Association of T Cell Subsets and Platelet/Lymphocyte Ratio with Long-Term Complications in Kidney Transplant Recipients.

BACKGROUND Infection and chronic rejection remain major issues for kidney transplant recipients (KTRs). The present study aimed to explore the association of CD4+/CD8+ T cell ratio (CD4+/CD8+) and platelet/lymphocyte ratio (PLR) with long-term infection and chronic renal insufficiency in KTRs. MATERIAL AND METHODS KTRs admitted to a single hospital from June 2014 to December 2021 were divided into infected (164) and non-infected (107) groups based on clinical data. The levels of CD4+/CD8+, PLR, neutrophil/lymphocyte ratio (NLR), and C-reactive Protein (CRP) in KTRs with long-term infection, and their correlation with chronic kidney insufficiency, were analyzed. Survival analysis was used to evaluate the risk factors for long-term infection and chronic kidney insufficiency. RESULTS Spearman correlation analysis showed that chronic kidney insufficiency was positively correlated with PLR, and negatively correlated with CRP and CD4+/CD8+ (P<0.05). PLR was positively correlated with CRP, procalcitonin, erythrocyte sedimentation rate, and NLR, but negatively with CD4+/CD8+. CD4+/CD8+ was correlated with CRP, NLR, and PLR (P<0.05). Survival analysis and survival curves showed that PLR and CD4+/CD8+ were risk factors for long-term infection and chronic kidney insufficiency in KTRs (P<0.05). CONCLUSIONS CD4+/CD8+ and PLR were associated with long-term complications, and were risk factors for long-term infection and chronic kidney insufficiency in KTRs.

Molecular and Spatial Signatures of Mouse Embryonic Endothelial Cells at Single-Cell Resolution.

BACKGROUND: Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized. METHODS: We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs). RESULTS: From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including Etv2+Bnip3+ EC progenitors. Spatial transcriptomic analysis of 15 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (Eln, Dkk2, Vegfc, and Egfl8), venous (Fam174b and Clec14a), and capillary (Kcne3) marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages. CONCLUSIONS: Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.

The yeast protein Ure2p triggers Tau pathology in a mouse model of tauopathy.

The molecular mechanisms that trigger Tau aggregation in Alzheimer's disease (AD) remain elusive. Fungi, especially Saccharomyces cerevisiae (S. cerevisiae), can be found in brain samples from patients with AD. Here, we show that the yeast protein Ure2p from S. cerevisiae interacts with Tau and facilitates its aggregation. The Ure2p-seeded Tau fibrils are more potent in seeding Tau and causing neurotoxicity in vitro. When injected into the hippocampus of Tau P301S transgenic mice, the Ure2p-seeded Tau fibrils show enhanced seeding activity compared with pure Tau fibrils. Strikingly, intracranial injection of Ure2p fibrils promotes the aggregation of Tau and cognitive impairment in Tau P301S mice. Furthermore, intranasal infection of S. cerevisiae in the nasal cavity of Tau P301S mice accelerates the aggregation of Tau. Together, these observations indicate that the yeast protein Ure2p initiates Tau pathology. Our results provide a conceptual advance that non-mammalian prions may cross-seed mammalian prion-like proteins.

Soluble TREM2 ameliorates tau phosphorylation and cognitive deficits through activating transgelin-2 in Alzheimer's disease.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane protein that is predominantly expressed by microglia in the brain. The proteolytic shedding of TREM2 results in the release of soluble TREM2 (sTREM2), which is increased in the cerebrospinal fluid of patients with Alzheimer's disease (AD). It remains unknown whether sTREM2 regulates the pathogenesis of AD. Here we identified transgelin-2 (TG2) expressed on neurons as the receptor for sTREM2. The microglia-derived sTREM2 binds to TG2, induces RhoA phosphorylation at S188, and deactivates the RhoA-ROCK-GSK3ß pathway, ameliorating tau phosphorylation. The sTREM2 (77-89) fragment, which is the minimal active sequence of sTREM2 to activate TG2, mimics the inhibitory effect of sTREM2 on tau phosphorylation. Overexpression of sTREM2 or administration of the active peptide rescues tau pathology and behavioral defects in the tau P301S transgenic mice. Together, these findings demonstrate that the sTREM2-TG2 interaction mediates the cross-talk between microglia and neurons. sTREM2 and its active peptide may be a potential therapeutic intervention for tauopathies including AD.

Time space and single-cell resolved tissue lineage trajectories and laterality of body plan at gastrulation.

Understanding of the molecular drivers of lineage diversification and tissue patterning during primary germ layer development requires in-depth knowledge of the dynamic molecular trajectories of cell lineages across a series of developmental stages of gastrulation. Through computational modeling, we constructed at single-cell resolution, a spatio-temporal transcriptome of cell populations in the germ-layers of gastrula-stage mouse embryos. This molecular atlas enables the inference of molecular network activity underpinning the specification and differentiation of the germ-layer tissue lineages. Heterogeneity analysis of cellular composition at defined positions in the epiblast revealed progressive diversification of cell types. The single-cell transcriptome revealed an enhanced BMP signaling activity in the right-side mesoderm of late-gastrulation embryo. Perturbation of asymmetric BMP signaling activity at late gastrulation led to randomization of left-right molecular asymmetry in the lateral mesoderm of early-somite-stage embryo. These findings indicate the asymmetric BMP activity during gastrulation may be critical for the symmetry breaking process.

Islet amyloid polypeptide triggers α-synuclein pathology in Parkinson's disease.

Pathologic aggregation and prion-like propagation of α-synuclein (α-syn) are the hallmarks of Parkinson's disease (PD). Emerging evidence shows that type 2 diabetes mellitus (T2DM) is a risk factor for PD. Interestingly, T2DM is characterized by the amyloid deposition of islet amyloid polypeptide (IAPP) in the pancreas. Although T2DM and PD share pathological similarities, the underlying molecular mechanisms bridging these two diseases remain unknown. Here, we report that IAPP co-deposits with α-syn in the brains of PD patients. IAPP interacts with α-syn and accelerates its aggregation. In addition, the IAPP-seeded α-syn fibrils show enhanced seeding activity and neurotoxicity compared with pure α-syn fibrils in vitro and in vivo. Strikingly, intravenous injection of IAPP fibrils into α-syn A53T transgenic mice or human SNCA transgenic mice accelerated the aggregation of α-syn and PD-like motor deficits. Taken together, these findings support that IAPP acts as a trigger of α-syn pathology in PD, and provide a mechanistic explanation for the increased risk and faster progression of PD in patients with T2DM.

Generation of functional posterior spinal motor neurons from hPSCs-derived human spinal cord neural progenitor cells.

Spinal motor neurons deficiency results in a series of devastating disorders such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and spinal cord injury (SCI). These disorders are currently incurable, while human pluripotent stem cells (hPSCs)-derived spinal motor neurons are promising but suffered from inappropriate regional identity and functional immaturity for the study and treatment of posterior spinal cord related injuries. In this study, we have established human spinal cord neural progenitor cells (hSCNPCs) via hPSCs differentiated neuromesodermal progenitors (NMPs) and demonstrated the hSCNPCs can be continuously expanded up to 40 passages. hSCNPCs can be rapidly differentiated into posterior spinal motor neurons with high efficiency. The functional maturity has been examined in detail. Moreover, a co-culture scheme which is compatible for both neural and muscular differentiation is developed to mimic the neuromuscular junction (NMJ) formation in vitro. Together, these studies highlight the potential avenues for generating clinically relevant spinal motor neurons and modeling neuromuscular diseases through our defined hSCNPCs.

Hsa_circ_0001162 Inhibition Alleviates High Glucose-Induced Human Podocytes Injury by the miR-149-5p/MMP9 Signaling Pathway.

Emerging evidences suggested that circular RNAs (circRNAs) are involved in diabetic nephropathy (DN). Accumulating evidence had suggested that the degree of podocyte is a major prognostic determinant of DN progression. However, the function and in-depth mechanisms of hsa_circ_0001162 in podocyte injury of DN remain unclear. Hsa_circ_0001162 expression was detected by real-time quantitative PCR (RT-qPCR) in peripheral blood of DN patients and high glucose-induced podocytes injury model. The cell counting kit 8, 5-ethynyl-2'-deoxyuridine, flow cytometry with Annexin V-FITC/PI staining, caspase-3 activity assay Kit, enzyme linked immunosorbent assay (ELISA), RT-qPCR and western blotting were used to evaluate the effect of hsa_circ_0001162 / miR-149-5p / MMP9 axis on high glucose-induced podocyte injury. Mechanistically, dual luciferase reporter was used to confirm the relationship of miR-149-5p and hsa_circ_0001162 or MMP9. Furthermore, RNA-pull down and immunoprecipitation assay were implemented to verify the potential regulatory effects of EIF4A3 on biogenesis of hsa_circ_0001162. Our results showed that hsa_circ_0001162 was highly expressed in peripheral blood of DN patients and high glucose-induced podocytes injury model, and the knockdown of hsa_circ_0001162 increased the proliferation, inhibited the apoptosis, and suppressed inflammatory response in high glucose-induced podocytes injury. Mechanism studies demonstrated that EIF4A3 bound with flanking sequences of hsa_circ_0001162 to promote hsa_circ_0001162 expression, upregulated hsa_circ_0001162 increased the MMP9 expression via sponging miR-149-5p, thus aggravating the high glucose-induced podocytes injury. Overall, our data demonstrated that knockdown of hsa_circ_0001162 inhibited high glucose-induced podocytes injury by regulating miR-149-5p/MMP9 axis, and intervention of hsa_circ_0001162/miR-149-5p/MMP9 axis may be a potentially promising therapeutic strategy for podocyte injury in DN patients.

DISC1 inhibits GSK3ß activity to prevent tau hyperphosphorylation under diabetic encephalopathy.

Diabetic encephalopathy (DE) is a common complication of type 2 diabetes (T2D), especially in those patients with long T2D history. Persistent high glucose (HG) stimulation leads to neuron damage and manifests like Alzheimer's disease's pathological features such as neurofilament tangle. However, the precise mechanism of high-glucose-induced tau hyperphosphorylation is not fully revealed. We here gave evidence that Disrupted in schizophrenia 1 protein (DISC1) could interact with glycogen synthase kinase 3ß (GSK3ß) and inhibit its activity to prevent tau hyperphosphorylation. By using DB/DB mice as animal model and HG-treated N2a cell as cell model, we found that DISC1 was downregulated both in vivo and in vitro, complicated with Tau hyperphosphorylation and GSK3ß activation. Further, we identified DISC1 interacted with GSK3ß by its 198th-237th amino acid residues. Overexpression of full length DISC1 but not mutated DISC1 lacking this domain could prevent HG induced tau hyperphosphorylation. Taken together, our work revealed DISC1 could be an important negative modulators of tau phosphorylation, and suggested that preservation of DISC1 could prevent HG induced neuron damage.

Maternal mRNA deadenylation and allocation via Rbm14 condensates facilitate vertebrate blastula development.

Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA-binding protein Rbm14 and maternal mRNAs co-phase separate into cytoplasmic condensates to facilitate vertebrate blastula-to-gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal γ-tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8- and 16-cell embryos. Their formation was dose-dependently stimulated by m6 A, but repressed by m5 C modification of the maternal mRNA. Furthermore, deadenylase Parn co-phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m6 A-modified maternal mRNAs, and by releasing the poly(A)-less transcripts upon regulated disassembly to allow their re-polyadenylation and translation or clearance.

Salidroside Ameliorates Ischemia-Induced Neuronal Injury through AMPK Dependent and Independent Pathways to Maintain Mitochondrial Quality Control.

Salidroside, an active ingredient in Rhodiola rosea, has potent protective activity against cerebral ischemia. However, the mechanisms underlying its pharmacological actions are poorly understood. In this study, we employed a mouse middle cerebral artery occlusion (MCAO) and cellular oxygen and glucose deprivation (OGD) models to test the hypothesis that salidroside may restore mitochondrial quality control in neurons by modulating the relevant signaling. The results indicated that salidroside mitigated almost 40% the ischemia-induced brain infarct volumes in mice and the OGD-decreased viability of neurons to ameliorate the mitochondrial functions. Furthermore, salidroside treatment alleviated the OGD- or ischemia-induced imbalance of mitochondrial fission and fusion, mitophagy and promoted mitochondrial biogenesis in neurons by attenuating the AMPK activity. Moreover, salidroside alleviated 50% the OGD-promoted mitochondrial calcium fluorescence intensity and 5% mitochondria-associated membrane (MAM) area by down-regulating GRP75 expression independent of the AMPK signaling. Finally, similar findings were achieved in primary mouse neurons. Collectively, these data indicate that salidroside effectively restores the mitochondria dynamics, facilitates mitochondrial biogenesis by attenuating the AMPK signaling, and maintains calcium homeostasis in neurons independent of the AMPK activity.

A synapsin â cleavage fragment contributes to synaptic dysfunction in Alzheimer's disease.

Synaptic dysfunction is a key feature of Alzheimer's disease (AD). However, the molecular mechanisms underlying synaptic dysfunction remain unclear. Here, we show that synapsin Ⅰ, one of the most important synaptic proteins, is fragmented by the cysteine proteinase asparagine endopeptidase (AEP). AEP cleaves synapsin at N82 in the brains of AD patients and generates the C-terminal synapsin Ⅰ (83-705) fragment. This fragment is abnormally distributed in neurons and induces synaptic dysfunction. Overexpression of AEP in the hippocampus of wild-type mice results in the production of the synapsin Ⅰ (83-705) fragment and induces synaptic dysfunction and cognitive deficits. Moreover, overexpression of the AEP-generated synapsin Ⅰ (83-705) fragment in the hippocampus of tau P301S transgenic mice and wild-type mice promotes synaptic dysfunction and cognitive deficits. These findings suggest a novel mechanism of synaptic dysfunction in AD.

Preserving mitochondrial function by inhibiting GRP75 ameliorates neuron injury under ischemic stroke.

Ischemic stroke is a life­threatening disease, which is closely related to neuron damage during ischemia. Mitochondrial dysfunction is essentially involved in the pathophysiological process of ischemic stroke. Mitochondrial calcium overload contributes to the development of mitochondrial dysfunction. However, the underlying mechanisms of mitochondrial calcium overload are far from being fully revealed. In the present study, middle cerebral artery obstruction (MCAO) was performed in vivo and oxygen and glucose deprivation (OGD) in vitro. The results indicated that both MCAO and OGD induced significant mitochondrial dysfunction in vivo and in vitro. The mitochondria became fragmented under hypoxia conditions, accompanied with upregulation of the heat shock protein 75 kDa glucose­regulated protein (GRP75). Inhibition of GRP75 was able to effectively ameliorate mitochondrial calcium overload and preserve mitochondrial function, which may provide evidence for further translational studies of ischemic diseases.

The Ribosome Biogenesis Factor Ltv1 Is Essential for Digestive Organ Development and Definitive Hematopoiesis in Zebrafish.

Ribosome biogenesis is a fundamental activity in cells. Ribosomal dysfunction underlies a category of diseases called ribosomopathies in humans. The symptomatic characteristics of ribosomopathies often include abnormalities in craniofacial skeletons, digestive organs, and hematopoiesis. Consistently, disruptions of ribosome biogenesis in animals are deleterious to embryonic development with hypoplasia of digestive organs and/or impaired hematopoiesis. In this study, ltv1, a gene involved in the small ribosomal subunit assembly, was knocked out in zebrafish by clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR associated protein 9 (Cas9) technology. The recessive lethal mutation resulted in disrupted ribosome biogenesis, and ltv1 Δ14/Δ14 embryos displayed hypoplastic craniofacial cartilage, digestive organs, and hematopoiesis. In addition, we showed that the impaired cell proliferation, instead of apoptosis, led to the defects in exocrine pancreas and hematopoietic stem and progenitor cells (HSPCs) in ltv1 Δ14/Δ14 embryos. It was reported that loss of function of genes associated with ribosome biogenesis often caused phenotypes in a P53-dependent manner. In ltv1 Δ14/Δ14 embryos, both P53 protein level and the expression of p53 target genes, Δ113p53 and p21, were upregulated. However, knockdown of p53 failed to rescue the phenotypes in ltv1 Δ14/Δ14 larvae. Taken together, our data demonstrate that LTV1 ribosome biogenesis factor (Ltv1) plays an essential role in digestive organs and hematopoiesis development in zebrafish in a P53-independent manner.

Protective Effect of Salidroside on Mitochondrial Disturbances via Reducing Mitophagy and Preserving Mitochondrial Morphology in OGD-induced Neuronal Injury.

Salidroside is the active ingredient extracted from Rhodiola rosea, and has been reported to show protective effects in cerebral ischemia, but the exact mechanisms of neuronal protective effects are still unrevealed. In this study, the protective effects of salidroside (1 µmol/L) in ameliorating neuronal injuries induced by oxygen-glucose deprivation (OGD), which is a classical model of cerebral ischemia, were clarified. The results showed that after 8 h of OGD, the mouse hippocampal neuronal cell line HT22 cells showed increased cell death, accompanied with mitochondrial fragmentation and augmented mitophagy. However, the cell viability of HT22 cells showed significant restoration after salidroside treatment. Mitochondrial morphology and mitochondrial function were effectively preserved by salidroside treatment. The protective effects of salidroside were further related to the prevention of mitochondrial over-fission. The results showed that mTOR could be recruited to the mitochondria after salidroside treatment, which might be responsible for inhibiting excessive mitophagy caused by OGD. Thus, salidroside was shown to play a protective role in reducing neuronal death under OGD by safeguarding mitochondrial function, which may provide evidence for further translational studies of salidroside in ischemic diseases.

ApoE4 (Δ272-299) induces mitochondrial-associated membrane formation and mitochondrial impairment by enhancing GRP75-modulated mitochondrial calcium overload in neuron.

BACKGROUND: Apolipoprotein E4 (apoE4) is a major genetic risk factor of Alzheimer's disease. Its C-terminal-truncated apoE4 (Δ272-299) has neurotoxicity by affecting mitochondrial respiratory function. However, the molecular mechanism(s) underlying the action of apoE4 (Δ272-299) in mitochondrial function remain poorly understood. METHODS: The impact of neuronal apoE4 (Δ272-299) expression on ER stress, mitochondrial-associated membrane (MAM) formation, GRP75, calcium transport and mitochondrial impairment was determined in vivo and in vitro. Furthermore, the importance of ER stress or GRP75 activity in the apoE4 (Δ272-299)-promoted mitochondrial dysfunction in neuron was investigated. RESULTS: Neuronal apoE4 (Δ272-299) expression induced mitochondrial impairment by inducing ER stress and mitochondrial-associated membrane (MAM) formation in vivo and in vitro. Furthermore, apoE4 (Δ272-299) expression promoted GRP75 expression, mitochondrial dysfunction and calcium transport into the mitochondria in neuron, which were significantly mitigated by treatment with PBA (an inhibitor of ER stress), MKT077 (a specific GRP75 inhibitor) or GRP75 silencing. CONCLUSIONS: ApoE4 (Δ272-299) significantly impaired neuron mitochondrial function by triggering ER stress, up-regulating GRP75 expression to increase MAM formation, and mitochondrial calcium overload. Our findings may provide new insights into the neurotoxicity of apoE4 (Δ272-299) against mitochondrial function and uncover new therapeutic targets for the intervention of Alzheimer's disease.

M-CSF, IL-6, and TGF-ß promote generation of a new subset of tissue repair macrophage for traumatic brain injury recovery.

Traumatic brain injury (TBI) leads to high mortality rate. We aimed to identify the key cytokines favoring TBI repair and found that patients with TBI with a better outcome robustly increased concentrations of macrophage colony-stimulating factor, interleukin-6, and transforming growth factor-ß (termed M6T) in cerebrospinal fluid or plasma. Using TBI mice, we identified that M2-like macrophage, microglia, and endothelial cell were major sources to produce M6T. Together with the in vivo tracking of mCherry+ macrophages in zebrafish models, we confirmed that M6T treatment accelerated blood-borne macrophage infiltration and polarization toward a subset of tissue repair macrophages that expressed similar genes as microglia for neuroprotection, angiogenesis and cell migration. M6T therapy in TBI mice and zebrafish improved neurological function while blocking M6T-exacerbated brain injury. Considering low concentrations of M6T in some patients with poor prognostic, M6T treatment might repair TBI via generating a previously unidentified subset of tissue repair macrophages.

PARP-1 and SIRT-1 are Interacted in Diabetic Nephropathy by Activating AMPK/PGC-1α Signaling Pathway.

INTRODUCTION: Diabetic nephropathy (DN) is a metabolic disorder characterized by the accumulation of extracellular matrix (ECM). This study aims to investigate whether exists an interplay between poly (ADP-ribose) polymerase 1 (PARP-1) and sirtuin 1 (SIRT-1) in DN via AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) signaling pathway. METHODS: Eight-week-old male obese leptin-resistant (db/db) mice and nondiabetic control male C57BLKs/J (db/m) mice were used in this study. Body weight and blood glucose were evaluated after 6 h of fasting, which continues for 4 weeks. The kidney tissues were dissected for Western blot, immunofluorescence (IF) assay. Besides, PARP activity assay, MTT assay, NAD+ qualification, Western blot and IF were also performed to detect the level and relation of PARP-1 and SIRT-1 in mouse mesangial cells (MCs) with or without high glucose followed by inhibiting or elevating PARP-1 and SIRT-1, respectively. RESULTS: Western blotting shows PARP-1 and ECM marker fibronectin (FN) are upregulated while SIRT-1 is downregulated in db/db mice (p<0.05) or in mouse MCs with high glucose (p<0.05), which are significantly restored by PARP-1 inhibitor (PJ34) (p<0.05) and SIRT-1 lentiviral transfected treatment (p<0.05), or worsened by SIRT-1 inhibitor EX527 (p<0.05). PJ34 treatment (p < 0.05) or SIRT-1 overexpression (p < 0.05) could increase PGC-1α and p-AMPK levels, concomitant with down expression of FN, however, were reversed in the presence of EX527 (p<0.05). DISCUSSION: Our results suggest an important relationship between PARP-1 and SIRT-1 through AMPK-PGC-1α pathway, indicating a potential therapeutic method for DN.

A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription.

OBJECTIVE: To simplify CRISPR/Cas9 genome editing in the industrial filamentous fungus Trichoderma reesei based on in vivo guide RNA (gRNA) transcription. RESULTS: Two putative RNA polymerase III U6 snRNA genes were identified in the genome of T. reesei QM6a by BLASTN using Myceliophthora. thermophila U6 snRNA gene as the template. The regions approximately 500 bp upstream of two U6 genes were efficient promoters for the in vivo expression of gRNA. The CRISPR system consisting of Cas9 and in vivo synthesized gRNA under control of the T. reesei U6 snRNA promoters was sufficient to cause a frameshift mutation in the ura5 gene via non-homologous end-joining-mediated events. CONCLUSIONS: We report a simple gene editing method using a CRISPR/Cas9-coupled in vivo gRNA transcription system in T. reesei.

Imbalance of Excitatory/Inhibitory Neuron Differentiation in Neurodevelopmental Disorders with an NR2F1 Point Mutation.

Recent studies have revealed an essential role for embryonic cortical development in the pathophysiology of neurodevelopmental disorders, including autism spectrum disorder (ASD). However, the genetic basis and underlying mechanisms remain unclear. Here, we generate mutant human embryonic stem cell lines (Mut hESCs) carrying an NR2F1-R112K mutation that has been identified in a patient with ASD features and investigate their neurodevelopmental alterations. Mut hESCs overproduce ventral telencephalic neuron progenitors (ventral NPCs) and underproduce dorsal NPCs, causing the imbalance of excitatory/inhibitory neurons. These alterations can be mainly attributed to the aberrantly activated Hedgehog signaling pathway. Moreover, the corresponding Nr2f1 point-mutant mice display a similar excitatory/inhibitory neuron imbalance and abnormal behaviors. Antagonizing the increased inhibitory synaptic transmission partially alleviates their behavioral deficits. Together, our results suggest that the NR2F1-dependent imbalance of excitatory/inhibitory neuron differentiation caused by the activated Hedgehog pathway is one precursor of neurodevelopmental disorders and may enlighten the therapeutic approaches.