The human FOXM1 homolog promotes basal progenitor cell proliferation and cortical folding in mouse.

Cortical expansion and folding are key processes in human brain development and evolution and are considered to be principal elements of intellectual ability. How cortical folding has evolved and is induced during embryo development is not well understood. Here, we show that the expression of human FOXM1 promotes basal progenitor cell proliferation and induces cortical thickening and folding in mice. Human-specific protein sequences further promote the generation of basal progenitor cells. Human FOXM1 increases the proliferation of neural progenitors by binding to the Lin28a promoter and increasing Lin28a expression. Furthermore, overexpression of LIN28A rescues the proliferation of human FOXM1 knockout neural progenitor cells. Together, our findings demonstrate that a human gene can increase the number of basal progenitor cells in mice, leading to brain size increase and gyrification, and may thus contribute to evolutionary brain development and cortical expansion.

Microglia homeostasis mediated by epigenetic ARID1A regulates neural progenitor cells response and leads to autism-like behaviors.

Microglia are resident macrophages of the central nervous system that selectively emerge in embryonic cortical proliferative zones and regulate neurogenesis by altering molecular and phenotypic states. Despite their important roles in inflammatory phagocytosis and neurodegenerative diseases, microglial homeostasis during early brain development has not been fully elucidated. Here, we demonstrate a notable interplay between microglial homeostasis and neural progenitor cell signal transduction during embryonic neurogenesis. ARID1A, an epigenetic subunit of the SWI/SNF chromatin-remodeling complex, disrupts genome-wide H3K9me3 occupancy in microglia and changes the epigenetic chromatin landscape of regulatory elements that influence the switching of microglial states. Perturbation of microglial homeostasis impairs the release of PRG3, which regulates neural progenitor cell self-renewal and differentiation during embryonic development. Furthermore, the loss of microglia-driven PRG3 alters the downstream cascade of the Wnt/ß-catenin signaling pathway through its interaction with the neural progenitor receptor LRP6, which leads to misplaced regulation in neuronal development and causes autism-like behaviors at later stages. Thus, during early fetal brain development, microglia progress toward a more homeostatic competent phenotype, which might render neural progenitor cells respond to environmental cross-talk perturbations.

MacroH2A1.2 deficiency leads to neural stem cell differentiation defects and autism-like behaviors.

The development of the nervous system requires precise regulation. Any disturbance in the regulation process can lead to neurological developmental diseases, such as autism and schizophrenia. Histone variants are important components of epigenetic regulation. The function and mechanisms of the macroH2A (mH2A) histone variant during brain development are unknown. Here, we show that deletion of the mH2A isoform mH2A1.2 interferes with neural stem cell differentiation in mice. Deletion of mH2A1.2 affects neurodevelopment, enhances neural progenitor cell (NPC) proliferation, and reduces NPC differentiation in the developing mouse brain. mH2A1.2-deficient mice exhibit autism-like behaviors, such as deficits in social behavior and exploratory abilities. We identify NKX2.2 as an important downstream effector gene and show that NKX2.2 expression is reduced after mH2A1.2 deletion and that overexpression of NKX2.2 rescues neuronal abnormalities caused by mH2A1.2 loss. Our study reveals that mH2A1.2 reduces the proliferation of neural progenitors and enhances neuronal differentiation during embryonic neurogenesis and that these effects are at least in part mediated by NKX2.2. These findings provide a basis for studying the relationship between mH2A1.2 and neurological disorders.

CD93 negatively regulates astrogenesis in response to MMRN2 through the transcriptional repressor ZFP503 in the developing brain.

Astrogenesis is repressed in the early embryonic period and occurs in the late embryonic period. A variety of external and internal signals contribute to the sequential differentiation of neural stem cells. Here, we discovered that immune-related CD93 plays a critical negative role in the regulation of astrogenesis in the mouse cerebral cortex. We show that CD93 expression is detected in neural stem cells and neurons but not in astrocytes and declines as differentiation proceeds. Cd93 knockout increases astrogenesis at the expense of neuron production during the late embryonic period. CD93 responds to the extracellular matrix protein Multimerin 2 (MMRN2) to trigger the repression of astrogenesis. Mechanistically, CD93 delivers signals to ß-Catenin through a series of phosphorylation cascades, and then ß-Catenin transduces these signals to the nucleus to activate Zfp503 transcription. The transcriptional repressor ZFP503 inhibits the transcription of glial fibrillary acidic protein (Gfap) by binding to the Gfap promoter with the assistance of Grg5. Furthermore, Cd93 knockout mice exhibit autism-like behaviors. Taken together, our results reveal that CD93 is a negative regulator of the onset of astrogenesis and provide insight into therapy for psychiatric disorders.

TCF20 dysfunction leads to cortical neurogenesis defects and autistic-like behaviors in mice.

Recently, de novo mutations of transcription factor 20 (TCF20) were found in patients with autism by large-scale exome sequencing. However, how TCF20 modulates brain development and whether its dysfunction causes ASD remain unclear. Here, we show that TCF20 deficits impair neurogenesis in mouse. TCF20 deletion significantly reduces the number of neurons, which leads to abnormal brain functions. Furthermore, transcriptome analysis and ChIP-qPCR reveal that the DNA demethylation factor TDG is a downstream target gene of TCF20. As a nonspecific DNA demethylation factor, TDG potentially affects many genes. Combined TDG ChIP-seq and GO analysis of TCF20 RNA-Seq identifies T-cell factor 4 (TCF-4) as a common target. TDG controls the DNA methylation level in the promoter area of TCF-4, affecting TCF-4 expression and modulating neural differentiation. Overexpression of TDG or TCF-4 rescues the deficient neurogenesis of TCF20 knockdown brains. Together, our data reveal that TCF20 is essential for neurogenesis and we suggest that defects in neurogenesis caused by TCF20 loss are associated with ASD.

H2A.Z.1 crosstalk with H3K56-acetylation controls gliogenesis through the transcription of folate receptor.

Astrocytes play crucial roles in the central nervous system, and defects in astrocyte function are closely related to many neurological disorders. Studying the mechanism of gliogenesis has important implications for understanding and treating brain diseases. Epigenetic regulations have essential roles during mammalian brain development. Here, we demonstrate that histone H2A.Z.1 is necessary for the specification of multiple neural precursor cells (NPCs) and has specialized functions that regulate gliogenesis. Depletion of H2A.Z.1 suppresses gliogenesis and results in reduced astrocyte differentiation. Additionally, H2A.Z.1 regulates the acetylation of H3K56 (H3K56ac) by cooperating with the chaperone of ASF1a. Furthermore, RNA-seq data indicate that folate receptor 1 (FOLR1) participates in gliogenesis through the JAK-STAT signaling pathway. Taken together, our results demonstrate that H2A.Z.1 is a key regulator of gliogenesis because it interacts with ASF1a to regulate H3K56ac and then directly affects the expression of FOLR1, which acts as a signal-transducing component of the JAK-STAT signaling pathway.

Robust partial synchronization of delay-coupled networks.

Networks of coupled systems may exhibit a form of incomplete synchronization called partial synchronization or cluster synchronization, which refers to the situation where only some, but not all, systems exhibit synchronous behavior. Moreover, due to perturbations or uncertainties in the network, exact partial synchronization in the sense that the states of the systems within each cluster become identical, cannot be achieved. Instead, an approximate synchronization may be observed, where the states of the systems within each cluster converge up to some bound, and this bound tends to zero if (the size of) the perturbations tends to zero. In order to derive sufficient conditions for this robustified notion of synchronization, which we refer to as practical partial synchronization, first, we separate the synchronization error dynamics from the network dynamics and interpret them in terms of a nonautonomous system of delay differential equations with a bounded additive perturbation. Second, by assessing the practical stability of this error system, conditions for practical partial synchronization are derived and formulated in terms of linear matrix inequalities. In addition, an explicit relation between the size of perturbation and the bound of the synchronization error is provided.

Coinfection with an intestinal helminth impairs host innate immunity against Salmonella enterica serovar Typhimurium and exacerbates intestinal inflammation in mice.

Salmonella enterica serovar Typhimurium is a Gram-negative food-borne pathogen that is a major cause of acute gastroenteritis in humans. The ability of the host to control such bacterial pathogens may be influenced by host immune status and by concurrent infections. Helminth parasites are of particular interest in this context because of their ability to modulate host immune responses and because their geographic distribution coincides with those parts of the world where infectious gastroenteritis is most problematic. To test the hypothesis that helminth infection may negatively regulate host mucosal innate immunity against bacterial enteropathogens, a murine coinfection model was established by using the intestinal nematode Heligmosomoides polygyrus and S. Typhimurium. We found that mice coinfected with S. Typhimurium and H. polygyrus developed more severe intestinal inflammation than animals infected with S. Typhimurium alone. The enhanced susceptibility to Salmonella-induced intestinal injury in coinfected mice was found to be associated with diminished neutrophil recruitment to the site of bacterial infection that correlated with decreased expression of the chemoattractants CXCL2/macrophage inflammatory protein 2 (MIP-2) and CXCL1/keratinocyte-derived chemokine (KC), poor control of bacterial replication, and exacerbated intestinal inflammation. The mechanism of helminth-induced inhibition of MIP-2 and KC expression involved interleukin-10 (IL-10) and, to a lesser extent, IL-4 and IL-13. Ly6G antibody-mediated depletion of neutrophils reproduced the adverse effects of H. polygyrus on Salmonella infection. Our results suggest that impaired neutrophil recruitment is an important contributor to the enhanced severity of Salmonella enterocolitis associated with helminth coinfection.

Helminth infection impairs autophagy-mediated killing of bacterial enteropathogens by macrophages.

Autophagy is an important mechanism used by macrophages to kill intracellular pathogens. The results reported in this study demonstrate that autophagy is also involved in the macrophage killing of the extracellular enteropathogen Citrobacter rodentium after phagocytosis. The process was significantly impaired in macrophages isolated from mice chronically infected with the helminth parasite Heligmosomoides polygyrus. The H. polygyrus-mediated inhibition of autophagy was Th2 dependent because it was not observed in macrophages isolated from helminth-infected STAT6-deficient mice. Moreover, autophagy of Citrobacter was inhibited by treating macrophages with IL-4 and IL-13. The effect of H. polygyrus on autophagy was associated with decreased expression and processing of L chain protein 3 (LC3), a key component of the autophagic machinery. The helminth-induced inhibition of LC3 expression and processing was STAT6 dependent and could be recapitulated by treatment of macrophages with IL-4 and IL-13. Knockdown of LC3 significantly inhibited autophagic killing of Citrobacter, attesting to the functional importance of the H. polygyrus-mediated downregulation of this process. These observations reveal a new aspect of the immunosuppressive effects of helminth infection and provide mechanistic insights into our earlier finding that H. polygyrus significantly worsens the in vivo course of Citrobacter infection.

BACH1 changes microglial metabolism and affects astrogenesis during mouse brain development.

Microglia are highly heterogeneous as resident immune cells in the central nervous system. Although the proinflammatory phenotype of microglia is driven by the metabolic transformation in the disease state, the mechanism of metabolic reprogramming in microglia and whether it affects surrounding astrocyte progenitors have not been well elucidated. Here, we illustrate the communication between microglial metabolism and astrogenesis during embryonic development. The transcription factor BTB and CNC homology 1 (Bach1) reduces lactate production by inhibiting two key enzymes, HK2 and GAPDH, during glycolysis. Metabolic perturbation of microglia reduces lactate-dependent histone modification enrichment at the Lrrc15 promoter. The microglia-derived LRRC15 interacts with CD248 to participate in the JAK/STAT pathway and influence astrogenesis. In addition, Bach1cKO-Cx3 mice exhibit abnormal neuronal differentiation and anxiety-like behaviors. Altogether, this work suggests that the maintenance of microglia metabolic homeostasis during early brain development is closely related to astrogenesis, providing insights into astrogenesis and related diseases.

BCAT1 controls embryonic neural stem cells proliferation and differentiation in the upper layer neurons.

The regulation of neural stem cell (NSC) proliferation and differentiation during brain development is a precisely controlled process, with the production of different neuronal subtypes governed by strict timelines. Glutamate is predominantly used as a neurotransmitter by the subtypes of neurons in the various layers of the cerebral cortex. The expression pattern of BCAT1, a gene involved in glutamate metabolism, in the different layers of neurons has yet to be fully understood. Using single-cell data, we have identified seven different states of NSCs and found that state 4 is closely associated with the development of projection neurons. By inferring the developmental trajectory of different neuronal subtypes from NSC subsets of this state, we discovered that BCAT1 is involved in the regulation of NSC proliferation and differentiation and is specifically highly expressed in layer II/III and IV neurons. Suppression of BCAT1 through shRNA resulted in a reduction in NSC proliferation and an abnormal development of layer II/III and IV neurons. These findings provide new insights into the role of BCAT1 in the regulation of NSC behavior and neuronal development.

Brain-specific Pd1 deficiency leads to cortical neurogenesis defects and depressive-like behaviors in mice.

Embryonic neurogenesis is tightly regulated by multiple factors to ensure the precise development of the cortex. Deficiency in neurogenesis may result in behavioral abnormalities. Pd1 is a well-known inhibitory immune molecule, but its function in brain development remains unknown. Here, we find brain specific deletion of Pd1 results in abnormal cortical neurogenesis, including enhanced proliferation of neural progenitors and reduced neuronal differentiation. In addition, neurons in Pd1 knockout mice exhibit abnormal morphology, both the total length and the number of primary dendrites were reduced. Moreover, Pd1cKO mice exhibit depressive-like behaviors, including immobility, despair, and anhedonia. Mechanistically, Pd1 regulates embryonic neurogenesis by targeting Pax3 through the ß-catenin signaling pathway. The constitutive expression of Pax3 partly rescues the deficiency of neurogenesis in the Pd1 deleted embryonic brain. Besides, the administration of ß-catenin inhibitor, XAV939, not only rescues abnormal brain development but also ameliorates depressive-like behaviors in Pd1cKO mice. Simultaneously, Pd1 plays a similar role in human neural progenitor cells (hNPCs) proliferation and differentiation. Taken together, our findings reveal the critical role and regulatory mechanism of Pd1 in embryonic neurogenesis and behavioral modulation, which could contribute to understanding immune molecules in brain development.

Endothelial Arid1a deletion disrupts the balance among angiogenesis, neurogenesis and gliogenesis in the developing brain.

The vascular system and the neural system processes occur simultaneously, the interaction among them is fundamental to the normal development of the central nervous system. Arid1a (AT-rich interaction domain 1A), which encodes an epigenetic subunit of the SWI/SNF chromatin-remodelling complex, is associated with promoter-mediated gene regulation and histone modification. However, the molecular mechanism of the interaction between cerebrovascular and neural progenitor cells (NPCs) remains unclear. To generate Arid1acKO-Tie2 mice, Arid1afl/fl mice were hybridized with Tie2-Cre mice. The Angiogenesis, neurogenesis and gliogenesis were studied by immunofluorescence staining and Western blotting. RNA-seq, RT-PCR, Western blotting, CO-IP and rescue experiments were performed to dissect the molecular mechanisms of Arid1a regulates fate determination of NPCs. We found that the absence of Arid1a results in increased the density of blood vessels, delayed neurogenesis and decreased gliogenesis, even after birth. Mechanistically, the deletion of Arid1a in endothelial cells causes a significant increase in H3k27ac and the secretion of maternal protein 2 (MATN2). In addition, matn2 alters the AKT/SMAD4 signalling pathway through its interaction with the NPCs receptor EGFR, leading to the decrease of SMAD4. SMAD complex further mediates the expression of downstream targets, thereby promoting neurogenesis and inhibiting gliogenesis. This study suggests that endothelial Arid1a tightly controls fate determination of NPCs by regulating the AKT-SMAD signalling pathway.

Human SERPINA3 induces neocortical folding and improves cognitive ability in mice.

Neocortex expansion and folding are related to human intelligence and cognition, but the molecular and cellular mechanisms underlying cortical folding remain poorly understood. Here, we report that the human gene SERPINA3 is linked to gyrification. Specifically, the overexpression of SERPINA3 induced neocortical folding, increased the abundance of neurons, and improved cognitive abilities. Further, SERPINA3 promoted proliferation of the outer radial glia (oRG, also referred to as the basal radial glia) and increased the number of upper-layer neurons. The downstream target Glo1 was determined to be involved in SERPINA3-induced gyrification. Moreover, SERPINA3 increased the proliferation of oRG by binding to the Glo1 promoter. Assessment of behavior performance showed enhanced cognitive abilities in SERPINA3 knock-in mice. Our findings will enrich the understanding of neocortical expansion and gyrification and provide insights into possible treatments for intellectual disability and lissencephaly syndrome.

Endothelial Cells Mediated by UCP2 Control the Neurogenic-to-Astrogenic Neural Stem Cells Fate Switch During Brain Development.

During mammalian cortical development, neural stem/progenitor cells (NSCs) gradually alter their characteristics, and the timing of generation of neurons and glial cells is strictly regulated by internal and external factors. However, whether the blood vessels located near NSCs affect the neurogenic-to-gliogenic transition remain unknown. Here, it is demonstrated that endothelial uncoupling protein 2 (UCP2) deletion reduces blood vessel diameter and affects the transition timing of neurogenesis and gliogenesis. Deletion of endothelial UCP2 results in a persistent increase in astrocyte production at the postnatal stage. Mechanistically, the endothelial UCP2/ROS/ERK1/2 pathway increases chymase-1 expression to enhance angiotensin II (AngII) secretion outside the brain endothelium. The endotheliocyte-driven AngII-gp130-JAK-STAT pathway also regulates gliogenesis initiation. Moreover, endothelial UCP2 knockdown decreases human neural precursor cell (hNPC) differentiation into neurons and accelerates hNPC differentiation into astrocytes. Altogether, this work provides mechanistic insights into how endothelial UCP2 regulates the neurogenic-to-gliogenic fate switch in the developing neocortex.

Endothelial cells regulated by RNF20 orchestrate the proliferation and differentiation of neural precursor cells during embryonic development.

The intimate communication between the vascular and nervous systems is critical for maintaining central nervous system (CNS) development. However, whether cerebrovascular endothelial cells (ECs) can orchestrate neural precursor cell (NPC) proliferation and differentiation, and the identity of the signals involved therein, is unclear. Here, we find that the development of ECs is often accompanied by DNA damage. RNF20, an E3 ubiquitin ligase, is required for the DNA damage response (DDR). The deletion of RNF20 causes the accumulation of DNA damage in ECs, which fails to secrete cartilage intermediate layer protein 2 (CILP2). Moreover, the loss of endothelium-derived CILP2 alters the downstream cascade signaling of Wnt signaling pathways through the interaction with Wnt3a, which disturbs the NPC fate and causes autism-like behaviors in mice. Therefore, the close and refined controlled neurovascular interactions ensure the normal operation of neurogenesis during embryonic development.

Expression and purification of functional Clostridium perfringens alpha and epsilon toxins in Escherichia coli.

The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.

Identification and characterization of myeloma-associated antigens in Trichinella spiralis.

To investigate the presence of myeloma-associated antigens in Trichinella spiralis and their anti-tumor effect, cross-immune responses between antigens of the myeloma cell SP2/0 versus positive sera to T. spiralis, and antigens of T. spiralis versus positive sera to myeloma cell SP2/0 were determined using T. spiralis and myeloma specific enzyme-linked immunosorbent assays (ELISA). The myeloma-associated antigens in T. spiralis were separated by ultrafiltration and 2-D electrophoresis, and the amino acid sequences and molecular weights were determined by spectrometry. An obvious reaction was found between a 33 kDa antigen and positive sera, and the major component of the antigen was tropomyosin (TM), which is an surface acidic protein with 284 amino acids. Mice were immunized with TM to determine the anti-tumor effect in vivo. The results showed that CD4(+), CD8(+) T lymphocyte, and CD19(+) B lymphocyte were significantly increased (P<0.05). The anti-tumor effects were significantly different between mice immunized with the antigens or adjuvant alone (P<0.05), while the difference between mice immunized with antigens and whole T. spiralis was not significant (P>0.05). The results indicated that TM identified in this study may play a role in eliciting cross-protective immunity.

[Prokaryotic expression and identification of S-dsRNA gene from Cryptosporidium parvum virus].

OBJECTIVE: To clone and express S-dsRNA gene of Cryptosporidium parvum virus, and investigate the reactogenicity of the recombinant. METHODS: Total RNA was extracted from Cryptosporidium parvum and S-dsRNA gene was amplified by RT-PCR. The PCR product was cloned into pET-28a(+) expression vector. The recombinant plasmid pET-28a(+)-S was transformed into E. coli BL21 (DE3) and induced with IPTG. The expression situation of recombinant protein was analyzed by SDS-PAGE. Its reactogenicity was examined by Western blotting analysis. RESULTS: pET-28a (+)-S was identified by PCR and double endonuclease digestion. SDS-PAGE result showed that the recombinant protein (M, 37,000) was expressed in the form of inclusion body. High level expression of recombinant protein was found at 1 mmol/L IPTG condition after incubation at 37 degrees C for 4 h and reached up to 72.6% of the total protein. The protein was recognized by the antisera from mice immunized with antigens from Cryptosporidium parvum oocysts. CONCLUSION: The S-dsRNA gene of Cryptosporidium parvum virus has been expressed with adequate reactogenicity.

Anti-aging and anti-oxidant activities of murine short interspersed nuclear element antisense RNA.

Short interspersed nuclear elements (SINEs) play a key role in regulating gene expression, and SINE RNAs are involved in age-related diseases. We investigated the anti-aging effects of a genetically engineered murine SINE B1 antisense RNA (B1as RNA) and explored its mechanism of action in naturally senescent BALB/c (≥14 months) and moderately senscent C57BL/6N (≥9 months) mice. After tail vein injection, B1as RNA was available in the blood of mice for approximately 30 min, persisted for approximately 2-4 h in most detected tissues and persisted approximately 48 h in lungs. We found that treatment with B1as RNA improved stamina and promoted hair re-growth in aged mice. Treatment with B1as RNA also partially rescued the increase in mitochondrial DNA copy number in liver and spleen tissues observed in aged and moderately senescent mice. Finally, treatment with B1as RNA increased the activities of superoxide dismutase and glutathione peroxidase in aged and moderately senescent mice, reduced these animals' malondialdehyde and reactive oxygen species levels, and modulated the expression of several aging-associated genes, including Sirtuin 1, p21, p16Ink4a, p15Ink4b and p19Arf, and anti-oxidant genes (Sesn1 and Sesn 2). These data suggest that B1as RNA inhibits the aging process by enhancing antioxidant activity, promoting the scavenging of free radicals, and modulating the expression of aging-associated genes. This is the first report describing the anti-aging activity of SINE antisense RNA, which may serve as an effective nucleic acid drug for the treatment of age-related diseases.