P53 Mice Research Articles

AAV9-MCT8 Delivery at Juvenile Stage Ameliorates Neurological and Behavioral Deficits in a Mouse Model of MCT8-Deficiency.

Background: Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor disability disorder that also manifests characteristic abnormal thyroid hormone (TH) levels. AHDS is caused by inactivating mutations in monocarboxylate transporter 8 (MCT8), a specific TH plasma membrane transporter widely expressed in the central nervous system (CNS). MCT8 mutations cause impaired transport of TH across brain barriers, leading to insufficient neural TH supply. There is currently no successful therapy for the neurological symptoms. Earlier work has shown that intravenous (IV), but not intracerebroventricular adeno-associated virus serotype 9 (AAV9) -based gene therapy given to newborn Mct8 knockout (Mct8-/y) male mice increased triiodothyronine (T3) brain content and partially rescued TH-dependent gene expression, suggesting a promising approach to treat this neurological disorder. Methods: The potential of IV delivery of AAV9 carrying human MCT8 was tested in the well-established Mct8-/y/Organic anion-transporting polypeptide 1c1 (Oatp1c1)-/ - double knockout (dKO) mouse model of AHDS, which, unlike Mct8-/y mice, displays both neurological and TH phenotype. Further, as the condition is usually diagnosed during childhood, treatment was given intravenously to P30 mice and psychomotor tests were carried out blindly at P120-P140 after which tissues were collected and analyzed. Results: Systemic IV delivery of AAV9-MCT8 at a juvenile stage led to improved locomotor and cognitive functions at P120-P140, which was accompanied by a near normalization of T3 content and an increased response of positively regulated TH-dependent gene expression in different brain regions examined (thalamus, hippocampus, and parietal cortex). The effects on serum TH concentrations and peripheral tissues were less pronounced, showing only improvement in the serum T3/reverse T3 (rT3) ratio and in liver deiodinase 1 expression. Conclusion: IV administration of AAV9, carrying the human MCT8, to juvenile dKO mice manifesting AHDS has long-term beneficial effects, predominantly on the CNS. This preclinical study indicates that this gene therapy has the potential to ameliorate the devastating neurological symptoms in patients with AHDS.

Correlated Activity in the Degenerate Retina Inhibits Focal Response to Electrical Stimulation.

Retinal prostheses have shown some clinical success in patients with retinitis pigmentosa and age-related macular degeneration. However, even after the implantation of a retinal prosthesis, the patient’s visual acuity is at best less than 20/420. Reduced visual acuity may be explained by a decrease in the signal-to-noise ratio due to the spontaneous hyperactivity of retinal ganglion cells (RGCs) found in degenerate retinas. Unfortunately, abnormal retinal rewiring, commonly observed in degenerate retinas, has rarely been considered for the development of retinal prostheses. The purpose of this study was to investigate the aberrant retinal network response to electrical stimulation in terms of the spatial distribution of the electrically evoked RGC population. An 8 × 8 multielectrode array was used to measure the spiking activity of the RGC population. RGC spikes were recorded in wild-type [C57BL/6J; P56 (postnatal day 56)], rd1 (P56), rd10 (P14 and P56) mice, and macaque [wild-type and drug-induced retinal degeneration (RD) model] retinas. First, we performed a spike correlation analysis between RGCs to determine RGC connectivity. No correlation was observed between RGCs in the control group, including wild-type mice, rd10 P14 mice, and wild-type macaque retinas. In contrast, for the RD group, including rd1, rd10 P56, and RD macaque retinas, RGCs, up to approximately 400–600 μm apart, were significantly correlated. Moreover, to investigate the RGC population response to electrical stimulation, the number of electrically evoked RGC spikes was measured as a function of the distance between the stimulation and recording electrodes. With an increase in the interelectrode distance, the number of electrically evoked RGC spikes decreased exponentially in the control group. In contrast, electrically evoked RGC spikes were observed throughout the retina in the RD group, regardless of the inter-electrode distance. Taken together, in the degenerate retina, a more strongly coupled retinal network resulted in the widespread distribution of electrically evoked RGC spikes. This finding could explain the low-resolution vision in prosthesis-implanted patients.

Fibroblast Senescence: A Risk Factor for Remodeling, Inflammation, and Arrhythmias in the Post‐MI Heart

ObjectivesCellular senescence is a stress response involving permanent replicative arrest. It is accompanied by a complex senescence‐associated secretory phenotype (SASP), which is characterized by the presence of pro‐inflammatory cytokines and chemokines, growth factors, and tissue‐remodeling metalloproteinases. Little is known regarding the role of senescence and the SASP in the context of arrhythmias post myocardial infarction (MI). Here, we characterized the arrhythmogenic cardiac consequences and molecular mechanisms underlying tissue remodeling changes in impaired or accelerated cardiac myofibroblast (CMF) senescence and the pro‐inflammatory SASP during post‐MI wound healing process in genetically engineered mice.Material‐MethodsImpaired or accelerated CMF‐specific senescence was achieved by using cre‐lox mouse models to delete p53 or MDM2 genes. MI surgery via direct ligation of the left anterior descending artery (LAD) was performed in 4‐month‐old wild‐type (WT), Pstn‐Cre(+/‐) p53(fl/fl), Pstn‐MerCreMer(+/‐) MDM2(fl/fl), and respective littermate control (LMC) mice of both sexes. Changes in cardiac structure and function post‐MI were assessed by echocardiography. Senescence‐associated β‐galactosidase (SA‐βgal) staining was applied to detect and quantify senescent cells in three zones: infarct zone (IZ), infarct border zone (IBZ), and remote zone (RZ). Immunofluorescence staining identified the types of senescent cells. Masson’s trichrome staining was used for fibrosis assessment. RT‐qPCR was performed to quantitate the expression of known senescence genes and canonical SASP factors.ResultsPstn‐Cre(+/‐) p53(fl/fl) mice displayed significantly decreased CMF senescence two weeks post‐MI in the scar compared to littermate control p53(fl/fl) and WT mice. We noticed a similar trend in the IBZ, but this did not reach statistical significance. Pstn‐MCM(+/‐) MDM2(fl/fl) mice showed significantly increased CMF senescence in the IZ compared littermate control MDM2(fl/fl) mice. We observed a similar trend in the IBZ, but this did not reach statistical significance. In all mice, we observed essentially no senescence in the RZ. Our data did not show any significant difference in fibrosis between any genotype in any zone. We did not observe any overall trend in differential expression in senescence and SASP genes between p53(fl/fl) and Pstn‐Cre(+/‐) p53(fl/fl) mice in any zone. There was no evidence of any sex‐specific differences in aforementioned results.ConclusionsA significant increase and persistence of CMF senescence likely contributes to pathological tissue remodeling during the post‐MI wound healing process. This is likely due to SASP‐induced pro‐arrhythmogenic chronic inflammation or via direct cell‐cell interactions between senescent CMFs and myocytes.

Increased Integration in Mutant Mice: An Analysis of the Patterns of Covariance through Ontogeny in Fgfr2 Mice

Mutations in the FGF/FGFR2 gene result in the premature fusion of fibrous and cartilaginous joints in the skull. This results in profound morphological changes in the skulls of both humans and mice with these mutations. Previous studies have indicated that morphological integration of the skull is expected to become more pronounced in dysmorphic syndromes, including FGFR2 syndromes which result in craniosynostosis. In this study, our objective was to test two hypotheses: 1) that the magnitude of morphological integration was greater in Fgfr2C342Y/+ mutant mice relative to wild‐type littermates; and 2) that the magnitude of morphological integration would increase through ontogeny.Our sample included Fgfr2C342Y/+ mutants and wild‐type littermates at postnatal day 14 (P14) and adult stages. We used micro‐computed tomography (microCT) scans to create image volumes of the skull. Virtual reconstructions of the cranium were produced in 3DSlicer software, using volume renders based on tissue densities to define the surface of the bone. We collected coordinate data for 38 biologically homologous landmarks with approximately equal distribution across face and neurocranium. All morphometric analyses were conducted using the geomorph package in R. The sample was subdivided by Age and Genotype. Landmark coordinates were divided into face and neurocranium modules. Coordinate data for each subset were superimposed with object symmetry using the bilat.symmetry() function. Covariance across modules was tested using the two.b.pls() function for each sample subset.Results confirmed our expectation that the magnitude of covariation between face and neurocranium increased in the mutant sample relative to the wild‐type sample at both timepoints. Surprisingly, within the mutant sample, integration between face and neurocranium was greater in the younger P14 mice than the older adults. Results of this study can be used to inform researchers about patterns of covariance that can be applied to better understand craniosynostosis syndromes that affect humans.

A Novel Role for Glucocerebrosidase 1 (GBA1) in Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, yet the pathological mechanisms driving disease onset and progression are not well understood. Glucocerebrosidase 1 (GBA1), the gene encoding the lysosomal enzyme glucoslyceramidase (GCase), has been identified as the most important genetic risk factor contributing to the development of PD. In this study, we aimed to investigate the relationship between inflammasome activation and GCase dysfunction in immune cells in PD. Primary microglial was generated from brains of P0‐ P2 mouse pups. After overnight serum cells was primed with 200 ng/mL of ultrapure LPS for 3 hr followed by treatment with either 40 µM of LTI‐291 or 200 µM of Condruitol β‐epoxide (CBE) for an hour. The cells was activated with 5 mM of adenosine triphosphate (ATP). The supernatant and cells were collected for IL‐1β and GBA assay, respectively. For in vivostudies, 8‐ 12 weeks old male C57BL/6J mice were injected with 20 μg of 6‐hydroxydopamine (6‐OHDA). Mice were treated with 12 mg/kg daily doses of LTI‐291 via oral gavage commencing 24 hours prior to surgery. On day 3 post‐surgery, brains were collected for analysis of protein expression. Our findings demonstrate that NLRP3 inflammasome activation increases IL‐1β production which significantly decreased in the presence of GCase activator, LTI‐291. This indicates the decreased inflammasome activation in conjunction with increased GCase activity. Similarly, GCase activity in LTI‐291 treated cells was significantly increased when compared to control and LPS primed samples. In vivo,6‐ OHDA mice had significant loss of tyrosine hydroxylase (TH), indicative of the substantial dopaminergic neuronal death which was rescued in LTI‐291 treated mice. Overall, these findings identify the potential role of NLRP3 inflammasome activation in driving GCase dysfunction, which could contribute to PD pathogenesis. Furthermore, we demonstrated that pharmacological augmentation of GCase activity was protective, reducing NLRP3 inflammasome activation in vitro and dopaminergic neuronal loss in vivo.

Sevoflurane-Induced Apoptosis in the Mouse Cerebral Cortex Follows Similar Characteristics of Physiological Apoptosis.

General anesthetics are capable of inducing neuronal apoptosis during the rapid synaptogenesis of immature mammalian brains. In this vulnerable time window, physiological apoptosis also occurs to eliminate excess and inappropriately integrated neurons. We previously showed that physiological and ketamine-induced apoptosis in mouse primary somatosensory cortex (S1) followed similar developmental patterns. However, since sevoflurane is more widely used in pediatric anesthesia, and targets mainly on different receptors, as compared with ketamine, it is important to determine whether sevoflurane-induced apoptosis also follows similar developmental patterns as physiological apoptosis or not. Mice at postnatal days 5 (P5) and P9 were anesthetized with 1.5% sevoflurane for 4 h, and the apoptotic neurons in S1 were quantitated by immunohistochemistry. The results showed that sevoflurane raised the levels of apoptosis in S1 without interfering with the developmental patterns of physiological apoptosis. The cells more vulnerable to both physiological and sevoflurane-induced apoptosis shifted from layer V pyramidal neurons at P5 to layers II–IV GABAergic neurons by P9. The magnitude of both sevoflurane-induced and physiological apoptosis was more attenuated at P9 than P5. To determine whether the Akt-FoxO1-PUMA pathway contributes to the developmental decrease in magnitude of both physiological and sevoflurane-induced apoptosis, Western blot was used to measure the levels of related proteins in S1 of P5 and P9 mice. We observed higher levels of antiapoptotic phosphorylated Akt (p-Akt) and phosphorylated FoxO1 (p-FoxO1), and lower levels of the downstream proapoptotic factor PUMA in control and anesthetized mice at P9 than P5. In addition, the Akt-FoxO1-PUMA pathway may also be responsible for sevoflurane-induced apoptosis. Together, these results suggest that magnitude, lamination pattern and cell-type specificity to sevoflurane-induced apoptosis are age-dependent and follow physiological apoptosis pattern. Moreover, The Akt-FoxO1-PUMA pathway may mediate the developmental decreases in magnitude of both physiological and sevoflurane-induced apoptosis in neonatal mouse S1.

Promoterless Gene Targeting Approach Combined to CRISPR/Cas9 Efficiently Corrects Hemophilia B Phenotype in Neonatal Mice.

Many inborn errors of metabolism require life-long treatments and, in severe conditions involving the liver, organ transplantation remains the only curative treatment. Non-integrative AAV-mediated gene therapy has shown efficacy in adult patients. However, treatment in pediatric or juvenile settings, or in conditions associated with hepatocyte proliferation, may result in rapid loss of episomal viral DNA and thus therapeutic efficacy. Re-administration of the therapeutic vector later in time may not be possible due to the presence of anti-AAV neutralizing antibodies. We have previously shown the permanent rescue of the neonatal lethality of a Crigler-Najjar mouse model by applying an integrative gene-therapy based approach. Here, we targeted the human coagulation factor IX (hFIX) cDNA into a hemophilia B mouse model. Two AAV8 vectors were used: a promoterless vector with two arms of homology for the albumin locus, and a vector carrying the CRISPR/SaCas9 and the sgRNA. Treatment of neonatal P2 wild-type mice resulted in supraphysiological levels of hFIX being stable 10 months after dosing. A single injection of the AAV vectors into neonatal FIX KO mice also resulted in the stable expression of above-normal levels of hFIX, reaching up to 150% of the human levels. Mice subjected to tail clip analysis showed a clotting capacity comparable to wild-type animals, thus demonstrating the rescue of the disease phenotype. Immunohistological analysis revealed clusters of hFIX-positive hepatocytes. When we tested the approach in adult FIX KO mice, we detected hFIX in plasma by ELISA and in the liver by western blot. However, the hFIX levels were not sufficient to significantly ameliorate the bleeding phenotype upon tail clip assay. Experiments conducted using a AAV donor vectors containing the eGFP or the hFIX cDNAs showed a higher recombination rate in P2 mice compared to adult animals. With this study, we demonstrate an alternative gene targeting strategy exploiting the use of the CRISPR/SaCas9 platform that can be potentially applied in the treatment of pediatric patients suffering from hemophilia, also supporting its application to other liver monogenic diseases. For the treatment of adult patients, further studies for the improvement of targeting efficiency are still required.

Antipiretik Ekstrak Etanol Daun Kemangi Aceh (Ocimi sancti folium) Pada Mencit

Traditional medicine or herbal medicine is widely used by ordinary people because it is considered safer than the use of modern medicine. This is because traditional medicine has fewer side effects than modern medicine. The purpose of this study was to observe the antipyretic effect of the ethanolic extract of basil leaves on mice. The experimental design in this study used a completely randomized design (CRD) where there were 4 treatments and 4 replications. The treatments in this study were control/P0 (not vaccinated and given extract), P1 (mouse vaccinated and given 0.5 ml of basil leaf extract), P2 (mouse vaccinated and given 0.7 ml of basil leaf extract), P3 (mouse vaccinated and given aquadest). The results showed that the body temperature of the mice that had increased due to the DPT vaccine had decreased after being given basil leaf extract. The P2 treatment, namely the extract treatment as much as 0.7 ml gave a significant effect on reducing the body temperature of mice

Oroxylin A attenuates cytokines in neonatal asthmatic mice by regulating Notch signaling pathway


 
 
 
 Purpose: To investigate the protective effect of oroxylin A against neonatal asthma in mice.
 Methods: Asthma was induced in P12 mice with ovalbumin (OVA) immediately after litter (day 0), and on day 14 of protocol. Oroxylin A was given daily in two doses (2 and 10 mg/kg, i.p.) from day 0 to 14, 2 h before the administration of OVA. The protective effect of oroxylin A against asthma was determined by evaluating lung function and levels of cytokines and IgE in OVA-challenged mice. Moreover, Western blot assay and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the effect of oroxylin A on Notch signaling pathway in OVA-challenged mice.
 Results: Oroxylin A improved lung function in OVA-challenged mice. There were significant (p < 0.01) reductions in levels of IgE and cytokines in the BALF of oroxylin A-treated group, when compared to OVA group of mice. Furthermore, treatment with oroxylin A significantly ameliorated the OVA-induced changes in protein expressions of Notch1, Jagged1, NICD, PTEN, PI3K and Akt in the CD+ cells of OVA-challenged mice (p < 0.01).
 Conclusion: These results indicate that oroxylin A improves lung function and reduces the level of cytokines in neonatal asthmatic rat by downregulating Notch signaling. Thus, oroxylin A may be clinically beneficial in the management of asthma.
 
 
 

K-Ras and p53 mouse model with molecular characteristics of human rhabdomyosarcoma and translational applications.

ABSTRACTRhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, with overall long-term survival rates of ∼65-70%. Thus, additional molecular insights and representative models are critical for identifying and evaluating new treatment modalities. Using MyoD-Cre-mediated introduction of mutant K-RasG12D and perturbations in p53, we developed a novel genetically engineered mouse model (GEMM) for RMS. The anatomic sites of primary RMS development recapitulated human disease, including tumors in the head, neck, extremities and abdomen. We confirmed RMS histology and diagnosis through Hematoxylin and Eosin staining, and positive immunohistochemical staining for desmin, myogenin, and phosphotungstic acid–Hematoxylin. Cell lines from GEMM tumors were established with the ability to engraft in immunocompetent mice with comparable histological and staining features as the primary tumors. Tail vein injection of cell lines had high metastatic potential to the lungs. Transcriptomic analyses of p53R172H/K-RasG12D GEMM-derived tumors showed evidence of high molecular homology with human RMS. Finally, pre-clinical use of these murine RMS lines showed similar therapeutic responsiveness to chemotherapy and targeted therapies as human RMS cell lines.

The Potential of Kebar Grass (Biophytum petersianum Klotzsch) on the Liver Histopathology of Lactating Mice (Mus musculus) Exposed to Carbofuran

This study aims to determine the potential of Kebar grass extract in reducing the impact of liver damage in mice (Mus musculus) during lactation exposed to carbofuran. The 42 lactation mice (Mus musculus) used in the study were divided into seven groups, each group consisting of six mice. Carbofuran, chewed grass, and vitamin C are exposed orally on days 1 to 14 after birth. This group consisted of C (aquadest control), P1 (carbofuran 0.0125 mg/day), P2 (carbofuran 0.00625 mg/day), P3 (carbofuran 0.0125 mg/day+extract of kebar grass 3.375 mg 0.2 ml), P4 (carbofuran 0.00625 mg/day+extract of kebar grass 3.375 mg 0.2 ml), P5 (carbofuran 0.0125 mg/day+vitamin C 5 mg 0.2 cc), and P6 (carbofuran 0.00625 mg/day+vitamin C 5 mg 0.2 cc). On the 15th day after birth, mice were sacrificed and their liver organs taken for microscopic examination with hematoxilin-eosin staining. The results of this study indicate that the liver histopathology of mice suffered damage characterized by an increase in the number of inflammation cell, degeneration, and necrosis in line with the carbofuran dose given during the lactation period (P1 and P2). The administration of kebar grass extract (P3 and P4) is more effective than vitamin C (P5 and P6) in reducing the amount of inflammation cell, degeneration, and hepatocyte necrosis caused by carbofuran administration in lactation mice. This can be seen in the amount of liver damage in group P3 and P4 mice less than the amount of liver damage in group P5 and P6.

The Effectivity of Thymus vulgaris Extract on IL-1 Level and Bacterial Count in The Heart of Balb-c Mice Infected with MRSA

Background: The increasing phenomenon resistance of bacteria to various types of antibiotics, for example MRSA (Methicillin Resistant Staphylococcus aureus) became a serious problem. Over the last few decades S. aureus has become the dominant cause of endocarditis. MRSA data in Indonesia are still rarely reported. This has led to the importance of extracting natural substances that have anti-bacterial effects or that modulate immune response such as Thymus vulgaris (TV). It was hypothesized that TV can overcome MRSA infection by increasing IL-1, which is the initial cytokine in a natural immune response stimulated by the presence of S. aureus. Objective: This study was to investigate the effect of Thymus vulgaris extract on IL-1 and bacterial counts in the heart of balb-c mice infected with MRSA. Methods: There were 30 male Balb-c mice randomly divided into six groups. Group K (mice infected with MRSA without treatment),P1(mice treated with TV extract 7 days later infected with MRSA), P2(mice infected and treated with vancomycin), P3(mice infected and treated with TV+vancomycin extract), P4(mice infected and treated with amoxicillin), and P5 (mice infected and treated with TV extract+amoxicillin). IL-1 and bacterial count was assessed by ELISA and microbiological culture. Results: The result showed that there was a significant increase in IL-1 between groups (p=0.001).The median decrease in the bacterial count in the heart was 0 in almost treatment group except for P4 group which its median was 26.000 (p=0.161). The correlation between IL-1 levels and MRSA count in the heart had a weak negative correlation (r=-0.182). Conclusion: Thymus vulgaris extract was effective to increase IL-1 levels significantly but was not proven to decrease MRSA count in the heart of balb-c mice.

High glutamate concentration reverses the inhibitory effect of microglial adenosine 2A receptor on NLRP3 inflammasome assembly and activation

NLRP3 inflammasome plays a crucial role in the innate immune system. Our group previously reported that the microglial adenosine 2A receptor (A2AR) regulates canonical neuroinflammation, which is affected by the glutamate concentration. However, the regulatory effect of A2AR on NLRP3 inflammasome and the effects of glutamate concentration remain unknown. Therefore, we aimed to investigate the regulatory effect of microglial A2AR on NLRP3 inflammasome assembly and activation as well as the effects of glutamate concentration on the inflammasome assembly and activation. Experiments were conducted on magnetically sorted primary microglia from P14 mice. The results showed that pharmacological A2AR activation ameliorated NLRP3 activation under no or low glutamate concentrations, but this effect was reversed by high glutamate concentrations. Moreover, the mRNA levels of NLRP3 inflammasome-related genes were not affected by A2AR activation or the glutamate concentration. We further demonstrated that A2AR activation inhibited the interaction between NLRP3 and caspase 1 under no or low glutamate concentrations while promoting their interaction under high glutamate concentrations. The oligomerization of ASC also showed a similar trend. In conclusion, our findings proved that the high glutamate concentration could reverse the inhibition of A2AR on NLRP3 inflammasome activation by modulating its assembly, which provides new insights into the regulatory effect of A2AR on neuroinflammation under different pathological conditions.

Tolerance Induced by Antigen-Loaded PLG Nanoparticles Affects the Phenotype and Trafficking of Transgenic CD4+ and CD8+ T Cells.

We have shown that PLG nanoparticles loaded with peptide antigen can reduce disease in animal models of autoimmunity and in a phase 1/2a clinical trial in celiac patients. Clarifying the mechanisms by which antigen-loaded nanoparticles establish tolerance is key to further adapting them to clinical use. The mechanisms underlying tolerance induction include the expansion of antigen-specific CD4+ regulatory T cells and sequestration of autoreactive cells in the spleen. In this study, we employed nanoparticles loaded with two model peptides, GP33–41 (a CD8 T cell epitope derived from lymphocytic choriomeningitis virus) and OVA323–339 (a CD4 T cell epitope derived from ovalbumin), to modulate the CD8+ and CD4+ T cells from two transgenic mouse strains, P14 and DO11.10, respectively. Firstly, it was found that the injection of P14 mice with particles bearing the MHC I-restricted GP33–41 peptide resulted in the expansion of CD8+ T cells with a regulatory cell phenotype. This correlated with reduced CD4+ T cell viability in ex vivo co-cultures. Secondly, both nanoparticle types were able to sequester transgenic T cells in secondary lymphoid tissue. Flow cytometric analyses showed a reduction in the surface expression of chemokine receptors. Such an effect was more prominently observed in the CD4+ cells rather than the CD8+ cells.

7, 8-Dihydroxyflavone, a TrkB receptor agonist, provides minimal protection against retinal vascular damage during oxygen-induced ischemic retinopathy.

Retinopathy of prematurity (ROP) is one of the main causes of blindness in children worldwide. Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin-related kinase B (TrkB), play critical protective roles in the development and function of neurons and vasculature. Lack of BDNF expression results in increased endothelial cell apoptosis and reduced endothelial cell-cell contact. Premature babies who develop ROP tend to have lower serum BDNF levels. BDNF expression is also significantly lower in mouse retinas following exposure to hyperoxia compared to those reared in room air. Specifically, BDNF promotes angiogenic tube formation of endothelial cells (EC), and it is considered an EC survival factor required for stabilization of intramyocardial vessels. We hypothesized that the activation of TrkB receptor protects retinal vasculature in the mice during oxygen-induced ischemic retinopathy (OIR), a model of ROP. To test this hypothesis, we treated neonatal mice with 7,8-dihydroxyflavone (DHF) (5 mg/kg body weight), a TrkB receptor agonist. We examined its potential protective effects on retinal vessel obliteration and neovascularization, two hallmarks of ROP and OIR. We found that retinas from DHF treated postnatal day 8 (P8) and P12 mice have similar levels of vessel obliteration as retinas from age-matched control mice subjected to OIR. Similarly, DHF showed no significant effect on mitigation of retinal neovascularization during OIR in P17 mice. Collectively, our studies demonstrate that the TrkB receptor agonist DHF provides no significant protective effects during OIR.

Tetraethylammonium chloride reduces anaesthetic-induced neurotoxicity in Caenorhabditis elegans and mice

BackgroundIf anaesthetics cause permanent cognitive deficits in some children, the implications are enormous, but the molecular causes of anaesthetic-induced neurotoxicity, and consequently possible therapies, are still debated. Anaesthetic exposure early in development can be neurotoxic in the invertebrate Caenorhabditis elegans causing endoplasmic reticulum (ER) stress and defects in chemotaxis during adulthood. We screened this model organism for compounds that alleviated neurotoxicity, and then tested these candidates for efficacy in mice. MethodsWe screened compounds for alleviation of ER stress induction by isoflurane in C. elegans assayed by induction of a green fluorescent protein (GFP) reporter. Drugs that inhibited ER stress were screened for reduction of the anaesthetic-induced chemotaxis defect. Compounds that alleviated both aspects of neurotoxicity were then blindly tested for the ability to inhibit induction of caspase-3 by isoflurane in P7 mice. ResultsIsoflurane increased ER stress indicated by increased GFP reporter fluorescence (240% increase, P<0.001). Nine compounds reduced induction of ER stress by isoflurane by 90–95% (P<0.001 in all cases). Of these compounds, tetraethylammonium chloride and trehalose also alleviated the isoflurane-induced defect in chemotaxis (trehalose by 44%, P=0.001; tetraethylammonium chloride by 23%, P<0.001). In mouse brain, tetraethylammonium chloride reduced isoflurane-induced caspase staining in the anterior cortical (–54%, P=0.007) and hippocampal regions (–46%, P=0.002). DiscussionTetraethylammonium chloride alleviated isoflurane-induced neurotoxicity in two widely divergent species, raising the likelihood that it may have therapeutic value. In C. elegans, ER stress predicts isoflurane-induced neurotoxicity, but is not its cause.

Development of Notch1 Positive T-Lineage Lymphomas or Splenic Marginal Zone Lymphomas with Pan-Hematopoietic or Pro-B Cell Specific Deletion of Trp53 with Distinct Differentially Dysregulated Pathways

Deletion of the tumor suppressor gene TP53 (Trp53 in mice) has been associated with the development of numerous human malignancies. TP53 acts as a central coordinator of the DNA damage response. In mice, pan-Trp53 deletion leads predominantly to the development of T-cell lymphomas, followed by B-cell lymphomas, sarcomas and teratomas. In order to dissect the role of Trp53 in the hematopoietic system, we created two different loss of function mouse models: Pan-hematopoietic Trp53 deletion using Vav1-Cre based deletion; and a B-cell-specific deletion was created using CD19-Cre.Vav1-p53CKO mice developed hematolymphoid malignancies with 100% penetrance by 12 months. Most malignancies observed were CD3e+ T-lineage lymphomas involving the thymus or spleen (37/45). Beyond 200 days, these mice predominantly developed mixed myeloid malignancies. The shift away from T-lineage malignancies in older mice may reflect aging-related decline of pre-malignant lymphoid progenitors and skewing to myeloid progenitors.Flow cytometric characterization of the T-lineage lymphomas identified a mix of tumors, including double-negative (CD4-CD8-), double-positive (CD4+CD8+), or single positive (CD4/CD8). In pre-malignant mice, Vav1-p53CKO thymocytes showed accelerated maturation with most of the cells in the DN4 stage, suggesting a bypass of the p53-dependent DN3 β-selection checkpoint. All T-lineage lymphomas showed overexpression of surface Notch1 as well as overexpression of Notch1 targets Hes1 and p21 at the transcript level. Consistent with prior data, normal murine thymocyte subsets showed high levels of expression of Notch1 target genes at the DN3 stage of development, which appears dysregulated in these T-lineage lymphomas. This Notch1 activation was found to be multifactorial with increased Mdm2 and decreased Numb levels seen in tumors. Overall, we demonstrate Notch1 activation and subsequent acceleration through the T-cell developmental stages in this model of pan-hematopoietic Trp53 deletion.The B-cell specific Trp53 knockout mice (CD19-P53CKO) (n=54) were followed up for up to 2 years. The majority (47/54) developed splenomegaly in an age-dependent manner. Histologic examination showed marginal zone expansion (6/54), frank low-grade marginal zone lymphoma (16/54) or diffuse splenic lymphoma (25/54). The disease was confined to the spleen in the case of lower-grade histology while higher grades correlated with liver and kidney involvement. Flow cytometric analysis of tumors showed B220+ CD19+ IgM+ cells. Interestingly, these tumors demonstrated low levels of Notch2 expression, which normally is highly expressed in marginal zone B-cells.In order to characterize pathogenesis, we sorted follicular and marginal zone B-cells from floxed P53 and pre-malignant CD19-P53CKO mice. RNA was isolated from all these fractions and the spleens of 5 CD19-P53KO mice with diffuse lymphoma and subjected to RNA-Seq. A comparison of the floxed p53 with the CD19-P53CKO fractions (follicular and marginal zone) revealed a highly similar transcriptome. On the other hand, p53-deficient lymphomas showed >10,000 genes significantly differentially expressed demonstrating the unique transcriptome which developed during malignant transformation.Pathway analysis of these genes using Gene Set Enrichment Analysis (GSEA) identified enrichment of PI3K, Rap1 and MAPK signaling pathways, which are associated with cellular proliferation. Overexpression of the PI3K pathway genes Ccne1, Sgk1, Mapk13 and Pik3cb were validated by qPCR in 10 independent tumor samples when compared to the splenic marginal zone fractions. In the B-cell lineage, Trp53 deficiency leads to the dysregulation of multiple genes involved in key cellular signaling pathways, including the PI3K/MAPK pathway.In summary, pan hematopoietic deletion of Trp53 led to T-lineage lymphoma in young mice and myeloid tumors in older mice; with activation of Notch1 signaling in the former. B-cell specific deletion of Trp53 led to splenic marginal zone and diffuse B-cell lymphoma with transcriptional dysregulation of key signaling molecules. Hence, tumorigenesis by Trp53 deletion is tightly linked to lineage and appears to dysregulate key signaling pathways that are operant in those lineages, potentially identifying novel strategies for therapeutic interventions in P53 dependent human hematolymphoid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Kir4.1 is coexpressed with stemness markers in activated astrocytes in the injured brain and a Kir4.1 inhibitor BaCl2 negatively regulates neurosphere formation in culture.

Astrocytes are activated in response to brain damage. Here, we found that expression of Kir4.1, a major potassium channel in astrocytes, is increased in activated astrocytes in the injured brain together with upregulation of the neural stem cell markers, Sox2 and Nestin. Expression of Kir4.1 was also increased together with that of Nestin and Sox2 in neurospheres formed from dissociated P7 mouse brains. Using the Kir4.1 blocker BaCl2 to determine whether Kir4.1 is involved in acquisition of stemness, we found that inhibition of Kir4.1 activity caused a concentration-dependent increase in sphere size and Sox2 levels, but had little effect on Nestin levels. Moreover, induction of differentiation of cultured neural stem cells by withdrawing epidermal growth factor and fibroblast growth factor from the culture medium caused a sharp initial increase in Kir4.1 expression followed by a decrease, whereas Sox2 and Nestin levels continuously decreased. Inhibition of Kir4.1 had no effect on expression levels of Sox2 or Nestin, or the astrocyte and neuron markers glial fibrillary acidic protein and β-tubulin III, respectively. Taken together, these results indicate that Kir4.1 may control gain of stemness but not differentiation of stem cells.

Repression of p53 function by SIRT5-mediated desuccinylation at Lysine 120 in response to DNA damage.

p53 is a classic tumor suppressor that functions in maintaining genome stability by inducing either cell arrest for damage repair or cell apoptosis to eliminate damaged cells in response to different types of stress. Posttranslational modifications (PTMs) of p53 are thought to be the most effective way for modulating of p53 activation. Here, we show that SIRT5 interacts with p53 and suppresses its transcriptional activity. Using mass spectrometric analysis, we identify a previously unknown PTM of p53, namely, succinylation of p53 at Lysine 120 (K120). SIRT5 mediates desuccinylation of p53 at K120, resulting in the suppression of p53 activation. Moreover, using double knockout mice (p53-/-Sirt5-/-), we validate that the suppression of p53 target gene expression and cell apoptosis upon DNA damage is dependent on cellular p53. Our study identifies a novel PTM of p53 that regulates its activation as well as reveals a new target of SIRT5 acting as a desuccinylase.

OS13.3.A Establishment of a novel system to specifically trace and ablate quiescent/slow cycling cells in high-grade glioma

Abstract BACKGROUND High-grade gliomas are the most common malignant brain tumors, with poor prognosis due to recurrence and tumor infiltration after surgical removal and chemotherapy. Quiescent/slow cycling stem cells have been proposed to be one of the main players of tumor relapse but their involvement in in the infiltration remain unclear. Despite they have been described in mouse models or after transcriptional profiling of human tumor samples, their direct visualization, targeting and ablation remains a challenge. MATERIAL AND METHODS Tumors were induced over-expressing oncogenic forms of MET and p53 in the subventricular zone (SVZ) of P2 mouse brain as well as human forebrain organoids. The co-expression with specific cell cycle sensors as well as lineage specific CreERT2 under control of stem cells promoters allowed to visualize and target glioma stem cells. RESULTS Here, we used a fluorescent cell cycle sensor to visualize quiescent tumor cells in mouse brain cancer and human cancer organoids. In particular, we characterized them within the tumor revealing the invasiveness capacity of slow cycling tumor cells. Furthermore, we generated a new system to specifically trace and ablate such cells. Indeed, lineage tracing experiments allowed to trace quiescent Prom1 progeny in the tumors after temozolomide treatment. In addition, the selective ablation of qProm1 in mouse brain cancer reduced tumor infiltration. Finally, time-lapse experiments showed that slow cycling cells are also able to infiltrate co-cultured human brain cancer organoids. CONCLUSION Overall, our data show that quiescent/slow cycling cells are key driver of tumor invasiveness, the major malignant feature of high-grade brain cancer.