Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys.

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

Single-cell transcriptome analyses reveal signals to activate dormant neural stem cells.

The scarcity of tissue-specific stem cells and the complexity of their surrounding environment have made molecular characterization of these cells particularly challenging. Through single-cell transcriptome and weighted gene co-expression network analysis (WGCNA), we uncovered molecular properties of CD133(+)/GFAP(-) ependymal (E) cells in the adult mouse forebrain neurogenic zone. Surprisingly, prominent hub genes of the gene network unique to ependymal CD133(+)/GFAP(-) quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133(+) ependymal neural stem cells (NSCs), lining not only the lateral but also the fourth ventricles and, together with basic fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the CNS, as well as signals abundant after injury for their activation.

Protective factors for children with autism spectrum disorder during COVID-19-related strict lockdowns: a Shanghai autism early developmental cohort study.

BACKGROUND: COVID-19 lockdowns increased the risk of mental health problems, especially for children with autism spectrum disorder (ASD). However, despite its importance, little is known about the protective factors for ASD children during the lockdowns. METHODS: Based on the Shanghai Autism Early Developmental Cohort, 188 ASD children with two visits before and after the strict Omicron lockdown were included; 85 children were lockdown-free, while 52 and 51 children were under the longer and the shorter durations of strict lockdown, respectively. We tested the association of the lockdown group with the clinical improvement and also the modulation effects of parent/family-related factors on this association by linear regression/mixed-effect models. Within the social brain structures, we examined the voxel-wise interaction between the grey matter volume and the identified modulation effects. RESULTS: Compared with the lockdown-free group, the ASD children experienced the longer duration of strict lockdown had less clinical improvement (ß = 0.49, 95% confidence interval (CI) [0.19-0.79], p = 0.001) and this difference was greatest for social cognition (2.62 [0.94-4.30], p = 0.002). We found that this association was modulated by parental agreeableness in a protective way (-0.11 [-0.17 to -0.05], p = 0.002). This protective effect was enhanced in the ASD children with larger grey matter volumes in the brain's mentalizing network, including the temporal pole, the medial superior frontal gyrus, and the superior temporal gyrus. CONCLUSIONS: This longitudinal neuroimaging cohort study identified that the parental agreeableness interacting with the ASD children's social brain development reduced the negative impact on clinical symptoms during the strict lockdown.

Identification and functional analysis of novel oncogene DDX60L in pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer. Approximately 80% of patients initially diagnosed with locally advanced or metastatic disease survive only 4-11 months after diagnosis. Tremendous efforts have been made toward understanding the biology of PDAC. RESULTS: In this study, we first utilized next-generation sequencing technique and existing microarray datasets to identify significant differentially expressed genes between PDAC and non-tumor adjacent tissue. By comparing top significant survival genes in PDAC Gene Expression Profiling Interactive Analysis database and PDAC transcriptome data from patients, our integrated analysis discovered five potential central genes (i.e., MYEOV, KCNN4, FAM83A, S100A16, and DDX60L). Subsequently, we analyzed the cellular functions of the potential novel oncogenes MYEOV and DDX60L, which are highly expressed in PDAC cells. Notably, the knockdown of MYEOV and DDX60L significantly inhibited the metastasis of cancer cells and induced apoptosis. Further RNA sequencing analyses showed that massive signaling pathways, particularly the TNF signaling pathway and nuclear factor-kappa B (NF-κB) signaling pathway, were affected in siRNA-treated cancer cells. The siDDX60L and siMYEOV significantly inhibited the expression of chemokine CXCL2, which may potentially affect the tumor microenvironment in PDAC tissues. CONCLUSIONS: The present findings identified the novel oncogene DDX60L, which was highly expressed in PDAC. Transcriptome profiling through siRNA knockdown of DDX60L uncovered its functional roles in the PDAC in humans.

NT3-chitosan enables de novo regeneration and functional recovery in monkeys after spinal cord injury.

Spinal cord injury (SCI) often leads to permanent loss of motor, sensory, and autonomic functions. We have previously shown that neurotrophin3 (NT3)-loaded chitosan biodegradable material allowed for prolonged slow release of NT3 for 14 weeks under physiological conditions. Here we report that NT3-loaded chitosan, when inserted into a 1-cm gap of hemisectioned and excised adult rhesus monkey thoracic spinal cord, elicited robust axonal regeneration. Labeling of cortical motor neurons indicated motor axons in the corticospinal tract not only entered the injury site within the biomaterial but also grew across the 1-cm-long lesion area and into the distal spinal cord. Through a combination of magnetic resonance diffusion tensor imaging, functional MRI, electrophysiology, and kinematics-based quantitative walking behavioral analyses, we demonstrated that NT3-chitosan enabled robust neural regeneration accompanied by motor and sensory functional recovery. Given that monkeys and humans share similar genetics and physiology, our method is likely translatable to human SCI repair.

Long noncoding RNA XIST regulates the EGF receptor to promote TGF-ß1-induced epithelial-mesenchymal transition in pancreatic cancer.

BACKGROUND: This study focuses on the lncRNA XIST (X inactive-specific transcript), an lncRNA involved in multiple human cancers, and investigates the functional significance of XIST and the molecular mechanisms underlying the epithelial-mesenchymal transition (EMT) in pancreatic cancer (PC). METHODS: Clinical specimens from 25 patients as well as 5 human PC cell lines were analyzed for XIST, YAP, and microRNA(miR)-34a by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. To investigate how XIST influences cell proliferation, invasiveness, and apoptosis in PC, we performed the CCK-8 assays, Transwell assays, and flow cytometry. Luciferase reporter assays, qRT-PCR, and Western blot were applied to prove that miR-34a directly binds to XIST. RESULTS: Up-regulation of XIST and Yes associated protein (YAP) and down-regulation of miR-34a were consistently observed in the clinical specimens and PC cell lines. Silencing XIST reduced the expression of YAP and suppressed transforming growth factor (TGF)-ß1-induced EMT, while over-expression of XIST increased the expression of YAP and promoted EMT. In addition, inhibition of epidermal growth factor receptor (EGFR) hampered the XIST-promoted EMT. The results from the luciferase reporter assays confirmed that miR-34a directly targets XIST and suggested that XIST regulates cell proliferation, invasiveness, and apoptosis in PC by sponging miR-34a. CONCLUSIONS: XIST promotes TGF-ß1-induced EMT by regulating the miR-34a-YAP-EGFR axis in PC.

FAM60A promotes cisplatin resistance in lung cancer cells by activating SKP2 expression.

Cisplatin is a widely used chemotherapeutic drug in lung cancer treatment. Most cancer patients eventually develop cisplatin resistance, resulting in a poor prognosis. Previously, we identified a novel marker, family with sequence similarity 60A (FAM60A), that was responsible for resistance in cisplatin-resistant human lung adenocarcinoma A549 (A549/DDP) cells. Here, we investigated the biological effects of FAM60A in A549/DDP cells and explored the underlying molecular mechanisms to understand its functional role in cisplatin resistance. Real-time quantitative PCR and western blot analysis were used to determine the expression levels of FAM60A in A549/DDP cells. FAM60A and SKP2 were knockdown with small-interfering RNA (siRNA). Cancer cell viability was analyzed with flow cytometry. The mRNA and protein expression levels of FAM60A increased significantly and dose-dependently in A549/DDP cells following cisplatin treatment. FAM60A overexpression up-regulated MDR1 expression, inhibited caspase 3, cleaved-caspase 3, and caspase 8 expression, and prevented cancer cell death. Microarray analysis of cells transfected with siRNA against the FAM60A transcript and control samples showed that SKP2 expression was positively regulated by FAM60A. SKP2 knockdown using a short-hairpin RNA reversed the functions induced by FAM60A. These results suggest that overexpression of FAM60A in A549/DDP cells led to SKP2 upregulation and enhanced cisplatin resistance in cancer cells. These provide new insights into chemoresistance and may contribute to reversing cisplatin resistance during lung cancer treatment.

The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice.

PRMT5 is a type II protein arginine methyltransferase with roles in stem cell biology, reprograming, cancer and neurogenesis. During embryogenesis in the mouse, it was hypothesized that PRMT5 functions with the master germline determinant BLIMP1 to promote primordial germ cell (PGC) specification. Using a Blimp1-Cre germline conditional knockout, we discovered that Prmt5 has no major role in murine germline specification, or the first global epigenetic reprograming event involving depletion of cytosine methylation from DNA and histone H3 lysine 9 dimethylation from chromatin. Instead, we discovered that PRMT5 functions at the conclusion of PGC reprograming I to promote proliferation, survival and expression of the gonadal germline program as marked by MVH. We show that PRMT5 regulates gene expression by promoting methylation of the Sm spliceosomal proteins and significantly altering the spliced repertoire of RNAs in mammalian embryonic cells and primordial cells.

Pancreatic cancer actionable genes in precision medicine and personalized surgery.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with an overall 5-year survival rate less than 5% due to the poor early diagnosis and lack of effective therapeutic options. The most effective therapy remains surgery, however post-operative survival could be enhanced with effective adjuvant therapy. The massive information gained from Omics techniques on PDAC at the beginning of the 21st century is a remarkable accomplishment. However, the information gained from the omics data, including next generation sequencing data, has yet to successfully affect care of patients suffering with PDAC. Therefore, we propose the development of an actionable genomic platform that matches a patient's PDAC clinically actionable genes with potential targeted adjuvant therapies. Using this platform, PDX1 has been identified as a potential actionable gene for PDAC, therefore, RNAi therapy, gene therapy and small inhibitory drugs, all targeting PDX1, serve as potential targeted adjuvant therapies. Preclinical studies support the hypothesis that identification of PDAC actionable genes could permit translation of a patient's genomic information into precision targeted adjuvant therapy for PDAC.

Selective demethylation and altered gene expression are associated with ICF syndrome in human-induced pluripotent stem cells and mesenchymal stem cells.

Immunodeficiency, centromeric instability and facial anomalies type I (ICF1) syndrome is a rare genetic disease caused by mutations in DNA methyltransferase (DNMT) 3B, a de novo DNA methyltransferase. However, the molecular basis of how DNMT3B deficiency leads to ICF1 pathogenesis is unclear. Induced pluripotent stem cell (iPSC) technology facilitates the study of early human developmental diseases via facile in vitro paradigms. Here, we generate iPSCs from ICF Type 1 syndrome patient fibroblasts followed by directed differentiation of ICF1-iPSCs to mesenchymal stem cells (MSCs). By performing genome-scale bisulfite sequencing, we find that DNMT3B-deficient iPSCs exhibit global loss of non-CG methylation and select CG hypomethylation at gene promoters and enhancers. Further unbiased scanning of ICF1-iPSC methylomes also identifies large megabase regions of CG hypomethylation typically localized in centromeric and subtelomeric regions. RNA sequencing of ICF1 and control iPSCs reveals abnormal gene expression in ICF1-iPSCs relevant to ICF syndrome phenotypes, some directly associated with promoter or enhancer hypomethylation. Upon differentiation of ICF1 iPSCs to MSCs, we find virtually all CG hypomethylated regions remained hypomethylated when compared with either wild-type iPSC-derived MSCs or primary bone-marrow MSCs. Collectively, our results show specific methylome and transcriptome defects in both ICF1-iPSCs and differentiated somatic cell lineages, providing a valuable stem cell system for further in vitro study of the molecular pathogenesis of ICF1 syndrome. GEO accession number: GSE46030.

Supramolecular nanosubstrate-mediated delivery for reprogramming and transdifferentiation of mammalian cells.

Supramolecular nanosubstrate-mediated delivery (SNSMD) leverages the power of molecular self-assembly and a nanostructured substrate platform for the low toxicity, highly efficient co-delivery of biological factors encapsulated in a nanovector. Human fibroblasts are successfully reprogrammed into induced pluripotent stems and transdifferentiated into induced neuronal-like cells.

KASH protein Syne-2/Nesprin-2 and SUN proteins SUN1/2 mediate nuclear migration during mammalian retinal development.

Nuclear movement relative to cell bodies is a fundamental process during certain aspects of mammalian retinal development. During the generation of photoreceptor cells in the cell division cycle, the nuclei of progenitors oscillate between the apical and basal surfaces of the neuroblastic layer (NBL). This process is termed interkinetic nuclear migration (INM). Furthermore, newly formed photoreceptor cells migrate and form the outer nuclear layer (ONL). In the current study, we demonstrated that a KASH domain-containing protein, Syne-2/Nesprin-2, as well as SUN domain-containing proteins, SUN1 and SUN2, play critical roles during INM and photoreceptor cell migration in the mouse retina. A deletion mutation of Syne-2/Nesprin-2 or double mutations of Sun1 and Sun2 caused severe reduction of the thickness of the ONL, mislocalization of photoreceptor nuclei and profound electrophysiological dysfunction of the retina characterized by a reduction of a- and b-wave amplitudes. We also provide evidence that Syne-2/Nesprin-2 forms complexes with either SUN1 or SUN2 at the nuclear envelope to connect the nucleus with dynein/dynactin and kinesin molecular motors during the nuclear migrations in the retina. These key retinal developmental signaling results will advance our understanding of the mechanism of nuclear migration in the mammalian retina.

RNA-Sequencing Reveals Gene Expression and Pathway Signatures in Umbilical Cord Blood Affected by Birth Delivery Mode.

Cesarean section (CS) confers increased risk of type I diabetes, asthma, inflammatory bowel disease, celiac disease, overweight and obesity, etc., in the offspring. However, the underlying mechanism remains unknown. To investigate the influence of CS on gene expression in cord blood, we have performed RNA-sequencing followed by single-gene analysis, gene set enrichment analysis, gene co-expression network analysis, and interacting genes/proteins analysis in eight full-term infants born by elective CS and eight matched vaginally delivered (VD) infants. Crucial genes identified above were further validated in another 20 CS and 20 VD infants. We found for the first time that mRNA expression of genes involved in immune response (IL12A, INFG, IL1B, TNF, MIF, IL4, CA1, IFI27, HLA-DOB and EPHB1) and metabolism (DLK1, CYP2A6 and GATM) were significantly influenced by CS. Notably, serum TNF-α and IFN-γ were remarkably up-regulated in the CS infants (p = 5.0 × 10-4 and 3.0 × 10-3, respectively) compared to the VD infants. It is biologically plausible that CS may exert adverse impacts on offspring health through influencing expression of genes in the above processes. These findings will help understand the potential underlying mechanisms of the adverse health impacts of CS and identify biomarkers for future health of offspring born with different delivery modes. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-022-00086-7.

Discovery and Validation of Novel Genes in a Large Chinese Autism Spectrum Disorder Cohort.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder that causes impairments in social communication and stereotypical behaviors, often accompanied by developmental delay or intellectual disability. A growing body of evidence suggests that ASD is highly heritable, and genetic studies have defined numerous risk genes. However, most studies have been conducted with individuals of European and Hispanic ancestry, and there is a lack of genetic analyses of ASD in the East Asian population. METHODS: We performed whole-exome sequencing on 772 Chinese ASD trios and combined the data with a previous study of 369 Chinese ASD trios, identifying de novo variants in 1141 ASD trios. We used single-cell RNA sequencing analysis to identify the cell types in which ASD-related genes were enriched. In addition, we validated the function of a candidate high-functioning autism gene in mouse models using genetic approaches. RESULTS: Our findings showed that ASD without developmental delay or intellectual disability carried fewer disruptive de novo variants than ASD with developmental delay or intellectual disability. Moreover, we identified 9 novel ASD candidate genes that were not present in the current ASD gene database. We further validated one such novel ASD candidate gene, SLC35G1, by showing that mice harboring a heterozygous deletion of Slc35g1 exhibited defects in interactive social behaviors. CONCLUSIONS: Our work nominates novel ASD candidate genes and emphasizes the importance of genome-wide genetic studies with ASD cohorts of different ancestries to reveal the comprehensive genetic architecture of ASD.

SH2B1 Tunes Hippocampal ERK Signaling to Influence Fluid Intelligence in Humans and Mice.

Fluid intelligence is a cognitive domain that encompasses general reasoning, pattern recognition, and problem-solving abilities independent of task-specific experience. Understanding its genetic and neural underpinnings is critical yet challenging for predicting human development, lifelong health, and well-being. One approach to address this challenge is to map the network of correlations between intelligence and other constructs. In the current study, we performed a genome-wide association study using fluid intelligence quotient scores from the UK Biobank to explore the genetic architecture of the associations between obesity risk and fluid intelligence. Our results revealed novel common genetic loci (SH2B1, TUFM, ATP2A1, and FOXO3) underlying the association between fluid intelligence and body metabolism. Surprisingly, we demonstrated that SH2B1 variation influenced fluid intelligence independently of its effects on metabolism but partially mediated its association with bilateral hippocampal volume. Consistently, selective genetic ablation of Sh2b1 in the mouse hippocampus, particularly in inhibitory neurons, but not in excitatory neurons, significantly impaired working memory, short-term novel object recognition memory, and behavioral flexibility, but not spatial learning and memory, mirroring the human intellectual performance. Single-cell genetic profiling of Sh2B1-regulated molecular pathways revealed that Sh2b1 deletion resulted in aberrantly enhanced extracellular signal-regulated kinase (ERK) signaling, whereas pharmacological inhibition of ERK signaling reversed the associated behavioral impairment. Our cross-species study thus provides unprecedented insight into the role of SH2B1 in fluid intelligence and has implications for understanding the genetic and neural underpinnings of lifelong mental health and well-being.

SUN1 is required for telomere attachment to nuclear envelope and gametogenesis in mice.

Prior to the pairing and recombination between homologous chromosomes during meiosis, telomeres attach to the nuclear envelope and form a transient cluster. However, the protein factors mediating meiotic telomere attachment to the nuclear envelope and the requirement of this attachment for homolog pairing and synapsis have not been determined in animals. Here we show that the inner nuclear membrane protein SUN1 specifically associates with telomeres between the leptotene and diplotene stages during meiotic prophase I. Disruption of Sun1 in mice prevents telomere attachment to the nuclear envelope, efficient homolog pairing, and synapsis formation in meiosis. Massive apoptotic events are induced in the mutant gonads, leading to the abolishment of both spermatogenesis and oogenesis. This study provides genetic evidence that SUN1-telomere interaction is essential for telomere dynamic movement and is required for efficient homologous chromosome pairing/synapsis during mammalian gametogenesis.

Molecular signature of primary retinal pigment epithelium and stem-cell-derived RPE cells.

Age-related macular degeneration (AMD) is characterized by the loss or dysfunction of retinal pigment epithelium (RPE) and is the most common cause of vision loss among the elderly. Stem-cell-based strategies, using human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs), may provide an abundant donor source for generating RPE cells in cell replacement therapies. Despite a significant amount of research on deriving functional RPE cells from various stem cell sources, it is still unclear whether stem-cell-derived RPE cells fully mimic primary RPE cells. In this report, we demonstrate that functional RPE cells can be derived from multiple lines of hESCs and hiPSCs with varying efficiencies. Stem-cell-derived RPE cells exhibit cobblestone-like morphology, transcripts, proteins and phagocytic function similar to human fetal RPE (fRPE) cells. In addition, we performed global gene expression profiling of stem-cell-derived RPE cells, native and cultured fRPE cells, undifferentiated hESCs and fibroblasts to determine the differentiation state of stem-cell-derived RPE cells. Our data indicate that hESC-derived RPE cells closely resemble human fRPE cells, whereas hiPSC-derived RPE cells are in a unique differentiation state. Furthermore, we identified a set of 87 signature genes that are unique to human fRPE and a majority of these signature genes are shared by stem-cell-derived RPE cells. These results establish a panel of molecular markers for evaluating the fidelity of human pluripotent stem cell to RPE conversion. This study contributes to our understanding of the utility of hESC/hiPSC-derived RPE in AMD therapy.

Dnmt3a regulates both proliferation and differentiation of mouse neural stem cells.

DNA methylation is known to regulate cell differentiation and neuronal function in vivo. Here we examined whether deficiency of a de novo DNA methyltransferase, Dnmt3a, affects in vitro differentiation of mouse embryonic stem cells (mESCs) to neuronal and glial cell lineages. Early-passage neural stem cells (NSCs) derived from Dnmt3a-deficient ESCs exhibited a moderate phenotype in precocious glial differentiation compared with wild-type counterparts. However, successive passaging to passage 6 (P6), when wild-type NSCs become gliogenic, revealed a robust phenotype of precocious astrocyte and oligodendrocyte differentiation in Dnmt3a(-/-) NSCs, consistent with our previous findings in the more severely hypomethylated Dnmt1(-/-) NSCs. Mass spectrometric analysis revealed that total levels of methylcytosine in Dnmt3a(-/-) NSCs at P6 were globally hypomethylated. Moreover, the Dnmt3a(-/-) NSC proliferation rate was significantly increased compared with control from P6 onward. Thus, our work revealed a novel role for Dnmt3a in regulating both the timing of neural cell differentiation and the cell proliferation in the paradigm of mESC-derived-NSCs.

Generation of an induced pluripotent stem cell line SJTUXHi001-A from an autism spectrum disorder patient carrying a heterozygous mutation in HDAC8 (p.P359S).

Mutations in the HDAC8 are considered to be a prominent cause of Cornelia de Lange syndrome 5, a leading cause of intellectual disability and social disability. Here, we report the generation of an induced pluripotent stem cell (iPSC) line from a 5-year-old girl diagnosed with autism spectrum disorder (ASD) who carries a heterozygous mutation in HDAC8 (c.1075C > T, p.Pro359Ser).

Generation of a human induced pluripotent stem cell line (SJTUXHi002-A) from an individual with autism spectrum disorder carrying a heterozygous mutation in GRIA2.

Herein, we report the generation of a human induced pluripotent stem cell (iPSC) line from an autism spectrum disorder (ASD) patient carrying the c.1998delT mutation in GRIA2 gene. The generated iPSC line exhibits normal karyotype, pluripotency markers and was able to differentiate into three germ layers. The iPSC line retained the GRIA2 mutation (c.1998delT), which could provide a valuable resource for investing pathogenic mechanisms underlying ASD and facilitating the development of targeted medicine.