Literature context: ling Technology, Cat# 9664, RRID:AB_2070042), or Î²-actin (Santa Cruz, Cat#
Alzheimer's disease (AD) is the most common form of dementia that is often accompanied by mood and emotional disturbances and seizures. There is growing body of evidence that neurons expressing γ-aminobutyric acid (GABA) play an important role in regulation of cognition, mood, and emotion as well as seizure susceptibility, but participation of GABAergic neuronal pathology in Alzheimer's disease (AD) is not understood well at present. Here, we report that transgenic mice expressing human amyloid precursor protein Swedish-Dutch-Iowa mutant (APPSweDI) exhibit early loss of neurons expressing GAD67, a GABA-synthesizing enzyme, in advance of the loss of pyramidal neurons in hippocampal CA1 region. The loss of GAD67+ neurons in APPSweDI mice accompanied with decreased spatial cognition as well as increased anxiety-like behaviors and kainic acid-induced seizure susceptibility at early phase. In the hippocampal CA1 region, GAD67+ neurons expressed high basal levels of neuronal nitric oxide synthase (nNOS) and nitrosative stress (nitrotyrosine). Similarly, GAD67+ neurons in primary cortical and hippocampal neuron cultures also expressed high basal levels of nNOS and degenerated in response to lower Aβ concentrations due to their high basal levels of nitrosative stress. Given the role of GABAergic neurons in cognitive and neuropsychiatric functions, this study reports the role of nNOS-mediated nitrosative stress in dysfunction of GABAergic neurons and its potential participation in early development of cognitive and neuropsychiatric symptoms in AD.
Literature context: Signaling Technology Cat# 9664, RRID:AB_2070042 Polyclonal rabbit anti-Rnf12 Me
A hallmark of naive pluripotency is the presence of two active X chromosomes in females. It is not clear whether prevention of X chromosome inactivation (XCI) is mediated by gene networks that preserve the naive state. Here, we show that robust naive pluripotent stem cell (nPSC) self-renewal represses expression of Xist, the master regulator of XCI. We found that nPSCs accumulate Xist on the male X chromosome and on both female X chromosomes as they become NANOG negative at the onset of differentiation. This is accompanied by the appearance of a repressive chromatin signature and partial X-linked gene silencing, suggesting a transient and rapid XCI-like state in male nPSCs. In the embryo, Xist is transiently expressed in males and in females from both X chromosomes at the onset of naive epiblast differentiation. In conclusion, we propose that XCI initiation is gender independent and triggered by destabilization of naive identity, suggesting that gender-specific mechanisms follow, rather than precede, XCI initiation.
Literature context: tibodiesCleaved caspase 3 (Cat# 9664) and insulin antibodies (Cat# 4
Lipotoxicity is associated with a high level of fatty acid accumulation in pancreatic β-cells. An overload of free fatty acids contributes to pancreatic β-cell apoptosis and dysfunction. Insulin secretion involves sequential ionic events upon glucose stimulation. ATP sensitive potassium (KATP) channels serve as glucose sensors and effectively initiate glucose-stimulated insulin secretion. This study investigated the effects of lipotoxicity on the trafficking of KATP channels in pancreatic β cells using chronic palmitic acid -injected mice and treated insulinoma cells. The chronic palmitic acid -injected mice displayed type II diabetic characteristics. The pancreatic sections of these mice exhibited a decrease in the expression of KATP channels. We then tested the time and dose effects of palmitic acid on the cell viability of INS-1 cells. We observed a significant decrease in the surface expression of KATP channels after 72 h of treatment with 0.4 mM palmitic acid. In addition, this treatment induced pancreatic β-cell apoptosis by increasing cleaved caspase 3 protein level. Our results demonstrated cotreatment with glibenclamide, the sulfonylurea compounds for type II diabetes mellitus, in palmitic acid -treated cells reduces cell death and recovers the glucose stimulated insulin secretion through increasing the surface expression of KATP channels. Importantly, glibenclamide also improved glucose tolerance, triglyceride concentration, and insulin sensitivity in the palmitic acid-injected mice. In conclusion, an increase in the surface expression of KATP channels restores insulin secretion, reduces pancreatic β-cell's apoptosis, highlighting correct trafficking of KATP channels is important in survival of β-cells during lipotoxicity.
Literature context: ignaling Technology clone 5A1E; RRID:AB_2070042 mouse Î±- Î±-MEK1 Cell Signaling
Hematopoietic stem cells (HSCs) sustain hematopoiesis throughout life. HSCs exit dormancy to restore hemostasis in response to stressful events, such as acute blood loss, and must return to a quiescent state to prevent their exhaustion and resulting bone marrow failure. HSC activation is driven in part through the phosphatidylinositol 3-kinase (PI3K)/AKT/mTORC1 signaling pathway, but less is known about the cell-intrinsic pathways that control HSC dormancy. Here, we delineate an ERK-dependent, rate-limiting feedback mechanism that controls HSC fitness and their re-entry into quiescence. We show that the MEK/ERK and PI3K pathways are synchronously activated in HSCs during emergency hematopoiesis and that feedback phosphorylation of MEK1 by activated ERK counterbalances AKT/mTORC1 activation. Genetic or chemical ablation of this feedback loop tilts the balance between HSC dormancy and activation, increasing differentiated cell output and accelerating HSC exhaustion. These results suggest that MEK inhibitors developed for cancer therapy may find additional utility in controlling HSC activation.
Literature context: aved caspase 3 mAb 1 : 500 (WB) RRID:AB_2070042 9664S Cell Signaling Technology
Menopause, a risk factor for brain dysfunction in women, is characterized by neuropsychological symptoms including depression and dementia, which are closely related to alterations in different brain regions after menopause. However, little is known about the variability of pathophysiologic changes associated with menopause in the brain. Here, we observed that menopause in rats induced by bilateral ovariectomy (OVX) showed depressive and dementia-related behaviors along with neuronal loss in the prefrontal cortex (PFC), hippocampus (HIP), hypothalamus (HYP) and amygdala (AMY) by Nissl staining. Meanwhile, by immunohistochemical staining, increased microglia in the HIP and AMY and increased astrocytes in the PFC, HYP and AMY were shown. By using quantitative proteomics, we identified 146 differentially expressed proteins in the brains of OVX rats, e.g., 20 in the PFC, 41 in the HIP, 17 in the HYP and 79 in the AMY, and performed further detection by Western blotting. A link between neuronal loss and apoptosis was suggested, as evidenced by increases in adenylate kinase 2 (AK2), B-cell lymphoma 2 associated X (Bax), cleaved caspase-3 and phosphorylated p53 and decreases in Huntingtin-interacting protein K (HYPK), hexokinase (HK), and phosphorylated B-cell lymphoma 2 (Bcl-2), and apoptosis might be triggered by endoplasmic reticulum stress (probed by increased glucose-regulated protein 78 (GRP78), cleaved caspase-12, phosphorylated protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE-1) and activating transcription factor 6 (ATF6)) and mitochondrial dysfunction (probed by increased cytochrome c and cleaved caspase-3 and decreased sideroflexin-1 (SFXN1) and NADH dehydrogenase (ubiquinone) 1 α subcomplex 11 (NDUFA11)). Activation of autophagy was also indicated by increased autophagy-related 7 (ATG7), γ-aminobutyric acid (GABA) receptor-associated protein-like 2 (GABARAPL2) and oxysterol-binding protein-related protein 1 (ORP1) and confirmed by increased microtubule-associated protein light chain 3 (LC3II/I), autophagy-related 5 (ATG5), and Beclin1 in the HIP and AMY. In the AMY, which is important in emotion, higher GABA transporter 3 (GAT3) and lower vesicular glutamate transporter 1 (VgluT1) levels indicated an imbalance between excitatory and inhibitory neurotransmission, and the increased calretinin and decreased calbindin levels suggested an adjustment of GABAergic transmission after OVX. In addition, cytoskeletal abnormalities including tau hyperphosphorylation, dysregulated Ca²+ signals and glutamic synaptic impairments were observed in the brains of OVX rats. Collectively, our study showed the changes in different brain regions related to depression and dementia during menopause. This article is protected by copyright. All rights reserved.
Literature context: Signaling technology Cat#9664; RRID:AB_2070042 Mouse monoclonal Î³H2AX clone JB
DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4-/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4-/-p53+/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.
Literature context: ase-3 Cell Signaling Cat# 9664; RRID:AB_2070042 Goat anti-Chicken IgY (H+L) Sec
Intestinal infection triggers potent immune responses to combat pathogens and concomitantly drives epithelial renewal to maintain barrier integrity. Current models propose that epithelial renewal is primarily driven by damage caused by reactive oxygen species (ROS). Here we found that in Drosophila, the Imd-NF-κB pathway controlled enterocyte (EC) shedding upon infection, via a mechanism independent of ROS-associated apoptosis. Mechanistically, the Imd pathway synergized with JNK signaling to induce epithelial cell shedding specifically in the context of bacterial infection, requiring also the reduced expression of the transcription factor GATAe. Furthermore, cell-specific NF-κB responses enabled simultaneous production of antimicrobial peptides (AMPs) and epithelial shedding in different EC populations. Thus, the Imd-NF-κB pathway is central to the intestinal antibacterial response by mediating both AMP production and the maintenance of barrier integrity. Considering the similarities between Drosophila Imd signaling and mammalian TNFR pathway, our findings suggest the existence of an evolutionarily conserved genetic program in immunity-induced epithelial shedding.
Literature context: RRID:AB_2070042 Rabbit anti-SQSTM1/p62 Thermo S
Obesity is a leading risk factor for cancer. However, understanding the crosstalk between adipocytes and tumor cells in vivo, independently of dietary contributions, is a major gap in the field. Here we used a prostate cancer (PCa) mouse model in which the signaling adaptor p62/Sqstm1 is selectively inactivated in adipocytes. p62 loss in adipocytes results in increased osteopontin secretion, which mediates tumor fatty acid oxidation and invasion, leading to aggressive metastatic PCa in vivo. Furthermore, p62 deficiency triggers in adipocytes a general shutdown of energy-utilizing pathways through mTORC1 inhibition, which supports nutrient availability for cancer cells. This reveals a central role of adipocyte's p62 in the symbiotic adipose tissue-tumor collaboration that enables cancer metabolic fitness.
Literature context: Signaling Technology Cat# 9664, RRID:AB_2070042 Mouse monoclonal anti Pax6 Deve
Recurrent mutations in chromatin modifiers are specifically prevalent in adolescent or adult patients with Sonic hedgehog-associated medulloblastoma (SHH MB). Here, we report that mutations in the acetyltransferase CREBBP have opposing effects during the development of the cerebellum, the primary site of origin of SHH MB. Our data reveal that loss of Crebbp in cerebellar granule neuron progenitors (GNPs) during embryonic development of mice compromises GNP development, in part by downregulation of brain-derived neurotrophic factor (Bdnf). Interestingly, concomitant cerebellar hypoplasia was also observed in patients with Rubinstein-Taybi syndrome, a congenital disorder caused by germline mutations of CREBBP. By contrast, loss of Crebbp in GNPs during postnatal development synergizes with oncogenic activation of SHH signaling to drive MB growth, thereby explaining the enrichment of somatic CREBBP mutations in SHH MB of adult patients. Together, our data provide insights into time-sensitive consequences of CREBBP mutations and corresponding associations with human diseases.
Literature context: Signaling Technology Cat# 9664 RRID:AB_2070042 Goat anti-IBA1 Abcam Cat# ab507
Mutations in triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to increased Alzheimer's disease (AD) risk. Neurobiological functions of TREM2 and its pathophysiological ligands remain elusive. Here we found that TREM2 directly binds to β-amyloid (Aβ) oligomers with nanomolar affinity, whereas AD-associated TREM2 mutations reduce Aβ binding. TREM2 deficiency impairs Aβ degradation in primary microglial culture and mouse brain. Aβ-induced microglial depolarization, K+ inward current induction, cytokine expression and secretion, migration, proliferation, apoptosis, and morphological changes are dependent on TREM2. In addition, TREM2 interaction with its signaling adaptor DAP12 is enhanced by Aβ, regulating downstream phosphorylation of SYK and GSK3β. Our data demonstrate TREM2 as a microglial Aβ receptor transducing physiological and AD-related pathological effects associated with Aβ.
Literature context: Signaling Technology Cat# 9664; RRID:AB_2070042 Rabbit anti-Î²-Catenin (D10A8) C
The underlying mechanisms of how self-renewing cells are controlled in regenerating tissues and cancer remain ambiguous. PCNA-associated factor (PAF) modulates DNA repair via PCNA. Also, PAF hyperactivates Wnt/β-catenin signaling independently of PCNA interaction. We found that PAF is expressed in intestinal stem and progenitor cells (ISCs and IPCs) and markedly upregulated during intestinal regeneration and tumorigenesis. Whereas PAF is dispensable for intestinal homeostasis, upon radiation injury, genetic ablation of PAF impairs intestinal regeneration along with the severe loss of ISCs and Myc expression. Mechanistically, PAF conditionally occupies and transactivates the c-Myc promoter, which induces the expansion of ISCs/IPCs during intestinal regeneration. In mouse models, PAF knockout inhibits Apc inactivation-driven intestinal tumorigenesis with reduced tumor cell stemness and suppressed Wnt/β-catenin signaling activity, supported by transcriptome profiling. Collectively, our results unveil that the PAF-Myc signaling axis is indispensable for intestinal regeneration and tumorigenesis by positively regulating self-renewing cells.
Literature context: Signaling Technology Cat#9664S; RRID:AB_2070042 Chicken polyclonal anti-GFP Ave
Phosphatidylinositol (PtdIns) transfer proteins (PITPs) stimulate PtdIns-4-P synthesis and signaling in eukaryotic cells, but to what biological outcomes such signaling circuits are coupled remains unclear. Herein, we show that two highly related StART-like PITPs, PITPNA and PITPNB, act in a redundant fashion to support development of the embryonic mammalian neocortex. PITPNA/PITPNB do so by driving PtdIns-4-P-dependent recruitment of GOLPH3, and likely ceramide transfer protein (CERT), to Golgi membranes with GOLPH3 recruitment serving to promote MYO18A- and F-actin-directed loading of the Golgi network to apical processes of neural stem cells (NSCs). We propose the primary role for PITP/PtdIns-4-P/GOLPH3/CERT signaling in NSC Golgi is not in regulating bulk membrane trafficking but in optimizing apically directed membrane trafficking and/or apical membrane signaling during neurogenesis.
Literature context: se-3 CST Cat# 9664; RRID:AB_2070042 Rabbit anti-human PARP CST Cat#
Carcinoma-associated fibroblasts (CAFs) are abundant and heterogeneous stromal cells in tumor microenvironment that are critically involved in cancer progression. Here, we demonstrate that two cell-surface molecules, CD10 and GPR77, specifically define a CAF subset correlated with chemoresistance and poor survival in multiple cohorts of breast and lung cancer patients. CD10+GPR77+ CAFs promote tumor formation and chemoresistance by providing a survival niche for cancer stem cells (CSCs). Mechanistically, CD10+GPR77+ CAFs are driven by persistent NF-κB activation via p65 phosphorylation and acetylation, which is maintained by complement signaling via GPR77, a C5a receptor. Furthermore, CD10+GPR77+ CAFs promote successful engraftment of patient-derived xenografts (PDXs), and targeting these CAFs with a neutralizing anti-GPR77 antibody abolishes tumor formation and restores tumor chemosensitivity. Our study reveals a functional CAF subset that can be defined and isolated by specific cell-surface markers and suggests that targeting the CD10+GPR77+ CAF subset could be an effective therapeutic strategy against CSC-driven solid tumors.
Literature context: RRID:AB_2070042 Rabbit anti-FGFR2 Abcam Cat# ab
The differentiation of alveolar epithelial type I (AT1) and type II (AT2) cells is essential for the lung gas exchange function. Disruption of this process results in neonatal death or in severe lung diseases that last into adulthood. We developed live imaging techniques to characterize the mechanisms that control alveolar epithelial cell differentiation. We discovered that mechanical forces generated from the inhalation of amniotic fluid by fetal breathing movements are essential for AT1 cell differentiation. We found that a large subset of alveolar progenitor cells is able to protrude from the airway epithelium toward the mesenchyme in an FGF10/FGFR2 signaling-dependent manner. The cell protrusion process results in enrichment of myosin in the apical region of protruded cells; this myosin prevents these cells from being flattened by mechanical forces, thereby ensuring their AT2 cell fate. Our study demonstrates that mechanical forces and local growth factors synergistically control alveolar epithelial cell differentiation.
Literature context: ell Signaling Technology #9664; RRID:AB_2070042 Polyclonal to PARP Cell Signali
TIA1 and TIAL1 encode a family of U-rich element mRNA-binding proteins ubiquitously expressed and conserved in metazoans. Using PAR-CLIP, we determined that both proteins bind target sites with identical specificity in 3' UTRs and introns proximal to 5' as well as 3' splice sites. Double knockout (DKO) of TIA1 and TIAL1 increased target mRNA abundance proportional to the number of binding sites and also caused accumulation of aberrantly spliced mRNAs, most of which are subject to nonsense-mediated decay. Loss of PRKRA by mis-splicing triggered the activation of the double-stranded RNA (dsRNA)-activated protein kinase EIF2AK2/PKR and stress granule formation. Ectopic expression of PRKRA cDNA or knockout of EIF2AK2 in DKO cells rescued this phenotype. Perturbation of maturation and/or stability of additional targets further compromised cell cycle progression. Our study reveals the essential contributions of the TIA1 protein family to the fidelity of mRNA maturation, translation, and RNA-stress-sensing pathways in human cells.
Literature context: 5)Cell Signaling TechnologyCat# 9664Rabbit monoclonal anti-HSP90Cell
KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.
Recent studies report that loss and dysfunction of mitochondria and peroxisomes contribute to the myelin and axonal damage in multiple sclerosis (MS). In this study, we investigated the efficacy of a combination of lovastatin and AMP-activated protein kinase (AMPK) activator (AICAR) on the loss and dysfunction of mitochondria and peroxisomes and myelin and axonal damage in spinal cords, relative to the clinical disease symptoms, using a mouse model of experimental autoimmune encephalomyelitis (EAE, a model for MS). We observed that lovastatin and AICAR treatments individually provided partial protection of mitochondria/peroxisomes and myelin/axons, and therefore partial attenuation of clinical disease in EAE mice. However, treatment of EAE mice with the lovastatin and AICAR combination provided greater protection of mitochondria/peroxisomes and myelin/axons, and greater improvement in clinical disease compared with individual drug treatments. In spinal cords of EAE mice, lovastatin-mediated inhibition of RhoA and AICAR-mediated activation of AMPK cooperatively enhanced the expression of the transcription factors and regulators (e.g. PPARα/β, SIRT-1, NRF-1, and TFAM) required for biogenesis and the functions of mitochondria (e.g. OXPHOS, MnSOD) and peroxisomes (e.g. PMP70 and catalase). In summary, these studies document that oral medication with a combination of lovastatin and AICAR, which are individually known to have immunomodulatory effects, provides potent protection and repair of inflammation-induced loss and dysfunction of mitochondria and peroxisomes as well as myelin and axonal abnormalities in EAE. As statins are known to provide protection in progressive MS (Phase II study), these studies support that supplementation statin treatment with an AMPK activator may provide greater efficacy against MS.
Literature context: Signaling Technology Cat#9664; RRID:AB_2070042 Mouse monoclonal anti-caspase-9
Evasion of apoptosis is a hallmark of cancer. Bcl-2 and p53 represent two important nodes in apoptosis signaling pathways. We find that concomitant p53 activation and Bcl-2 inhibition overcome apoptosis resistance and markedly prolong survival in three mouse models of resistant acute myeloid leukemia (AML). Mechanistically, p53 activation negatively regulates the Ras/Raf/MEK/ERK pathway and activates GSK3 to modulate Mcl-1 phosphorylation and promote its degradation, thus overcoming AML resistance to Bcl-2 inhibition. Moreover, Bcl-2 inhibition reciprocally overcomes apoptosis resistance to p53 activation by switching cellular response from G1 arrest to apoptosis. The efficacy, together with the mechanistic findings, reveals the potential of simultaneously targeting these two apoptosis regulators and provides a rational basis for clinical testing of this therapeutic approach.
Literature context: Rabbit; monoclonal 1:1000 (W) RRID:AB_2070042
Inflammation can interfere with endometrial receptivity. We examined how interleukin 1β (IL-1β) affects expression of the uterine gap junction protein connexin 43 (Cx43), which is known to be critical for embryonic implantation. We used an in vitro model of human endometrial stromal cells (ESCs), Western blotting, and a combination of validated, selective kinase inhibitors to evaluate five canonical IL-1β signaling pathways. Cx43 and two other markers of ESC differentiation (prolactin and VEGF) were inhibited predominantly via IL-1β-activated ERK1/2 and p38 MAP kinase cascades. The findings were corroborated using small interfering RNA to silence critical genes in either pathway. By contrast, upregulation of endogenous pro-IL-1α and pro-IL-1β following recombinant IL-1β treatment was mediated via the Jun N-terminal kinase pathway. The clinicopharmacological significance of our findings is that multiple signaling cascades may need to be neutralized to reverse deleterious effects of IL-1β on human endometrial function.
Literature context: 664 Rabbit; monoclonal 1:500 RRID:AB_2070042
The balances of mitochondrial dynamic changes, mitochondrial morphology, and mitochondrial number are critical in cell metabolism. Once they are disturbed, disorders in these processes generally cause diseases or even death in animals. We performed large-scale genetic screenings in fruit flies and discovered the mitoguardin gene (Miga) that encodes for a mitochondrial outer membrane protein. To examine the physiological functions of its mammalian homologs Miga1 and Miga2, we generated Miga1 and Miga2 single- and double-knockout mouse strains and found that the knockout mice were viable, but the females were subfertile. The ovarian phenotypes of these mice suggested that the MIGA1/2 proteins play an important role in ovulation and ovarian steroidogenesis. In vivo and in vitro analyses of Miga1/2-knockout granulosa cells showed severe defects in luteinization and steroidogenesis and disordered mitochondrial morphology and function in response to gonadotropins. This is a report of genes involved in mitochondrial fusion and morphology-regulating mitochondrial functions during ovulation and luteinization. These results suggest a mechanism of gonadotropin-regulated ovarian endocrine functions and provide clues for therapeutic treatments of infertile females.
Literature context: Caspase 3 Cell Signaling #9664; RRID:AB_2070042 Chemicals, Peptides, and Recomb
Glioblastoma (GBM) is a devastating malignancy with few therapeutic options. We identify PRMT5 in an in vivo GBM shRNA screen and show that PRMT5 knockdown or inhibition potently suppresses in vivo GBM tumors, including patient-derived xenografts. Pathway analysis implicates splicing in cellular PRMT5 dependency, and we identify a biomarker that predicts sensitivity to PRMT5 inhibition. We find that PRMT5 deficiency primarily disrupts the removal of detained introns (DIs). This impaired DI splicing affects proliferation genes, whose downregulation coincides with cell cycle defects, senescence and/or apoptosis. We further show that DI programs are evolutionarily conserved and operate during neurogenesis, suggesting that they represent a physiological regulatory mechanism. Collectively, these findings reveal a PRMT5-regulated DI-splicing program as an exploitable cancer vulnerability.
Literature context: ti-cleaved caspase 3 (1 : 200) (RRID:AB_2070042) (Cell Signaling Technology, Be
Both lysophosphatidic acid (LPA) and antidepressants have been shown to affect neuronal survival and differentiation, but whether LPA signalling participates in the action of antidepressants is still unknown. In this study, we examined the role of LPA receptors in the regulation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activity and neuronal survival by the tetracyclic antidepressants, mianserin and mirtazapine in hippocampal neurons. In HT22 immortalized hippocampal cells, antidepressants and LPA induced a time- and concentration-dependent stimulation of ERK1/2 phosphorylation. This response was inhibited by either LPA1 and LPA1/3 selective antagonists or siRNA-induced LPA1 down-regulation, and enhanced by LPA1 over-expression. Conversely, the selective LPA2 antagonist H2L5186303 had no effect. Antidepressants induced cyclic AMP response element binding protein phosphorylation and this response was prevented by LPA1 blockade. ERK1/2 stimulation involved pertussis toxin-sensitive G proteins, Src tyrosine kinases and fibroblast growth factor receptor (FGF-R) activity. Tyrosine phosphorylation of FGF-R was enhanced by antidepressants through LPA1 . Serum withdrawal induced apoptotic death, as indicated by increased annexin V staining, caspase activation and cleavage of poly-ADP-ribose polymerase. Antidepressants inhibited the apoptotic cascade and this protective effect was curtailed by blockade of either LPA1 , ERK1/2 or FGF-R activity. Moreover, in primary mouse hippocampal neurons, mianserin acting through LPA1 increased phospho-ERK1/2 and protected from apoptosis induced by removal of growth supplement. These data indicate that in neurons endogenously expressed LPA1 receptors mediate intracellular signalling and neuroprotection by tetracyclic antidepressants.
Literature context: ge, UK), and cleaved caspase 3 (RRID:AB_2070042) (Cell Signaling Technology). A
Proteasome inhibitors have been frequently used in treating hematologic and solid tumors. They are administered individually or in combination with other regimens, to prevent severe side effects and resistance development. Because they have been shown to be efficient and are pharmaceutically available, we tested the first Food and Drug Administration-approved proteasome inhibitor bortezomib alone and in combination with another proteasome inhibitor, salinosporamid A, in pheochromocytoma cells. Pheochromocytomas/Paragangliomas (PHEOs/PGLs) are neuroendocrine tumors for which no definite cure is yet available. Therefore, drugs with a wide spectrum of mechanisms of action are being tested to identify suitable candidates for PHEO/PGL treatment. In the current study, we show that bortezomib induces PHEO cell death via the apoptotic pathway in vitro and in vivo. The combination of bortezomib with salinosporamid A exhibits additive effect on these cells and inhibits proliferation, cell migration and invasion, and angiogenesis more potently than bortezomib alone. Altogether, we suggest these proteasome inhibitors, especially bortezomib, could be potentially tested in PHEO/PGL patients who might benefit from treatment with either the inhibitors alone or in combination with other treatment options.
Literature context: ell Signaling 9664; RRID:AB_2070042 Rat anti-PDGFRa BD Biosciences
YAP/TAZ are the major mediators of mammalian Hippo signaling; however, their precise function in the gastrointestinal tract remains poorly understood. Here we dissect the distinct roles of YAP/TAZ in endodermal epithelium and mesenchyme and find that, although dispensable for gastrointestinal epithelial development and homeostasis, YAP/TAZ function as the critical molecular switch to coordinate growth and patterning in gut mesenchyme. Our genetic analyses reveal that Lats1/2 kinases suppress expansion of the primitive mesenchymal progenitors, where YAP activation also prevents induction of the smooth muscle lineage through transcriptional repression of Myocardin. During later development, zone-restricted downregulation of YAP/TAZ provides the positional cue and allows smooth muscle cell differentiation induced by Hedgehog signaling. Taken together, our studies identify the mesenchymal requirement of YAP/TAZ in the gastrointestinal tract and highlight the functional interplays between Hippo and Hedgehog signaling underlying temporal and spatial control of tissue growth and specification in developing gut.
Literature context: RRID:AB_2070042 Cleaved PARP1 (Asp214) Antibody
We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.
Literature context: Signaling Technology Cat# 9664; RRID:AB_2070042 Cleaved Caspase-8 (Asp387) (D5B
Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.
Literature context: AT# 9664; RRID:AB_2070042 Mouse anti
Injured skeletal muscle regenerates, but with age or in muscular dystrophies, muscle is replaced by fat. Upon injury, muscle-resident fibro/adipogenic progenitors (FAPs) proliferated and gave rise to adipocytes. These FAPs dynamically produced primary cilia, structures that transduce intercellular cues such as Hedgehog (Hh) signals. Genetically removing cilia from FAPs inhibited intramuscular adipogenesis, both after injury and in a mouse model of Duchenne muscular dystrophy. Blocking FAP ciliation also enhanced myofiber regeneration after injury and reduced myofiber size decline in the muscular dystrophy model. Hh signaling through FAP cilia regulated the expression of TIMP3, a secreted metalloproteinase inhibitor, that inhibited MMP14 to block adipogenesis. A pharmacological mimetic of TIMP3 blocked the conversion of FAPs into adipocytes, pointing to a strategy to combat fatty degeneration of skeletal muscle. We conclude that ciliary Hh signaling by FAPs orchestrates the regenerative response to skeletal muscle injury.
Literature context: ng 9664s; RRID:AB_2070042 Goat polyc
Activation of the FGF signaling pathway during preimplantation development of the mouse embryo is known to be essential for differentiation of the inner cell mass and the formation of the primitive endoderm (PrE). We now show using fluorescent reporter knockin lines that Fgfr1 is expressed in all cell populations of the blastocyst, while Fgfr2 expression becomes restricted to extraembryonic lineages, including the PrE. We further show that loss of both receptors prevents the development of the PrE and demonstrate that FGFR1 plays a more prominent role in this process than FGFR2. Finally, we document an essential role for FGFRs in embryonic stem cell (ESC) differentiation, with FGFR1 again having a greater influence than FGFR2 in ESC exit from the pluripotent state. Collectively, these results identify mechanisms through which FGF signaling regulates inner cell mass lineage restriction and cell commitment during preimplantation development.
Literature context: 3 (1:300, Cell Signaling, 9664; AB_2070042); rabbit anti-AQP3 (1:400, Life
Acinar cells play an essential role in the secretory function of exocrine organs. Despite this requirement, how acinar cells are generated during organogenesis is unclear. Using the acini-ductal network of the developing human and murine salivary gland, we demonstrate an unexpected role for SOX2 and parasympathetic nerves in generating the acinar lineage that has broad implications for epithelial morphogenesis. Despite SOX2 being expressed by progenitors that give rise to both acinar and duct cells, genetic ablation of SOX2 results in a failure to establish acini but not ducts. Furthermore, we show that SOX2 targets acinar-specific genes and is essential for the survival of acinar but not ductal cells. Finally, we illustrate an unexpected and novel role for peripheral nerves in the creation of acini throughout development via regulation of SOX2. Thus, SOX2 is a master regulator of the acinar cell lineage essential to the establishment of a functional organ.
Literature context: echnology RRID:AB_2070042 Rabbit mon
Diarrhea is a host response to enteric pathogens, but its impact on pathogenesis remains poorly defined. By infecting mice with the attaching and effacing bacteria Citrobacter rodentium, we defined the mechanisms and contributions of diarrhea and intestinal barrier loss to host defense. Increased permeability occurred within 2 days of infection and coincided with IL-22-dependent upregulation of the epithelial tight junction protein claudin-2. Permeability increases were limited to small molecules, as expected for the paracellular water and Na+ channel formed by claudin-2. Relative to wild-type, claudin-2-deficient mice experienced severe disease, including increased mucosal colonization by C. rodentium, prolonged pathogen shedding, exaggerated cytokine responses, and greater tissue injury. Conversely, transgenic claudin-2 overexpression reduced disease severity. Chemically induced osmotic diarrhea reduced colitis severity and C. rodentium burden in claudin-2-deficient, but not transgenic, mice, demonstrating that claudin-2-mediated protection is the result of enhanced water efflux. Thus, IL-22-induced claudin-2 upregulation drives diarrhea and pathogen clearance.
Literature context: Cat#9664; RRID:AB_2070042 Rabbit ant
Early postnatal mammals, including human babies, can perform only basic motor tasks. The acquisition of skilled behaviors occurs later, requiring anatomical changes in neural circuitry to support the development of coordinated activation or suppression of functionally related muscle groups. How this circuit reorganization occurs during postnatal development remains poorly understood. Here we explore the connectivity between corticospinal (CS) neurons in the motor cortex and muscles in mice. Using trans-synaptic viral and electrophysiological assays, we identify the early postnatal reorganization of CS circuitry for antagonistic muscle pairs. We further show that this synaptic rearrangement requires the activity-dependent, non-apoptotic Bax/Bak-caspase signaling cascade. Adult Bax/Bak mutant mice exhibit aberrant co-activation of antagonistic muscle pairs and skilled grasping deficits but normal reaching and retrieval behaviors. Our findings reveal key cellular and molecular mechanisms driving postnatal motor circuit reorganization and the resulting impacts on muscle activation patterns and the execution of skilled movements.
Literature context: t # 9664; RRID:AB_2070042 Rabbit ant
Intimate communication between neural and vascular cells is critical for normal brain development and function. Germinal matrix (GM), a key primordium for the brain reward circuitry, is unique among brain regions for its distinct pace of angiogenesis and selective vulnerability to hemorrhage during development. A major neonatal condition, GM hemorrhage can lead to cerebral palsy, hydrocephalus, and mental retardation. Here we identify a brain-region-specific neural progenitor-based signaling pathway dedicated to regulating GM vessel development. This pathway consists of cell-surface sphingosine-1-phosphate receptors, an intracellular cascade including Gα co-factor Ric8a and p38 MAPK, and target gene integrin β8, which in turn regulates vascular TGF-β signaling. These findings provide insights into region-specific specialization of neurovascular communication, with special implications for deciphering potent early-life endocrine, as well as potential gut microbiota impacts on brain reward circuitry. They also identify tissue-specific molecular targets for GM hemorrhage intervention.
Literature context: 664Â Rabbit, monoclonalÂ 1 in 200Â AB_2070042Â Peptide/Protein TargetÂ Antigen
Androgens are essential for the normal function of mature antral follicles but also have a role in the early stages of follicle development. Polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility, is characterized by androgen excess and aberrant follicle development that includes accelerated early follicle growth. We have examined the effects of testosterone and dihydrotestosterone (DHT) on development of isolated mouse preantral follicles in culture with the specific aim of investigating interaction with follicle-stimulating hormone (FSH), the steroidogenic pathway, and growth factors of the TGFβ superfamily that are known to have a role in early follicle development. Both testosterone and DHT stimulated follicle growth and augmented FSH-induced growth and increased the incidence of antrum formation among the granulosa cell layers of these preantral follicles after 72 hours in culture. Effects of both androgens were reversed by the androgen receptor antagonist flutamide. FSH receptor expression was increased in response to both testosterone and DHT, as was that of Star, whereas Cyp11a1 was down-regulated. The key androgen-induced changes in the TGFβ signaling pathway were down-regulation of Amh, Bmp15, and their receptors. Inhibition of Alk6 (Bmpr1b), a putative partner for Amhr2 and Bmpr2, by dorsomorphin resulted in augmentation of androgen-stimulated growth and modification of androgen-induced gene expression. Our findings point to varied effects of androgen on preantral follicle growth and function, including interaction with FSH-activated growth and steroidogenesis, and, importantly, implicate the intrafollicular TGFβ system as a key mediator of androgen action. These findings provide insight into abnormal early follicle development in PCOS.
Literature context: lso 9664P RRID:AB_2070042), anti-cle
Regeneration of skeletal muscle in adults is mediated by satellite stem cells. Accumulation of misfolded proteins triggers endoplasmic reticulum stress that leads to unfolded protein response (UPR). The UPR is relayed to the cell through the activation of PERK, IRE1/XBP1, and ATF6. Here, we demonstrate that levels of PERK and IRE1 are increased in satellite cells upon muscle injury. Inhibition of PERK, but not the IRE1 arm of the UPR in satellite cells inhibits myofiber regeneration in adult mice. PERK is essential for the survival and differentiation of activated satellite cells into the myogenic lineage. Deletion of PERK causes hyper-activation of p38 MAPK during myogenesis. Blocking p38 MAPK activity improves the survival and differentiation of PERK-deficient satellite cells in vitro and muscle formation in vivo. Collectively, our results suggest that the PERK arm of the UPR plays a pivotal role in the regulation of satellite cell homeostasis during regenerative myogenesis.
Literature context: Cat# 9664 RRID:AB_2070042), GAPDH (C
While angiotensin II (ang II) has been implicated in the pathogenesis of cardiac cachexia (CC), the molecules that mediate ang II's wasting effect have not been identified. It is known TNF-α level is increased in patients with CC, and TNF-α release is triggered by ang II. We therefore hypothesized that ang II induced muscle wasting is mediated by TNF-α. Ang II infusion led to skeletal muscle wasting in wild type (WT) but not in TNF alpha type 1 receptor knockout (TNFR1KO) mice, suggesting that ang II induced muscle loss is mediated by TNF-α through its type 1 receptor. Microarray analysis identified cholesterol 25-hydroxylase (Ch25h) as the down stream target of TNF-α. Intraperitoneal injection of 25-hydroxycholesterol (25-OHC), the product of Ch25h, resulted in muscle loss in C57BL/6 mice, accompanied by increased expression of atrogin-1, MuRF1 and suppression of IGF-1/Akt signaling pathway. The identification of 25-OHC as an inducer of muscle wasting has implications for the development of specific treatment strategies in preventing muscle loss.
Literature context: No. 9664; RRID:AB_2070042 Goat anti-
Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.
Literature context: at# 9664, RRID:AB_2070042) and NeuN
The robust neuroprotective effects of transarterial regional hypothermia have been demonstrated in the typical transient middle cerebral artery occlusion (tMCAO) model, but have not yet been tested in other ischemic stroke models, even though clinical ischemic conditions are diverse. In order to clarify these effects in a different ischemic stroke model, we employed a rat model of permanent MCAO (pMCAO) with transient collateral hypoperfusion (tCHP), which was achieved by direct MCA ligation through craniotomy and 1-h bilateral common carotid artery occlusion at the beginning of pMCAO. The infusion of 20ml/kg of 4°C cold saline (CS) or 37°C warm saline (WS) into the ipsilateral internal carotid artery (ICA) was performed for 15min in intra- or post-tCHP. Neurological scores, infarct/edema volumes, and neuronal apoptosis and reactive gliosis were compared between the CS and WS groups and a non-infusion control group after 48h of reperfusion. Although brain temperatures were only reduced by 2-3°C for 15min, the CS group had significantly better neurological scores, smaller infarct/edema volumes, and less penumbral neuronal apoptosis and reactive gliosis than the control and WS groups. The post-tCHP CS group exhibited prominent neuroprotective effects, even though infarct volumes and neuronal apoptosis were reduced less than those in the intra-tCHP CS group. In conclusion, we demonstrated the neuroprotective effects of transarterial regional hypothermia in an ischemic model of pMCAO with tCHP. Even though MCAO is persistent, cold infusion via the ICA is neuroprotective for the penumbra, suggesting the wider therapeutic application of this therapy.
Literature context: (RRID:AB_2070042)
When unperturbed, somatic stem cells are poised to affect immediate tissue restoration upon trauma. Yet, little is known regarding the mechanistic basis controlling initial and homeostatic 'scaling' of stem cell pool sizes relative to their target tissues for effective regeneration. Here, we show that TEAD1-expressing skeletal muscle of transgenic mice features a dramatic hyperplasia of muscle stem cells (i.e. satellite cells, SCs) but surprisingly without affecting muscle tissue size. Super-numeral SCs attain a 'normal' quiescent state, accelerate regeneration, and maintain regenerative capacity over several injury-induced regeneration bouts. In dystrophic muscle, the TEAD1 transgene also ameliorated the pathology. We further demonstrate that hyperplastic SCs accumulate non-cell-autonomously via signal(s) from the TEAD1-expressing myofiber, suggesting that myofiber-specific TEAD1 overexpression activates a physiological signaling pathway(s) that determines initial and homeostatic SC pool size. We propose that TEAD1 and its downstream effectors are medically relevant targets for enhancing muscle regeneration and ameliorating muscle pathology.
Literature context: leaved caspase-3 (Asp175) (Cat. 9664), 1:2,000 cleaved caspase-7 (As
Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors.
Literature context: ignaling, RRID:AB_2070042), IBA1: ra
Inflammation is an important factor contributing to developmental brain injury in preterm infants. Although tumor necrosis factor-inducible gene 6 protein (TSG-6) has immunomodulatory effects in several inflammatory conditions of adult animals, nothing is currently known about the role of TSG-6 in the developing brain, its impact on perinatal inflammation and its therapeutic potential. The aim of the current work was 1) to characterize the developmental expression of TSG-6 in the newborn rat brain, 2) to evaluate the impact of LPS exposure on TSG-6 expression and 3) to assess the therapeutic potential of exogenous TSG-6 administration. Brain hemispheres of healthy Wistar rats (postnatal day 1-postnatal day 15 (P1-P15)) were evaluated with regard to the physiological expression of TSG-6. LPS-treated rats (0.25mg/kg LPS i.p. on P3) were analyzed for inflammation-induced changes in TSG-6 and cytokine expression. To evaluate whether exogenous recombinant human (rh)TSG-6 affects inflammation-induced brain injury, newborn Wistar rats, exposed to LPS on P3, were treated with rhTSG-6 i.p. (four repetitive doses of 2.25mg/kg every 12h, first dose 3h before LPS injection). PCR, Western blotting and multiplex ELISA were performed according to standard protocols. TSG-6 is physiologically expressed in the developing brain with a linear increase in expression from P1 to P15 at the mRNA level. At P6, regional differences in TSG-6 expression in the cortex, thalamus and striatum were detected at mRNA and protein level. Furthermore, TSG-6 gene expression was significantly increased by inflammation (induced by LPS treatment). Combined treatment with LPS and TSG-6 vs. LPS exposure alone, resulted in significant down-regulation of cleaved caspase-3, a marker of apoptosis and neuronal plasticity. In addition, several inflammatory serum markers were decreased after TSG-6 treatment. Finally, TSG-6 is physiologically expressed in the developing brain. Changes of TSG-6 expression associated with inflammation suggest a role of TSG-6 in neuroinflammation. Reduction of cleaved caspase-3 by TSG-6 treatment demonstrates the putative neuroprotective potential of exogenous TSG-6 administration in inflammation-induced developmental brain injury.
Cortical interneurons are generated predominantly in the medial ganglionic eminence (MGE) and migrate through the ventral and dorsal telencephalon before taking their final positions within the developing cortical plate. Previously we demonstrated that interneurons from Robo1 knockout (Robo1(-/-)) mice contain reduced levels of neuropilin 1 (Nrp1) and PlexinA1 receptors, rendering them less responsive to the chemorepulsive actions of semaphorin ligands expressed in the striatum and affecting their course of migration (Hernandez-Miranda et al.  J. Neurosci. 31:6174-6187). Earlier studies have highlighted the importance of Nrp1 and Nrp2 in interneuron migration, and here we assess the role of PlexinA1 in this process. We observed significantly fewer cells expressing the interneuron markers Gad67 and Lhx6 in the cortex of PlexinA1(-/-) mice compared with wild-type littermates at E14.5 and E18.5. Although the level of apoptosis was similar in the mutant and control forebrain, proliferation was significantly reduced in the former. Furthermore, progenitor cells in the MGE of PlexinA1(-/-) mice appeared to be poorly anchored to the ventricular surface and showed reduced adhesive properties, which may account for the observed reduction in proliferation. Together our data uncover a novel role for PlexinA1 in forebrain development.
Glutathione (GSH) is the one of the most abundant intracellular antioxidants. Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH. Our prior work showed that GSH plays antiapoptotic roles in ovarian follicles. We hypothesized that Gclm(-/-) mice have accelerated ovarian aging due to ovarian oxidative stress. We found significantly decreased ovarian GSH concentrations and oxidized GSH/oxidized glutathione redox potential in Gclm(-/-) vs Gclm(+/+) ovaries. Prepubertal Gclm(-/-) and Gclm(+/+) mice had similar numbers of ovarian follicles, and as expected, the total number of ovarian follicles declined with age in both genotypes. However, the rate of decline in follicles was significantly more rapid in Gclm(-/-) mice, and this was driven by accelerated declines in primordial follicles, which constitute the ovarian reserve. We found significantly increased 4-hydroxynonenal immunostaining (oxidative lipid damage marker) and significantly increased nitrotyrosine immunostaining (oxidative protein damage marker) in prepubertal and adult Gclm(-/-) ovaries compared with controls. The percentage of small ovarian follicles with increased granulosa cell proliferation was significantly higher in prepubertal and 2-month-old Gclm(-/-) vs Gclm(+/+) ovaries, indicating accelerated recruitment of primordial follicles into the growing pool. The percentages of growing follicles with apoptotic granulosa cells were increased in young adult ovaries. Our results demonstrate increased ovarian oxidative stress and oxidative damage in young Gclm(-/-) mice, associated with an accelerated decline in ovarian follicles that appears to be mediated by increased recruitment of follicles into the growing pool, followed by apoptosis at later stages of follicular development.
Insulinoma is the main type of functional pancreatic neuroendocrine tumors. The functional microRNAs (miRNAs) regulating tumor growth and progression in insulinomas are still unknown. We conducted the miRNA expression profile analysis using miRNA quantitative RT-PCR array and identified 114 differentially expressed miRNAs in human insulinomas compared with normal pancreatic islets. Forty-one differentially expressed miRNAs belonged to 7 miRNA families, and 28 miRNAs in 3 of the families localized in the epigenetically regulated imprinted chromosome 14q32 region. We validated the most significant differentially expressed miRNA cluster miR-144/451 in another 8 human normal islet samples and 25 insulinomas. Our data showed that the overexpression of miR-144/451 in mouse pancreatic β-cells promoted cell proliferation by targeting the β-cell regulator phosphatase and tensin homolog deleted on chromosome ten/v-akt murine thymoma viral oncogene homolog pathway and cyclin-dependent kinase inhibitor 2D. Our findings highlight the importance of functional miRNAs in insulinomas.
LH receptor (LHR) expression in the ovary is regulated by the RNA binding protein, (LHR mRNA binding protein [LRBP]), which has been identified as being mevalonate kinase. This study examined the role of microRNA miR-122 in LRBP-mediated LHR mRNA expression. Real-time PCR analysis of ovaries from pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-primed female rats treated with hCG to down-regulate LHR expression showed that an increase in miR-122 expression preceded LHR mRNA down-regulation. The expression of miR-122 and its regulation was confirmed using fluorescent in situ hybridization of the frozen ovary sections using 5'-fluorescein isothiocyanate-labeled miR-122 locked nucleic acid probe. The increased expression of miR-122 preceded increased expression of LRBP mRNA and protein, and these increases were followed by LHR mRNA down-regulation. Inhibition of protein kinase A (PKA) and ERK1/2 signaling pathways by H89 and UO126, respectively, attenuated the hCG-mediated up-regulation of miR-122 levels. This was also confirmed in vitro using human granulosa cells. These results suggest the possibility that hCG-mediated miR-122 expression is mediated by the activation of cAMP/PKA/ERK signaling pathways. Inhibition of miR-122 by injection of the locked nucleic acid-conjugated antagomir of miR-122 abrogated the hCG-mediated increases in LRBP protein expression. Because it has been previously shown that miR-122 regulates sterol regulatory element-binding proteins (SREBPs) and SREBPs, in turn, regulate LRBP expression, the role of SREBPs in miR-122-mediated increase in LRBP expression was then examined. The levels of active forms of both SREBP-1a and SREBP-2 were increased in response to hCG treatment, and the stimulatory effect was sustained up to 4 hours. Taken together, our results suggest that hCG-induced down-regulation of LHR mRNA expression is mediated by activation of cAMP/PKA/ERK pathways to increase miR-122 expression, which then increases LRBP expression through the activation of SREBPs.
Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are 2 closely related TGF-β ligands implicated as key regulators of follicle development and fertility. Animals harboring mutations of these factors often exhibit a blockage in follicle development beyond the primary stage and therefore little is known about the role of these ligands during subsequent (preantral) stages. Preantral follicles isolated from immature mice were cultured with combinations of BMP15, GDF9, and activin receptor-like kinase (ALK) inhibitors. Individually, GDF9 and BMP15 promoted follicle growth during the first 24 hours, whereas BMP15 subsequently (48-72 h) caused follicle shrinkage and atresia with increased granulosa cell apoptosis. Inhibition of ALK6 prevented the BMP15-induced reduction in follicle size and under basal conditions promoted a rapid increase in granulosa cell proliferation, suggesting BMP15 signals through ALK6, which in turn acts to restrain follicle growth. In the presence of GDF9, BMP15 no longer promoted atresia and in fact follicle growth was increased significantly more than with either ligand alone. This cooperative effect was accompanied by differential expression of Id1-3, Smad6-7, and Has2 and was blocked by the same ALK5 inhibitor used to block GDF9 signaling. Immunostaining for SMAD2/3 and SMAD1/5/8, representing the 2 main branches of TGF-β signaling, supported the fact that both canonical pathways have the potential to be active in growing follicles, whereas primordial follicles only express SMAD2/3. Overall results highlight differential effects of the 2 main TGF-β signaling pathways during preantral follicle growth.
Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) is a well-known pro-inflammatory chemokine and its role in ischemic stroke remains controversial. We examined the significance of RANTES in ischemic stroke and aimed to elucidate the direct effect of RANTES on neurons. Plasma concentrations of major C-C chemokines, including RANTES, and neurotrophic factors were examined in 171 ischemic stroke patients and age- and gender- matched healthy subjects. Plasma concentrations of RANTES at day 0 after onset were significantly elevated in stroke patients, compared with controls, and were highly correlated with those of BDNF, EGF, and VEGF. In a mouse middle cerebral artery occlusion model (MCAO), plasma RANTES was significantly elevated and the expression of RANTES was markedly upregulated in neurons particularly in peri-infarct areas. The expression of CCR3 and CCR5, receptors for RANTES, was also induced in neurons, while another receptor, CCR1, was observed in vascular cells, in peri-infarct areas after MCAO. We examined the effects of RANTES on differentiated PC12 cells, a model of neuronal cells. Treatment with RANTES induced the activation of Akt and Erk1/2, and attenuated the cleavage of caspase-3 in the cells. RANTES increased the expression of BDNF, EGF, and VEGF in the cells. Moreover, RANTES maintained the number of cells under serum free conditions. The RANTES-mediated upregulation of neurotrophic factors and cell survival were significantly attenuated by the inhibition of Akt or Erk1/2. Taken together, RANTES is an interesting chemokine that is produced from neurons after ischemic stroke and has the potential to protect neurons directly or indirectly through the production of neurotrophic factors in peri-infarct areas.
Cuprizone (bis-cyclohexanone oxaldihydrazone) was previously shown to induce demyelination in white matter enriched brain structures. In the present study we used the cuprizone demyelination model in transgenic mice expressing the enhanced green fluorescent protein (GFP) under the 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) promoter. The use of these particular transgenic mice allows easy detection of cells belonging to the entire oligodendroglial (OLG) lineage, ranging from OLG precursors to mature myelinating OLGs. We were able to evaluate the precise extent of oligodendroglial cell damage and recovery within the murine adult central nervous system (CNS) after inducing demyelination by acute cuprizone intoxication. A generalized loss of GFP+ cells was observed after cuprizone exposure and correlated with a decline in myelin basic protein (MBP) expression. OLGs were depleted in many brain areas that were previously thought to be unaffected by cuprizone treatment. Thus, in addition to the well-known cuprizone effects on the medial corpus callosum, we also found a loss of GFP+ cells in most brain structures, particularly in the caudatus putamen, cortex, anterior commissure, olfactory bulb, hippocampus, optic chiasm, brainstem, and cingulum. Loss of GFP+ cells was accompanied by extensive astrogliosis and microglial activation, although neurons were not affected. Interestingly, cuprizone-treated animals showed both activation of GFAP expression and a higher proliferation rate in subventricular zone cells. A week after cuprizone removal from the diet, GFP+ oligodendroglial cells began repopulating the damaged structures. GFP expression precedes that of MBP and allows OLG detection before myelin restoration.
Here we analyze the role of the Lhx6 lim-homeobox transcription factor in regulating the development of subsets of neocortical, hippocampal, and striatal interneurons. An Lhx6 loss-of-function allele, which expresses placental alkaline phosphatase (PLAP), allowed analysis of the development and fate of Lhx6-expressing interneurons in mice lacking this homeobox transcription factor. There are Lhx6+;Dlx+ and Lhx6-;Dlx+ subtypes of tangentially migrating interneurons. Most interneurons in Lhx6(PLAP/PLAP) mutants migrate to the cortex, although less efficiently, and exhibit defects in populating the marginal zone and superficial parts of the neocortical plate. By contrast, migration to superficial parts of the hippocampus is not seriously affected. Furthermore, whereas parvalbumin+ and somatostatin+ interneurons do not differentiate, NPY+ interneurons are present; we suggest that these NPY+ interneurons are derived from the Lhx6-;Dlx+ subtype. Striatal interneurons show deficits distinct from pallial interneurons, including a reduction in the NPY+ subtype. We provide evidence that Lhx6 mediates these effects through promoting expression of receptors that regulate interneuron migration (ErbB4, CXCR4, and CXCR7), and through promoting the expression of transcription factors either known (Arx) or implicated (bMaf, Cux2, and NPAS1) in controlling interneuron development.
Apoptosis of olfactory sensory neurons (OSNs) induced by olfactory bulbectomy (OBX) leads to the activation of resident macrophages within the olfactory epithelium (OE). These macrophages phagocytose degenerating OSNs and secrete chemokines, which recruit additional macrophages into the OE, and cytokines/growth factors, which regulate basal cell proliferation and differentiation and maturation of OSNs. In this study we apply for the first time the use of liposome-encapsulated clodronate to selectively deplete macrophages during the OSN degeneration/regeneration cycle in order to elucidate the role(s) of macrophages in regulating cellular mechanisms that lead to apoptosis and neurogenesis. Mice were injected intranasally and intravenously with either liposome-encapsulated clodronate or empty liposomes prior to and after OBX or sham OBX. At 48 hours after surgery the numbers of macrophages in the OE of both sham and OBX clodronate-treated mice were significantly reduced compared to liposome-treated controls (38% and 35%, respectively, P < 0.05). The reduction in macrophage numbers was accompanied by significant decreases in OE thickness (22% and 21%, P < 0.05), the number of mOSNs (1.2- and 1.9-fold, P < 0.05), and basal cell proliferation (7.6- and 3.8-fold, P < 0.005) in sham and OBX mice, respectively, compared to liposome-treated controls. In OBX mice there was also increased immunoreactivity for active caspase-3 in the OE and olfactory nerves of clodronate-treated OBX mice compared to liposome-treated controls. These results indicate that macrophages modulate the OSN population in the normal and target-ablated murine OE by influencing neuronal survival and basal cell proliferation, resulting in neurogenesis and replacement of mature OSNs.