MDA-9/Syntenin in the tumor and microenvironment defines prostate cancer bone metastasis.

Bone metastasis is a frequent and incurable consequence of advanced prostate cancer (PC). An interplay between disseminated tumor cells and heterogeneous bone resident cells in the metastatic niche initiates this process. Melanoma differentiation associated gene-9 (mda-9/Syntenin/syndecan binding protein) is a prometastatic gene expressed in multiple organs, including bone marrow-derived mesenchymal stromal cells (BM-MSCs), under both physiological and pathological conditions. We demonstrate that PDGF-AA secreted by tumor cells induces CXCL5 expression in BM-MSCs by suppressing MDA-9-dependent YAP/MST signaling. CXCL5-derived tumor cell proliferation and immune suppression are consequences of the MDA-9/CXCL5 signaling axis, promoting PC disease progression. mda-9 knockout tumor cells express less PDGF-AA and do not develop bone metastases. Our data document a previously undefined role of MDA-9/Syntenin in the tumor and microenvironment in regulating PC bone metastasis. This study provides a framework for translational strategies to ameliorate health complications and morbidity associated with advanced PC.

Spatial transcriptomics unravels palmitoylation and zonation-dependent gene regulation by AEG-1 in mouse liver.

Obesity-induced metabolic dysfunction-associated steatohepatitis (MASH) leads to hepatocellular carcinoma (HCC). Astrocyte-elevated gene-1/Metadherin (AEG-1/MTDH) plays a key role in promoting MASH and HCC. AEG-1 is palmitoylated at residue cysteine 75 (Cys75) and a knock-in mouse representing mutated Cys75 to serine (AEG-1-C75S) showed activation of MASH- and HCC-promoting gene signature when compared to wild-type littermates (AEG-1-WT). The liver consists of three zones, periportal, mid-lobular, and pericentral, and zone-specific dysregulated gene expression impairs metabolic homeostasis in the liver, contributing to MASH and HCC. Here, to elucidate how palmitoylation influences AEG-1-mediated gene regulation in regard to hepatic zonation, we performed spatial transcriptomics (ST) in the livers of AEG-1-WT and AEG-1-C75S littermates. ST identified six different clusters in livers and using zone- and cell-type-specific markers we attributed specific zones and cell types to specific clusters. Ingenuity Pathway Analysis (IPA) of differentially expressed genes in each cluster unraveled activation of pro-inflammatory and MASH- and HCC-promoting pathways, mainly in periportal and pericentral hepatocytes, in AEG-1-C75S liver compared to AEG-1-WT. Interestingly, in AEG-1-C75S liver, the mid-lobular zone exhibited widespread inhibition of xenobiotic metabolism pathways and inhibition of PXR/RXR and LXR/RXR activation, versus AEG-1-WT. In conclusion, AEG-1-C75S mutant exhibited zone-specific differential gene expression, which might contribute to metabolic dysfunction and dysregulated drug metabolism leading to MASH and HCC.

SPTLC3 Is Essential for Complex I Activity and Contributes to Ischemic Cardiomyopathy.

BACKGROUND: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). METHODS: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. RESULTS: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. CONCLUSIONS: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.

Melanoma differentiation associated gene-9/syndecan binding protein promotes hepatocellular carcinoma.

BACKGROUND AND AIMS: The oncogene Melanoma differentiation associated gene-9/syndecan binding protein (MDA-9/SDCBP) is overexpressed in many cancers, promoting aggressive, metastatic disease. However, the role of MDA-9 in regulating hepatocellular carcinoma (HCC) has not been well studied. APPROACH AND RESULTS: To unravel the function of MDA-9 in HCC, we generated and characterized a transgenic mouse with hepatocyte-specific overexpression of MDA-9 (Alb/MDA-9). Compared with wild-type (WT) littermates, Alb/MDA-9 mice demonstrated significantly higher incidence of N-nitrosodiethylamine/phenobarbital-induced HCC, with marked activation and infiltration of macrophages. RNA sequencing (RNA-seq) in naive WT and Alb/MDA-9 hepatocytes identified activation of signaling pathways associated with invasion, angiogenesis, and inflammation, especially NF-κB and integrin-linked kinase signaling pathways. In nonparenchymal cells purified from naive livers, single-cell RNA-seq showed activation of Kupffer cells and macrophages in Alb/MDA-9 mice versus WT mice. A robust increase in the expression of Secreted phosphoprotein 1 (Spp1/osteopontin) was observed upon overexpression of MDA-9. Inhibition of NF-κB pathway blocked MDA-9-induced Spp1 induction, and knock down of Spp1 resulted in inhibition of MDA-9-induced macrophage migration, as well as angiogenesis. CONCLUSIONS: Alb/MDA-9 is a mouse model with MDA-9 overexpression in any tissue type. Our findings unravel an HCC-promoting role of MDA-9 mediated by NF-κB and Spp1 and support the rationale of using MDA-9 inhibitors as a potential treatment for aggressive HCC.

Insights into the Mechanisms of Action of MDA-7/IL-24: A Ubiquitous Cancer-Suppressing Protein.

Melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24), a secreted protein of the IL-10 family, was first identified more than two decades ago as a novel gene differentially expressed in terminally differentiating human metastatic melanoma cells. MDA-7/IL-24 functions as a potent tumor suppressor exerting a diverse array of functions including the inhibition of tumor growth, invasion, angiogenesis, and metastasis, and induction of potent "bystander" antitumor activity and synergy with conventional cancer therapeutics. MDA-7/IL-24 induces cancer-specific cell death through apoptosis or toxic autophagy, which was initially established in vitro and in preclinical animal models in vivo and later in a Phase I clinical trial in patients with advanced cancers. This review summarizes the history and our current understanding of the molecular/biological mechanisms of MDA-7/IL-24 action rendering it a potent cancer suppressor.

Human DENND1A.V2 Drives Cyp17a1 Expression and Androgen Production in Mouse Ovaries and Adrenals.

The DENND1A locus is associated with polycystic ovary syndrome (PCOS), a disorder characterized by androgen excess. Theca cells from ovaries of PCOS women have elevated levels of a DENND1A splice variant (DENND1A.V2). Forced expression of this variant in normal theca cells increases androgen biosynthesis and CYP17A1 expression, whereas knockdown of the transcript in PCOS theca cells reduced androgen production and CYP17A1 mRNA. We attempted to create a murine model of PCOS by expressing hDENND1A.V2 using standard transgenic approaches. There is no DENND1A.V2 protein equivalent in mice, and the murine Dennd1a gene is essential for viability since Dennd1a knockout mice are embryonically lethal, suggesting that Dennd1a is developmentally critical. Three different hDENND1A.V2 transgenic mice lines were created using CMV, Lhcgr, and TetOn promoters. The hDENND1A.V2 mice expressed hDENND1A.V2 transcripts. While hDENND1A.V2 protein was not detectable by Western blot analyses, appropriate hDENND1A.V2 immunohistochemical staining was observed. Corresponding Cyp17a1 mRNA levels were elevated in ovaries and adrenals of CMV transgenic mice, as were plasma steroid production by theca interstitial cells isolated from transgenic ovaries. Even though the impact of robust hDENND1A.V2 expression could not be characterized, our findings are consistent with the notion that elevated hDENND1A.V2 has a role in the hyperandrogenemia of PCOS.

StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis.

How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain (StarD)5 leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the major cell type that expresses StarD5. ER stress increases StarD5 expression in mouse hepatocytes, which results in an increase in accessible PM cholesterol in WT but not in StarD5-/- hepatocytes. StarD5-/- mice store higher levels of cholesterol and triglycerides, which leads to altered expression of cholesterol-regulated genes. In vitro, a recombinant GST-StarD5 protein transfers cholesterol between synthetic liposomes. StarD5 overexpression leads to a marked increase in PM cholesterol. Phasor analysis of 6-dodecanoyl-2-dimethylaminonaphthalene fluorescence lifetime imaging microscopy data revealed an increase in PM fluidity in StarD5-/- macrophages. Taken together, these studies show that StarD5 is a stress-responsive protein that regulates PM cholesterol and intracellular cholesterol homeostasis.

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro.

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43def ), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.

A novel role of astrocyte elevated gene-1 (AEG-1) in regulating nonalcoholic steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) is the most prevalent cause of chronic liver disease in the Western world. However, an optimum therapy for NASH is yet to be established, mandating more in-depth investigation into the molecular pathogenesis of NASH to identify novel regulatory molecules and develop targeted therapies. Here, we unravel a unique function of astrocyte elevated gene-1(AEG-1)/metadherin in NASH using a transgenic mouse with hepatocyte-specific overexpression of AEG-1 (Alb/AEG-1) and a conditional hepatocyte-specific AEG-1 knockout mouse (AEG-1ΔHEP ). Alb/AEG-1 mice developed spontaneous NASH whereas AEG-1ΔHEP mice were protected from high-fat diet (HFD)-induced NASH. Intriguingly, AEG-1 overexpression was observed in livers of NASH patients and wild-type (WT) mice that developed steatosis upon feeding HFD. In-depth molecular analysis unraveled that inhibition of peroxisome proliferator-activated receptor alpha activity resulting in decreased fatty acid ß-oxidation, augmentation of translation of fatty acid synthase resulting in de novo lipogenesis, and increased nuclear factor kappa B-mediated inflammation act in concert to mediate AEG-1-induced NASH. Therapeutically, hepatocyte-specific nanoparticle-delivered AEG-1 small interfering RNA provided marked protection from HFD-induced NASH in WT mice. CONCLUSION: AEG-1 might be a key molecule regulating initiation and progression of NASH. AEG-1 inhibitory strategies might be developed as a potential therapeutic intervention in NASH patients. (Hepatology 2017;66:466-480).

Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis.

Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity.

Astrocyte elevated gene-1 and c-Myc cooperate to promote hepatocarcinogenesis in mice.

UNLABELLED: Astrocyte elevated gene-1 (AEG-1) and c-Myc are overexpressed in human hepatocellular carcinoma (HCC) functioning as oncogenes. AEG-1 is transcriptionally regulated by c-Myc, and AEG-1 itself induces c-Myc by activating the Wnt/ß-catenin-signaling pathway. We now document the cooperation of AEG-1 and c-Myc in promoting hepatocarcinogenesis by analyzing hepatocyte-specific transgenic mice expressing either AEG-1 (albumin [Alb]/AEG-1), c-Myc (Alb/c-Myc), or both (Alb/AEG-1/c-Myc). Wild-type and Alb/AEG-1 mice did not develop spontaneous HCC. Alb/c-Myc mice developed spontaneous HCC without distant metastasis, whereas Alb/AEG-1/c-Myc mice developed highly aggressive HCC with frank metastasis to the lungs. Induction of carcinogenesis by N-nitrosodiethylamine significantly accelerated the kinetics of tumor formation in all groups. However, in Alb/AEG-1/c-Myc, the effect was markedly pronounced with lung metastasis. In vitro analysis showed that Alb/AEG-1/c-Myc hepatocytes acquired increased proliferation and transformative potential with sustained activation of prosurvival and epithelial-mesenchymal transition-signaling pathways. RNA-sequencing analysis identified a unique gene signature in livers of Alb/AEG-1/c-Myc mice that was not observed when either AEG-1 or c-Myc was overexpressed. Specifically, Alb/AEG-1/c-Myc mice overexpressed maternally imprinted noncoding RNAs (ncRNAs), such as Rian, Meg-3, and Mirg, which are implicated in hepatocarcinogenesis. Knocking down these ncRNAs significantly inhibited proliferation and invasion by Alb/AEG-1/c-Myc hepatocytes. CONCLUSION: Our studies reveal a novel cooperative oncogenic effect of AEG-1 and c-Myc that might explain the mechanism of aggressive HCC. Alb/AEG-1/c-Myc mice provide a useful model to understand the molecular mechanism of cooperation between these two oncogenes and other molecules involved in hepatocarcinogenesis. This model might also be of use for evaluating novel therapeutic strategies targeting HCC.

Astrocyte elevated gene-1 promotes hepatocarcinogenesis: novel insights from a mouse model.

UNLABELLED: Astrocyte elevated gene-1 (AEG-1) is a key contributor to hepatocellular carcinoma (HCC) development and progression. To enhance our understanding of the role of AEG-1 in hepatocarcinogenesis, a transgenic mouse with hepatocyte-specific expression of AEG-1 (Alb/AEG1) was developed. Treating Alb/AEG-1, but not wild-type (WT) mice, with N-nitrosodiethylamine resulted in multinodular HCC with steatotic features and associated modulation of expression of genes regulating invasion, metastasis, angiogenesis, and fatty acid synthesis. Hepatocytes isolated from Alb/AEG-1 mice displayed profound resistance to chemotherapeutics and growth factor deprivation with activation of prosurvival signaling pathways. Alb/AEG-1 hepatocytes also exhibited marked resistance toward senescence, which correlated with abrogation of activation of a DNA damage response. Conditioned media from Alb/AEG-1 hepatocytes induced marked angiogenesis with elevation in several coagulation factors. Among these factors, AEG-1 facilitated the association of factor XII (FXII) messenger RNA with polysomes, resulting in increased translation. Short interfering RNA-mediated knockdown of FXII resulted in profound inhibition of AEG-1-induced angiogenesis. CONCLUSION: We uncovered novel aspects of AEG-1 functions, including induction of steatosis, inhibition of senescence, and activation of the coagulation pathway to augment aggressive hepatocarcinogenesis. The Alb/AEG-1 mouse provides an appropriate model to scrutinize the molecular mechanism of hepatocarcinogenesis and to evaluate the efficacy of novel therapeutic strategies targeting HCC.

ADAM10 overexpression shifts lympho- and myelopoiesis by dysregulating site 2/site 3 cleavage products of Notch.

Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that ADAM10 is a critical regulator of Notch and that its deletion is embryonically lethal, we generated mice that overexpress ADAM10 (ADAM10 transgenic [A10Tg]) at early stages of lympho- and myeloid development. Transgene expression resulted in abrogated B cell development, delayed T cell development in the thymus, and unexpected systemic expansion of CD11b(+)Gr-1(+) cells, also known as myeloid-derived suppressor cells. Mixed bone marrow reconstitution assays demonstrated that transgene expression altered hematopoiesis via a cell-intrinsic mechanism. Consistent with previously reported observations, we hypothesized that ADAM10 overexpression dysregulated Notch by uncoupling the highly regulated proteolysis of Notch receptors. This was confirmed using an in vitro model of hematopoiesis via culturing A10Tg hematopoietic Lineage(-)Sca-1(+)c-Kit(+) cells with OP-9 stromal cells in the presence or absence of Delta-like 1, a primary ligand for Notch. Blockade of the site 2 (S2) and site 3 (S3) cleavage of the Notch receptor demonstrated differential effects on hematopoiesis. OP9-DL1 cultures containing the ADAM10 inhibitor (S2 cleavage site) enhanced and rescued B cell development from wild-type and A10Tg Lineage(-)Sca-1(+)c-Kit(+) cells, respectively. In contrast, blockade of γ-secretase at the S3 cleavage site induced accumulation of the S2 product and consequently prevented B cell development and resulted in myeloid cell accumulation. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical to hematopoietic cell-fate determination.

Osteoclast-derived IGF1 induces RANKL production in osteocytes and contributes to pagetic lesion formation.

We previously reported that measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) of patients with Paget disease (PD) or targeted to the OCL lineage in MVNP-transgenic mice (MVNP mice) increases IGF1 production in osteoclasts (OCL-IGF1) and leads to development of PD OCLs and pagetic bone lesions (PDLs). Conditional deletion of Igf1 in OCLs of MVNP mice fully blocked development of PDLs. In this study, we examined whether osteocytes (OCys), key regulators of normal bone remodeling, contribute to PD. OCys in PDLs of patients and of MVNP mice expressed less sclerostin, and had increased RANKL expression compared with OCys in bones from WT mice or normal patients. To test whether increased OCL-IGF1 is sufficient to induce PDLs and PD phenotypes, we generated TRAP-Igf1 (T-Igf1) transgenic mice to determine whether increased IGF1 expression in the absence of MVNP in OCLs is sufficient to induce PDLs and pagetic OCLs. We found that T-Igf1 mice at 16 months of age developed PD OCLs, PDLs, and OCys, with decreased sclerostin and increased RANKL, similar to MVNP mice. Thus, pagetic phenotypes could be induced by OCLs expressing increased IGF1. OCL-IGF1 in turn increased RANKL production in OCys to induce PD OCLs and PDLs.

Cytoplasmic-delivery of polyinosine-polycytidylic acid inhibits pancreatic cancer progression increasing survival by activating Stat1-CCL2-mediated immunity.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer without effective therapies and with poor prognosis, causing 7% of all cancer-related fatalities in the USA. Considering the lack of effective therapies for this aggressive cancer, there is an urgent need to define newer and more effective therapeutic strategies. Polyinosine-polycytidylic acid (pIC) is a synthetic double-stranded RNA (dsRNA) which directly activates dendritic cells and natural killer cells inhibiting tumor growth. When pIC is delivered into the cytoplasm using polyethyleneimine (PEI), pIC-PEI, programmed-cell death is induced in PDAC. Transfection of [pIC]PEI into PDAC cells inhibits growth, promotes toxic autophagy and also induces apoptosis in vitro and in vivo in animal models. METHODS: The KPC transgenic mouse model that recapitulates PDAC development in patients was used to interrogate the role of an intact immune system in vivo in PDAC in response to [pIC]PEI. Antitumor efficacy and survival were monitored endpoints. Comprehensive analysis of the tumor microenvironment (TME) and immune cells, cytokines and chemokines in the spleen, and macrophage polarization were analyzed. RESULTS: Cytosolic delivery of [pIC]PEI induces apoptosis and provokes strong antitumor immunity in vivo in immune competent mice with PDAC. The mechanism underlying the immune stimulatory properties of [pIC]PEI involves Stat1 activation resulting in CCL2 and MMP13 stimulation thereby provoking macrophage polarization. [pIC]PEI induces apoptosis via the AKT-XIAP pathway, as well as macrophage differentiation and T-cell activation via the IFNγ-Stat1-CCL2 signaling pathways in PDAC. In transgenic tumor mouse models, [pIC]PEI promotes robust and profound antitumor activity implying that stimulating the immune system contributes to biological activity. The [pIC]PEI anti-PDAC effects are enhanced when used in combination with a standard of care (SOC) treatment, that is, gemcitabine. CONCLUSIONS: In summary, [pIC]PEI treatment is non-toxic toward normal pancreatic cells while displaying strong cytotoxic and potent immune activating activities in PDAC, making it an attractive therapeutic when used alone or in conjunction with SOC therapeutic agents, potentially providing a safe and effective treatment protocol with translational potential for the effective therapy of PDAC.

Blunted Amphetamine-induced Reinforcing Behaviors and Transporter Downregulation in Knock-in Mice Carrying Alanine Mutations at Threonine-258 and Serine-259 of Norepinephrine Transporter.

Altered amine transporter function, phosphorylation, and association with interacting proteins are evident in animals with a history of psychostimulant exposure. Our previous studies have shown that the Thr258/Ser259 motif in the norepinephrine transporter (NET) is involved in amphetamine (AMPH)-mediated NET regulation and behavior. However, the neurobiological consequences of in vivo Thr258/Ser259-dependent NET regulation in an intact animal model are unclear. Therefore, we generated a viable construct-valid NET-Thr258Ala/Ser259Ala (NET-T258A/S259A) mouse model using CRISPR/Cas9 technology by replacing Thr258/Ser259 motif with Ala258/Ala259 motif. NET-T258A/S259A mice have a birth rate consistent with Mendelian inheritance ratios. Both male and female homozygous NET-T258A/S259A mice are viable, display normal growth and general health, and exhibit normal body weight (sex-dependent) and total activity in the open field similar to their wild-type (WT) littermates. NET-T258A/S259A mice showed reduced NET function in the prefrontal cortex (PFC) compared to WT mice while NET function in the nucleus accumbens (NAc) remained unchanged. Compared to respective WT counterparts, NET-T258A/S259A males but not females showed significantly reduced locomotor activation in response to acute AMPH administration and significantly reduced AMPH-induced conditioned place preference (CPP). When tested in the males only, acute AMPH administration inhibited NET function and surface expression in the WT NAc but not in the NET-T258A/S259A NAc while AMPH administration inhibited DAT function and surface expression in the NAc of both WT and NET-T258A/S259A mice. Collectively, our findings reveal that the mice carrying the T258A/S259A mutation in NET gene display brain region-specific differences in NET functional expression and blunted response to AMPH.

Dissecting the Balance Between Metabolic and Oncogenic Functions of Astrocyte-Elevated Gene-1/Metadherin.

Obesity is an enormous global health problem, and obesity-induced nonalcoholic steatohepatitis (NASH) is contributing to a rising incidence and mortality for hepatocellular carcinoma (HCC). Increase in de novo lipogenesis and decrease in fatty acid ß-oxidation (FAO) underlie hepatic lipid accumulation in NASH. Astrocyte-elevated gene-1/metadherin (AEG-1) overexpression contributes to both NASH and HCC. AEG-1 harbors an LXXLL motif through which it blocks activation of peroxisome proliferator activated receptor α (PPARα), a key regulator of FAO. To better understand the role of LXXLL motif in mediating AEG-1 function, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, we generated a mouse model (AEG-1-L24K/L25H) in which the LXXLL motif in AEG-1 was mutated to LXXKH. We observed increased activation of PPARα in AEG-1-L24K/L25H livers providing partial protection from high-fat diet-induced steatosis. Interestingly, even with equal gene dosage levels, compared with AEG-1-wild-type livers, AEG-1-L24K/L25H livers exhibited increase in levels of lipogenic enzymes, mitogenic activity and inflammation, which are attributes observed when AEG-1 is overexpressed. These findings indicate that while LXXLL motif favors steatotic activity of AEG-1, it keeps in check inflammatory and oncogenic functions, thus maintaining a homeostasis in AEG-1 function. AEG-1 is being increasingly appreciated as a viable target for ameliorating NASH and NASH-HCC, and as such, in-depth understanding of the functions and molecular attributes of this molecule is essential. Conclusion: The present study unravels the unique role of the LXXLL motif in mediating the balance between the metabolic and oncogenic functions of AEG-1.

Novelty-induced hyperactivity and suppressed cocaine induced locomotor activation in mice lacking threonine 53 phosphorylation of dopamine transporter.

Dopamine (DA) transporter (DAT) is dynamically regulated by several protein kinases and the Thr53 phosphorylation of DAT (pT53-DAT) is documented in heterologous cell models and in rat brain. However, the role of endogenous pT53-DAT in living animals has never been addressed. Here we generated and studied the pT53-lacking DAT mouse model (DAT-Ala53) by CRISPR/Cas9 technology. DAT-Ala53 mice showed normal growth, body weight, body temperature, grip strength, and sucrose preference while pT53-DAT was completely absent. However, DAT-Ala53 mice showed hyperlocomotion, pronounced vertical exploratory behavior, and stereotypy in a novel environment compared to wild-type littermates (WT). DAT-Ala53 mice displayed unaltered levels of monoamines, glutamate, and GABA in the striatum compared to WT. There were also no significant differences between DAT-Ala53 mice and WT in tyrosine hydroxylase (TH) and phospho-TH levels, or in total and surface DAT levels, or in DA-transport kinetic parameters Vmax and Km. Immunohistochemical and colocalization analyses of TH and DAT in caudate-putamen and nucleus accumbens revealed no significant differences between DAT-Ala53 and WT mice. Interestingly, cocaine's potency to inhibit striatal DA transport and cocaine-induced locomotor activation were significantly reduced in the DAT-Ala53 mice. Also, ERK1/2 inhibitors completely failed to inhibit striatal DA uptake in DAT-Ala53 mice. Collectively, our findings reveal that the mice lacking pT53-DAT display novelty-induced hyperactive phenotype despite having normal transporter protein expression, DA-transport kinetics and DA-linked markers. The results also reveal that the lack of endogenous pT53-DAT renders DAT resistant to ERK1/2 inhibition and also less susceptible to cocaine inhibition and cocaine-evoked locomotor stimulation.

The oncogenicity of tumor-derived mutant p53 is enhanced by the recruitment of PLK3.

p53 mutations with single amino acid changes in cancer often lead to dominant oncogenic changes. Here, we have developed a mouse model of gain-of-function (GOF) p53-driven lung cancer utilizing conditionally active LSL p53-R172H and LSL K-Ras-G12D knock-in alleles that can be activated by Cre in lung club cells. Mutation of the p53 transactivation domain (TAD) (p53-L25Q/W26S/R172H) eliminating significant transactivation activity resulted in loss of tumorigenicity, demonstrating that transactivation mediated by or dependent on TAD is required for oncogenicity by GOF p53. GOF p53 TAD mutations significantly reduce phosphorylation of nearby p53 serine 20 (S20), which is a target for PLK3 phosphorylation. Knocking out PLK3 attenuated S20 phosphorylation along with transactivation and oncogenicity by GOF p53, indicating that GOF p53 exploits PLK3 to trigger its transactivation capability and exert oncogenic functions. Our data show a mechanistic involvement of PLK3 in mutant p53 pathway of oncogenesis.

In vitro complementation of Tdp1 deficiency indicates a stabilized enzyme-DNA adduct from tyrosyl but not glycolate lesions as a consequence of the SCAN1 mutation.

A homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1) has been implicated in hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), an autosomal recessive neurodegenerative disease. However, it is uncertain how the H493R mutation elicits the specific pathologies of SCAN1. To address this question, and to further elucidate the role of TDP1 in repair of DNA end modifications and general physiology, we generated a Tdp1 knockout mouse and carried out detailed behavioral analyses as well as characterization of repair deficiencies in extracts of embryo fibroblasts from these animals. While Tdp1(-/-) mice appear phenotypically normal, extracts from Tdp1(-/-) fibroblasts exhibited deficiencies in processing 3'-phosphotyrosyl single-strand breaks and 3'-phosphoglycolate double-strand breaks (DSBs), but not 3'-phosphoglycolate single-strand breaks. Supplementing Tdp1(-/-) extracts with H493R TDP1 partially restored processing of 3'-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on phosphoglycolate (PG) termini on 3' overhangs of double-strand breaks; these remained completely unprocessed. Altogether, these results suggest that for 3'-phosphoglycolate overhang lesions, the SCAN1 mutation confers loss of function, while for 3'-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate.