GDNF Expression Research Articles

Crocin inhibits the activation of mouse hepatic stellate cells via the lnc-LFAR1/MTF-1/GDNF pathway

ABSTRACT Crocin is the main monomer of saffron, which is a momentous component of traditional Chinese medicine Lang Qing A Ta. Here, we tried to probe into the role of crocin in liver fibrosis. Hematoxylin-eosin staining and Sirius Red staining were used to observe the pathological changes of liver tissues. After hepatic stellate cells (HSCs) were isolated from liver tissues, lnc-LFAR1, MTF-1, GDNF, and α-SMA expressions were detected by qRT-PCR and western blot. Immunohistochemistry and immunofluorescence were used to detect α-SMA expression. Chromatin immunoprecipitation was used to analyze the binding of MTF-1 to the GDNF promoter. Moreover, the dual-luciferase reporter gene, RNA pull-down, and RNA immunoprecipitation were used to clarify the interaction between MTF-1 and GDNF, lnc-LFAR1 and MTF-1. The degree of liver fibrosis was more severe in the mice from the liver fibrosis model, while the liver fibrosis was alleviated by the injection of crocin. lnc-LFAR1, GDNF, and α-SMA were up-regulated, and MTF-1 was down-regulated in liver fibrosis tissues and cells, while these trends were reversed after the injection of crocin. Besides, lnc-LFAR1 negatively regulated MTF-1 expression, and positively regulated GDNF and α-SMA expressions, and MTF-1 was enriched in the promoter region of GDNF. Furthermore, the cellular direct interactions between MTF-1 and GDNF, lnc-LFAR1 and MTF-1 were verified. In vivo experiments confirmed the relief of crocin on liver fibrosis. Our research expounded that crocin restrained the activation of HSCs through the lnc-LFAR1/MTF-1/GDNF axis, thereby ameliorating liver fibrosis.

CD74 Promotes Perineural Invasion of Cancer Cells and Mediates Neuroplasticity Via the AKT/EGR-1/GDNF Axis in Pancreatic Ductal Adenocarcinoma

Background: Perineural invasion (PNI) is a common feature of pancreatic ductal adenocarcinoma (PDAC) and is one of the important causes of local recurrence in resected pancreatic cancer, but the molecular mechanism remains largely unexplored. Methods: Here, we used immunohistochemistry staining to determine the expression of CD74. Then the in vivo PNI model, in vitro neuroplasticity assay, cell proliferation assay, wound healing and Transwell-based invasion assay were performed to examine the function of CD74 in pancreatic cancer cell lines. ChIP assay and Luciferase reporter assay were used to illustrate the mechanism underlying CD74 induced GDNF expression. Findings: We confirmed that the expression level of CD74 was an independent predictor of PNI and poor prognosis for PDAC. Moreover, we found that upregulation of CD74 on PDAC enhanced its migration and invasive capabilities and potentiated the secretion of neurotrophic factor GDNF to promote the neuroplasticity. Mechanistically, CD74 promoted GDNF production via the AKT/EGR-1/GDNF axis in PDAC. Interpretation: Taken together, our findings suggest a supportive role of CD74 in the PNI of PDAC, and deepen our understanding of how cancer cells promote neuroplasticity in the microenvironment of PDAC. Funding: This work was supported by grants from National Natural Science Foundation of China No.81401931 (Jun-Feng Zhang), 81602414(Jian-Yu Yang), 81702739 (Xue-Liang Fu), 81802317(Min-Wei Yang), and additional grants from Science and Technology funds of School of Medicine, Shanghai Jiao Tong University No. 14XJ10022 (Jun-Feng Zhang), Scientific research project of Shanghai municipal health and Family Planning Commission No. 201740218 (Jun-Feng Zhang). Conflict of Interest: The authors have no conflicts of interest to declare. Ethical Approval: All the patients provided written informed consent before admission, and the study was approved by the Research Ethics Committee of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University.

Enhancer II-targeted dsRNA decreases GDNF expression via histone H3K9 trimethylation to inhibit glioblastoma progression

BackgroundGlial cell line-derived neurotrophic factor (GDNF) is expressed in both astrocytes and glioblastoma (GBM) cells. GDNF expression is significantly increased in GBM, and inhibiting its expression can retard GBM progression. However, there is no known method for specific inhibition of GDNF in GBM cells. MethodsPromoter-targeted dsRNA-induced transcriptional gene silencing or activation was recently achieved in human cells. This approach has the potential to specifically regulate gene transcription via epigenetic modifications. In this study, we designed six candidate dsRNAs targeting the enhancer or silencer in GDNF gene promoter II to check their effects on GDNF transcription and GBM progression. ResultsAmong these dsRNAs, enhancer II-targeted dsRNA significantly inhibited U251 GBM progression by downregulating GDNF (P < 0.05), while silencer II-targeted dsRNA exerted an opposite effect. Moreover, enhancer II-targeted dsRNA did not significantly change GDNF expression in human astrocytes (HA) and the proliferation and migration of HA cells (P > 0.05). Bisulfate PCR and chromatin immunoprecipitation analyses revealed that both DNA methylation and trimethylation of histone 3 at lysine 9 (H3K9me3) at silencer II-targeted region significantly increased, and H3K9me3 at enhancer II-targeted region significantly decreased, in U251 cells compared with HA cells in non-intervention condition (P < 0.05). Both enhancer II- and silencer II-targeted dsRNA significantly increased H3K9me3 methylation rather than DNA at the targeted site in U251 cells (P < 0.05). The expression and activity of histone methyltransferase SETDB1 increased dramatically in U251 cells compared with HA cells, and it was recruited to enhancer II targeting region after enhancer II-targeted dsRNA treatment in U251 cells (P < 0.05). ConclusionsOur results demonstrate that a promoter-targeted dsRNA can silence or promote gene transcription depending on its targeted site in different cis-acting elements in the gene promoter. Targeted inhibition of GDNF by enhancer II-targeted dsRNA may be explored as a novel treatment for GBM.

CARF promotes spermatogonial self-renewal and proliferation through Wnt signaling pathway

Collaborator of ARF (CARF) regulates cell proliferative fate through both p53-dependent and -independent mechanisms. Recently, we reported a new function of CARF as a positive regulator of Wnt signaling. Despite these findings, the physiological function of CARF has not been well studied. Here, we generated CARF knockout mice and found that male CARF−/− mice exhibited significantly impaired fertility and Sertoli-cell-only (SCO) syndrome phenotypes. Further studies revealed that loss of CARF in Sertoli cells led to decreased GDNF expression, which hindered spermatogonial stem cells (SSCs) self-renewal. Meanwhile, CARF loss in undifferentiated spermatogonia impaired their proliferation. These two mechanisms together led to SCO syndrome phenotypes, which could be functionally rescued by pharmacological or genetic reactivation of Wnt signaling. Finally, we identified CARFS351F as a potential pathogenic mutation in an SCO patient. Overall, our findings reveal important roles of CARF in spermatogonial self-renewal and proliferation through the Wnt signaling pathway.

CircFOXK2 Promotes Tumor Growth and Metastasis of Pancreatic Ductal Adenocarcinoma via Complexing with RNA Binding Proteins and Sponging MiR-942

Abstract Objective Circular RNA (circRNA) is a novel class of non-coding RNAs that regulate gene expression. However, the role of circRNAs in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. Design We performed circRNA sequencing of non-tumor HPDE and PDAC cells. We investigated the functions of circFOXK2 in PDAC by gain-of-function and loss-of-function assays. Bioinformatics analysis, luciferase assay and microRNA pulldown assays were performed to identify circFOXK2 interacting-miRNAs. To further investigate the mechanism, we performed circRNA-pulldown and mass spectrometry to identify circFOXK2-interacting proteins in PDAC. Results We identified 169 differentially expressed circRNAs in PDAC cells. We validated that one of the circRNAs circFOXK2 was significantly up-regulated in PDAC cells and in 63 % of primary tumor (53 out of 84). Gain-of-function and loss-of-function assays demonstrated that circFOXK2 promoted PDAC cell growth, migration and invasion. CircFOXK2 was also involved in cell cycle progression and apoptosis. circFOXK2 functioned as sponge for miR-942, and in turn promoted the expression of miR-942 targets ANK1, GDNF and PAX6. Furthermore, circFOXK2 interacted with 94 proteins, which were involved in cell adhesion and mRNA splicing. Among these circFOXK2-interacting proteins, YBX1 and hnRNPK were validated by RNA immunoprecipitation. Importantly, circFOKX2 interacted with YBX1 and hnRNPK targets NUF2 and PDXK in PDAC cells. Knockdown of circFOXK2 reduced the binding of YBX1 and hnRNPK to NUF2 and PDXK, and in turn decreased their expressions in PDAC cells. Conclusion We identified that circFOXK2 promoted PDAC cells growth and metastasis. Also, circFOXK2 complexed with YBX1 and hnRNPK to promote the expressions of oncogenic proteins. Significance of this study What is already known on this subject? Differentially expressed circRNAs are involved in carcinogenesis of many cancers. CircRNAs function as microRNA sponges to regulate gene expression. The roles of circRNAs in PDAC progression is largely unknown. What are the new findings? circFOXK2 is upregulated in PDAC primary tumors. circFOXK2 promotes PDAC tumor growth and liver metastasis. circFOXK2 functions as sponges for miR-942 to promote the expressions of oncogenic ANK1, GDNF and PAX6. circFOXK2 complexes with YBX1 and hnRNPK to promote the expressions of oncogenic proteins in PDAC. How might it impact on clinical practice in the foreseeable future? circFOXK2 upregulation in PDAC may function as a novel biomarker for diagnosis. circFOXK2 may be a novel therapeutic target in treating PDAC.

Effect of long-term treatment with a mixture of pyrethroids on the expression of genes that govern male germ cell production in rats.

Humans are exposed to pyrethroid-based pesticides through agricultural produce. In this study, male Wistar rats were orally treated for 9 to 12 months with a mixture of pyrethroids that is equivalent to one-fifth (high dose; HD) or one-twenty fifth (low dose; LD) of the amount of pyrethroids present in the cereals and rice consumed by an average Indian. In rats treated for 9 months, the spermatogenesis-associated genes Abp, Ar, Cd9, Dax1, Dazap1, Ddx3y, Gdnf, Gfra1, Grth, Inhb, Ovol1, P1, Plzf, Pygo2, Scf, Tgfb1, Tp1, Tp2, and Vim1 were downregulated in both LD and HD groups. In rats treated for 12 months Gdnf, Hsf2, Inhb, Tgfb1, Thy1, and Ybx2 expression was downregulated in both LD and HD groups. Steroidogenesis-associated genes 17-β-Hsd, Gata4, Hmgcr, Hmgcs1, Pde4b, and Tspo gene expression were reduced in both LD- and HD-treated groups treated for 9 months. In 12-month-treated rats, Creb1 expression decreased in both LD and HD groups. The epigenetic reprogramming-associated genes, Dnmt1, Dnmt3a, Dnmt3b, Hdac10, Hp1bp3, Kat3a Kat3b, Mch2ta, Ncoa7, and Sirt1 were downregulated in both HD and LD groups of 9-months-treated rats. In rats treated for 12 months, Hdac10, Mch2ta, Ncoa7, and Sirt1 messenger RNA levels decreased in both the HD and LD groups. Thus, we demonstrate that long-term exposure to a mixture of pyrethroids caused aberrations in the transcriptome of factors involved in sperm production and development.

Expression Analysis of GDNF/RET Signaling Pathway in Human AD-MSCs Grown in HEK 293 Conditioned Medium (HEK293-CM).

Mesenchymal stem cells have been considered as the suitable source for the repair of kidney lesions. The study and identification of novel approaches could improve the efficiency of these cells in the recovery of kidney. In the present study, the effect of HEK 293 conditioned medium (HEK293-CM) was evaluated on the expression of GDNF/RET signaling pathway and their downstream genes in the human adipose-derived mesenchymal stem cells (AD-MSCs). For this purpose, the human AD-MSCs were cultured in the medium containing HEK293-CM. After the RNA extraction and cDNA synthesis, the expression level of GFRA1, GDNF, SPRY1, ETV4, ETV5, and CRLF1 genes were determined by SYBR Green Real time PCR. The obtained results indicated that the GDNF and GFRA1 expression enhanced in the AD-MSCs following treatment with 10% HEK293-CM-5%FBS as compared to the untreated AD-MSCs. These results were consistent with the decreased expression of SPRY1. The significant increased expression of ETV4, ETV5, and CRLF1 genes also showed that HEK293-CM activated the GDNF/RET signaling pathway in the AD-MSCs (P < 0.05). The obtained data suggested that the treatment with HEK293-CM activated the GDNF/RET signaling pathway in the human AD-MSCs.

A Novel Anti-inflammatory Phenotype Transformed by Repetitive Low-dose Lipopolysaccharide in Primary Peritoneal Tissue-resident Macrophages.

Our previous studies suggested that oral administration of lipopolysaccharide (LPS) regulates the progression of various diseases via transformation of tissue-resident macrophages (MΦ). Recently, we characterized microglia transformed by repetitive low-dose LPS treatment (REPELL-microglia) in vitro, and this response was similar to that observed in response to oral administration of LPS in vivo. Here, we examined the characteristics of peritoneal tissue-resident MΦ (pMΦ) transformed by repetitive low-dose LPS treatment (REPELL-pMΦ). Primary pMΦ were treated with low-dose LPS (1 ng/ml) three times; subsequently, phagocytic activity and gene expression were evaluated. REPELL-pMΦ exhibited high phagocytic activity and elevated expression of Arg1, Gipr, Gdnf, and Fpr2. The gene expression profiles observed in REPELL-pMΦ were distinct from those of REPELL-microglia. REPELL-pMΦ have the potential to promote clearance of xenobiotics and to suppress inflammation. The present study also demonstrates the diversity of tissue-resident MΦ transformation that reflect their tissue origin.

MMP-9 Processing of Intestinal Smooth Muscle-derived GDNF is Required for Neurotrophic Action on Enteric Neurons

The neurotrophin GDNF guides development of the enteric nervous system (ENS) in embryogenesis and directs survival and axon outgrowth in postnatal myenteric neurons in vitro. GDNF expression in intestinal smooth muscle cells is dynamic, with upregulation by inflammatory cytokines in vitro or intestinal inflammation in vivo, but the role of post-translational proteolytic cleavage is undefined. In a co-culture model of myenteric neurons, smooth muscle and glia, inhibition of serine or cysteine protease activity was ineffective against the >2-fold increase in axon density caused by TNFα. However, inhibitors of metalloproteinases (MMP) identified an essential role of MMP-9, and qPCR and western blotting showed that pro-inflammatory cytokines increased both mRNA and protein expression for MMP-9, in both cellular lysates and conditioned medium (CM). Inhibition of MMP-9 prevented the cytokine-induced increase in mature GDNF in CM or cellular lysates of co-cultures or cell lines of intestinal smooth muscle cells (ISMC) from adult rat colon. Western blotting showed parallel upregulation of mature GDNF and MMP-9 vs control in ISMC isolated on Day 2 of TNBS-induced colitis. Nonetheless, transfection of GDNF plasmid into HEK-293 cells as a carrier system, or directly into the co-culture model, conveyed a strong neurotrophic effect that was MMP-9 dependent. We conclude that MMP-9 activity is required for the neurotrophic effects of GDNF on myenteric neurons in vitro. However, the coordinated upregulation of GDNF and MMP-9 in intestinal smooth muscle by inflammatory cytokines provides a supportive, target cell-derived environment that limits inflammatory damage to the ENS.

Localized delivery of CRISPR/dCas9 via layer-by-layer self-assembling peptide coating on nanofibers for neural tissue engineering

The clustered regularly interspaced short palindromic repeat (CRISPR) systems have a wide variety of applications besides precise genome editing. In particular, the CRISPR/dCas9 system can be used to control specific gene expression by CRISPR activation (CRISPRa) or interference (CRISPRi). However, the safety concerns associated with viral vectors and the possible off-target issues of systemic administration remain huge concerns to be safe delivery methods for CRISPR/Cas9 systems. In this study, a layer-by-layer (LbL) self-assembling peptide (SAP) coating on nanofibers is developed to mediate localized delivery of CRISPR/dCas9 systems. Specifically, an amphiphilic negatively charged SAP− is first coated onto PCL nanofibers through strong hydrophobic interactions, and the pDNA complexes and positively charged SAP+-RGD are then absorbed via electrostatic interactions. The SAPcoated scaffolds facilitate efficient loading and sustained release of the pDNA complexes, while enhancing cell adhesion and proliferation. As a proof of concept, the scaffolds are used to activate GDNF expression in mammalian cells, and the secreted GDNF subsequently promotes neurite outgrowth of rat neurons. These promising results suggest that the LbL self-assembling peptide coated nanofibers can be a new route to establish a bioactive interface, which provides a simple and efficient platform for the delivery of CRISPR/dCas9 systems for regenerative medicine.

Molecular targets for endogenous glial cell line-derived neurotrophic factor modulation in striatal parvalbumin interneurons

Administration of recombinant glial cell line-derived neurotrophic factor into the putamen has been tested in preclinical and clinical studies to evaluate its neuroprotective effects on the progressive dopaminergic neuronal degeneration that characterizes Parkinson’s disease. However, intracerebral glial cell line-derived neurotrophic factor infusion is a challenging therapeutic strategy, with numerous potential technical and medical limitations. Most of these limitations could be avoided if the production of endogenous glial cell line-derived neurotrophic factor could be increased. Glial cell line-derived neurotrophic factor is naturally produced in the striatum from where it exerts a trophic action on the nigrostriatal dopaminergic pathway. Most of striatal glial cell line-derived neurotrophic factor is synthesized by a subset of GABAergic interneurons characterized by the expression of parvalbumin. We sought to identify molecular targets specific to those neurons and which are putatively associated with glial cell line-derived neurotrophic factor synthesis. To this end, the transcriptomic differences between glial cell line-derived neurotrophic factor-positive parvalbumin neurons in the striatum and parvalbumin neurons located in the nearby cortex, which do not express glial cell line-derived neurotrophic factor, were analysed. Using mouse reporter models, we have defined the genomic signature of striatal parvalbumin interneurons obtained by fluorescence-activated cell sorting followed by microarray comparison. Short-listed genes were validated by additional histological and molecular analyses. These genes code for membrane receptors (Kit, Gpr83, Tacr1, Tacr3, Mc3r), cytosolic proteins (Pde3a, Crabp1, Rarres2, Moxd1) and a transcription factor (Lhx8). We also found the proto-oncogene cKit to be highly specific of parvalbumin interneurons in the non-human primate striatum, thus highlighting a conserved expression between species and suggesting that specific genes identified in mouse parvalbumin neurons could be putative targets in the human brain. Pharmacological stimulation of four G-protein-coupled receptors enriched in the striatal parvalbumin interneurons inhibited Gdnf expression presumably by decreasing cyclic adenosine monophosphate formation. Additional experiments with pharmacological modulators of adenylyl cyclase and protein kinase A indicated that this pathway is a relevant intracellular route to induce Gdnf gene activation. This preclinical study is an important step in the ongoing development of a specific pro-endo-glial cell line-derived neurotrophic factor pharmacological strategy to treat Parkinson’s disease.

EZH2 expression and its role in spermatogonial stem cell self-renewal in goats

Enhancer of zeste homolog 2 (EZH2) is a histone H3 lysine 27 (H3K27) methyltransferase that plays vital roles in mouse spermatogenesis. However, the expression pattern and role of EZH2 in goat spermatogonial stem cells (SSCs) is unknown. In the present study, we investigated EZH2 expression in the testis of postpubertal goats and its effect on the biological characteristics of goat SSCs. We found that EZH2 mRNA (P < 0.01) and protein (P < 0.05) expression was increased in the testes of postpubertal goats compared to that of prepubertal goats. Moreover, EZH2 was more highly expressed in goat SSCs than in Leydig cells (P < 0.01) and Sertoli cells (P < 0.01) as determined by qPCR, Western blot, and immunofluorescence. Compared to a negative control (NC), cell proliferation (P < 0.01) and viability (P < 0.01) were decreased in SSCs in which EZH2 was knocked down, and the G2/M phase of the cell cycle was blocked (P < 0.01), as determined by Edu staining, CCK-8 assay, and flow cytometry analysis. Additionally, the expression of CASP3, CASP9, and BAX was significantly increased (P < 0.01) while BCL2 expression was decreased (P < 0.01) in EZH2 knockdown SSCs. Notably, the expression of GDNF, a SSCs marker gene, and DAZL, a spermatogenesis-related gene, were significantly decreased (P < 0.01) while GFRA1 expression was significantly up-regulated (P < 0.01) in EZH2 knockdown SSCs. Our data suggest that EZH2 plays a pivotal role in the self-renewal of goat SSCs, and knockdown of EZH2 might impair spermatogenesis in goats.

FBXW7alpha Promotes the Recovery of Traumatic Spinal Cord.

White matter damage and neuronal cell death are incurred by spinal cord injury (SCI). FBXW7α, an important mediator of cell division and growth was investigated to explore its role in repairing the traumatic spinal cord in rats. Underlying mechanisms such as oxidative stress and inflammasomes signaling were also studied. Spinal cord injury in rats was established by longitudinal surgical incision from the lower to mid-thoracic vertebrae on the backside, followed by 20-g weight placed on the exposed Th12 surface for 30 min. AAV-delivered FBXW7α and -sh-FBXW7α were intrathecally injected into the rat spinal cord. Indices of oxidation, neurotrophic factors, and pyroptosis were measured by Western blot, Elisa, and RT-PCR. We found the overexpression of FBXW7α in spinal cord rescue neuronal death triggered by the injury. Specifically, the nutritional condition, oxidative stress, and pyroptosis were improved. A synchronization of BNDF and GDNF expression patterns in various groups indicated the secretion of neurotrophic factors affect the outcome of SCI. The SOD1, CAT, and GSH-px were suppressed after trauma but all restored in response to FBXW7α overexpression. Inflammasomes-activated pyroptosis was incurred after the injury, and relevant biomarkers such as GSDMD, caspase-1, caspase- 11, IL-1β, and IL-18 were down-regulated after the introduction of FBXW7α into the injured cord. Additionally, up-regulating FBXW7α also repaired the mitochondria dysfunction. Our data indicate FBXW7α probably serves as an important molecular target for the therapy of spinal cord injury.

Analysis of structure and gene expression in developing kidneys of male and female rats exposed to low protein diets in utero.

A maternal low protein (LP) diet in rodents often results in low nephron endowment and renal pathophysiology in adult life, with outcomes often differing between male and female offspring. Precisely how a maternal LP diet results in low nephron endowment is unknown. We conducted morphological and molecular studies of branching morphogenesis and nephrogenesis to identify mechanisms and timepoints that might give rise to low nephron endowment. Sprague-Dawley rats were fed a normal protein (19.4% protein, NP) or LP (9% protein) diet for 3 weeks prior to mating and throughout gestation. Embryonic day 14.25 (E14.25) kidneys from males and females were either cultured for 2 days after which branching morphogenesis was quantified, or frozen for gene expression analysis. Real-time PCR was used to quantify expression of key nephrogenesis and branching morphogenesis genes at E14.25 and 17.25. At E17.25, nephron number was determined in fixed tissue. There was no effect of either maternal diet or sex on branching morphogenesis. Nephron number at E17.25 was 14% lower in male and female LP offspring than in NP controls. At E14.25 expression levels of genes involved in branching morphogenesis (Gfrα1, Bmp4, Gdnf) and nephrogenesis (Hnf4a, Pax2, Wnt4) were similar in the dietary groups, but significant differences between sexes were identified. At E17.25, expression of Gfrα1, Gdnf, Bmp4, Pax2 and Six2 was lower in LP offspring than NP offspring, in both male and female offspring. These findings provide new insights into how a LP diet leads to low nephron endowment and renal sexual dimorphism.

Quercetin treatment reduces the severity of renal dysplasia in a beta-catenin dependent manner.

Renal dysplasia, the major cause of childhood renal failure, is characterized by defective branching morphogenesis and nephrogenesis. Beta-catenin, a transcription factor and cell adhesion molecule, is markedly increased in the nucleus of kidney cells in human renal dysplasia and contributes to its pathogenesis by altering target genes that are essential for kidney development. Quercetin, a naturally occurring flavonoid, reduces nuclear beta-catenin levels and reduces beta-catenin transcriptional activity. In this study, we utilized wild type and dysplastic mouse kidney organ explants to determine if quercetin reduces beta-catenin activity during kidney development and whether it improves the severity of renal dysplasia. In wild type kidney explants, quercetin treatment resulted in abnormal branching morphogenesis and nephrogenesis in a dose dependent manner. In wild type embryonic kidneys, quercetin reduced nuclear beta-catenin expression and decreased expression of beta-catenin target genes Pax2, Six2, and Gdnf, which are essential for kidney development. Our RDB mouse model of renal dysplasia recapitulates the overexpression of beta-catenin and histopathological changes observed in human renal dysplasia. RDB kidneys treated with quercetin resulted in improvements in the overall histopathology, tissue organization, ureteric branching morphogenesis, and nephrogenesis. Quercetin treatment also resulted in reduced nuclear beta-catenin and reduced Pax2 expression. These improvements were associated with the proper organization of vimentin, NCAM, and E-cadherin, and a 45% increase in the number of developing and maturing nephrons. Further, our results show that in human renal dysplasia, beta-catenin, vimentin, and e-cadherin also have abnormal expression patterns. Taken together, these data demonstrate that quercetin treatment reduces nuclear beta-catenin and this is associated with improved epithelial organization of developing nephrons, resulting in increased developing nephrons and a partial rescue of renal dysplasia.

Combining timed GDNF and ChABC gene therapy to promote long-distance regeneration following ventral root avulsion and repair.

Ventral root avulsion leads to severe motoneuron degeneration and prolonged distal nerve denervation. After a critical period, a state of chronic denervation develops as repair Schwann cells lose their pro-regenerative properties and inhibitory factors such as CSPGs accumulate in the denervated nerve. In rats with ventral root avulsion injuries, we combined timed GDNF gene therapy delivered to the proximal nerve roots with the digestion of inhibitory CSPGs in the distal denervated nerve using sustained lentiviral-mediated chondroitinase ABC (ChABC) enzyme expression. Following reimplantation of lumbar ventral roots, timed GDNF-gene therapy enhanced motoneuron survival up to 45weeks and improved axonal outgrowth, electrophysiological recovery, and muscle reinnervation. Despite a timed GDNF expression period, a subset of animals displayed axonal coils. Lentiviral delivery of ChABC enabled digestion of inhibitory CSPGs for up to 45weeks in the chronically denervated nerve. ChABC gene therapy alone did not enhance motoneuron survival, but led to improved muscle reinnervation and modest electrophysiological recovery during later stages of the regeneration process. Combining GDNF treatment with digestion of inhibitory CSPGs did not have a significant synergistic effect. This study suggests a delicate balance exists between treatment duration and concentration in order to achieve therapeutic effects.

Role of pyroglutamylated RFamide peptide43 in germ cell dynamics in mice testes in relation to energy metabolism

QRFP is a neuropeptide that regulates glucose homeostasis and increases insulin sensitivity in tissues. We have previously shown that QRFP and its receptor (GPR103) are predominantly expressed in germ cells and Sertoli cells, respectively, in mice testes. In the present study, we report that QRFP caused an increase in PCNA and a decrease in p27Kip1 expressions in the testis under both in vivo and ex vivo conditions. Besides, via an in vivo study, cell cycle analysis by FACS showed an increase in 2C cells and a decrease in 1C cells. QRFP also induced expression of GDNF and phosphorylation of Akt and ERK-1/2. Together these results suggest that QRFP has a proliferative effect on germ cells in mice testes, since it caused a proportional increase in the mitotic activity and the number of spermatogonial cells. Further, observations of increased expressions of STAT-3 and Neurog3 in treated mice suggest that QRFP treatment regulates priming of undifferentiated spermatogonia to undergo differentiation, while a decrease in c-Kit expression indicate that spermatogonia at this time point are in an undifferentiated state. In addition, QRFP administration also caused an increase in intratesticular levels of glucose and lactate, and in LDH activity accompanied by increased expressions of GLUT-3 and LDH-C in the testis. Also, the phosphorylation of IR-β and expressions of p-Akt and p-mTOR were increased under ex vivo conditions in testicular tissue. In conclusion, our findings suggest that QRFP treatment caused proliferation of germ cells independently from the hypothalamic-pituitary axis via regulation of testicular energy metabolism.

CircFOXK2 Promotes Growth and Metastasis of Pancreatic Ductal Adenocarcinoma by Complexing with RNA-Binding Proteins and Sponging MiR-942

The detailed biological functions of circular RNA (circRNA) are largely unexplored. Using circRNA sequencing, we identified 169 differentially expressed circRNA in pancreatic ductal adenocarcinoma (PDAC) cells compared with nontumor human pancreatic ductal epithelial cells. Among them, circFOXK2 was validated with significant upregulation in PDAC cells and 63% of primary tumors (53 of 84). circFOXK2 promoted cell growth, migration, and invasion and was involved in cell-cycle progression and apoptosis. circFOXK2 contained multiple miRNA binding sites, functioning as a sponge for miR-942, which in turn promoted expression of ANK1, GDNF, and PAX6. A novel and highly specific circRNA-pulldown followed by mass spectrometry analysis identified 94 circFOXK2-interacting proteins, which were involved in cell adhesion, mRNA splicing, and structural molecule activity. Of these, circFOKX2 interactions with YBX1 and hnRNPK enhanced expression of oncogenes NUF2 and PDXK. Knockdown of circFOXK2 reduced binding of YBX1 and hnRNPK to NUF2 and PDXK, in turn decreasing their expression. Collectively, our findings demonstrate that circFOXK2 in complex with YBX1 and hnRNPK promotes expression of oncogenic proteins that contribute to PDAC progression. SIGNIFICANCE: This study reveals a prominent role for the circRNA circFOXK2 in PDAC progression, suggesting that circFOXK2 might be a novel diagnostic marker for PDAC.

Lauric acid promotes neuronal maturation mediated by astrocytes in primary cortical cultures

Previous studies have suggested the potential efficacy of middle chain fatty acids (MCFAs) in the treatment of mood disorders and cognitive dysfunction. MCFAs are metabolized to ketone bodies in astrocytes; however, their effects on neuronal development including neurotrophic factor level are not well-understood. In the present study, we examined the effect of MCFAs on the mRNA expression of growth factors and cytokines in primary cultures of cortical astrocytes. The effect of MCFAs on neuron-astrocyte interaction in neuronal maturation was also determined using co-culture and astrocyte-conditioned medium. Lauric acid (LA) typically increased the mRNA expression of glial-derived neurotrophic factor (Gdnf), interleukin-6 (Il6), and C–C motif chemokine 2 (Ccl2) in astrocytes. LA-induced phosphorylation of extracellular signal-regulated kinase contributed to these changes. In primary cultures of cortical neurons containing astrocytes, LA enhanced the presynaptic protein levels. Astrocyte-conditioned medium after LA treatment also enhanced the presynaptic protein levels in the cortical neuron cultures. These results suggest that LA increase the mRNA expression of GDNF and cytokines in astrocytes, and thereby, enhances the presynaptic maturation.

Nerve impulse transmission pathway-focused genes expression analysis in patients with primary hypothyroidism and autoimmune thyroiditis.

Thyroid hormones have important actions in the adult brain. They regulate genes expression in myelination, differentiation of neuronal and glial cells, and neuronal viability and function. We used the pathway-specific real-time PCR array (Neurotrophins and Receptors RT2 Profiler PCR Array, QIAGEN, Germany) to identify and verify nerve impulse transmission pathway-focused genes expression in peripheral white blood cells of patients with postoperative hypothyroidism, hypothyroidism as a result of autoimmune thyroiditis (AIT) and AIT with elevated serum an anti-thyroglobulin (anti-Tg) and anti-thyroid peroxidase (anti-TPO) antibodies. It was shown that patients with postoperative hypothyroidism and hypothyroidism resulting from AIT had significantly lower expression of BDNF and CBLN1. In patients with AIT with elevated serum anti-Tg and anti-TPO antibodies, the expression of GDNF was significantly down-regulated and the expression of PNOC was up-regulated. The expression levels of MEF2C and NTSR1 were decreased in the group of patients with postoperative hypothyroidism and AIT, correspondingly. The results of this study demonstrate that AIT and hypothyroidism can affect the expression of mRNA nerve impulse transmission genes in gene specific manner and that these changes in gene expressions can be playing a role in the development of neurological complications associated with thyroid pathology. Detection of the transcriptional activity of nerve impulse transmission genes in peripheral white blood cells can be used as an important minimally invasive prognostic marker of the risk for developing neurological complications comorbid with thyroid pathology.