant toDlx2-induced neuronal transdifferentiation. In contrast, the neurogenic competence ofInk4a/Arf-deficient astroglia is both greatly increased and does not diminish through serial cell culture passage. Electrophysiological analysis further demonstrates the neuronal identity of cells induced fromInk4a/Arf-null astroglia, and short hairpin RNA-mediated acute knockdown of p16Ink4a and p19Arf p16Ink4aand p19Arfindicates that these gene products function postnatally as a barrier to cellular transdifferentiation. Finally, we found that mouse fibroblasts deficient forInk4a/Arfalso exhibit greatly enhanced transcription factor-induced neuronal induction. These data indicate thatInk4a/Arfis a potent barrier to direct neuronal transdifferentiation and further suggest that this locus functions normally in the progressive developmental restriction of postnatal astrocytes.

SummaryThe ventricular-subventricular zone (V-SVZ) is an extensive germinal niche containing neural stem cells (NSCs) in the walls of the lateral ventricles of the adult brain. How the adult brain’s neural activity influences the behavior of adult NSCs remains largely unknown. We show that serotonergic (5HT) axons originating from a small group of neurons in the raphe form an extensive plexus on most of the ventricular walls. Electron microscopy revealed intimate contacts between 5HT axons and NSCs (B1) or ependymal cells (E1) and these cells were labeled by a transsynaptic viral tracer injected into the raphe. B1 cells express the 5HT receptors 2C and 5A. Electrophysiology showed that activation of these receptors in B1 cells induced small inward currents. Intraventricular infusion of 5HT2C agonist or antagonist increased or decreased V-SVZ proliferation, respectively. These results indicate that supraependymal 5HT axons directly interact with NSCs to regulate neurogenesis via 5HT2C.

Directed differentiation from human pluripotent stem cells (hPSCs) has seen significant progress in recent years. However, most differentiated populations exhibit immature properties of an early embryonic stage, raising concerns about their ability to model and treat disease. Here, we report the directed differentiation of hPSCs into medial ganglionic eminence (MGE)-like progenitors and their maturation into forebrain type interneurons. We find that early-stage progenitors progress via a radial glial-like stem cell enriched in the human fetal brain. Both invitro and posttransplantation into the rodent cortex, the MGE-like cells develop into GABAergic interneuron subtypes with mature physiological properties along a prolonged intrinsic timeline of up to 7months, mimicking endogenous human neural development. MGE-derived cortical interneuron deficiencies are implicated in a broad range of neurodevelopmental and degenerative disorders, highlighting the importance of these results for modeling human neural development and disease.