P53 Mice Research Articles

Cntn6 deficiency impairs allocentric navigation in mice.

Introduction CNTN6 is an immunoglobulin domain‐containing cell adhesion molecule that belongs to the contactin family. It is involved in the development of the nervous system. We aim to determine the effect of Cntn6 deficiency on the allocentric navigation in mice.MethodsWe recorded the travel distance and escape time of wild‐type and Cntn6 mutant male and female mice in the Morris water maze task according to the protocol.ResultsThere was hardly any Cntn6 expression in the hippocampus of postnatal day 0 (P0) mice, while obvious Cntn6 expression was present in the hippocampal CA1 region of the P7 mice. During the acquisition period of Morris water maze task (Day 1 to 4), Cntn6 −/− male mice failed to shorten the escape time to reach platform on the third day, while the travel distance to platform was not significantly different. There was no significant difference in both escape time and travel distance to the platform among all female subjects. In the probe trial test (Day 5), spatial memory of the female mutant mice was mildly affected, while Cntn6 −/− male mice were normal. In the spatial relearning test (Day 7 to 10), Cntn6 −/− male mice showed no difference in escape time to the platform compared to the wild‐type male mice, while Cntn6 deficient female mice required shorter escape time to travel to the platform on day 7, day 8, and day 10.Conclusions Cntn6 is expressed in the developing hippocampus in mice. Cntn6 deficiency affects spatial learning and memory, indicating that Cntn6 plays a role in the development of hippocampus and affects allocentric navigation of the animals.

Endocardial cell heterogeneity at the origin of valvular cell clones

Valves are affected in 1/3 of cardiac congenital diseases. Cardiac valves derive from the process of endothelial-mesenchymal transition (EMT) of endocardial cells in the atrioventricular canal (AVC) and in the outflow tract of embryonic mouse hearts at E9.5 and E10.5, respectively. The origin and fate of endocardial cells as well as how EMT is specifically triggered in a subset of the latter are still poorly understood. To determine the extent of endocardial cells and valvular cells heterogeneity and in turn to assess whether endocardial cells are already committed to a valvular specific cell type. Clonal analysis: a genetic labeling of endocardial valve progenitors was performed using Brainbow and CAGERT2Cre mouse strains. Single cell sequencing: Tie2Cre/− male was bred with RosatdTomato females. AVC cells were dissected out from E9.5 embryos. Mitral valve leaflets were dissected out from both E16.5 and P0 mice. All cells were enzymatically dissociated and tomato+ cells sorted out by FACS and used for single cell RNA sequencing using the 10X genomics technology. Preliminary results point to a cell heterogeneity in each cell population so far investigated. A full panel of both clonal analysis and single cell RNA analysis will be presented.

Does p53 Inhibition Suppress Myocardial Ischemia-Reperfusion Injury?

p53 is well known as a regulator of apoptosis and autophagy. In addition, a recent study showed that p53 is a modulator of the opening of the mitochondrial permeability transition pore (mPTP), a trigger event of necrosis, but the role of p53 in necrosis induced by myocardial ischemia-reperfusion (I/R) remains unclear. The aim of this study was to determine the role of p53 in acute myocardial I/R injury in perfused mouse hearts. In male C57BL6 mice between 12 and 15 weeks of age, 2 types of p53 inhibitors were used to suppress p53 function during I/R: pifithrin-α, an inhibitor of transcriptional functions of p53, and pifithrin-μ, an inhibitor of p53 translocation from the cytosol to mitochondria. Neither infusion of these inhibitors before ischemia nor infusion for the first 30-minute period of reperfusion reduced infarct size after 20-minute ischemia/120-minute reperfusion. Infarct sizes were similar in p53 heterozygous knockout mice (p53+/-) and wild-type mice (WT), but recovery of rate pressure product (RRP) 120 minutes after reperfusion was higher in p53+/- than in WT. The protein expression of p53 in WT was negligible under baseline conditions, during ischemia, and at 10 minutes after the start of reperfusion, but it became detectable at 120 minutes after reperfusion. In conclusion, upregulation of p53 during the late phase of reperfusion plays a significant role in contractile dysfunction after reperfusion, although p53 is not involved in cardiomyocyte necrosis during ischemia or in the early phase of reperfusion.

Abnormal Ribosome Biogenesis in Aortic Medial Smooth Muscle Cells Partly Induce P53 Dependence Apoptosis is Relate to Aortic Dissection

Background: Ribosome biogenesis is related to cell proliferation and contractility. Aortic smooth muscle cells showed proliferation and apoptosis in aortic dissection. Theoretically, ribosome biogenesis should be enhanced in aortic smooth muscle cell at proliferative state but suppressed at apoptosis state. It still needs to elucidate the ribosome biogenesis status and possible mechanism in aortic dissection. Methods: The BOP1 level, indicator of rRNA processing, was detected in clinical aortic samples. The protein synthesis, motility, apoptosis and related protein were investigated upon knockdown BOP1 expression, over-express BOP1 or cx-5461 treatment. PFTI± was used to deplete p53 function. BAPN-base mouse AD model was to explore the ribosome biogenesis status in vivo. Administration mouse with cx-5461 was used to interference ribosome biogenesis in vivo. P53(-/-) mice were used to explore the function of p53 in BAPN-base AD model. Findings: BOP1 was decreased in aortic sample of AD group. Knockdown BOP1 slowed-down protein synthesis rate and cell mobility in HASMCs. cx-5461 lead HASMCs apoptosis, which partly attenuated by depletion p53. Moreover, cx-5461 aggravated the severity of AD in vivo. cx-5461 cause proliferative- suppression and apoptosis in aortic smooth muscle cells. P53(-/-) mice showed extended life-span and lower AD incidence rate with significant increased proliferation and low apoptosis rate of aortic smooth muscle cells. Conclusion: Our finding indicated impaired ribosome biogenesis in aortic smooth muscle cells, results in p53 dependent proliferative-inhibition and apoptosis is involved in aortic dissection. Clinical Trial Registry Number: ChiCTR1800014384 Funding Statement: National Natural Science Foundation of China (NSFC)81570428; National Natural Science Foundation of China (NSFC)81600367. Conflicts of Interest: There were no potential conflicts of interest to be disclosed. Ethics Approval Statement: This study protocol was approved by the Clinical Research Ethics Committees of Renmin Hospital of Wuhan University of China and registered at the Chinese Clinical Trial Registry (ChiCTR), and informed written consent was obtained from each subject.

P.3.015 - Selective inhibitors of GSK-3β: a suitable therapy for Down syndrome?

Introduction Intellectual disability (ID) is the most invalidating feature of Down syndrome (DS), a condition caused by triplication of HSA21. ID is mainly due to early-occurring neurogenesis defects. Despite intense efforts, there are currently no pharmacotherapies for DS. Recent evidence in the Ts65Dn model of DS shows that overactivation of GSK3β, a kinase strongly involved in neurogenesis [1], plays a key role in neurogenesis impairment in DS [2]. Several inhibitors of GSK3β have been developed during the last few years, as a strategy for treating psychiatric disorders. Interestingly, recent evidence in adult rats shows that tideglusib (NP031112), a selective non-ATP competitive GSK3β inhibitor, increases neurogenesis in the hippocampus [3], a region fundamental for long-term memory. Aim Based on these premises, the goal of the current study was to establish whether treatment with tideglusib restores neurogenesis in the hippocampal dentate gyrus (DG) of Ts65Dn mice and whether treatment leads to memory improvement. Methods In vivo study. Four-months-old Euploid (Eu) and Ts65Dn (Ts) mice were i.p. injected with saline (sham), vehicle (corn oil) or vehicle+tideglusib (50 mg/kg) for one month and memory was tested with the Morris Water Maze (MWM). Eu and Ts pups were injected with saline, vehicle or vehicle+tideglusib (50 mg/kg) from postnatal day 3 (P3) to P15. In vitro study. Cultures of NPCs from the subventricular zone of P2 mice were treated with i) tideglusib (0.1-10.0 µM), ii) each of the two main components of corn oil: Oleic Acid (OA; 100 µM) and Linoleic Acid (LA; 100 µM); iii) tideglusib+OA or tideglusib+LA. Statistics. In vivo: three-way mixed ANOVA (learning phase of MWM) and two-way ANOVA (probe test and immunohistochemistry) followed by post hoc Fisher LSD test. In vitro: one-way ANOVA followed by post hoc Tukey test. Results Ts were severely impaired in various learning and memory parameters (p Conclusion The finding that tideglusib does not improve neurogenesis and behavior indicates that it is not a suitable treatment for DS. The finding that the fatty acids present in the vehicle (OA and LA) have a pro-neurogenic effect suggests that agonists of fatty acid receptors may represent a good strategy to ameliorate brain development in individuals with DS.

HCN channels contribute to the sensitivity of intravenous anesthetics in developmental mice.

It is widely accepted that the induction dose of anesthetics is higher in infants than in adults, although the relevant molecular mechanism remains elusive. We previously showed neuronal hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to hypnotic actions of propofol and ketamine. Interestingly, the expression of HCN channels in neocortex significantly changes during postnatal periods. Thus, we postulated that changes in HCN channels expression might contribute to sensitivity to intravenous anesthetics. Here we showed the EC50 for propofol- and ketamine-induced loss-of-righting reflex (LORR) was significantly lower for P35 than for P14 mice. Cerebrospinal fluid concentrations of propofol and ketamine were significantly higher in P14 mice than in P35 mice, with similar propofol- and ketamine-induced anesthesia at the LORR EC50. Western blotting indicated that the expression of HCN channels in neocortex changed significantly from P14 to P35 mice. In addition, the amplitude of HCN currents in the neocortical layer 5 pyramidal neurons and the inhibition of propofol and ketamine on HCN currents dramatically increased with development. Logistic regression analysis indicated that the changes of HCN channels were correlated with the age-related differences of propofol- and ketamine-induced anesthesia. These data reveal that the change of HCN channels expression with postnatal development may contribute to sensitivity to the hypnotic actions of propofol and ketamine in mice.

Neuroprotection of the hypoxic-ischemic mouse brain by human CD117+CD90+CD105+ amniotic fluid stem cells

Human amniotic fluid contains two morphologically-distinct sub-populations of stem cells with regenerative potential, spindle-shaped (SS-hAFSCs) and round-shaped human amniotic fluid stem cells (RS-hAFSCs). However, it is unclear whether morphological differences correlate with functionality, and this lack of knowledge limits their translational applications. Here, we show that SS-hAFSCs and RS-hAFSCs differ in their neuro-protective ability, demonstrating that a single contralateral injection of SS-hAFSCs into hypoxic-ischemic P7 mice conferred a 47% reduction in hippocampal tissue loss and 43–45% reduction in TUNEL-positive cells in the hippocampus and striatum 48 hours after the insult, decreased microglial activation and TGFβ1 levels, and prevented demyelination. On the other hand, RS-hAFSCs failed to show such neuro-protective effects. It is possible that SS-hAFSCs exert their neuroprotection via endoglin-dependent inhibition of TGFβ1 signaling in target cells. These findings identify a sub-population of CD117+CD90+CD105+ stem cells as a promising source for the neuro-protection of the developing brain.

Abstract 16: Oligodendrocytes Provide Trophic Support to Vasculature Following Ischemic Stroke in Neonatal Mice

Introduction: Incidence of perinatal arterial ischemic stroke (PAIS) is surprisingly high, approaching that of the elderly. It has been postulated that the developing brain has a greater degree of plasticity and capacity for repair compared to the adult, thereby enhancing functional recovery. To address this issue, we utilized the recently developed intraluminal filament mouse model of ischemic stroke in postnatal day 10 mice. The aim of this study was to examine the cellular responses to ischemic stroke in the neonatal brain that contribute to the enhanced functional recovery. Method: Middle cerebral artery occlusion (45 minutes) was performed on adult and p10 mice using the intraluminal filament method. Immunohistochemistry was performed to identify oligodendrocyte precursors (PDGFRα/Olig2+), mature oligodendrocytes (CC1/Olig2+), vascular endothelial cells (Glut-1) and pericytes (CD13), and TUNEL to identify injured cells. Blood brain barrier integrity was examined by staining for fibrinogen. A protein array was conducted to examine extracellular protein levels. In situ hybridization was used to confirm source of IGF-1. Results: Oligodendrocytes were remarkably resistant to ischemic insult in the neonatal brain up to 7 days after injury; no change in oligodendrocyte precursor or mature oligodendrocyte cell count following neonatal stroke, compared to the significant loss of oligodendrocytes following adult stroke. In contrast, large numbers of TUNEL+ neurons were observed in both groups following stroke. In contrast to well-established vascular injury in adult stroke, no change in vascular integrity (Glut-1 positive vessel length/number or fibrinogen leakage) was detected following neonatal stroke. Unbiased analysis of extracellular protein levels in the adult and neonatal brain following stroke identified IGF-1 as a protein uniquely up-regulated following neonatal stroke. In situ hybridization revealed increased IGF-1 mRNA levels in oligodendrocytes following neonatal stroke. Conclusion: These results suggest that ischemia-resistant developing oligodendrocytes provide trophic support (IGF-1) to the vasculature following neonatal stroke.

Influence of sevoflurane exposure on mitogen-activated protein kinases and Akt/GSK-3β/CRMP-2 signaling pathways in the developing rat brain.

Prolonged exposure to volatile anesthetics causes neurodegeneration in developing animal brains. However, their underlying mechanisms of action remain unclear. The current study investigated the expression of proteins associated with the mitogen-activated protein kinases (MAPK) and protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/collapsin response mediator protein 2 (CRMP-2) signaling pathways in the cortices of neonatal mice following exposure to sevoflurane. Seven-day-old (P7) neonatal C57BL/6 mice were randomly divided into 2 groups and either exposed to 2.6% sevoflurane or air for 6 h. Terminal deoxyribonucleotide transferase mediated dUTP nick end labeling (TUNEL) staining, as well as the expression of activated caspase-3 and α-fodrin, was used to detect neuronal apoptosis in the cortices of mice. MAPK signaling pathways were investigated by detecting the expression of phosphorylated (p-) extracellular signal-regulated kinase 1/2 (ERK1/2), p-cyclic adenosine monophosphate response element-binding protein (CREB), p-p38, p-nuclear factor (NF-κB) and p-c-Jun N-terminal kinase (p-JNK). Akt/GSK-3β/CRMP-2 signaling pathways were assessed by detecting the expression of p-Akt, p-GSK-3β and p-CRMP-2 in the cortices of P7 mice 2 h following exposure to sevoflurane. The results demonstrated that sevoflurane significantly increased the apoptosis of cells in the retrosplenial cortex (RS), frontal cortex (FC) and parietal association cortex (PtA), increased the expression of cleaved caspase-3 expression and promoted the formation of 145 kDa and 120 kDa fragments from α-fodrin. Sevoflurane inhibited the phosphorylation of ERK1/2 and CREB, stimulated the phosphorylation of p38 and NF-κB, but did not significantly affect the phosphorylation of JNK. Furthermore, sevoflurane inhibited the phosphorylation of Akt, decreased the phosphorylation of GSK-3β at ser9 and increased the phosphorylation of CRMP2 at Thr514. These results suggest that multiple signaling pathways, including ERK1/2, P38 and Akt/GSK-3β/CRMP-2 may be involved in sevoflurane-induced neuroapoptosis in the developing brain.

Skeletal Muscle Fibre-Specific Knockout of p53 Does Not Reduce Mitochondrial Content or Enzyme Activity.

Tumour protein 53 (p53) has been implicated in the regulation of mitochondrial biogenesis in skeletal muscle, with whole-body p53 knockout mice displaying impairments in basal mitochondrial content, respiratory capacity, and enzyme activity. This study aimed to determine the effect of skeletal muscle-specific loss of p53 on mitochondrial content and enzyme activity. Mitochondrial protein content, enzyme activity and mRNA profiles were assessed in skeletal muscle of 8-week-old male muscle fibre-specific p53 knockout mice (p53 mKO) and floxed littermate controls (WT) under basal conditions. p53 mKO and WT mice displayed similar content of electron transport chain proteins I-V and citrate synthase enzyme activity in skeletal muscle. In addition, the content of proteins regulating mitochondrial morphology (MFN2, mitofillin, OPA1, DRP1, FIS1), fatty acid metabolism (β-HAD, ACADM, ACADL, ACADVL), carbohydrate metabolism (HKII, PDH), energy sensing (AMPKα2, AMPKβ2), and gene transcription (NRF1, PGC-1α, and TFAM) were comparable in p53 mKO and WT mice (p > 0.05). Furthermore, p53 mKO mice exhibited normal mRNA profiles of targeted mitochondrial, metabolic and transcriptional proteins (p > 0.05). Thus, it appears that p53 expression in skeletal muscle fibres is not required to develop or maintain mitochondrial protein content or enzyme function in skeletal muscle under basal conditions.

Degeneration of short-wavelength cone cells in rd12 mice

Background Retinitis pigmentosa (RP) is one of the causes of congenital blindness.It is well known that the degeneration process of rod cells is difficult to detect in RP.Retinal degeneration 12 (rd12) mice is a new, spontaneously arising mouse model for human Leber congenital amaurosis (LCA), and it is helpful for us to explore the pathogenesis and determine the treating target of RP. Objective This study was to investigate the natural disease process of short-length sensitive cone cells in rd12 mice, a LCA Rpe65rd12 (B6[A]-Rpe65rd12/J) mouse. Methods The rd12 mice at postnatal (P) 14, P21, P35 and P90 were selected (5 mice for each), and the wild-type C57BL/6J (B6) mice with matched ages were included as controls.Photopic full-field electroretinogram (ERG) was recorded with Roland Q450SC UV visual physiology instrument.Cone response was recorded using single white light-emitting diode (LED) stimulation with the flash intensity of 1.00 cds/m2 and 1.96 cds/m2, and short wave-length sensitive cone response was recorded using ultraviolet light ([363±6]nm) stimulation with the flash intensity of 2.0 mWs/m2 and 3.0 mW/m2.The mice were sacrificed and retinal whole-mounts were prepared.The distribution and number of cone cells and UV-sensitive cone cells were detected by FITC-peanut agglutinin (FITC-PNA) and Cy3 immunofluorescence stainning, respectively. Results In P14 rd12 mice, the ERG responses of overall cone cells presented the negative waveform and the latency was delayed, and UV-sensitive cone response was unrecordable.The b-wave amplitude of overall cone cells reduced by 75% in P21 rd12 mice compared with wild-type B6 mice, and the mean latency of b-wave in the P21 rd12 mice was significantly longer than that in the wild-type B6 mice ([102.80±11.39]ms vs.[43.40±5.60]ms) (t=-8.106, P=0.001). The mean b-wave amplitudes of UV-sensitive cone cells were (59.60±36.00), (82.40±12.22) and (68.43±17.63)μV in the wild-type B6 mice, and those in the rd12 mice were unrecordable.Immunofluorescence showed that a lager number of cone cells with green fluorescence were seen, and the expression of opsin with red fluorescence was displayed in the UV-sensitive cone cells of nasal lateral on retinal ventral side in P14 wild-type B6 mice; while only a few opsin positive-response cells were seen in P14, P21 and P35 rd12 mice. Conclusions In rd12 mice, the functional abnormality and quantitative reduction of cone cells appear in the early postnatal days, and the loss of UV-sensitive cone cells is earlier and more obvious. Key words: Retinal degeneration/congenital; Eye proteins/metabolism; Photoreceptor/pathology; Electroretinography; Fluorescent antibody technique; Leber congenital amaurosis; Mice, inbred C57BL; Disease models, animal

Spatiotemporal changes in the distribution of LHFPL5 in mice cochlear hair bundles during development and in the absence of PCDH15.

Mechanosensory transduction by vertebrate hair cells depends on a protein complex at the tips of shorter stereocilia associated with mechanoelectrical transduction channels activated by tip links in the hair bundle. In mammalian hair cells, this complex includes transmembrane channel-like protein subunit 1 (TMC1), lipoma HMGIC fusion partner-like 5 protein (LHFPL5) and protocadherin 15 (PCDH15), a lower-end component of the tip link. TMC1 interacts with LHFPL5 and PCDH15 but how the complex develops to maturity, and the relationships between these proteins, remains uncertain. Here we evaluate the spatiotemporal development of LHFPL5 distributions in mouse cochlear hair bundles by immunofluorescence and immunogold transmission electron microscopy, from postnatal day 0 (P0) through P21 in wild type and PCDH15-deficient mice. At P0, hair bundles contain many short microvilli-like processes which we term unranked stereocilia, and a subset of lengthening rows, adjacent to a kinocilium. LHFPL5 is distributed throughout the bundle, including on stereocilia tips and the kinocilium. At P3, 4-to-6 rows of ranked stereocilia are evident, total LHFPL5 expression peaks, and LHFPL5 is localised to ranked stereocilia tips of all rows and to lower shaft/ankle links. By P12, the bundle has a mature pattern with 3 ranked rows but virtually no unranked stereocilia or kinocilium; LHFPL5 expression has declined and become restricted to the tips of shorter stereocilia. Throughout development from P0, expression of LHFPL5 is greater overall on apical than basal bundles, but there is, on average, an equal amount of labelling per labelled tip. In P3 mice lacking PCDH15, LHFPL5 labelling is not at the tips but is primarily on unranked stereocilia and lower lateral links. These data show that LHFPL5 is already present in the MET apparatus at P0 but requires PCDH15 at P3 to remain there. Shaft/ankle link localisation suggests it interacts with link proteins other than PCDH15.

Defining the Pathways of Urogenital Schistosomiasis-Associated Urothelial Carcinogenesis through Transgenic and Bladder Wall Egg Injection Models.

Urogenital schistosomiasis (infection with Schistosoma haematobium) is a major cause of bladder carcinogenesis. However, the exact mechanisms of the sequelae leading up to the development of bladder cancer are poorly understood, mainly because of a dearth of tractable mouse models. We developed a mouse model of urogenital schistosomiasis through intramural injection of parasite eggs into the bladder wall to mimic the trapping of parasite eggs in the bladder. This approach recapitulates many of the sequelae observed in infected humans. Here, we describe procedures for utilizing this surgical technique in combination with well-established transgenic mouse strains to dissect the role of cancer-related genes in the initiation and establishment of bladder carcinogenesis. The described method utilizes CRE-mediated flox activity to render mice p53 haploinsufficient before challenging them with bladder wall egg injection. These techniques are potentially amenable to studying the role of other pro-carcinogenic and cancer suppressor gene(s) in urogenital schistosomiasis-associated urothelial carcinogenesis.

PENGARUH AIR REBUSAN DAUN MAMPELAS (Teteracera indica) TERHADAP KADAR KOLESTEROL DARAH MENCIT

Tetracera indica Merr. locally known as Mampelas is commonly used in traditional remedies. It believed to have the potential to cure hypercholesterolemia. The potential use of this plant to lower blood cholesterol level was understudied; therefore this study aimed to determine the effect of the infusion of fresh leaves of Tetracera indica to the blood cholesterol levels of mice. This research was true experimental design, a post-test only design with nonequivalent (control) groups. This study used 25 male mice divided into five categories, i.e., P0 mice were administered with 0 g of infusion, P1 mice with 2 g infusion, P2 mice with 5 g infusion, P3 mice with 10 g infusion, and P4 mice without Tetracera indica infusion but 10 mg of simvastatin. The treatment in all categories were given continuously for seven days. The results showed the mean of the blood cholesterol levels were 169.4 ± 59.37 mg/dL in P0, 121.8 ± 7.62 mg/dL in P1, 136.2 ± 15.69 mg/dL in P2, 110.6 ± 37 , 01 mg/dL in P3 , and 179,0 ± 35,24 mg/dL in P4. Kruskal Wallis test was calculated to determine the difference of blood cholesterol level among all groups, and it reported p < 0.05. The results of this study showed that infusion of Tetracera indica leaf was significantly lowered the level of blood cholesterol in mice.

3. Lactobacillus casei Fermented Milk as a Treatment for Diabetes in Mice (Mus musculus)

The study aimed to find out the effect of Lactobacillus casei fermented milk as diabetic drug (Therapeutic). Mice were 3-4 month old, male and body weight ranged 20-30 g. This Research used Completely Randomized Design with factorial pattern, consisted of dose and duration of feeding. Treatments were divided into four treatment, P1 (no treatment), P2 (mice were induced with alloxan), P3 (mice were induced with alloxan and fed with fermented milk L. casei with 0.5 mL/mice) and P4 (mice were induced with alloxan and fed with Lactobacillus casei fermented milk 1 mL/mice). Provision of L. casei fermented milk provided at libitium and L. casei fermented milk given for 7 and 14 days. Blood were collected from Vena lateralis and dropped on the strip test Easy Touch GGHb. The results showed that not only dose administration of L. casei fermented milk had significantly decrease (P0.01) of glucose levels in mice, but also duration of treatment. It can be concluded that the administration of fermented milk L. casei can be used as a complement therapy for diabetic mice

Transcriptome profiling of mouse brains with qkI-deficient oligodendrocytes reveals major alternative splicing defects including self-splicing

The qkI gene encodes a family of RNA binding proteins alternatively spliced at its 3′ end, giving rise to three major spliced isoforms: QKI-5, QKI-6 and QKI-7. Their expression is tightly regulated during brain development with nuclear QKI-5 being the most abundant during embryogenesis followed by QKI-6 and QKI-7 that peak during myelination. Previously, we generated a mouse conditional qkI allele where exon 2 is excised using Olig2-Cre resulting in QKI-deficient oligodendrocytes (OLs). These mice have dysmyelination and die at the third post-natal week. Herein, we performed a transcriptomic analysis of P14 mouse brains of QKI-proficient (QKIFL/FL;-) and QKI-deficient (QKIFL/FL;Olig2-Cre) OLs. QKI deficiency results in major global changes of gene expression and RNA processing with >1,800 differentially expressed genes with the top categories being axon ensheathment and myelination. Specific downregulated genes included major myelin proteins, suggesting that the QKI proteins are key regulators of RNA metabolism in OLs. We also identify 810 alternatively spliced genes including known QKI targets, MBP and Nfasc. Interestingly, we observe in QKIFL/FL;Olig2-Cre a switch in exon 2-deficient qkI mRNAs favoring the expression of the qkI-5 rather than the qkI-6 and qkI-7. These findings define QKI as regulators of alternative splicing in OLs including self-splicing.

In vivo genetic manipulation of inner ear connexin expression by bovine adeno-associated viral vectors

We have previously shown that in vitro transduction with bovine adeno–associated viral (BAAV) vectors restores connexin expression and rescues gap junction coupling in cochlear organotypic cultures from connexin–deficient mice that are models DFNB1 nonsyndromic hearing loss and deafness. The aims of this study were to manipulate inner ear connexin expression in vivo using BAAV vectors, and to identify the optimal route of vector delivery. Injection of a BAAV vector encoding a bacterial Cre recombinase via canalostomy in adult mice with floxed connexin 26 (Cx26) alleles promoted Cre/LoxP recombination, resulting in decreased Cx26 expression, decreased endocochlear potential, increased hearing thresholds, and extensive loss of outer hair cells. Injection of a BAAV vector encoding GFP-tagged Cx30 via canalostomy in P4 mice lacking connexin 30 (Cx30) promoted formation of Cx30 gap junctions at points of contacts between adjacent non-sensory cells of the cochlear sensory epithelium. Levels of exogenous Cx30 decayed over time, but were still detectable four weeks after canalostomy. Our results suggest that persistence of BAAV-mediated gene replacement in the cochlea is limited by the extensive remodeling of the organ of Corti throughout postnatal development and associated loss of non-sensory cells.

Abstract 2063: The farnesyltransferase inhibitor tipifarnib causes dramatic tumor regression and increases survival in murine HrasG12V driven aggressive thyroid cancers: Consequent adaptive and acquired resistance mechanisms inform combination treatments with improved responses

Abstract Of the three RAS oncoproteins, only HRAS is delocalized and functionally inactivated by farnesyltransferase inhibition (FTI), an approach that has yet to be exploited clinically. We treated a murine model of poorly differentiated and anaplastic thyroid cancer (Tpo-cre/HrasG12V/p53flox/flox; Hras;p53) with the FTI tipifarnib, and observed sustained tumor regression and increased survival; however, tumors eventually recurred. Following HRAS delocalization by tipifarnib in vitro, ERK phosphorylation was only inhibited transiently in HRAS-mutant cell lines, which was associated with increased GTP loading of wild-type RAS proteins in the setting of RTK ligand stimulation. This adaptive reactivation of RAS-MAPK signaling was abrogated by selective RTK (i.e. EGFR, FGFR) inhibitors, or by MEK inhibitors. Importantly, tipifarnib combined with the MEK inhibitor AZD6244 led to improved responses in Hras;p53 mouse tumors, whereas combination with the EGFR/FGFR inhibitors erlotinib and ponatinib did not, suggesting heterogeneity of upstream inputs. In order to identify acquired resistance mechanisms, tumor-bearing Hras;p53 mice were treated with tipifarnib until resistance developed (6 months). Whole exome sequencing of resistant tumors identified a truncating NF1 mutation and an activating mutation in GNAS at high allelic frequency. Upon NF1 knockdown in the human HRAS- mutant cell line C643, tipifarnib failed to inhibit pERK in vitro and caused resistance to tipifarnib in xenografts. By contrast, activating GNAS mutations transduced into Hras;p53 mouse tumor cell lines produced tipifarnib-resistant xenografts that activated the GNAS-cAMP-CREB pathway and demonstrated signs of redifferentiation. These data show that pharmacological targeting of RAS in a genetically accurate, mouse model of a RAS-driven virulent cancer leads to objective major responses and improved survival. We identified adaptive and acquired resistance mechanisms, and show that combined treatment with selective MEK inhibitors are beneficial. These data should also inform a currently enrolling clinical trial of tipifarnib for HRAS-mutant malignancies. Citation Format: Brian R. Untch, Vanessa Dos Anjos, Maria ER Garcia-Rendueles, Jeff A. Knauf, Alan L. Ho, James A. Fagin. The farnesyltransferase inhibitor tipifarnib causes dramatic tumor regression and increases survival in murine HrasG12V driven aggressive thyroid cancers: Consequent adaptive and acquired resistance mechanisms inform combination treatments with improved responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2063. doi:10.1158/1538-7445.AM2017-2063

Abstract 5244: Partial knockdown of RLIP76 prevents cancer susceptibility of p53 null mice

Abstract P53 is a stress-responsive, genome protective, tumor suppressor whose functions are lost or altered in nearly all neoplasia. P53 homozygous knockout mice (p53-/-) develop spontaneous lymphoma and other malignancies in nearly 100% of cases by 6 months of life. In stark contrast, homozygous gene knockout of the stress-responsive, anti-apoptotic, mercapturic acid pathway transporter protein, RLIP76, results in marked protection from chemical carcinogenesis. P53 expression and activation is regulated by intermediary metabolites of this pathway, and p53 regulates the expression of key enzymes of this pathway. P53 can directly bind to RLIP76 through a CDK-interaction domain, and inhibit its transport activity as well as endocytosis stimulatory activity. These results indicate that RLIP76 is an effector of p53 in a manner analogous to its known function as the first known effector of the Ral pathways that regulate membrane plasticity, motility and invasion. This translates into reduced kinase signaling down-stream of membrane-receptor/ligand interactions. An inverse relationship between RLIP76 and p53 protein and mRNA expression has been observed in-vitro, and is confirmed by analysis of human cancer genomic analyses in the COH database. RLIP76 depletion causes apoptosis in cancer cells independent of p53 status, strengthening the view that RLIP76 is key down-stream effector of p53. We reasoned that if RLIP76 is necessary for cancer to develop in p53-deficient animals, its suppression will reduce the incidence of malignancy in 12-week old p53-/- mice treated with COHSA007 phosphorothioated antisense, 200 μg i.p.q.2wk till age 8 months. Results of our studies confirmed these predictions, since 4/4 scrambled antisense treated p53-/- mice developed T-cell lymphoma whereas 8/8 COHSA007 treated p53-/- mice survived to the end of the experiment without development of any malignancy. These findings demonstrate that RLIP76 is required for malignancy in the p53-/- mice. Taken in context of our previous studies showing that RLIP76-/- mice are highly resistant to inflammation and carcinogenesis in chemical carcinogenesis models of the skin and lung, the present findings strongly support the idea of an existential role of RLIP76 in the malignant phenotype, and strengthen the rationale for using targeted systemic depletion of RLIP76 for treatment of human neoplasia (Supported in part by the Department of Defense grant (W81XWH-16-1-0641) and USPHS grant CA 77495). Citation Format: Sharad S. Singhal, Jyotsana Singhal, Joshua Tompkins, David Horne, Xiwei Wu, Arthur Riggs, Sanjay Awasthi. Partial knockdown of RLIP76 prevents cancer susceptibility of p53 null mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2017-5244

Abstract 2578: Investigating the role of mutant p53 in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer characterized by a high rate of metastasis, limited therapeutic options, and a poor prognosis. However, there is limited information regarding the molecular mechanisms underlying the metastatic properties of ESCC. p53 is one of the most commonly mutated genes in ESCC, and our group has shown that esophageal cells lines expressing a mutation in human p53 shows signs of malignancy and increased invasion in 3D organotypic culture. A mouse model of mutant p53 (R172H) in ESCC is lacking in the field. To elucidate the role of mutant p53 in ESCC we developed a novel mouse model utilizing a genetic and carcinogenic approach. L2cre;p53-/- and p53R172H/- mice were generated and treated with 4NQO in their drinking water for 16 weeks, which resulted in the development of ESCC. Compared to wildtype mice, p53R172H/- mice and p53-/- mice exhibited a decreased tumor latency time, increased tumor frequency and a more severe tumor diagnosis. However, p53R172H/- mice and p53-/- mice displayed similar tumorigenic properties. RNA-seq was performed on cell lines established from wildtype and p53 mouse models and reveled different gene expression profiles between wildtype, p53R172H/-, and p53-/- cells. p53R172H/- cells displayed an increase in mesenchymal and decrease in epithelial marker expression, supporting the idea that they are undergoing EMT. In addition, several endocytic recycling related genes, including Rab11-fip1, Rab25, and Myo5b were downregulated in mutant and null p53 compared to wildtype cells. Further examination of the differing genetic profiles in our mouse models can provide novel insight into the role of mutant p53 in ESCC tumorigenesis and lead to the identification of new therapeutic targets. Citation Format: Ashley Lento, Apple Long, Veronique Giroux, Qiaosi Tang, Morgan Sammons, Andres Klein-Szanto, Shelly Berger, Anil Rustgi. Investigating the role of mutant p53 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2578. doi:10.1158/1538-7445.AM2017-2578