Null Animals Research Articles

DNA polymerase β deficiency in the p53 null cerebellum leads to medulloblastoma formation

Defects in DNA damage response or repair mechanisms during neurogenesis result in genomic instability, which is causative for several neural defects. These include brain tumors, particularly medulloblastoma, which occurs in the cerebellum with a high incidence in children. We generated an animal model with defective base excision repair during brain development through selective inactivation of DNA polymerase β (Polb) in neuroprogenitor cells. All of Polb conditional knockout mice developed medulloblastoma in a p53 null background, similar to the Xrcc1 and p53 double deficient animal model. XRCC1 is a scaffolding protein which is involved in DNA damage repair and binds to POLB. In both animal models, the histopathological characteristics of the medulloblastoma were similar to those of human classic medulloblastoma. Brain tumor development was slower in the Polb and p53 double null animals than in the Xrcc1 and p53 double knockout animals. Molecular marker analysis suggested that Polb- and Xrcc1-deficient medulloblastomas belonged to the SHHα subtype, underscoring the important role of genomic stability in preventing this devastating pediatric cerebellar tumor.

CDK-11-Cyclin L is required for gametogenesis and fertility in C. elegans

CDK11, a member of the cyclin-dependent kinase family, has been implicated in a diverse array of functions including transcription, RNA processing, sister chromatid cohesion, spindle assembly, centriole duplication and apoptosis. Despite its involvement in many essential functions, little is known about the requirements for CDK11 and its partner Cyclin L in a developing multicellular organism. Here we investigate the function of CDK11 and Cyclin L during development of the nematode Caenorhabditis elegans. Worms express two CDK11 proteins encoded by distinct loci: CDK-11.1 is essential for normal male and female fertility and is broadly expressed in the nuclei of somatic and germ line cells, while CDK-11.2 is nonessential and is enriched in hermaphrodite germ line nuclei beginning in mid pachytene. Hermaphrodites lacking CDK-11.1 develop normally but possess fewer mature sperm and oocytes and do not fully activate the RAS-ERK pathway that is required for oocyte production in response to environmental cues. Most of the sperm and eggs that are produced in cdk-11.1 null animals appear to complete development normally but fail to engage in sperm-oocyte signaling suggesting that CDK-11.1 is needed at multiple points in gametogenesis. Finally, we find that CDK-11.1 and CDK-11.2 function redundantly during embryonic and postembryonic development and likely do so in association with Cyclin L. Our results thus define multiple requirements for CDK-11-Cyclin L during animal development.

MIR205HG is a Long Noncoding RNA with Distinct Functions in the Thymus versus the Anterior Pituitary

Abstract MicroRNAs (miRNAs) are processed from primary RNA transcripts (pri-miRNAs) encoded in host genes. Several miRNAs including miR-205 are present within long noncoding RNAs (lncRNAs). MiR-205 is an epithelial-specific miRNA that supports thymopoiesis by positively regulating Forkhead Box N1 (Foxn1) expression. We assessed whether the host gene for miR-205, MIR205HG, has independent functions as a lncRNA. The more severe stress-induced thymic atrophy reported in miR-205-deficient mice is also evident in MIR205HG knockout lines. However, MIR205HG knockouts have a small stature phenotype. A new set of miR-205KI mice did not have this phenotype. The smaller mouse size of the MIR205HG null animals is partly a consequence of reduced levels of endocrine hormones produced by the anterior pituitary. In addition, the MIR205HG null animals had abnormal development of the lacrimal and harderian glands that produce eye secretions. Transcriptome analyses revealed that MIR205HG regulates gene expression in the anterior pituitary unlike miR-205. In contrast, transcripts regulated by miR-205 and MIR205HG in the epithelial cells of the skin and thymus overlap significantly. These data indicate that MIR205HG has a specific function as a lncRNA in the anterior pituitary in addition to its primary role as the host gene for miR-205 in thymic epithelial cells.

Bacterial and Pneumocystis Infections in the Lungs of Gene-Knockout Rabbits with Severe Combined Immunodeficiency.

Using the CRISPR/Cas9 gene-editing technology, we recently produced a number of rabbits with mutations in immune function genes, including FOXN1, PRKDC, RAG1, RAG2, and IL2RG. Seven founder knockout rabbits (F0) and three male IL2RG null (−/y) F1 animals demonstrated severe combined immunodeficiency (SCID), characterized by absence or pronounced hypoplasia of the thymus and splenic white pulp, and absence of immature and mature T and B-lymphocytes in peripheral blood. Complete blood count analysis showed severe leukopenia and lymphocytopenia accompanied by severe neutrophilia. Without prophylactic antibiotics, the SCID rabbits universally succumbed to lung infections following weaning. Pathology examination revealed severe heterophilic bronchopneumonia caused by Bordetella bronchiseptica in several animals, but a consistent feature of lung lesions in all animals was a severe interstitial pneumonia caused by Pneumocystis oryctolagi, as confirmed by histological examination and PCR analysis of Pneumocystis genes. The results of this study suggest that these SCID rabbits could serve as a useful model for human SCID to investigate the disease pathogenesis and the development of gene and drug therapies.

Hscb, a Mitochondrial Iron-Sulfur Cluster Assembly Co-Chaperone, Is a Novel Candidate Gene for Congenital Sideroblastic Anemia

Congenital sideroblastic anemias (CSAs) are uncommon inherited diseases resulting from defects in heme biosynthesis, mitochondrial translation or mitochondrial iron-sulfur cluster (ISC) assembly. CSAs are characterized by pathological mitochondrial iron deposits in bone marrow erythroblasts. Recently, mutations in mitochondrialheat shock protein 70 (HSPA9), a critical chaperone involved in mitochondrial ISC assembly, have been reported as a cause of non-syndromic CSA. Human heat shock cognate protein 20 (HSCB), a highly conserved mitochondrial co-chaperone, is the primary binding partner of HSPA9. HSCB allows the transfer of nascent ISC to HSPA9 and stimulates its ATPase activity, promoting ISC transfer to target proteins.To identify novel genes responsible for CSA, we performed whole exome sequencing on more than 75 CSA probands and their family members. In one patient, a young woman, with pancytopenia characterized by a normocytic anemia with numerous bone marrow ringed sideroblasts, we identified two variants in HSCB : a paternally-inherited promoter variant (c.-134C>A) predicted to disrupt a conserved ETS transcription factor binding site, and a maternally-inherited frameshift (c.259dup, p.T87fs*27). A fibroblast cell-line derived from the proband showed a decrease in HSCB expression, but normal HSPA9 expression compared to healthy, unrelated controls. Impairment of ETS1-dependent transcriptional activation of the promoter variant was demonstrated in K562 cells transfected with an HSCB-luciferase reporter construct.K562 cells were also employed to determine if reduced expression of HSCB could result in impaired erythroid metabolism, maturation, or proliferation. K562 cells infected with shRNA directed against HSCB were deficient in multiple mitochondrial respiratory complexes, had abnormal iron metabolism and a defect of protein lipoylation, all consistent with defective ISC metabolism. In addition, both IRP1 and IRP2 expression were decreased and cell surface transferrin receptor 1 (TFR1) expression was enhanced, suggesting disturbed cellular iron metabolism. Nevertheless, cells lacking HSCB partially retained an ability to respond to iron chelation and iron overload. Cells lacking HSCB lose their ability to hemoglobinize in response to sodium butyrate treatment (Figure 1A). This defect was confirmed in vivo using a morpholino strategy in zebrafish, as fish lacking HSCB are also unable to hemoglobize (Fig 1B).We generated an Hscb conditional mouse to better elucidate the underlying pathophysiology of the disease. Heterozygous (Hscb+/-) animals have no discernable phenotype; however, null animals die prior to embryonic day E7.5. Thus, to avoid this lethality, we employed Vav-cre animals (Tg(Vav1-cre)1Graf) to evaluate the loss of HSCB specifically in the hematopoietic compartment. Hscbc/- Vav-cre+ pups are pale and growth retarded compared to control littermates and die at approximately p10 with severe pancytopenia. To assess the loss of HSCB specifically in the erythroid lineage, we bred conditional animals to EpoR-cre (Eportm1(EGFP/cre)Uk) mice. Hscbc/- EpoR-cre+ mice die at approximately E12.5 due to a complete failure of erythropoiesis (Figure 1C). Finally, temporally inducible, hematopoietic-specific deletion animals were generated by transplantation of fetal livers from Mx-Cre (Tg(Mx1-cre)1Cgn) positive Hscbc/- animals. After polyinosinic:polycytidylic acid (pIpC) induction, global defects of hematopoiesis were observed in Mx-Cre+ animals, leading to their death 3-weeks post-induction from profound pancytopenia. A transient siderocytosis was seen in the peripheral blood between days 6-8 post-pIpC. Flow cytometry using FSC-TER119-CD44 gating strategy confirmed the defect in erythropoiesis.Taken together, these data demonstrate that HSCB is essential for hematopoiesis; both whole animal and in vitro cell culture models recapitulate the patient's phenotype, suggesting that the two patient mutations are likely disease-causing. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DPFF-1 transcription factor deficiency causes the aberrant activation of MPK-1 and meiotic defects in the Caenorhabditis elegans germline.

The d4 family of transcription factors consists of three members in mammals. DPF1/neuro-d4 is expressed mainly in neurons and the peripheral nervous system, and is important for brain development. DPF2/requiem/ubi-d4 is expressed ubiquitously and presumably functions as an apoptotic factor, especially during the deprivation of trophic factors. DPF3/cer-d4 is expressed in neurons and in the heart, and is important for heart development and function in zebrafish. In Drosophila, there is only one member, dd4, whose function is still unknown, but it is expressed in many tissues and is particularly abundant in the brain of developing embryos and in adults. Here, we present DPFF-1, the only member of this family of proteins in the nematode C. elegans. DPFF-1 is similar to its mammalian homolog DPF2/requiem/ubi-d4 because it is ubiquitously expressed during embryogenesis and in adult tissues, and because it is important for the induction of germ cell apoptosis during stress. Here, we show that dpff-1 null mutant animals produce less progeny than wild-type nematodes, presumably due to meiotic defects. Gonads of dpff-1 deficient animals showed more germ cells in pachytene and overexpressed the P-MPK-1 signal. Additionally, these animals presented higher levels of p53-induced germ cell apoptosis than wild-type animals. Furthermore, we observed that dpff-1 deficient animals are more sensitive to heat shock. This is the first report showing that the d4 family of transcription factors could be involved in meiosis and stress protection.

The Long 3'UTR mRNA of CaMKII Is Essential for Translation-Dependent Plasticity of Spontaneous Release in Drosophila melanogaster.

A null mutation of the Drosophila calcium/calmodulin-dependent protein kinase II gene (CaMKII) was generated using homologous recombination. Null animals survive to larval and pupal stages due to a large maternal contribution of CaMKII mRNA, which consists of a short 3'-untranslated region (UTR) form lacking regulatory elements that guide local translation. The selective loss of the long 3'UTR mRNA in CaMKII-null larvae allows us to test its role in plasticity. Development and evoked function of the larval neuromuscular junction are surprisingly normal, but the resting rate of miniature excitatory junctional potentials (mEJPs) is significantly lower in CaMKII mutants. Mutants also lack the ability to increase mEJP rate in response to spaced depolarization, a type of activity-dependent plasticity shown to require both transcription and translation. Consistent with this, overexpression of miR-289 in wild-type animals blocks plasticity of spontaneous release. In addition to the defects in regulation of mEJP rate, CaMKII protein is largely lost from synapses in the mutant. All phenotypes are non-sex-specific and rescued by a fosmid containing the entire wild-type CaMKII locus, but only viability and CaMKII localization are rescued by genomic fosmids lacking the long 3'UTR. This suggests that synaptic CaMKII accumulates by two distinct mechanisms: local synthesis requiring the long 3'UTR form of CaMKII mRNA and a process that requires zygotic transcription of CaMKII mRNA. The origin of synaptic CaMKII also dictates its functionality. Locally translated CaMKII has a privileged role in regulation of spontaneous release, which cannot be fulfilled by synaptic CaMKII from the other pool.SIGNIFICANCE STATEMENT As a regulator of synaptic development and plasticity, CaMKII has important roles in both normal and pathological function of the nervous system. CaMKII shows high conservation between Drosophila and humans, underscoring the usefulness of Drosophila in modeling its function. Drosophila CaMKII-null mutants remain viable throughout development, enabling morphological and electrophysiological characterization. Although the structure of the synapse is normal, maternally contributed CaMKII does not localize to synapses. Zygotic production of CaMKII mRNA with a long 3'-untranslated region is necessary for modulating spontaneous neurotransmission in an activity-dependent manner, but not for viability. These data argue that regulation of CaMKII localization and levels by local transcriptional processes is conserved. This is the first demonstration of distinct functions for Drosophila CaMKII mRNA variants.

A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster.

By covalently conjugating to target proteins, ubiquitin-like modifiers (UBLs) act as important regulators of target protein localization and activity, thereby playing a critical role in the orchestration of cellular biology. The most ancient and one of the least studied UBLs is Urm1, a dual-function protein that in parallel to performing similar functions as its prokaryotic ancestors in tRNA modification, also has adopted the capacity to conjugate to cellular proteins analogous to ubiquitin and other UBL modifiers. In order to increase the understanding of Urm1 and its role in multicellular organisms, we have used affinity purification followed by mass spectrometry to identify putative targets of Urm1 conjugation (urmylation) at three developmental stages of the Drosophila melanogaster lifecycle. Altogether we have recovered 79 Urm1-interacting proteins in Drosophila, which include the already established Urm1 binding partners Prx5 and Uba4, together with 77 candidate urmylation targets that are completely novel in the fly. Among these, the majority was exclusively identified during either embryogenesis, larval stages or in adult flies. We further present biochemical evidence that four of these proteins are covalently conjugated by Urm1, whereas the fifth verified Urm1-binding protein appears to interact with Urm1 via non-covalent means. Besides recapitulating the previously established roles of Urm1 in tRNA modification and during oxidative stress, functional clustering of the newly identified Urm1-associated proteins further positions Urm1 in protein networks that control other types of cellular stress, such as immunological threats and DNA damage. In addition, the functional characteristics of several of the candidate targets strongly match the phenotypes displayed by Urm1n123 null animals, including embryonic lethality, reduced fertility and shortened lifespan. In conclusion, this identification of candidate targets of urmylation significantly increases the knowledge of Urm1 and presents an excellent starting point for unravelling the role of Urm1 in the context of a complex living organism.

Locomotion Behavior Is Affected by the GαS Pathway and the Two-Pore-Domain K+ Channel TWK-7 Interacting in GABAergic Motor Neurons in Caenorhabditis elegans.

Adjusting the efficiency of movement in response to environmental cues is an essential integrative characteristic of adaptive locomotion behavior across species. However, the modulatory molecules and the pathways involved are largely unknown. Recently, we demonstrated that in Caenorhabditis elegans, a loss-of-function of the two-pore-domain potassium (K2P) channel TWK-7 causes a fast, coordinated, and persistent forward crawling behavior in which five central aspects of stimulated locomotion-velocity, direction, wave parameters, duration, and straightness-are affected. Here, we isolated the reduction-of-function allele cau1 of the C. elegans gene kin-2 in a forward genetic screen and showed that it phenocopies the locomotor activity and locomotion behavior of twk-7(null) animals. Kin-2 encodes the negative regulatory subunit of protein kinase A (KIN-1/PKA). Consistently, we found that other gain-of-function mutants of the GαS-KIN-1/PKA pathway resemble kin-2(cau1) and twk-7(null) in locomotion phenotype. Using the powerful genetics of the C. elegans system in combination with cell type-specific approaches and detailed locomotion analyses, we identified TWK-7 as a putative downstream target of the GαS-KIN-1/PKA pathway at the level of the γ-aminobutyric acid (GABA)ergic D-type motor neurons. Due to this epistatic interaction, we suggest that KIN-1/PKA and TWK-7 may share a common pathway that is probably involved in the modulation of both locomotor activity and locomotion behavior during forward crawling.

Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components.

Amyotrophic Lateral Sclerosis (ALS) is the most common adult-onset motor neuron disease and familial forms can be caused by numerous dominant mutations of the copper-zinc superoxide dismutase 1 (SOD1) gene. Substantial efforts have been invested in studying SOD1-ALS transgenic animal models; yet, the molecular mechanisms by which ALS-mutant SOD1 protein acquires toxicity are not well understood. ALS-like phenotypes in animal models are highly dependent on transgene dosage. Thus, issues of whether the ALS-like phenotypes of these models stem from overexpression of mutant alleles or from aspects of the SOD1 mutation itself are not easily deconvolved. To address concerns about levels of mutant SOD1 in disease pathogenesis, we have genetically engineered four human ALS-causing SOD1 point mutations (G37R, H48R, H71Y, and G85R) into the endogenous locus of Drosophila SOD1 (dsod) via ends-out homologous recombination and analyzed the resulting molecular, biochemical, and behavioral phenotypes. Contrary to previous transgenic models, we have recapitulated ALS-like phenotypes without overexpression of the mutant protein. Drosophila carrying homozygous mutations rendering SOD1 protein enzymatically inactive (G85R, H48R, and H71Y) exhibited neurodegeneration, locomotor deficits, and shortened life span. The mutation retaining enzymatic activity (G37R) was phenotypically indistinguishable from controls. While the observed mutant dsod phenotypes were recessive, a gain-of-function component was uncovered through dosage studies and comparisons with age-matched dsod null animals, which failed to show severe locomotor defects or nerve degeneration. We conclude that the Drosophila knock-in model captures important aspects of human SOD1-based ALS and provides a powerful and useful tool for further genetic studies.

GDE2 is essential for neuronal survival in the postnatal mammalian spinal cord

BackgroundGlycerophosphodiester phosphodiesterase 2 (GDE2) is a six-transmembrane protein that cleaves glycosylphosphatidylinositol (GPI) anchors to regulate GPI-anchored protein activity at the cell surface. In the developing spinal cord, GDE2 utilizes its enzymatic function to regulate the production of specific classes of motor neurons and interneurons; however, GDE2’s roles beyond embryonic neurogenesis have yet to be defined.MethodUsing a panel of histological, immunohistochemical, electrophysiological, behavioral, and biochemistry techniques, we characterized the postnatal Gde2−/− mouse for evidence of degenerative neuropathology. A conditional deletion of Gde2 was used to study the temporal requirements for GDE2 in neuronal survival. Biochemical approaches identified deficits in the processing of GPI-anchored GDE2 substrates in the SOD1G93A mouse model of familial Amyotrophic Lateral Sclerosis that shows robust motor neuron degeneration.ResultsHere we show that GDE2 expression continues postnatally, and adult mice lacking GDE2 exhibit a slow, progressive neuronal degeneration with pathologies similar to human neurodegenerative disease. Early phenotypes include vacuolization, microgliosis, cytoskeletal accumulation, and lipofuscin deposition followed by astrogliosis and cell death. Remaining motor neurons exhibit peripheral motor unit restructuring causing behavioral motor deficits. Genetic ablation of GDE2 after embryonic neurogenesis is complete still elicits degenerative pathology, signifying that GDE2’s requirement for neuronal survival is distinct from its involvement in neuronal differentiation. Unbiased screens identify impaired processing of Glypican 4 and 6 in Gde2 null animals, and Glypican release is markedly reduced in SOD1G93A mice.ConclusionsThis study identifies a novel function for GDE2 in neuronal survival and implicates deregulated GPI-anchored protein activity in pathways mediating neurodegeneration. These findings provide new molecular insight for neuropathologies found in multiple disease settings, and raise the possibility of GDE2 hypofunctionality as a component of neurodegenerative disease.

Decreased Anxiety-Related Behaviour but Apparently Unperturbed NUMB Function in Ligand of NUMB Protein-X (LNX) 1/2 Double Knockout Mice.

NUMB is a key regulator of neurogenesis and neuronal differentiation that can be ubiquitinated and targeted for proteasomal degradation by ligand of numb protein-X (LNX) family E3 ubiquitin ligases. However, our understanding of LNX protein function in vivo is very limited. To examine the role of LNX proteins in regulating NUMB function in vivo, we generated mice lacking both LNX1 and LNX2 expression in the brain. Surprisingly, these mice are healthy, exhibit unaltered levels of NUMB protein and do not display any neuroanatomical defects indicative of aberrant NUMB function. Behavioural analysis of LNX1/LNX2 double knockout mice revealed decreased anxiety-related behaviour, as assessed in the open field and elevated plus maze paradigms. By contrast, no major defects in learning, motor or sensory function were observed. Given the apparent absence of major NUMB dysfunction in LNX null animals, we performed a proteomic analysis to identify neuronal LNX-interacting proteins other than NUMB that might contribute to the anxiolytic phenotype observed. We identified and/or confirmed interactions of LNX1 and LNX2 with proteins known to have presynaptic and neuronal signalling functions, including the presynaptic active zone constituents ERC1, ERC2 and LIPRIN-αs (PPFIA1, PPFIA3), as well as the F-BAR domain proteins FCHSD2 (nervous wreck homologue) and SRGAP2. These and other novel LNX-interacting proteins identified are promising candidates to mediate LNX functions in the central nervous system, including their role in modulating anxiety-related behaviour.

NOX isoforms in the development of abdominal aortic aneurysm

Oxidative stress plays an important role in the formation of abdominal aortic aneurysm (AAA), and we have recently established a causal role of uncoupled eNOS in this severe human disease. We have also shown that activation of NADPH oxidase (NOX) lies upstream of uncoupled eNOS. Therefore, identification of the specific NOX isoforms that are required for eNOS uncoupling and AAA formation would ultimately lead to novel therapies for AAA. In the present study, we used the Ang II infused hph-1 mice to examine the roles of NOX isoforms in the development of AAA. We generated double mutants of hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4. After two weeks of Ang II infusion, the incidence rate of AAA substantially dropped from 76.5% in Ang II infused hph-1 mice (n=34) to 11.1%, 15.0%, 9.5% and 0% in hph-1-NOX1 (n=27), hph-1-NOX2 (n=40), hph-1-p47phox (n=21), and hph-1-NOX4 (n=33) double mutant mice, respectively. The size of abdominal aortas of the four double mutant mice, determined by ultrasound analyses, was significantly smaller than the hph-1 mice. Aortic nitric oxide and H4B bioavailabilities were markedly improved in the double mutants, while superoxide production and eNOS uncoupling activity were substantially diminished. These effects seemed attributed to an endothelial specific restoration of dihydrofolate reductase expression and activity, deficiency of which has been shown to induce eNOS uncoupling and AAA formation in both Ang II-infused hph-1 and apoE null animals. In addition, over-expression of human NOX4 N129S or T555S mutant newly identified in aneurysm patients increased hydrogen peroxide production, further implicating a relationship between NOX and human aneurysm. Taken together, these data indicate that NOX isoforms 1, 2 or 4 lies upstream of dihydrofolate reductase deficiency and eNOS uncoupling to induce AAA formation. These findings may promote development of novel therapeutics for the treatment of the disease by inhibiting NOX signaling.

Abstract 1857: Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis

Abstract Introduction: Ulcerative colitis (UC) is a highly morbid, chronic inflammatory disease characterized by mucosal ulceration of the colonic mucosa and is associated with the higher risk of colitis-associated cancer (CAC). The exact mechanism(s) causing UC progression to CAC is currently unknown. The balanced activity of transsulfuration pathway enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), and their production of endogenous hydrogen sulfide gas, are critical to maintenance of the colonic homeostasis. CSE activity is suggested to be important for wound healing and mucosal protection. Consistent with this, we have previously showed a decrease CSE expression in human colonic mucosa obtained from patients with chronic UC, compared to normal colonic mucosa by immunocytochemistry. By contrast, increased CBS expression is implicated in the progression of sporadic colorectal carcinoma. However, the role of these enzymes in CAC is unknown. We hypothesize that the dysregulation in CSE/CBS expression and activity is important to the progression from UC to CAC. Methods: CSE null mice and wild type Sv129/B6 (control) mice were used in azoxymethane-dextran sodium sulfate (AOM-DSS) colon cancer model which mimics human CAC. The disease development was followed up to day 80. Confocal microscopy and Western blot was used to assess the gene expression during cancer development. Size, number, and time interval to tumor formation, as well inflammation were assessed. Results: Abrogation of CSE expression using CSE null animals in AOM-DSS model of CAC accelerated the time to tumor development and resulted in an increase in both tumor size (p<0.001) and number (p<0.001) compared to wild-type controls. CBS protein expression was increased within the colonic tumor when compared to the normal margin in AOM-DSS treated animals by Western blot analysis and tissue immunostaining. Interestingly, CAC liver metastases, an exceedingly rare finding in this mouse model, were identified. Conclusion: Taken together, our human and murine data suggest that dysregulation in the transsulfuration pathway enzymes CSE and CBS expression/activity may be critical contributor to the CAC development in UC and may serve as a potential biomarker for disease progression in the future. Citation Format: Paul Johnson, Ches’Que M. Phillips, Carl Grim, John R. Zatarain, Aakash H. Gajjar, Suimin Qiu, Rui Wang, Celia Chao, Iryna V. Pinchuk, Mark R. Hellmich. Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1857.

Opiates Modulate Noxious Chemical Nociception through a Complex Monoaminergic/Peptidergic Cascade.

The ability to detect noxious stimuli, process the nociceptive signal, and elicit an appropriate behavioral response is essential for survival. In Caenorhabditis elegans, opioid receptor agonists, such as morphine, mimic serotonin, and suppress the overall withdrawal from noxious stimuli through a pathway requiring the opioid-like receptor, NPR-17. This serotonin- or morphine-dependent modulation can be rescued in npr-17-null animals by the expression of npr-17 or a human κ opioid receptor in the two ASI sensory neurons, with ASI opioid signaling selectively inhibiting ASI neuropeptide release. Serotonergic modulation requires peptides encoded by both nlp-3 and nlp-24, and either nlp-3 or nlp-24 overexpression mimics morphine and suppresses withdrawal. Peptides encoded by nlp-3 act differentially, with only NLP-3.3 mimicking morphine, whereas other nlp-3 peptides antagonize NLP-3.3 modulation. Together, these results demonstrate that opiates modulate nociception in Caenorhabditis elegans through a complex monoaminergic/peptidergic cascade, and suggest that this model may be useful for dissecting opiate signaling in mammals. Opiates are used extensively to treat chronic pain. In Caenorhabditis elegans, opioid receptor agonists suppress the overall withdrawal from noxious chemical stimuli through a pathway requiring an opioid-like receptor and two distinct neuropeptide-encoding genes, with individual peptides from the same gene functioning antagonistically to modulate nociception. Endogenous opioid signaling functions as part of a complex, monoaminergic/peptidergic signaling cascade and appears to selectively inhibit neuropeptide release, mediated by a α-adrenergic-like receptor, from two sensory neurons. Importantly, receptor null animals can be rescued by the expression of the human κ opioid receptor, and injection of human opioid receptor ligands mimics exogenous opiates, highlighting the utility of this model for dissecting opiate signaling in mammals.

Commentary: Oxygen regulation of breathing through an olfactory receptor activated by lactate.

Commentary: Oxygen regulation of breathing through an olfactory receptor activated by lactate.

Circadian factors BMAL1 and RORα control HIF-1α transcriptional activity in nucleus pulposus cells: implications in maintenance of intervertebral disc health.

BMAL1 and RORα are major regulators of the circadian molecular oscillator. Since previous work in other cell types has shown cross talk between circadian rhythm genes and hypoxic signaling, we investigated the role of BMAL1 and RORα in controlling HIF-1-dependent transcriptional responses in NP cells that exist in the physiologically hypoxic intervertebral disc. HIF-1-dependent HRE reporter activity was further promoted by co-transfection with either BMAL1 or RORα. In addition, stable silencing of BMAL1 or inhibition of RORα activity resulted in decreased HRE activation. Inhibition of RORα also modulated HIF1α-TAD activity. Interestingly, immunoprecipitation studies showed no evidence of BMAL1, CLOCK or RORα binding to HIF-1α in NP cells. Noteworthy, stable silencing of BMAL1 as well as inhibition of RORα decreased expression of select HIF-1 target genes including VEGF, PFKFB3 and Eno1. To delineate if BMAL1 plays a role in maintenance of disc health, we studied the spinal phenotype of BMAL1-null mice. The lumbar discs of null mice evidenced decreased height, and several parameters associated with vertebral trabecular bone quality were also affected in nulls. In addition, null animals showed a higher ratio of cells to matrix in NP tissue and hyperplasia of the annulus fibrosus. Taken together, our results indicate that BMAL1 and RORα form a regulatory loop in the NP and control HIF-1 activity without direct interaction. Importantly, activities of these circadian rhythm molecules may play a role in the adaptation of NP cells to their unique niche.

MiR-8 modulates cytoskeletal regulators to influence cell survival and epithelial organization in Drosophila wings

The miR-200 microRNA family plays important tumor suppressive roles. The sole Drosophila miR-200 ortholog, miR-8 plays conserved roles in Wingless, Notch and Insulin signaling – pathways linked to tumorigenesis, yet homozygous null animals are viable and often appear morphologically normal. We observed that wing tissues mosaic for miR-8 levels by genetic loss or gain of function exhibited patterns of cell death consistent with a role for miR-8 in modulating cell survival in vivo. Here we show that miR-8 levels impact several actin cytoskeletal regulators that can affect cell survival and epithelial organization. We show that loss of miR-8 can confer resistance to apoptosis independent of an epithelial to mesenchymal transition while the persistence of cells expressing high levels of miR-8 in the wing epithelium leads to increased JNK signaling, aberrant expression of extracellular matrix remodeling proteins and disruption of proper wing epithelial organization. Altogether our results suggest that very low as well as very high levels of miR-8 can contribute to hallmarks associated with cancer, suggesting approaches to increase miR-200 microRNAs in cancer treatment should be moderate.

Hoxa10 null animals exhibit reduced platelet biogenesis.

The transcription factor HOXA10 is an important regulator of myelopoiesis. Engineered over-expression of Hoxa10 in mice results in a myeloproliferative disorder that progresses to acute myeloid leukaemia (AML) over time, and in humans over-expression is associated with poor outcomes in AML. Here, we report that loss of Hoxa10 expression in mice results in reduced platelet count and platelet production, but does not affect clotting efficiency. About 40% fewer platelets were found in Hoxa10 null animals in comparison to wild type littermates. We found a nearly 50% reduction in the percentage of reticulated platelets in Hoxa10 null mice, suggesting deficient platelet production. Furthermore, Hoxa10 null animals recovered less efficiently from induced thrombocytopenia, supporting our hypothesis of defective platelet production. This also correlated with reduced colony formation potential of stem and progenitor cells seeded in megakaryocyte-enhancing conditions invitro. Together, our results indicate that HOXA10 is important for megakaryopoiesis and platelet biogenesis.

Roles of VAMP7 in Drosophila Synaptic Transmission

Soluble N-ethylmaleimide-sensitive factor Attachment Receptor (SNARE) proteins control the fusion of intracellular membrane compartments. The vesicle-associated membrane proteins (VAMPs) are a family of SNAREs essential to establish the interaction with target SNAREs like Syntaxin and SNAP. Tetanus-toxin Insensitive VAMP (TI-VAMP) is different from the Synaptobrevin VAMP family which majorly controls synaptic vesicle (SVs) fusion. Ti-VAMP or VAMP7 has a longing domain necessary to sort proteins to internal compartments. In neurons, VAMP7 is thought to regulate SV trafficking and spontaneous fusion. In addition, VAMP7 participates in neurite extension and SV cycling through a reserve pool, however, its function is still under investigation. Here, we characterized a VAMP7 loss of function Drosophila animal (VAMP-7−/−) generated by imprecise p-element excision. Null animals survive until third instar larval stage in low density condition and display sluggish phenotype. Basal synaptic transmission analyses by two-electrode voltage-clamp indicate unaltered spontaneous and evoked SV fusion. Additionally, exo/endocytosis imaging with FM 1-43 revealed normal exo/endo-cycling pool but altered reserve pool formation. Neuronal driven expression of Drosophila VAMP7 tagged with GFP display punctate morphology at nerve terminals.