Lactate dehydrogenases promote glioblastoma growth and invasion via a metabolic symbiosis.

Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy.

Hepatocyte proteomes reveal the role of protein disulfide isomerase 4 in alpha 1-antitrypsin deficiency.

BACKGROUND & AIMS: A single point mutation in the Z-variant of alpha 1-antitrypsin (Z-AAT) alone can lead to both a protein folding and trafficking defect, preventing its exit from the endoplasmic reticulum (ER), and the formation of aggregates that are retained as inclusions within the ER of hepatocytes. These defects result in a systemic AAT deficiency (AATD) that causes lung disease, whereas the ER-retained aggregates can induce severe liver injury in patients with ZZ-AATD. Unfortunately, therapeutic approaches are still limited and liver transplantation represents the only curative treatment option. To overcome this limitation, a better understanding of the molecular basis of ER aggregate formation could provide new strategies for therapeutic intervention. METHODS: Our functional and omics approaches here based on human hepatocytes from patients with ZZ-AATD have enabled the identification and characterisation of the role of the protein disulfide isomerase (PDI) A4/ERP72 in features of AATD-mediated liver disease. RESULTS: We report that 4 members of the PDI family (PDIA4, PDIA3, P4HB, and TXNDC5) are specifically upregulated in ZZ-AATD liver samples from adult patients. Furthermore, we show that only PDIA4 knockdown or alteration of its activity by cysteamine treatment can promote Z-AAT secretion and lead to a marked decrease in Z aggregates. Finally, detailed analysis of the Z-AAT interactome shows that PDIA4 silencing provides a more conducive environment for folding of the Z mutant, accompanied by reduction of Z-AAT-mediated oxidative stress, a feature of AATD-mediated liver disease. CONCLUSIONS: PDIA4 is involved in AATD-mediated liver disease and thus represents a therapeutic target for inhibition by drugs such as cysteamine. PDI inhibition therefore represents a potential therapeutic approach for treatment of AATD. LAY SUMMARY: Protein disulfide isomerase (PDI) family members, and particularly PDIA4, are upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. PDI inhibition upon cysteamine treatment leads to improvements in features of AATD and hence represents a therapeutic approach for treatment of AATD-mediated liver disease.

The Autophagy Pathway: A Critical Route in the Disposal of Alpha 1-Antitrypsin Aggregates That Holds Many Mysteries.

The maintenance of proteome homeostasis, or proteostasis, is crucial for preserving cellular functions and for cellular adaptation to environmental challenges and changes in physiological conditions. The capacity of cells to maintain proteostasis requires precise control and coordination of protein synthesis, folding, conformational maintenance, and clearance. Thus, protein degradation by the ubiquitin-proteasome system (UPS) or the autophagy-lysosomal system plays an essential role in cellular functions. However, failure of the UPS or the autophagic process can lead to the development of various diseases (aging-associated diseases, cancer), thus both these pathways have become attractive targets in the treatment of protein conformational diseases, such as alpha 1-antitrypsin deficiency (AATD). The Z alpha 1-antitrypsin (Z-AAT) misfolded variant of the serine protease alpha 1-antitrypsin (AAT) is caused by a structural change that predisposes it to protein aggregation and dramatic accumulation in the form of inclusion bodies within liver hepatocytes. This can lead to clinically significant liver disease requiring liver transplantation in childhood or adulthood. Treatment of mice with autophagy enhancers was found to reduce hepatic Z-AAT aggregate levels and protect them from AATD hepatotoxicity. To date, liver transplantation is the only curative therapeutic option for patients with AATD-mediated liver disease. Therefore, the development and discovery of new therapeutic approaches to delay or overcome disease progression is a top priority. Herein, we review AATD-mediated liver disease and the overall process of autophagy. We highlight the role of this system in the regulation of Z-variant degradation and its implication in AATD-medicated liver disease, including some open questions that remain challenges in the field and require further elucidation. Finally, we discuss how manipulation of autophagy could provide multiple routes of therapeutic benefit in AATD-mediated liver disease.

Deciphering the complex role of thrombospondin-1 in glioblastoma development.

We undertook a systematic study focused on the matricellular protein Thrombospondin-1 (THBS1) to uncover molecular mechanisms underlying the role of THBS1 in glioblastoma (GBM) development. THBS1 was found to be increased with glioma grades. Mechanistically, we show that the TGFß canonical pathway transcriptionally regulates THBS1, through SMAD3 binding to the THBS1 gene promoter. THBS1 silencing inhibits tumour cell invasion and growth, alone and in combination with anti-angiogenic therapy. Specific inhibition of the THBS1/CD47 interaction using an antagonist peptide decreases cell invasion. This is confirmed by CD47 knock-down experiments. RNA sequencing of patient-derived xenograft tissue from laser capture micro-dissected peripheral and central tumour areas demonstrates that THBS1 is one of the gene with the highest connectivity at the tumour borders. All in all, these data show that TGFß1 induces THBS1 expression via Smad3 which contributes to the invasive behaviour during GBM expansion. Furthermore, tumour cell-bound CD47 is implicated in this process.

VEGF modulates synaptic activity in the developing spinal cord.

Although it has been documented that the nervous and the vascular systems share numerous analogies and are closely intermingled during development and pathological processes, interactions between the two systems are still poorly described. In this study, we investigated whether vascular endothelial growth factor (VEGF), which is a key regulator of vascular development, also modulates neuronal developmental processes. We report that VEGF enhances the gamma-aminobutyric acid (GABA)/glycinergic but not glutamatergic synaptic activity in embryonic spinal motoneurons (MNs), without affecting MNs excitability. In response to VEGF, the frequency of these synaptic events but not their amplitude was increased. Blocking endogenous VEGF led to an opposite effect by decreasing frequency of synaptic events. We found that this effect occurred specifically at early developmental stages (E13.5 and E15.5) and vanished at the prenatal stage E17.5. Furthermore, VEGF was able to increase vesicular inhibitory amino acid transporter density at the MN membrane. Inhibition of single VEGF receptors did not modify electrophysiological parameters indicating receptor combinations or an alternative pathway. Altogether, our findings identify VEGF as a modulator of the neuronal activity during synapse formation and highlight a new ontogenic role for this angiogenic factor in the nervous system.

Genetic analysis of the biosynthesis of 2-methoxy-3-isobutylpyrazine, a major grape-derived aroma compound impacting wine quality.

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.

Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit.

Changes in carbohydrate metabolism during grape berry development play a central role in shaping the final composition of the fruit. The present work aimed to identify metabolic switches during grape development and to provide insights into the timing of developmental regulation of carbohydrate metabolism. Metabolites from central carbon metabolism were measured using high-pressure anion-exchange chromatography coupled to tandem mass spectrometry and enzymatic assays during the development of grape berries from either field-grown vines or fruiting cuttings grown in the greenhouse. Principal component analysis readily discriminated the various stages of berry development, with similar trajectories for field-grown and greenhouse samples. This showed that each stage of fruit development had a characteristic metabolic profile and provided compelling evidence that the fruit-bearing cuttings are a useful model system to investigate regulation of central carbon metabolism in grape berry. The metabolites measured showed tight coordination within their respective pathways, clustering into sugars and sugar-phosphate metabolism, glycolysis, and the tricarboxylic acid cycle. In addition, there was a pronounced shift in metabolism around veraison, characterized by rapidly increasing sugar levels and decreasing organic acids. In contrast, glycolytic intermediates and sugar phosphates declined before veraison but remained fairly stable post-veraison. In summary, these detailed and comprehensive metabolite analyses revealed the timing of important switches in primary carbohydrate metabolism, which could be related to transcriptional and developmental changes within the berry to achieve an integrated understanding of grape berry development. The results are discussed in a meta-analysis comparing metabolic changes in climacteric versus non-climacteric fleshy fruits.

Over-expression of VvWRKY1 in grapevines induces expression of jasmonic acid pathway-related genes and confers higher tolerance to the downy mildew.

Most WRKY transcription factors activate expression of defence genes in a salicylic acid- and/or jasmonic acid-dependent signalling pathway. We previously identified a WRKY gene, VvWRKY1, which is able to enhance tolerance to fungal pathogens when it is overexpressed in tobacco. The present work analyzes the effects of VvWRKY1 overexpression in grapevine. Microarray analysis showed that genes encoding defence-related proteins were up-regulated in the leaves of transgenic 35S::VvWRKY1 grapevines. Quantitative RT-PCR analysis confirmed that three genes putatively involved in jasmonic acid signalling pathway were overexpressed in the transgenic grapes. The ability of VvWRKY1 to trans-activate the promoters of these genes was demonstrated by transient expression in grape protoplasts. The resistance to the causal agent of downy mildew, Plasmopara viticola, was enhanced in the transgenic plants. These results show that VvWRKY1 can increase resistance of grapevine against the downy mildew through transcriptional reprogramming leading to activation of the jasmonic acid signalling pathway.

The experimental renal cell carcinoma model in the chick embryo.

The clear cell subtype of renal carcinoma (CCRCC) is highly vascularized and despite a slow progression rate, it is potentially a highly aggressive tumor. Although a doubling of median progression-free survival in CCRCC patients treated by targeted therapies has been observed, the fact that tumors escape after anti-VEGF treatment suggests alternative pathways. The chick chorioallantoic membrane (CAM) is a well-established model, which allows in vivo studies of tumor angiogenesis and the testing of anti-angiogenic molecules. However, only a few data exist on CCRCC grafted onto CAM. We aimed to validate herein the CAM as a suitable model for studying the development of CCRCC and the interactions with the surrounding stroma. Our study uses both CCRCC cell lines and fresh tumor samples after surgical resection. We demonstrate that in both cases CCRCC can be grafted onto the CAM, to survive and to induce an angiogenic process. We further provide insights into the transcriptional regulation of the model by performing a differential analysis of tumor-derived and stroma-derived transcripts.

A transcriptomic study of grapevine (Vitis vinifera cv. Cabernet-Sauvignon) interaction with the vascular ascomycete fungus Eutypa lata.

Eutypa dieback is a vascular disease that may severely affect vineyards throughout the world. In the present work, microarrays were made in order (i) to improve our knowledge of grapevine (Vitis vinifera cv. Cabernet-Sauvignon) responses to Eutypa lata, the causal agent of Eutypa dieback; and (ii) to identify genes that may prevent symptom development. Qiagen/Operon grapevine microarrays comprising 14,500 probes were used to compare, under three experimental conditions (in vitro, in the greenhouse, and in the vineyard), foliar material of infected symptomatic plants (S(+)R(+)), infected asymptomatic plants (S(-)R(+)), and healthy plants (S(-)R(-)). These plants were characterized by symptom notation after natural (vineyard) or experimental (in vitro and greenhouse) infection, re-isolation of the fungus located in the lignified parts, and the formal identification of E. lata mycelium by PCR. Semi-quantitative real-time PCR experiments were run to confirm the expression of some genes of interest in response to E. lata. Their expression profiles were also studied in response to other grapevine pathogens (Erysiphe necator, Plasmopara viticola, and Botrytis cinerea). (i) Five functional categories of genes, that is those involved in metabolism, defence reactions, interaction with the environment, transport, and transcription, were up-regulated in S(+)R(+) plants compared with S(-)R(-) plants. These genes, which cannot prevent infection and symptom development, are not specific since they were also up-regulated after infection by powdery mildew, downy mildew, and black rot. (ii) Most of the genes that may prevent symptom development are associated with the light phase of photosynthesis. This finding is discussed in the context of previous data on the mode of action of eutypin and the polypeptide fraction secreted by Eutypa.

The basic helix-loop-helix transcription factor MYC1 is involved in the regulation of the flavonoid biosynthesis pathway in grapevine.

Previous results indicated that in grapevine (Vitis vinifera), regulation of the flavonoid pathway genes by MYB transcription factors depends on their interaction with basic helix-loop-helix proteins (bHLHs). The present study describes the first functional characterization of a bHLH factor from grapevine named VvMYC1. This transcription factor is phylogenetically related to Arabidopsis bHLH proteins, which participate in the control of flavonoid biosynthesis and epidermal cell fate. Transient promoter and yeast two-hybrid assays demonstrated that VvMYC1 physically interacts with MYB5a, MYB5b, MYBA1/A2, and MYBPA1 to induce promoters of flavonoid pathway genes involved in anthocyanin and/or proanthocyanidin (PA) synthesis. Additionally, transient promoter assays revealed that VvMYC1 is involved in feedback regulation of its own expression. Transcript levels of VvMYC1 during berry development correlate with the synthesis of anthocyanins and PAs in skins and seeds of berries, suggesting that VvMYC1 is involved in the regulation of anthocyanins and PA synthesis in these organs. Likewise, transient expression of VvMYC1 and VvMYBA1 induces anthocyanin synthesis in grapevine suspension cells. These results suggest that VvMYC1 is part of the transcriptional cascade controlling anthocyanin and PA biosynthesis in grapevine.

The grapevine transcription factor WRKY2 influences the lignin pathway and xylem development in tobacco.

Previous work has shown that transgenic tobacco plants constitutively over-expressing the Vitis vinifera L. transcription factor VvWRKY2 exhibit reduced susceptibility to necrotrophic fungal pathogens, suggesting that this transcription factor plays a role in grapevine response to phytopathogens. The work presented here characterizes the modifications in cell wall structure observed in the stems and petioles of these transgenic plants. Histochemical stainings of stem and petiole cross-sections using phloroglucinol or Maüle reagents revealed a delay in xylem formation, particularly in the petioles, and differences in lignin composition. Evaluation of lignin quantity and quality showed a decrease in the syringyl/guaiacyl ratio in both stem and petioles. Expression analysis using RT-PCR and potato microarrays showed that tobacco plants over-expressing VvWRKY2 exhibited altered expression of genes involved in lignin biosynthesis pathway and cell wall formation. The ability of VvWRKY2 to activate the promoter of the VvC4H gene, which is involved in the lignin biosynthetic pathway, was confirmed by transient transcriptional activation assays in tobacco protoplasts. Moreover, in situ hybridization revealed that VvWRKY2 is specifically expressed in cells undergoing lignification in young grapevine stems. Together, these results confirm that VvWRKY2 plays a role in regulating lignification in grapevine, possibly in response to biotic or abiotic stresses.

[Identification and characterization of "rd22" dehydration responsive gene in grapevine (Vitis vinifera L.)]. / Identification et caractérisation d'un gène de réponse à la déshydratation "rd22" chez la vigne (Vitis vinifera L.).

To identify and isolate genes related to abiotic stresses (salinity and drought) tolerance in grapevine, a candidate gene approach was developed and allowed isolating a full-length cDNA of rd22 gene from the Cabernet Sauvignon variety. The latter, named Vvrd22, is a dehydration-responsive gene that is usually induced by the application of exogenous ABA. Details of the physicochemical parameters and structural properties (molecular mass, secondary structure, conserved domains and motives, putative post-translational modification sites...) of the encoded protein have also been elucidated. The expression study of Vvrd22 was carried out at the berry growth stages and at the level of plant organs and tissues as well as under both drought and salt stresses. The results showed that Vvrd22 is constitutively expressed at a low level in all analyzed tissues. Moreover, salt stress induced Vvrd22 expression, particularly for the tolerant variety (Razegui), contrary to the sensitive one (Syrah), which did not display any expression variation during the stress, which means that Vvrd22 is involved in salt stress response and that its expression level depends on regulatory mechanisms that are efficient only for the tolerant variety. On the other hand, under drought stress, Vvrd22 is induced in an identical manner for both tolerant and sensitive varieties. In addition, stress signal molecules such as ABA (lonely applied or in combination with sucrose) induced Vvrd22 expression, even at a low level. A minimal knowledge about the role and the functionality of this gene is necessary and constitutes a prerequisite condition before starting and including Vvrd22 in any program of improvement of grapevine's abiotic stress tolerance.

Identification of grapevine aquaporins and expression analysis in developing berries.

Aquaporins are membrane water channels that play critical roles in controlling the water content of cells and tissues. In this work, nine full-length cDNAs encoding putative aquaporins were isolated from grape berry cDNA libraries. A phylogenetic analysis conducted with 28 aquaporin genes identified in the grapevine genome and previously characterized aquaporins from Arabidopsis indicates that three cDNAs encode putative tonoplast aquaporins (TIPs) whereas six cDNAs belong to the plasma membrane aquaporin subfamily (PIPs). Specific probes designed on the 3' untranslated regions of each cDNA were used for the preparation of cDNA macroarray filters and in situ hybridization experiments. Macroarray data indicate that expression levels of most TIP and PIP genes depend on grape berry developmental stages and point out to a global decrease of aquaporin gene expression during berry ripening. In young berries, high expression of aquaporin genes was preferentially observed in dividing and elongating cells and in cells involved in water and solutes transport. Taken together, the data provided in this paper indicate that aquaporins are implicated in various physiological aspects of grape berry development.

Isogene specific oligo arrays reveal multifaceted changes in gene expression during grape berry (Vitis vinifera L.) development.

The transition from a green, hard, and acidic pericarp to a sweet, soft, coloured, and sugar-rich ripe fruit occurs in many unrelated fruit species. High throughput identification of differentially expressed genes in grape berry has been achieved by the use of 50-mers oligoarrays bearing a set of 3,200 Unigenes from Vitis vinifera to compare berry transcriptome at nine developmental stages. Analysis of transcript profiles revealed that most activations were triggered simultaneously with softening, occurring within only 24 h for an individual berry, just before any change in colouration or water, sugar, and acid content can be detected. Although most dramatically induced genes belong to unknown functional categories, numerous changes occur in the expression of isogenes involved in primary and secondary metabolism during ripening. Focusing on isogenes potentially significant in development regulation (hormonal control of transcription factor) revealed a possible role for several hormones (cytokinin, gibberellin, or jasmonic acid). Transcription factor analysis revealed the induction of RAP2 and WRKY genes at véraison, suggesting increasing biotic and abiotic stress conditions during ripening. This observation was strengthened by an increased expression of multiple transcripts involved in sugar metabolism and also described as induced in other plant organs during stress conditions. This approach permitted the identification of new isogenes as possible control points: a glutathione S-transferase exhibits the same expression profile as anthocyanin accumulation and a new putative sugar transporter is induced in parallel with sugar import.

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.