The role of the thyroid in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disease in women of childbearing age and can cause metabolic disorder, infertility, and increased anxiety and depression; as a result, it can seriously affect the physical and mental health of fertile women. PCOS is a highly clinically heterogeneous disease with unclear etiology and pathogenesis, which increases the difficulty of treatment. The thyroid gland has complex regulatory effects on metabolism, reproduction, and emotion, and produces hormones that act on almost all cells of the human body. The clinical manifestations of PCOS are similar to some thyroid diseases. Furthermore, some thyroid diseases, such as subclinical hypothyroidism (SCH), not only increase the incidence rate of PCOS, but also exacerbate its associated metabolic abnormalities and reproductive disorders. Interestingly, PCOS also increases the incidence of some thyroid diseases. However, the role of the thyroid in PCOS remains unclear. This review is intended to thoroughly explore the critical role of the thyroid in PCOS by summarizing the comorbidity of PCOS and thyroid diseases and their combined role in metabolic disorders, related metabolic diseases, and reproductive disorders; and by analyzing the potential mechanism through which the thyroid influences the development and progression of PCOS and its symptoms. We hope this review will provide a valuable reference for the role of the thyroid in PCOS.

Modeling disrupted synapse formation in wolfram syndrome using hESCs-derived neural cells and cerebral organoids identifies Riluzole as a therapeutic molecule.

Dysregulated neurite outgrowth and synapse formation underlie many psychiatric disorders, which are also manifested by wolfram syndrome (WS). Whether and how the causative gene WFS1 deficiency affects synapse formation remain elusive. By mirroring human brain development with cerebral organoids, WFS1-deficient cerebral organoids not only recapitulate the neuronal loss in WS patients, but also exhibit significantly impaired synapse formation and function associated with reduced astrocytes. WFS1 deficiency in neurons autonomously delays neuronal differentiation with altered expressions of genes associated with psychiatric disorders, and impairs neurite outgrowth and synapse formation with elevated cytosolic calcium. Intriguingly, WFS1 deficiency in astrocytes decreases the expression of glutamate transporter EAAT2 by NF-κB activation and induces excessive glutamate. When co-cultured with wildtype neurons, WFS1-deficient astrocytes lead to impaired neurite outgrowth and increased cytosolic calcium in neurons. Importantly, disrupted synapse formation and function in WFS1-deficient cerebral organoids and impaired neurite outgrowth affected by WFS1-deficient astrocytes are efficiently reversed with Riluzole treatment, by restoring EAAT2 expression in astrocytes. Furthermore, Riluzole rescues the depressive-like behavior in the forced swimming test and the impaired recognition and spatial memory in the novel object test and water maze test in Wfs1 conditional knockout mice. Altogether, our study provides novel insights into how WFS1 deficiency affects synapse formation and function, and offers a strategy to treat this disease.

ZnT8 loss-of-function accelerates functional maturation of hESC-derived ß cells and resists metabolic stress in diabetes.

Human embryonic stem cell-derived ß cells (SC-ß cells) hold great promise for treatment of diabetes, yet how to achieve functional maturation and protect them against metabolic stresses such as glucotoxicity and lipotoxicity remains elusive. Our single-cell RNA-seq analysis reveals that ZnT8 loss of function (LOF) accelerates the functional maturation of SC-ß cells. As a result, ZnT8 LOF improves glucose-stimulated insulin secretion (GSIS) by releasing the negative feedback of zinc inhibition on insulin secretion. Furthermore, we demonstrate that ZnT8 LOF mutations endow SC-ß cells with resistance to lipotoxicity/glucotoxicity-triggered cell death by alleviating endoplasmic reticulum (ER) stress through modulation of zinc levels. Importantly, transplantation of SC-ß cells with ZnT8 LOF into mice with preexisting diabetes significantly improves glycemia restoration and glucose tolerance. These findings highlight the beneficial effect of ZnT8 LOF on the functional maturation and survival of SC-ß cells that are useful as a potential source for cell replacement therapies.

Dnmt3a is required for the tumor stemness of B16 melanoma cells.

The relationship of carcinogenesis and DNA methyltransferases has attracted extensive attention in tumor research. We reported previously that inhibition of de novo DNA methyltransferase 3a (Dnmt3a) in murine B16 melanoma cells significantly suppressed tumor growth and metastasis in xenografted mouse model. Here, we further demonstrated that knockdown of Dnmt3a enhanced the proliferation in anchor-independent conditions of B16 cells, but severely disrupted its multipotent differentiation capacity in vitro. Furthermore, transforming growth factor ß1, a key trigger in stem cell differentiation and tumor cell epithelial-mesenchymal transition (EMT), mainly induced apoptosis, but not EMT in Dnmt3a-deficient B16 cells. These data suggested that Dnmt3a is required for maintaining the tumor stemness of B16 cells and it assists B16 cells to escape from death during cell differentiation. Thus it is hypothesized that not only extraordinary self-renewal ability, but also the capacity of multipotent differentiation is necessary for the melanoma tumorigenesis. Inhibition of multipotent differentiation of tumor cells may shed light on the tumor treatment.

POT1 inhibits the efficiency but promotes the fidelity of nonhomologous end joining at non-telomeric DNA regions.

Robust DNA double strand break (DSB) repair and stabilized telomeres help maintain genome integrity, preventing the onset of aging or tumorigenesis. POT1 is one of the six factors in the shelterin complex, which protects telomeres from being recognized as DNA damages. TRF1 and TRF2, two other shelterin proteins, have been shown to participate in DNA DSB repair at non-telomeric regions, but whether POT1, which binds to single strand telomeric DNA at chromosomal ends, is involved in DNA DSB repair has not been assessed. Here we found that POT1 arrives at DNA damage sites upon the occurrence of DNA DSBs. It suppresses the efficiency of nonhomologous end joining (NHEJ), the major pathway for fixing DNA DSBs in mammals, but surprisingly promotes NHEJ fidelity. Mechanistic studies indicate that POT1 facilitates the recruitment of Artemis, which is a nuclease and promotes fidelity of NHEJ, to DNA damage sites. In addition, we found that overexpression of POT1 inhibits the protein stability of Lig3, which is the major regulator of alternative NHEJ (alt-NHEJ), therefore suppressing the efficiency of alt-NHEJ. Taken together we propose that POT1 is a key factor regulating the balance between the efficiency and fidelity of NHEJ at non-telomeric DNA regions.

Fate tracing of hepatocytes in mouse liver.

Hepatocytes perform most of the functions of the liver and are considered terminally differentiated cells. Recently, it has been suggested that hepatocytes might have the potential to transdifferentiate or dedifferentiate under physiological or pathological conditions in vivo. Epithelial-mesenchymal transition of hepatocytes in liver fibrosis has also been proposed. However, these findings have not been fully confirmed. In this study, hepatocytes were genetically labelled for cell fate tracing using lacZ via the tamoxifen-induced CreERT/loxP system. After induction with tamoxifen, alb + cells were permanently marked by lacZ expression, and all progeny lacZ + cells were derived from a single source with no interference. We did not observe transdifferentiation or dedifferentiation of hepatocytes into cholangiocytes or hepatic progenitor cells under conditions of liver homeostasis or following a 2/3 partial hepatectomy. Meanwhile, lacZ/OPN-positive cells were observed in livers of 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed mice, and lacZ/alpha-smooth muscle actin-positive cells were detected in carbon tetrachloride-induced chronic liver injury models. These results suggested that some existing differentiated alb + cells might have the potential of transdifferentiation/dedifferentiation or epithelial-to-mesenchymal transition in vivo in some liver injury models, but the proportion of these alb + cells in liver was very low, and their significance and actual function during the pathological process remains to be elucidated.

N-myc is a key switch regulating the proliferation cycle of postnatal cerebellar granule cell progenitors.

N-myc plays an important role in early cerebellar development; however, the role of N-myc in postnatal cerebellar development is still unknown. In this study, inducible and reversible N-myc mouse models (Nmyc(TRE/TRE):tTS and Nmyc(EGFP/TRE):tTS) are used to regulate and track the expression of endogenous N-myc in vivo. Loss of N-myc at the neonatal stage results in reduced proliferation of granule cell precursors (GCPs) and reduced cerebellar volume/mass. Restoration of N-myc expression no later than postnatal day 4 can rescue the cerebellar developmental defect caused by the absence of N-myc after birth. During cerebellar postnatal development, N-myc acts as a key switch, regulating the proliferation cycle of postnatal granule cell progenitors. Loss of N-myc significantly impairs the Sonic hedgehog signalling pathway, and disrupts the expression of cell cycle effectors with a significant reduction of Ccnd2. More importantly, N-myc negatively regulates the expression of microRNA-9 during postnatal cerebellar development. Our findings demonstrate that over-expression of miR-9 can inhibit the proliferation of GCPs. The regulation of these factors by N-myc is at least partly responsible for the switch role of N-myc in the proliferation cycle of GCPs.

Parkin overexpression ameliorates hippocampal long-term potentiation and ß-amyloid load in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a severe decline of memory performance. A widely studied AD mouse model is the APPswe/PSEN1ΔE9 (APP/PS1) strain, as mice exhibit amyloid plaques as well as impaired memory capacities. To test whether restoring synaptic plasticity and decreasing ß-amyloid load by Parkin could represent a potential therapeutic target for AD, we crossed APP/PS1 transgenic mice with transgenic mice overexpressing the ubiquitin ligase Parkin and analyzed offspring properties. Overexpression of Parkin in APP/PS1 transgenic mice restored activity-dependent synaptic plasticity and rescued behavioral abnormalities. Moreover, overexpression of Parkin was associated with down-regulation of APP protein expression, decreased ß-amyloid load and reduced inflammation. Our data suggest that Parkin could be a promising target for AD therapy.

Overexpression of parkin ameliorates dopaminergic neurodegeneration induced by 1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice.

Mutations in the parkin gene are currently thought to be the most common cause of recessive familial Parkinsonism. Parkin functions as an E3 ligase to regulate protein turnover, and its function in mitochondrial quality control has been reported recently. Overexpression of parkin has been found to prevent neuronal degeneration under various conditions both in vivo and in vitro. Here, we generated a transgenic mouse model in which expression of wild type parkin was driven by neuron-specific enolase (NSE) promoter. We reported that both young and old parkin transgenic mice exhibited less reduction of striatal TH protein and number of TH positive neurons in the substantia nigra induced by 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP), compared to wild type littermates. MPTP-induced mitochondrial impairment in the substantia nigra was improved in young parkin transgenic mice. Decreased striatal α-synuclein was demonstrated in old parkin transgenic mice. These results provide reliable evidence from the transgenic mouse model for parkin that overexpression of parkin may attenuate dopaminergic neurodegeneration induced by MPTP through protection of mitochondria and reduction of α-synuclein in the nigrostriatal pathway.

Neuronal conditional knockout of NRSF decreases vulnerability to seizures induced by pentylenetetrazol in mice.

Neuron restrictive silencer factor (NRSF), also known as repressor element-1 silencing transcription factor, has been reported to modulate neuronal excitability and acts as endogenous anticonvulsant in kainic acid-induced or kindling-evoked seizure activity. However, whether NRSF functions in pentylenetetrazol (PTZ)-induced seizure activity has never been studied. To investigate the role of endogenous NRSF in the epileptogenesis induced by PTZ, in our experiment, NRSF neuronal conditional knockout mice (NRSF cKO) were adopted, in which NRSF was specifically deleted in neurons by the Cre-loxP system. Seizure threshold for PTZ, including the dose-response convulsions and the threshold dose, was compared between NRSF cKO and control mice. The threshold dose of PTZ that induced clonic and tonic seizures was significantly higher in NRSF cKO mice compared with the control. Similarly, the median lethal dose (LD(50)) of PTZ in NRSF cKO mice was also considerably higher than that of the control mice. These results revealed that NRSF cKO mice are of higher resistance to convulsions induced by PTZ. Our work first demonstrated the function of NRSF in PTZ-induced seizure and provided new evidence for differential pathways in diverse types of seizure.

Identification of a Smad4/YY1-recognized and BMP2-responsive transcriptional regulatory module in the promoter of mouse GABA transporter subtype I (Gat1) gene.

GABAergic dysfunction is implicated in a variety of neurodevelopmental and psychiatric disorders. The mechanisms underlying GABAergic differentiation, however, are not well understood. GABA transporter 1 (Gat1; Slc6a1) is an essential component of the GABAergic system, and its ectopic mRNA expression may be responsible for GABAergic malfunction under different pathological conditions. Thus, monitoring the transcriptional regulation of gat1 may help to elucidate the mechanisms that govern the differentiation of GABAergic neurons. In this study, we identified a promoter region that is sufficient to recapitulate endogenous gat1 expression in transgenic mice. A 46 bp cis-regulator in the promoter sequence was responsible for the stimulation of bone morphogenetic protein-2 (BMP2) on gat1 expression in cortical cortex. Furthermore, our study demonstrated that Smad4 and YY1 are physically bound to the element and mediate both the negative and positive regulatory effects in which BMP2 can affect the balance. In summary, we have identified a Smad4/YY1-based bidirectional regulation model for GABAergic gene transcription and demonstrated a molecular cue important for the differentiation of GABAergic neurons.

Functional imaging of interleukin 1 beta expression in inflammatory process using bioluminescence imaging in transgenic mice.

BACKGROUND: Interleukin 1 beta (IL-1beta) plays an important role in a number of chronic and acute inflammatory diseases. To understand the role of IL-1beta in disease processes and develop an in vivo screening system for anti-inflammatory drugs, a transgenic mouse line was generated which incorporated the transgene firefly luciferase gene driven by a 4.5-kb fragment of the human IL-1beta gene promoter. Luciferase gene expression was monitored in live mice under anesthesia using bioluminescence imaging in a number of inflammatory disease models. RESULTS: In a LPS-induced sepsis model, dramatic increase in luciferase activity was observed in the mice. This transgene induction was time dependent and correlated with an increase of endogenous IL-1beta mRNA and pro-IL-1beta protein levels in the mice. In a zymosan-induced arthritis model and an oxazolone-induced skin hypersensitivity reaction model, luciferase expression was locally induced in the zymosan injected knee joint and in the ear with oxazolone application, respectively. Dexamethasone suppressed the expression of luciferase gene both in the acute sepsis model and in the acute arthritis model. CONCLUSION: Our data suggest that the transgenic mice model could be used to study transcriptional regulation of the IL-1beta gene expression in the inflammatory process and evaluation the effect of anti-inflammatory drug in vivo.

Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice.

Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgenic animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibody preparation.

Reduced anxiety and depression-like behaviors in mice lacking GABA transporter subtype 1.

Gamma-aminobutyric acid (GABA) transporter subtype 1 (GAT1), which transports extracellular GABA into presynaptic neurons, plays an important regulatory role in the function of GABAergic systems. However, the contributions of the GAT1 in regulating mental status are not fully understood. In this paper, we observed the behavioral alterations of GAT1 knockout (GAT1(-/-)) mice using several depression- and anxiety-related models (eg, the forced-swimming test and the tail-suspension test for testing depression-related behaviors; the open-field test, the dark-light exploration test, the emergence test, and the elevated plus maze (EPM) test for anxiety-related behaviors). Here we found that GAT1(-/-) mice showed a lower level of depression- and anxiety-like behaviors in comparison to wild-type mice. Furthermore, GAT1(-/-) mice exhibited measurable insensitivity to selected antidepressants and anxiolytics such as fluoxetine, amitriptyline, buspirone, diazepam, and tiagabine in the tail-suspension test and/or the EPM test. Moreover, the basal level of corticosterone was found to be significantly lower in GAT1(-/-) mice. These results showed that the absence of GAT1 affects mental status through enhancing the GABAergic system, as well as modifying the serotonergic system and the hypothalamic-pituitary-adrenal (HPA) activity in mice.

Mice with genetically altered GABA transporter subtype I (GAT1) expression show altered behavioral responses to ethanol.

It is widely accepted that the GABAergic system plays an important role in the action of ethanol in vivo. GABA transporter subtype 1 (GAT1) constructs high affinity reuptake sites in the CNS and regulates GABAergic transmissions. In this study, mice lacking the GAT1 were developed by homologous recombination. Both hetero- and homozygous GAT1 mutant mice were tested for ethanol, saccharin or quinine consumption, ethanol-conditioned place preference, ethanol-conditioned taste aversion, ethanol-simulated motor activity, and ethanol-induced sedation/hypnosis. The GAT1(-/-) mice showed decreased ethanol aversion and ethanol reward, and insensitivity to both the sedative/hypnotic and the motor stimulant effects of ethanol, along with increased avoidance of quinine preference and consumption. GAT1(+/-) mice showed significantly increased consumption of ethanol and saccharin, however, enhanced the rewarding and preference effect of ethanol, increased avoidance of quinine, and higher sensitivity to the motor stimulant effect of ethanol. These results demonstrate that GAT1, perhaps in a bi-directional way, modulates some behavioral effects of ethanol. The GAT1 mutant mice provided us a very useful model to investigate the mechanisms of ethanol action in vivo.