Publications by authors named "Francesca De Giorgi"

34 Publications

New mechanistic insights to PLOD1-mediated human vascular disease.

Transl Res 2021 Aug 13. Epub 2021 Aug 13.

The Dorothy Davis Heart and Lung Research Institute and the Frick Center for Heart Failure and Arrhythmia, The Ohio State University, Columbus, Ohio; The Ohio State University College of Medicine and Wexner Medical Center, Division of Cardiovascular Medicine, Department of Internal Medicine, Columbus, Ohio. Electronic address:

Heritable thoracic aortic disease and familial thoracic aortic aneurysm/dissection are important causes of human morbidity/mortality, most without identifiable genetic cause. In a family with familial thoracic aortic aneurysm/dissection, we identified a missense p. (Ser178Arg) variant in PLOD1 segregating with disease, and evaluated PLOD1 enzymatic activity, collagen characteristics and in human aortic vascular smooth muscle cells, studied the effect on function. Comparison with homologous PLOD3 enzyme indicated that the pathogenic variant may affect the N-terminal glycosyltransferase domain, suggesting unprecedented PLOD1 activity. In vitro assays demonstrated that wild-type PLOD1 is capable of processing UDP-glycan donor substrates, and that the variant affects the folding stability of the glycosyltransferase domain and associated enzymatic functions. The PLOD1 substrate lysine was elevated in the proband, however the enzymatic product hydroxylysine and total collagen content was not different, albeit despite collagen fibril narrowing and preservation of collagen turnover. In VSMCs overexpressing wild-type PLOD1, there was upregulation in procollagen gene expression (secretory function) which was attenuated in the variant, consistent with loss-of-function. In comparison, si-PLOD1 cells demonstrated hypercontractility and upregulation of contractile markers, providing evidence for phenotypic switching. Together, the findings suggest that the PLOD1 product is preserved, however newly identified glucosyltransferase activity of PLOD1 appears to be affected by folding stability of the variant, and is associated with compensatory vascular smooth muscle cells phenotypic switching to support collagen production, albeit with less robust fibril girth. Future studies should focus on the impact of PLOD1 folding/variant stability on the tertiary structure of collagen and ECM interactions.
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http://dx.doi.org/10.1016/j.trsl.2021.08.002DOI Listing
August 2021

Neuro-telehealth for fragile patients in a tertiary referral neurological institute during the COVID-19 pandemic in Milan, Lombardy.

Neurol Sci 2021 Jul 30;42(7):2637-2644. Epub 2021 Apr 30.

Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy.

Background: Lombardy was severely hit by the COVID-19 pandemic since February 2020 and the Health System underwent rapid reorganization. Outpatient clinics were stopped for non-urgent patients: it became a priority to manage hundreds of fragile neurological patients who suddenly had less reference points. In Italy, before the pandemic, Televisits were neither recognized nor priced.

Methods: At the Fondazione IRCCS Istituto Neurologico C. Besta, we reorganized outpatient clinics to deliver Neuro-telemedicine services, including Televisits and Teleneurorehabilitation, since March 2020. A dedicated Working Group prepared the procedure, tested the system, and designed satisfaction questionnaires for adults and children.

Results: After a pilot phase, we prepared a procedure for Telemedicine outpatient clinics which was approved by hospital directions. It included prescription, booking, consenting, privacy and data protection, secure connection with patients (Teams Microsoft 365), electronic report preparation and delivery, reporting, and accountability of the services. During the March-September 2020 period, we delivered 3167 Telemedicine services, including 1618 Televisits, to 1694 patients (972 adults, 722 children) with a wide range of chronic neurological disorders. We successfully administered different clinical assessment and scales. Satisfaction among patients and caregivers was very high.

Conclusions: During the dramatic emergency, we were able to take care of more than 1600 patients by organizing Neuro-telehealth in a few weeks, lessening the impact of the pandemic on fragile patients with chronic neurological disorders; this strategy is now stably embedded in our care pathways. In Italy, Telehealth is at present recognized and priced and is becoming a stable pillar of the health system.
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http://dx.doi.org/10.1007/s10072-021-05252-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086222PMC
July 2021

Collagen hydroxylysine glycosylation: non-conventional substrates for atypical glycosyltransferase enzymes.

Biochem Soc Trans 2021 04;49(2):855-866

The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology, University of Pavia, via Ferrata 9/A, 27100 Pavia, Italy.

Collagen is a major constituent of the extracellular matrix (ECM) that confers fundamental mechanical properties to tissues. To allow proper folding in triple-helices and organization in quaternary super-structures, collagen molecules require essential post-translational modifications (PTMs), including hydroxylation of proline and lysine residues, and subsequent attachment of glycan moieties (galactose and glucose) to specific hydroxylysine residues on procollagen alpha chains. The resulting galactosyl-hydroxylysine (Gal-Hyl) and less abundant glucosyl-galactosyl-hydroxylysine (Glc-Gal-Hyl) are amongst the simplest glycosylation patterns found in nature and are essential for collagen and ECM homeostasis. These collagen PTMs depend on the activity of specialized glycosyltransferase enzymes. Although their biochemical reactions have been widely studied, several key biological questions about the possible functions of these essential PTMs are still missing. In addition, the lack of three-dimensional structures of collagen glycosyltransferase enzymes hinders our understanding of the catalytic mechanisms producing this modification, as well as the impact of genetic mutations causing severe connective tissue pathologies. In this mini-review, we summarize the current knowledge on the biochemical features of the enzymes involved in the production of collagen glycosylations and the current state-of-the-art methods for the identification and characterization of this important PTM.
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http://dx.doi.org/10.1042/BST20200767DOI Listing
April 2021

Overexpression of α-Synuclein by Oligodendrocytes in Transgenic Mice Does Not Recapitulate the Fibrillar Aggregation Seen in Multiple System Atrophy.

Cells 2020 10 29;9(11). Epub 2020 Oct 29.

CNRS, Institut des Maladies Neurodégénératives, UMR 5293, 33076 Bordeaux, France.

The synucleinopathy underlying multiple system atrophy (MSA) is characterized by the presence of abundant amyloid inclusions containing fibrillar α-synuclein (α-syn) aggregates in the brains of the patients and is associated with an extensive neurodegeneration. In contrast to Parkinson's disease (PD) where the pathological α-syn aggregates are almost exclusively neuronal, the α-syn inclusions in MSA are principally observed in oligodendrocytes (OLs) where they form glial cytoplasmic inclusions (GCIs). This is intriguing because differentiated OLs express low levels of α-syn, yet pathogenic amyloid α-syn seeds require significant amounts of α-syn monomers to feed their fibrillar growth and to eventually cause the buildup of cytopathological inclusions. One of the transgenic mouse models of this disease is based on the targeted overexpression of human α-syn in OLs using the PLP promoter. In these mice, the histopathological images showing a rapid emergence of S129-phosphorylated α-syn inside OLs are considered as equivalent to GCIs. Instead, we report here that they correspond to the accumulation of phosphorylated α-syn monomers/oligomers and not to the appearance of the distinctive fibrillar α-syn aggregates that are present in the brains of MSA or PD patients. In spite of a propensity to co-sediment with myelin sheath contaminants, the phosphorylated forms found in the brains of the transgenic animals are soluble (>80%). In clear contrast, the phosphorylated species present in the brains of MSA and PD patients are insoluble fibrils (>95%). Using primary cultures of OLs from PLP-αSyn mice we observed a variable association of S129-phosphorylated α-syn with the cytoplasmic compartment, the nucleus and with membrane domains suggesting that OLs functionally accommodate the phospho-α-syn deriving from experimental overexpression. Yet and while not taking place spontaneously, fibrillization can be seeded in these primary cultures by challenging the OLs with α-syn preformed fibrils (PFFs). This indicates that a targeted overexpression of α-syn does not model GCIs in mice but that it can provide a basis for seeding aggregation using PFFs. This approach could help establishing a link between α-syn aggregation and the development of a clinical phenotype in these transgenic animals.
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http://dx.doi.org/10.3390/cells9112371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693764PMC
October 2020

Novel self-replicating α-synuclein polymorphs that escape ThT monitoring can spontaneously emerge and acutely spread in neurons.

Sci Adv 2020 Oct 2;6(40). Epub 2020 Oct 2.

CNRS, Institut des Maladies Neurodégénératives, UMR 5293, Bordeaux, France.

The conformational strain diversity characterizing α-synuclein (α-syn) amyloid fibrils is thought to determine the different clinical presentations of neurodegenerative diseases underpinned by a synucleinopathy. Experimentally, various α-syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe thioflavin T (ThT). We report that, concurrent with classical ThT-positive products, fibrillization in saline also gives rise to polymorphs invisible to ThT (τ). The generation of τ fibril polymorphs is stochastic and can skew the apparent fibrillization kinetics revealed by ThT. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. They present a yet undescribed atomic organization and show an exacerbated propensity toward self-replication in cortical neurons, and in living mice, their injection into the substantia nigra pars compacta triggers a synucleinopathy that spreads toward the dorsal striatum, the nucleus accumbens, and the insular cortex.
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http://dx.doi.org/10.1126/sciadv.abc4364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852382PMC
October 2020

Obesity treatment within the Italian national healthcare system tertiary care centers: what can we learn?

Eat Weight Disord 2021 Apr 25;26(3):771-778. Epub 2020 May 25.

Department of Experimental Medicine, Section of Medical Pathophysiology, Food Science and Endocrinology, Sapienza University of Rome, Rome, Italy.

Introduction: The prevalence of obesity is soaring all over the world, and Italy is reaching the same pace. Similar to other countries, the Italian healthcare system counts on a three-tier model for obesity care, and each region has freedom in the implementation of guidelines. No national record is currently available to monitor the actual situation throughout the country.

Purpose: To provide a report of the current status on the availability of specialized public obesity care services in Italy.

Methods: Regional prevalence of obesity was extrapolated from publicly available data. Data on facilities for the management of obesity were retrieved from records provided by national scientific societies. Whenever possible, data was verified through online research and direct contact.

Results: We report a north-south and east-west gradient regarding the presence of obesity focused facilities, with an inverse correlation with the regional prevalence of obesity (R = 0.25, p = 0.03). Medical-oriented centers appear homogeneous in the multidisciplinary approach, the presence of a bariatric surgery division, the availability of support materials and groups, with no major difference on follow-up frequency. Surgery-oriented centers have a more capillary territorial distribution than the medically oriented, but not enough data was retrieved to provide a thorough description of their characteristics.

Conclusion: Obtaining a clear picture of the situation and providing consistent care across the country is a challenging task due to the decentralized organization of regions. We provide a first sketch, reporting that the model is applied unevenly, and we suggest feasible actions to improve the situation in our country and elsewhere.

Level Of Evidence: Level V, narrative review.
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http://dx.doi.org/10.1007/s40519-020-00936-1DOI Listing
April 2021

Reduced oligodendrocyte exosome secretion in multiple system atrophy involves SNARE dysfunction.

Brain 2020 06;143(6):1780-1797

Department of Pathology, University of Washington School of Medicine, 325 9th Ave, HMC Box 359635, Seattle, WA 98104, USA.

Transportation of key proteins via extracellular vesicles has been recently implicated in various neurodegenerative disorders, including Parkinson's disease, as a new mechanism of disease spreading and a new source of biomarkers. Extracellular vesicles likely to be derived from the brain can be isolated from peripheral blood and have been reported to contain higher levels of α-synuclein (α-syn) in Parkinson's disease patients. However, very little is known about extracellular vesicles in multiple system atrophy, a disease that, like Parkinson's disease, involves pathological α-syn aggregation, though the process is centred around oligodendrocytes in multiple system atrophy. In this study, a novel immunocapture technology was developed to isolate blood CNPase-positive, oligodendrocyte-derived enriched microvesicles (OEMVs), followed by fluorescent nanoparticle tracking analysis and assessment of α-syn levels contained within the OEMVs. The results demonstrated that the concentrations of OEMVs were significantly lower in multiple system atrophy patients, compared to Parkinson's disease patients and healthy control subjects. It is also noted that the population of OEMVs involved was mainly in the size range closer to that of exosomes, and that the average α-syn concentrations (per vesicle) contained in these OEMVs were not significantly different among the three groups. The phenomenon of reduced OEMVs was again observed in a transgenic mouse model of multiple system atrophy and in primary oligodendrocyte cultures, and the mechanism involved was likely related, at least in part, to an α-syn-mediated interference in the interaction between syntaxin 4 and VAMP2, leading to the dysfunction of the SNARE complex. These results suggest that reduced OEMVs could be an important mechanism related to pathological α-syn aggregation in oligodendrocytes, and the OEMVs found in peripheral blood could be further explored for their potential as multiple system atrophy biomarkers.
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http://dx.doi.org/10.1093/brain/awaa110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296853PMC
June 2020

Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis.

Nat Catal 2019 Nov;2(12):1115-1123

Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.

D-Apiose is a -branched pentose sugar important for plant cell wall development. Its biosynthesis as UDP-D-apiose involves decarboxylation of the UDP-D-glucuronic acid precursor coupled to pyranosyl-to-furanosyl sugar ring contraction. This unusual multistep reaction is catalyzed within a single active site by UDP-D-apiose/UDP-D-xylose synthase (UAXS). Here, we decipher the UAXS catalytic mechanism based on crystal structures of the enzyme from , molecular dynamics simulations expanded by QM/MM calculations, and mutational-mechanistic analyses. Our studies show how UAXS uniquely integrates a classical catalytic cycle of oxidation and reduction by a tightly bound nicotinamide coenzyme with retro-aldol/aldol chemistry for the sugar ring contraction. They further demonstrate that decarboxylation occurs only after the sugar ring opening and identify the thiol group of Cys100 in steering the sugar skeleton rearrangement by proton transfer to and from the C3'. The mechanistic features of UAXS highlight the evolutionary expansion of the basic catalytic apparatus of short-chain dehydrogenases/reductases for functional versatility in sugar biosynthesis.
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http://dx.doi.org/10.1038/s41929-019-0382-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914363PMC
November 2019

TDP-43 extracted from frontotemporal lobar degeneration subject brains displays distinct aggregate assemblies and neurotoxic effects reflecting disease progression rates.

Nat Neurosci 2019 01 17;22(1):65-77. Epub 2018 Dec 17.

Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of people with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTLD cases into at least four subtypes, which are correlated with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for biochemical isolation of pathological TDP-43. By combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43 that become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from brain forms stable assemblies of distinct densities and morphologies that are associated with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies showed differential neurotoxicity and seeding that were correlated with disease duration of FTLD subjects. Our data are consistent with the notion that disease heterogeneity could originate from alternate pathological TDP-43 conformations, which are reminiscent of prion strains.
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http://dx.doi.org/10.1038/s41593-018-0294-yDOI Listing
January 2019

Author Correction: Molecular architecture of the multifunctional collagen lysyl hydroxylase and glycosyltransferase LH3.

Nat Commun 2018 09 20;9(1):3912. Epub 2018 Sep 20.

The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9/A, 27100, Pavia, Italy.

The previously published version of this Article contained an error in Figure 3. In panel a, the residues His667 and Asp669 were incorrectly labelled as His627 and Asp629. The error has been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-06481-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6147860PMC
September 2018

Overweight and obese patients with nickel allergy have a worse metabolic profile compared to weight matched non-allergic individuals.

PLoS One 2018 28;13(8):e0202683. Epub 2018 Aug 28.

Department of Experimental Medicine, Section of Medical Pathophysiology, Food Science and Endocrinology, Sapienza University of Rome, Rome, Italy.

Background: A lack of balance between energy intake and expenditure due to overeating or reduced physical activity does not seem to explain entirely the obesity epidemic we are facing, and further factors are therefore being evaluated. Nickel (Ni) is a ubiquitous heavy metal implied in several health conditions. Regarding this, the European Food Safety Authority has recently released an alert on the possible deleterious effects of dietary Ni on human health given the current levels of Ni dietary intake in some countries. Pre-clinical studies have also suggested its role as an endocrine disruptor and have linked its exposure to energy metabolism and glucose homeostasis dysregulation. Ni allergy is common in the general population, but preliminary data suggest it being even more widespread among overweight patients.

Objectives: The aim of this study has been to evaluate the presence of Ni allergy and its association with the metabolic and endocrine profile in overweight and obese individuals.

Methods: We have evaluated 1128 consecutive overweight and obese outpatients. 784 were suspected of being allergic to Ni and 666 were assessed for it. Presence of Ni allergy and correlation with body mass index (BMI), body composition, metabolic parameters and hormonal levels were evaluated.

Results: We report that Ni allergy is more frequent in presence of weight excess and is associated with worse metabolic parameters and impaired Growth Hormone secretion.

Conclusions: We confirm that Ni allergy is more common in obese patients, and we report for the first time its association with worse metabolic parameters and impaired function of the GH-IGF1 axis in human subjects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0202683PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112671PMC
February 2019

Molecular architecture of the multifunctional collagen lysyl hydroxylase and glycosyltransferase LH3.

Nat Commun 2018 08 8;9(1):3163. Epub 2018 Aug 8.

The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9/A, 27100, Pavia, Italy.

Lysyl hydroxylases catalyze hydroxylation of collagen lysines, and sustain essential roles in extracellular matrix (ECM) maturation and remodeling. Malfunctions in these enzymes cause severe connective tissue disorders. Human lysyl hydroxylase 3 (LH3/PLOD3) bears multiple enzymatic activities, as it catalyzes collagen lysine hydroxylation and also their subsequent glycosylation. Our understanding of LH3 functions is currently hampered by lack of molecular structure information. Here, we present high resolution crystal structures of full-length human LH3 in complex with cofactors and donor substrates. The elongated homodimeric LH3 architecture shows two distinct catalytic sites at the N- and C-terminal boundaries of each monomer, separated by an accessory domain. The glycosyltransferase domain displays distinguishing features compared to other known glycosyltransferases. Known disease-related mutations map in close proximity to the catalytic sites. Collectively, our results provide a structural framework characterizing the multiple functions of LH3, and the molecular mechanisms of collagen-related diseases involving human lysyl hydroxylases.
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http://dx.doi.org/10.1038/s41467-018-05631-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082870PMC
August 2018

In vitro α-synuclein neurotoxicity and spreading among neurons and astrocytes using Lewy body extracts from Parkinson disease brains.

Neurobiol Dis 2017 Jul 11;103:101-112. Epub 2017 Apr 11.

Univ. de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France; CNRS, Institut des Maladies Neurodégénératives, UMR 5293, F-33000 Bordeaux, France; Motac Neuroscience, UK-M15 6WE Manchester, UK. Electronic address:

Synucleinopathies are a group of diseases characterized by the presence of intracellular protein aggregates containing α-synuclein (α-syn). While α-syn aggregates have been shown to induce multimodal cellular dysfunctions, uptake and transport mechanisms remain unclear. Using high-content imaging on cortical neurons and astrocytes, we here define the kinetics of neuronal and astrocytic abnormalities induced by human-derived α-syn aggregates grounding the use of such system to identify and test putative therapeutic compounds. We then aimed at characterizing uptake and transport mechanisms using primary cultures of cortical neurons and astrocytes either in single well or in microfluidic chambers allowing connection between cells and cell-types. We report that astrocytes take up α-syn-aggregates far more efficiently than neurons through an endocytic event. We also highlight that active α-syn transport occurs between cells and any cell-types. Of special interest regarding the disease, we also show that uptake and spreading of α-syn from astrocytes to neurons can lead to neuronal death. Altogether, we here show that patients-derived α-synuclein aggregates, which are taken up by neurons and astrocytes, induce a differential endogenous response in the two cell types including a peculiar astrocytic toxic gain-of-function that leads to neuronal death.
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http://dx.doi.org/10.1016/j.nbd.2017.04.011DOI Listing
July 2017

Identification and super-resolution imaging of ligand-activated receptor dimers in live cells.

Sci Rep 2013 ;3:2387

LP2N, University of Bordeaux, UMR 5298, F-33405 Talence, France.

Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.
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http://dx.doi.org/10.1038/srep02387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737505PMC
February 2014

HIPK1 drives p53 activation to limit colorectal cancer cell growth.

Cell Cycle 2013 Jun 15;12(12):1879-91. Epub 2013 May 15.

INSERM U916, Institut Bergonié, Université de Bordeaux, Bordeaux, France.

HIPK1 (homeodomain interacting protein kinase 1) is a serine/threonine kinase that belongs to the CMGC superfamily. Emerging data point to the role of HIPK1 in cancer, but it is still not clear whether it acts as a tumor suppressor or promoter. Here we identified HIPK1 as a kinase that is significantly overexpressed in colorectal cancer (CRC) and whose expression is stage-dependent. Being abundantly expressed at the onset of the disease, the HIPK1 level gradually decreased as tumor stage progressed. To further uncover how this factor regulates tumorigenesis and establish whether it constitutes an early factor necessary for neoplastic transformation or for cellular defense, we studied the effect of its overexpression in vitro by investigating various cancer-related signaling cascades. We found that HIPK1 mostly regulates the p53 signaling pathway both in HCT116 and HeLa cells. By phosphorylating p53 on its serine-15, HIPK1 favored its transactivation potential, which led to a rise in p21 protein level and a decline in cell proliferation. Assuming that HIPK1 could impede CRC growth by turning on the p53/p21 pathway, we then checked p21 mRNA levels in patients. Interestingly, p21 transcripts were only increased in a subset of patients expressing high levels of HIPK1. Unlike the rest of the cohort, the majority of these patients hosted a native p53 protein, meaning that such a pro-survival pathway (HIPK1+ > p53 > p21) is active in patients, and that HIPK1 acts rather as a tumor suppressor.
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http://dx.doi.org/10.4161/cc.24927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3735702PMC
June 2013

Synthesis and evaluation of apoptosis induction of thienopyrimidine compounds on KRAS and BRAF mutated colorectal cancer cell lines.

Bioorg Med Chem 2012 Nov 24;20(22):6724-31. Epub 2012 Sep 24.

Service Pharmacie, Hôpital Saint-André, CHU de Bordeaux, 1 rue Jean Burguet, Bordeaux, France.

Monoclonal antibodies (MoAb) and tyrosine kinase inhibitors (TKI) targeting the EGFR (Epidermal Growth Factor Receptor) pathways are currently used in colorectal cancer treatment. Despite the improvement of median overall survival, resistance is observed notably due to KRAS and BRAF gene mutations. We synthesized four series of thienopyrimidines whose scaffold is structurally close to TKI used in clinical practice. We evaluated apoptosis induced by these compounds using flow cytometry on KRAS and BRAF mutated cell lines. Our results confirm that the mutated cell lines (HCT116 and HT29) are more resistant to apoptosis than the non-mutated cell line (Hela). Interestingly, among the 13 compounds tested, three of them (5b, 6b and 6d) and gefitinib exhibited a noteworthy pro-apoptotic effect, especially on mutated cell lines with an IC(50) value between 70 and 110μM. These three compounds seem particularly attractive for the development of novel treatments for colorectal cancer patients harboring EGFR pathway mutations.
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http://dx.doi.org/10.1016/j.bmc.2012.09.034DOI Listing
November 2012

Non-antioxidant properties of α-tocopherol reduce the anticancer activity of several protein kinase inhibitors in vitro.

PLoS One 2012 8;7(5):e36811. Epub 2012 May 8.

INSERM U916, VINCO, Institut Bergonié, Université Victor Segalen Bordeaux 2, Bordeaux, France.

The antioxidant properties of α-tocopherol have been proposed to play a beneficial chemopreventive role against cancer. However, emerging data also indicate that it may exert contrasting effects on the efficacy of chemotherapeutic treatments when given as dietary supplement, being in that case harmful for patients. This dual role of α-tocopherol and, in particular, its effects on the efficacy of anticancer drugs remains poorly documented. For this purpose, we studied here, using high throughput flow cytometry, the direct impact of α-tocopherol on apoptosis and cell cycle arrest induced by different cytotoxic agents on various models of cancer cell lines in vitro. Our results indicate that physiologically relevant concentrations of α-tocopherol strongly compromise the cytotoxic and cytostatic action of various protein kinase inhibitors (KI), while other classes of chemotherapeutic agents or apoptosis inducers are unaffected by this vitamin. Interestingly, these anti-chemotherapeutic effects of α-tocopherol appear to be unrelated to its antioxidant properties since a variety of other antioxidants were completely neutral toward KI-induced cell cycle arrest and cell death. In conclusion, our data suggest that dietary α-tocopherol could limit KI effects on tumour cells, and, by extent, that this could result in a reduction of the clinical efficacy of anti-cancer treatments based on KI molecules.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0036811PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348137PMC
September 2012

The polyphenolic ellagitannin vescalagin acts as a preferential catalytic inhibitor of the α isoform of human DNA topoisomerase II.

Mol Pharmacol 2012 Jul 23;82(1):134-41. Epub 2012 Apr 23.

Institut National de la Santé et de la Recherche Médicale U916 and Université de Bordeaux, Institut Bergonié, Bordeaux, France.

Polyphenolic ellagitannins are natural compounds that are often associated with the therapeutic activity of plant extracts used in traditional medicine. They display cancer-preventing activity in animal models by a mechanism that remains unclear. Potential targets have been proposed, including DNA topoisomerases II (Top2). Top2α and Top2β, the two isoforms of the human Top2, play a crucial role in the regulation of replication, transcription, and chromosome segregation. They are the target of anticancer agents used in the clinic such as anthracyclines (e.g., doxorubicin) or the epipodophyllotoxin etoposide. It was recently shown that the antitumor activity of etoposide was due primarily to the inhibition of Top2α, whereas inhibition of Top2β was responsible for the development of secondary malignancies, pointing to the need for more selective Top2α inhibitors. Here, we show that the polyphenolic ellagitannin vescalagin preferentially inhibits the decatenation activity of Top2α in vitro, by a redox-independent mechanism. In CEM cells, we also show that transient small interfering RNA-mediated down-regulation of Top2α but not of Top2β conferred a resistance to vescalagin, indicating that the α isoform is a preferential target. We further confirmed that Top2α inhibition was due to a catalytic inhibition of the enzyme because it did not induce DNA double-strand breaks in CEM-treated cells but prevented the formation of Top2α- rather than Top2β-DNA covalent complexes induced by etoposide. To our knowledge, vescalagin is the first example of a catalytic inhibitor for which cytotoxicity is due, at least in part, to the preferential inhibition of Top2α.
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http://dx.doi.org/10.1124/mol.111.077537DOI Listing
July 2012

Tissue microarray cytometry reveals positive impact of homeodomain interacting protein kinase 2 in colon cancer survival irrespective of p53 function.

Am J Pathol 2011 May;178(5):1986-98

INSERM U916, Institut Bergonié, Université Victor Segalen Bordeaux 2, Bordeaux, France.

The human p53 gene is a tumor suppressor mutated in half of colon cancers. Although p53 function appears important for proliferation arrest and apoptosis induced by cancer therapeutics, the prognostic significance of p53 mutations remains elusive. This suggests that p53 function is modulated at a posttranslational level and that dysfunctions affecting its modulators can have a prognostic impact. Among p53 modulators, homeodomain interacting protein kinase (HIPK) 2 emerges as a candidate "switch" governing p53 transition from a cytostatic to a proapoptotic function. Thus, we investigated the possible prognostic role of HIPK2 on a retrospective series of 80 colon cancer cases by setting up a multiplexed cytometric approach capable of exploring correlative protein expression at the single tumor cell level on TMA. Crossing the data with quantitative PCR and p53 gene sequencing and p53 functional assays, we observed the following: despite a strong impact on p21 transcription, the presence of disabling p53 mutations has no prognostic value, and the increased expression of the HIPK2 protein in tumor cells compared with paired normal tissue cells has a strong impact on survival. Unexpectedly, HIPK2 effect does not appear to be mediated by p53 function because it is also observed in p53-disabling mutated backgrounds. Thus, our results point to a prominent and p53-independent role of HIPK2 in colon cancer survival.
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http://dx.doi.org/10.1016/j.ajpath.2011.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3081143PMC
May 2011

Synthesis and study of antiproliferative activity of novel thienopyrimidines on glioblastoma cells.

Eur J Med Chem 2010 Jun 18;45(6):2473-9. Epub 2010 Feb 18.

Service Pharmacie, Hôpital Saint-André, CHU de Bordeaux, INSERM U916, Institut Bergonié, Université Victor Segalen Bordeaux 2, 229 cours de l'Argonne, 33 076 Bordeaux, France.

The receptor tyrosine kinases (for example EGFR, PDGFR, VEGFR) are a transmembrane protein family which plays a crucial role in tumor growth, survival, metastasis dissemination and angiogenesis. During the past 10 years, many tyrosine kinase inhibitors (TKIs) have been approved for cancer treatment (imatinib, gefitinib, erlotinib, sunitinib, sorafenib). These compounds generally possess a pyrrolo- or pyrimido- pyrimidine scaffold or approaching molecular structure. We synthesized 10 thienopyrimidine compounds (including 5 newly synthesized) whose scaffold is very similar to the agents cited above. The cytotoxicity of these agents was evaluated using a MTT assay and a flow cytometry technique on glioblastoma cell lines. Two compounds showed a similar cytotoxicity to the standard anti-EGFR gefitinib (IC50: gefitinib=51.9 microM, 6b=61.8 microM, 6c=41.2 microM), suggesting a blockade of the EGFR pathway by binding to the TK receptor.
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http://dx.doi.org/10.1016/j.ejmech.2010.02.032DOI Listing
June 2010

Recombinant differential anchorage probes that tower over the spatial dimension of intracellular signals for high content screening and analysis.

Anal Chem 2009 Dec;81(23):9590-8

INSERM U916, Institut Bergonié, Université Victor Segalen, 229 cours de l'Argonne, 33076 Bordeaux, France.

Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into "all-or-none" fluorescence intensity changes (differential anchorage probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound characterization, and drug profiling.
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http://dx.doi.org/10.1021/ac9015227DOI Listing
December 2009

Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis.

Cell Res 2009 Dec 11;19(12):1363-76. Epub 2009 Aug 11.

INSERM U916, Université Bordeaux 2, Institut Bergonié, 33076 Bordeaux, France.

Voltage-dependent anion channel (VDAC)1 is the main channel of the mitochondrial outer membrane (MOM) and it has been proposed to be part of the permeability transition pore (PTP), a putative multiprotein complex candidate agent of the mitochondrial permeability transition (MPT). Working at the single live cell level, we found that overexpression of VDAC1 triggers MPT at the mitochondrial inner membrane (MIM). Conversely, silencing VDAC1 expression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-MIM crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and Bcl-X(L), indicative of PTP operation. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome c release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDAC1-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.
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http://dx.doi.org/10.1038/cr.2009.98DOI Listing
December 2009

Anticancer drugs exert differential apoptotic and cytotoxic effects on glioblastoma primary cultures with various EGFR and bcl-2 profiles.

J Exp Ther Oncol 2009 ;8(2):105-16

Laboratoire de Biologie Cellulaire, Travail, Environnement EA 3672, Université Victor Segalen, Bordeaux 2, 146 rue Léo Saignat, 33 076 Bordeaux, France.

The aim of this study was to determine the apoptotic and cytotoxic effects induced on glioblastoma cells by various anticancer agents that possess different mechanisms of action (alkylating drugs, anti-EGFR (Epidermal Growth Factor receptor), proteasome inhibitor). Primary cell cultures were obtained from patients who underwent surgery for their glioblastoma. The cytotoxic effects of drugs were determined by MTT (dimethylthiazolyl diphenyl tetrazolium bromide) assay and apoptosis was evaluated by measuring mitochondrial potential by flow cytometry. Biological markers (EGFR, bcl-2) were studied by a immunoblotting technique to find out predictive markers of response. We found a large interindividual sensitivity, thus confirming the interest of the primary cultures. New proteasome inhibitor bortezomib had considerable cytotoxic and apoptotic potential in glioblastoma, even at very low concentrations. Moreover, the characterization of patients' cells for EGFR and bcl-2 status could constitute an interest, with the evaluation of other markers, in the study of expected chemotherapy response.
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March 2010

An intracellular wave of cytochrome c propagates and precedes Bax redistribution during apoptosis.

J Cell Sci 2008 Nov 7;121(Pt 21):3515-23. Epub 2008 Oct 7.

INSERM U916, Université Bordeaux 2, Institut Bergonié, 229 cours de l'Argonne, 33000 Bordeaux, France.

Bax is considered to be pivotal in inducing cytochrome c release (CCR) from mitochondria during apoptosis. Indeed, Bax redistributes to the mitochondrial outer membrane (MOM) upon activation and forms homo-multimers that are capable of permeabilizing the MOM. Our attempts to image this sequence of events in single live cells resulted in unexpected observations. Bax redistribution exhibited two distinct components: an early minor redistribution that was silent in terms of homo-multimerization and a major late redistribution that was synchronous with the formation of Bax multimers, but that proceeded belatedly, i.e. only after caspase 3/7 (C3/7) had already been activated. Intriguingly, neither of these two components of redistribution correlated with CCR, which turned out to be spatially organized, propagating as a traveling wave at constant velocity. Strikingly, propagation of the CCR wave (1) preceded signs of in situ Bax conformational activation; (2) appeared to be independent of autocatalytic loops involving a positive feedback of either C3/7, Ca(2+) mobilization or mitochondrial permeability transition; and (3) was triggered by diffuse stimulation with the synthetic Bak activator BH3I-1 but then proceeded independently of Bak activation. Thus, the CCR wave not only questions the exact role of Bax redistribution in cell death, but also indicates the existence of yet unidentified positive-feedback loops that ensure a spatiotemporal control of apoptosis at the subcellular scale.
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http://dx.doi.org/10.1242/jcs.029587DOI Listing
November 2008

Cytotoxic and apoptotic effects of bortezomib and gefitinib compared to alkylating agents on human glioblastoma cells.

J Exp Ther Oncol 2008 ;7(2):99-111

Laboratoire de Biologie Cellulaire, Laboratoire Santé, Travail, Environnement EA 3672, Université Victor Segalen, 146 rue Léo Saignat, 33 076 Bordeaux, France.

Glioblastoma is a malignant astrocytic tumor with a median survival of about 12 months for which new therapeutic strategies are required. We therefore examined the cytotoxicity of anticancer drugs with different mechanisms of action on two human glioblastoma cell lines expressing various levels of EGFR (epidermal growth factor receptor). Apoptosis induced by these anticancer agents was evaluated by flow cytometry. The cytotoxicity of alkylating drugs followed a dose-effect curve and cytotoxicity index values were lower with carboplatin than with BCNU and temozolomide. Anti-EGFR gefitinib (10 microM) cytotoxicity on DBTRG.05-MG expressing high levels of EGFR was significantly higher than on U87-MG expressing low levels of EGFR. Carboplatin and temozolomide cytotoxicity was potentiated with the addition of gefitinib on DBTRG.05-MG. Among the anticancer agents tested, the proteasome inhibitor bortezomib was the most cytotoxic with very low IC50 on the two cell lines. Moreover, all anticancer drugs tested induced apoptosis in a concentration-dependent manner. Bortezomib proved to be a more potent inductor of apoptosis than gefitinib and alkylating agents. These results show the efficacy of bortezomib and of the association between conventional chemotherapy and gefitinib on glioblastoma cells and therefore suggest the interest of these molecules in the treatment of glioblastoma.
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January 2009

Spatial relational memory requires hippocampal adult neurogenesis.

PLoS One 2008 Apr 9;3(4):e1959. Epub 2008 Apr 9.

INSERM U862, Institut F. Magendie, Bordeaux, France.

The dentate gyrus of the hippocampus is one of the few regions of the mammalian brain where new neurons are generated throughout adulthood. This adult neurogenesis has been proposed as a novel mechanism that mediates spatial memory. However, data showing a causal relationship between neurogenesis and spatial memory are controversial. Here, we developed an inducible transgenic strategy allowing specific ablation of adult-born hippocampal neurons. This resulted in an impairment of spatial relational memory, which supports a capacity for flexible, inferential memory expression. In contrast, less complex forms of spatial knowledge were unaltered. These findings demonstrate that adult-born neurons are necessary for complex forms of hippocampus-mediated learning.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001959PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2396793PMC
April 2008

High content analysis of gamma-secretase activity reveals variable dominance of presenilin mutations linked to familial Alzheimer's disease.

Biochim Biophys Acta 2008 Aug 3;1783(8):1551-60. Epub 2008 Apr 3.

Department Biomedical Sciences and CNR Institute of Neuroscience, University of Padua, Via G. Colombo, 3, 35121 Padua, Italy.

gamma-Secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP), Notch and other cellular substrates and is considered a prime pharmacological target in the development of therapeutics for Alzheimer's disease (AD). We describe here an efficient, new, simple, sensitive and rapid assay to quantify gamma-secretase activity in living cells by flow cytometry using two membrane-bound fluorescent probes, APP-GFP or C99-GFP, as substrates for gamma-secretase. The principle of the assay is based on the fact that the soluble intracellular domain of GFP-tagged APP (AICD-GFP) is released from the membrane into the cytosol following gamma-secretase cleavage. Using this feature, enzymatic activity of gamma-secretase could be deduced from the extent of the membrane retention of the probe observed after plasma membrane permeabilization and washout of the cleaved fraction. By applying two well-known gamma-secretase inhibitors (DAPT and L-685,458), we validated our assay showing that the positional GFP-based probes for gamma-secretase activity behave properly when expressed in different cell lines, providing the basis for the further development of a high-throughput and high content screening for AD targeted drug discovery. Moreover, by co-expression of different familial AD-linked mutated forms of presenilin--the key component of the gamma-secretase complex--in cells devoid of any endogenous gamma-secretase, our method allowed us to evaluate in situ the contribution of different presenilin variants to the modulation of the enzyme.
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http://dx.doi.org/10.1016/j.bbamcr.2008.03.012DOI Listing
August 2008

Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway.

Mol Cancer Res 2008 Apr;6(4):604-13

Centre National de la Recherche Scientifique-Unite Mixte de Recherche 5164, 33076 Bordeaux, France.

Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is known to protect tumor cells from apoptosis and more specifically from the Fas-mediated apoptotic signal. The antitumoral agent edelfosine sensitizes leukemic cells to death by inducing the redistribution of the apoptotic receptor Fas into plasma membrane subdomains called lipid rafts. Herein, we show that inhibition of the PI3K signal by edelfosine triggers a Fas-mediated apoptotic signal independently of the Fas/FasL interaction. Furthermore, similarly to edelfosine, blockade of the PI3K activity, using specific inhibitors LY294002 and wortmannin, leads to the clustering of Fas whose supramolecular complex is colocalized within the lipid rafts. These findings indicate that the antitumoral agent edelfosine down-modulates the PI3K signal to sensitize tumor cells to death through the redistribution of Fas into large platform of membrane rafts.
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http://dx.doi.org/10.1158/1541-7786.MCR-07-0331DOI Listing
April 2008

Label-free optical imaging of mitochondria in live cells.

Opt Express 2007 Oct;15(21):14184-93

The far-field optical imaging of mitochondria of live cells without the use of any label is demonstrated. It uses a highly sensitive photothermal method and has a resolution comparable to confocal fluorescence setups. The morphological states of mitochondria were followed under different physiological treatments, and the role of cytochrome c was ruled out as the main origin of the photothermal signals. This label free optical method provides a high contrast imaging of live mitochondria and should find many applications in biosciences.
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http://dx.doi.org/10.1364/oe.15.014184DOI Listing
October 2007
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