Role of the deubiquitinating enzyme UCH-L1 in mitochondrial function

International audienc

HERC5 downregulation in non-small cell lung cancer is associated with altered energy metabolism and metastasis

Abstract Background Metastasis is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC) patients. We previously showed that low HERC5 expression predicts early tumor dissemination and a dismal prognosis in NSCLC patients. Here, we performed functional studies to unravel the mechanism underlying the “metastasis-suppressor” effect of HERC5, with a focus on mitochondrial metabolism pathways. Methods We assessed cell proliferation, colony formation potential, anchorage-independent growth, migration, and wound healing in NSCLC cell line models with HERC5 overexpression (OE) or knockout (KO). To study early tumor cell dissemination, we used these cell line models in zebrafish experiments and performed intracardial injections in nude mice. Mass spectrometry (MS) was used to analyze protein changes in whole-cell extracts. Furthermore, electron microscopy (EM) imaging, cellular respiration, glycolytic activity, and lactate production were used to investigate the relationships with mitochondrial energy metabolism pathways. Results Using different in vitro NSCLC cell line models, we showed that NSCLC cells with low HERC5 expression had increased malignant and invasive properties. Furthermore, two different in vivo models in zebrafish and a xenograft mouse model showed increased dissemination and metastasis formation (in particular in the brain). Functional enrichment clustering of MS data revealed an increase in mitochondrial proteins in vitro when HERC5 levels were high. Loss of HERC5 leads to an increased Warburg effect, leading to improved adaptation and survival under prolonged inhibition of oxidative phosphorylation. Conclusions Taken together, these results indicate that low HERC5 expression increases the metastatic potential of NSCLC in vitro and in vivo. Furthermore, HERC5-induced proteomic changes influence mitochondrial pathways, ultimately leading to alterations in energy metabolism and demonstrating its role as a new potential metastasis suppressor gene

Neuronal Growth and Behavioral Alterations in Mice Deficient for the Psychiatric Disease-Associated Negr1 Gene

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of β-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders

Neuronal Growth and Behavioral Alterations in Mice Deficient for the Psychiatric Disease-Associated Negr1 Gene

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of β-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders

<i>Negr1</i>-I87N is a loss-of-function mutation.

<p><b>A–C.</b> NSC-34 cells expressing EGFP alone (control) or together with <i>Negr1</i>-WT or <i>Negr1</i>-I87N mutants at 0 min (t1) and following 60 min (t2) of cell aggregation. <b>D.</b> Histogram showing cell aggregation expressed as the ratio of 0 min (t1) and 60 min (t2) time points. EGFP+empty vector (control): 1.06±0.05; NEGR1-WT+EGFP: 2.11±0.2; EGFP+NEGR1-I87N: 0.93±0.2. Data represent means ± sd calculated from three independent experiments. <b>E–G.</b> Confocal images showing hypothalamic neurons immunostained for the neuronal marker βIII-tubulin (red) cultured together with transfected NSC-34 cells (green). NSC34 cells were transfected with (E) pEGFP together with an empty pcDNA-vector (control), (F) pEGFP+pcDNA3-NEGR1-WT and (G) EGFP+NEGR1-I87N. <b>H.</b> Mean neurite lengths of hypothalamic neurons relative to control (set to 100%). Error bars represent SEM from three independent experiments (∼400 neurites per condition). Two-tailed Student's test; *<i>P</i><0.05; **<i>P</i><0.01; *** <i>P</i><0.001. Scale: 100 µm (A), 20 µm (E).</p

<i>Negr1</i>-I87N mutants display altered body mass and composition.

<p><b>A–D</b>. Body mass (A), Lean mass (B), fat mass (C) of wild type (female, n = 28; male, n = 22), heterozygous (female, n = 52; male, n = 51) and homozygous (female, n = 28; male, n = 24) <i>Negr1</i>-I87N mice fed on high-fat diet measured across 18 weeks. Data shown are mean +/− SEM within each sex-genotype group at each time point.</p

<i>Negr1</i>–I87N mice have unchanged energy expenditure relative to lean mass and reduced physical activity.

<p><b>A,B,F,G</b>. Energy expenditure (A, F) normalised to lean mass (B, G) over a 22-hr period during light and dark phases in 16-week old wild-type (female, n = 23; male, n = 21) and homozygotes (female, n = 20; male, n = 22) females (A–B) and males (F–G). <b>C–E, H–J</b>. Physical activity as measured by the average number of beambreak counts in various different dimensions (C, H), average speed (D, I) and total distance travelled (E, J) in 16-week old wild-type (female, n = 23; male, n = 21) and homozygotes (female, n = 20; male, n = 22) females (C–E) and males (H–J). All data are presented as mean ± SEM. Two-tailed Student's <i>t</i>-test was carried out between groups, *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. Bonferroni correction was applied to adjust for multiple measurements (A,B,F,G).</p

Repeated measures ANOVA—results for <i>Negr1</i>-I87N experiment.

v<p>‘Heterozygote – WT’ denotes the mean difference between the heterozygote and WT genotypic classes (i.e. in the notation developed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041537#s4" target="_blank">Materials and Methods</a>).</p>vi<p>‘Homozygote – WT’ denotes the mean difference between the heterozygote and WT genotypic classes (i.e. in the notation developed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041537#s4" target="_blank">Materials and Methods</a>).</p>vii<p>Standard error.</p>viii<p>Nominal p-values for the test of the null hypothesis of no genotypic effect (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041537#s4" target="_blank">Materials and Methods</a>).</p

Ablation of NEGR1 causes reduction in food intake.

<p><b>A,B.</b> Food intake over 24 hrs measured at 16-weeks in <i>Negr1</i>-I87N female (A) (WT, n = 23; hom, n = 20) and male (B) (WT, n = 21; hom, n = 22). All data are presented as mean ± SEM. Student's <i>t</i>-test (2-tailed) was carried out between groups, *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

Ablation of NEGR1 causes reduction in fatty droplets.

<p><b>A</b>,<b>B</b>. Number of triglyceride droplets classified into ranges of size in diameter (µm) in hepatocytes of female <i>Negr1</i>-I87N mice (n = 3 mice per genotype). All data are presented as mean ± SEM. Student's <i>t</i>-test (2-tailed) was carried out between groups, *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p